Through ultramicroscopic analysis, we hardly observed the phenotype of autophagy in the 1000TTO group, and the lipid droplet morphology was intact compared with a small number of autophagolysosomes existing in lipid droplets in the CT group. In sharp contrast, the 100TTO group had a large number of autophagosomes in lipid droplets accompanied by the breakdown of lipid droplets. These results showed that the 100TTO group enhanced autophagy, possibly decomposing lipids through autophagy, while the 1000TTO group inhibited autophagy and caused lipid deposition in the hepatopancreas. Degraded lipids by enhanced mitophagy and lipophagy were commonly found in other studies [58,59], but there was seldom research on essential oils affecting lipid metabolism by regulating autophagy.

Autophagy is a process by which a membrane derived from the organelle or lysosome surrounds the material for degradation to form an autophagosome [30]. At the molecular level, the mRNA results showed that 100 mg/kg TTO activates autophagy by initiating autophagic membrane phagocytosis and regulating autophagy-related gene expression. The 100 mg/kg TTO enhanced autophagosome membrane initiation and the expansion of mRNA expression, and 1000 mg/kg TTO inhibited the generation and expansion of autophagosome membrane mRNA expression. Meanwhile, the WB analysis showed that 100TTO intervention up-regulated the expression of two autophagy marker proteins (*Beclin1* and *Lc3*) and increased the expression level of autophagy initiation protein *Ulk1* and the degrading cell protein *atg7* [60]. In addition, we found that 100TTO increased the gene expression of the upstream activator proteins *Ulk1* and *Ampk*, and the 1000TTO group increased the expression of the upstream autophagy-inhibiting protein *mTOR* [61]. Moreover, a higher expression of *p62* was found in the 1000TTO group, indicating that the autophagy was suppressed, as the high intracellular expression of *p62* occurs when autophagy is blocked [62]. The above results indicated that the 100TTO treatment activated the autophagy upstream essential proteins to enhance the formation of the autophagy membrane. In contrast, the 1000TTO group down-regulated the expression of autophagyrelated proteins to inhibit autophagy.

Studies have shown that oxidative stress could impair cell metabolic function to obtain abnormal liver lipid deposition, and the abnormal lipid deposition is accompanied by severe oxidative stress [8,63]. The study found that TTO levels have antioxidant properties [64]. In addition, consuming moderate levels of TTO may enhance host antioxidant capacity as a cofactor for antioxidant enzymes to increase defense against oxidative stress [23,65]. Based on these premises, we evaluated the oxidative stress status of each group. Our study found that the 100 mg/kg TTO diet enhances antioxidant enzyme activity and reduces peroxide production in the hepatopancreas compared to the control. On the contrary, 1000TTO did not improve the activity of antioxidant enzymes while it induced a significant increase in the levels of MDA and PCO, indicating that oxidative damage occurs as MDA and PCO are both marker products of oxidative damage [66]. The *Keap1*-*Nrf2* pathway has a central role in the defense against oxidative stress in vertebrates [67,68]. Studies in crustaceans have also found that oxidative stress activates the *Keap1*-*Nrf2*-mediated antioxidant pathway [69]. The activation of this pathway is mainly caused by ROS-stimulated *Nrf2* uncoupling from *Keap1* into the nucleus and binding to AREs to achieve antioxidant purposes. Our study found that the addition of 100TTO activated the *Keap1*-*Nrf2* pathway expression compared to the control group, and 1000TTO significantly increased the level of *Nrf2* expression compared to the 100TTO group. Moreover, 1000TTO enormously increased ROS production and caused mitochondrial damage accompanied by increased lipid peroxidation products (LPO and 4-HNE). This is consistent with previous findings that state that the sharp up-regulation of *Nrf2* always accompanies a vastly improved level of ROS, which may be compensatory stress [70]. Another study has

shown that oxidative stress disrupted the morphology and function of mitochondria [71]. In addition, the *NF-κB* signaling pathway was found to have bidirectional regulation effects on ROS regulation [72]. *Relish* was an *NF-κB* homologous protein in invertebrate animals and was found to have oxidative stress-regulating effects [73,74]. Our results showed that 100TTO significantly increased *Relish* and its downstream factor *Imd*, whereas 1000TTO did not activate *Relish*-*Imd* expression. The above results show that 100TTO improves the antioxidant capacity by activating the *Keap1*-*Nrf2* and *Relish-Imd* signaling pathways, while the 1000TTO group only activates *Keap1*-*Nrf2* but without activating the *Relish*-*Imd* antioxidant pathway, which may also cause oxidation stress finally. Therefore, we speculated that excess TTO levels stimulate ROS production and impair the antioxidant system, thereby inducing oxidative stress that mediates excess TTO-induced mitochondrial dysfunction. Similarly, other studies have shown that high levels of essential oils cause an increase in ROS, and excess ROS induce oxidative stress damage [75,76].

The above results suggest that different TTO levels simultaneously affected oxidative stress status, autophagy levels, and the lipid metabolism process. However, the concrete underlying molecular link among them is unclear. In view of this, Pearson correlation analysis was performed to explore the correlation between the expression levels of the two oxidative stress regulatory pathways *Keap1*-*Nrf2* and *Relish*-*Imd*, lipid metabolism regulatory factors, and autophagy indicators. Through Pearson correlation analysis, two key node factors are determined; one is *Nrf2*, and the other is *Relish*.

Firstly, we found that *Nrf2* and *Pparγ* were significantly positively correlated. Martinez-Lopez et al. [77] pointed out that *Nrf2* translocates to the nucleus and activates many genes via ARE transport following oxidative stress. Oxidative stress promoted the binding of *Nrf2* to the *Pparγ* promoter and increased *Pparγ*-mediated adipogenesis [78]. At the same time, *Nrf2* was also positively correlated with *Fasn* and *Srebp1* in this study; this is consistent with previous conclusions that the activation of *Nrf2* under oxidative stress promotes adipogenesis by activating *Srebp1*, and the increase in *Fasn* expression directly causes the increase in fat synthesis [79,80]. In addition, *Nrf2* was negatively correlated with lipolysis genes *Cpt1*, *Hsl*, and *Ampkα* in this study. Past studies have found that the inhibition of lipolysis promotes adipocyte lipid accumulation [81]. Therefore, it is demonstrated that the high expression of *Nrf2* leads to increased lipid synthesis. In terms of autophagy, *Nrf2* was found to be positively correlated with autophagy-related genes, including autophagy marker *p62* and upstream regulatory genes (*Akt* and *mtor*), and negatively correlated with *Beclin1* and *Lc3*. *Nrf2* hyperactivation leads to the accumulation of *p62* and the inhibition of autophagy [82]. *Nrf2* positively regulates *Akt* expression under oxidative stress [83], and the increase in *Akt* activates the *Akt*-*mtor* pathway expression [84]. The increase in *Akt* expression will induce the dephosphorylation of *Lc3* [85]. The activation of mtor inhibits autophagy and reduces marker *Beclin1* expression [62].

The above results demonstrate that *Nrf2* is an important node between oxidative stress, autophagy, and lipid metabolism, and affects lipid deposition in the hepatopancreas simultaneously through antioxidant and lipid droplet autophagy pathways. This also explains why the 1000TTO group caused liver cell damage and excessive lipid deposition, accompanied by the high expression of *Nrf2* and inhibited autophagy and lipolysis. In addition, the down-regulation of hepatic lipid deposition in the 100TTO group may be regulated by other factors.

In terms of the other key node factor, *Relish*, correlation analysis data found that *Relish* was positively correlated with key lipolytic genes *Atgl* and *Cpt1*. Although few studies have shown that *Relish* enhances lipolysis, one study found that *Relish* was inhibited when the body fat increased in shrimp [35]. In addition, *Relish* was positively correlated with *Ulk1*, *Beclin1*, and *Lc3*, and negatively correlated with *p62*. Research has shown that *Relish* increases the activation of *atg1*(*Ulk1*) and the level of autophagy, and an increase in marker *Lc3* inevitably accompanies the increased expression of *Relish* [86]. When autophagy is blocked, the *p62* protein accumulates in the cytoplasm, so the negative correlation between *Relish* and *p62* in this study means the activation of autophagy [87].

The Ampk-mtor pathway is critical for regulating autophagy [88]. *Imd* was found negatively correlated with *mtor*, and *Relish* was positively correlated with *Ampkα*. Studies have shown that *Imd* inhibits the expression of *mTOR* [89], and mtor acts as an upstream pathway of autophagy; its inhibition will activate autophagy. Moreover, *Ampkα* acts as an essential gene for lipolysis [90]. Therefore, the *Relish*-*Imd* signal may enhance lipolytic ability by regulating *Ampk*-*mtor* to stimulate autophagy activation. *Pparγ* as an essential fat synthesis gene was negatively correlated with *Relish* in this study. A similar study also found that increased expression of *Relish* downregulates lipid synthesis through the regulation of *Pparγ* [91]. Therefore, the 100TTO group may activate autophagy by increasing the expression of *Relish* to regulate the autophagy initiation factor and downregulating *Pparγ* to promote lipolysis.

#### **5. Conclusions**

In conclusion, this study demonstrates that 100 mg/kg TTO enhances antioxidant activity and induces autophagy through the *Relish*-*Imd* pathway to enhance lipid droplet breakdown and maintain lipid homeostasis. However, 1000 mg/kg TTO treatment will overexpress *Nrf2*, thus inducing the transcriptional activation of *Pparγ* and other genes to promote fat synthesis and inhibit autophagy, eventually causing lipid peroxidation and oxidative stress. This putative mechanism that can explain our results is shown in Figure 9. Our study supplies a new perspective focus on *Nrf2*, *Relish*, and the "antioxidantautophagy-lipid metabolism" axis to reveal the regulation mechanisms of lipid metabolism by tea tree oil. Next, we will design the agonists and inhibitors of *Nrf2* and *Relish* and verify the precise mechanism of TTO to regulate the lipid metabolism of crustaceans based on the "antioxidant-autophagy-lipid-metabolism" axis at the tissue or cell level in vitro.

**Figure 9.** The mechanism of 100TTO and 1000TTO regulates lipid metabolism based on antioxidant

factors *Relish* and *Nrf2*-autophagy axis. The blue arrows indicate the regulation effects of 100TTO; the red arrows indicate the regulation effects of 1000TTO. Arrows up indicate significant up-regulation or improvement, and arrows down indicate significant down-regulation or inhibition. Thick arrows indicate extremely significant regulation.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11112260/s1. Table S1: Primers used in the study.

**Author Contributions:** M.L. contributed to the areas of experimental design, sampling, data analysis, and write-up; B.L. and P.X. contributed to the experimental design and manuscript review; C.S., Q.Z., B.L. and P.X. contributed to the feeding and cultivating of experimental prawns, sampling, and statistics, and X.Z. contributed to feed production, the feeding of prawns, and sampling. X.Z. was also responsible for language review and revision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Project of National Key R&D Program of China (2019YFD09 00200), China Agriculture Research System of MOF and MARA (CARS-48), the National Natural Science Foundation of China (32002404), Central Public-interest Scientific Institution Basal Research Fund, and CAFS (2020TD59). The authors would like to express their sincere thanks to the personnel of these teams for their kind assistance.

**Institutional Review Board Statement:** The care and use of animals followed the Animal Research Institute Committee guidelines of Nanjing Agricultural University, China. This study has been approved by the Committee of the Animal Research Institute of Nanjing Agricultural University, China (SYXK (Su) 2019-0136, approved 21 May 2017).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Acknowledgments:** The authors would like to express sincere thanks to the personnel of the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences for their kind assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Article* **High Dietary Histamine Induces Digestive Tract Oxidative Damage in Juvenile Striped Catfish (***Pangasianodon hypophthalmus***)**

**Yu Liu 1,2,3,†, Xinlangji Fu 1,2,3,†, Huajing Huang 1,2,3, Jiongting Fan 1,2,3, Hang Zhou 1,2,3, Junming Deng 1,2,3,4,\* and Beiping Tan 1,2,3,4,\***

**Abstract:** A 56-day feeding trial investigated the effects of dietary histamine on the antioxidant capacity, gastric and intestinal barrier functions, and growth performance of striped catfish (*Pangasianodon hypophthalmus*). Seven isonitrogenous (34.0% crude protein) and isolipidic (10.5% crude lipid) diets were formulated with supplemental 0, 15, 30, 60, 120, 240, and 480 mg/kg of histamine, named H0, H15, H30, H60, H120, H240, and H480 group, respectively. Results showed that the weight gain rate, specific growth rate, relative intestinal length in the H240 and H480 groups, and the condition factors in the H480 group were significantly lower than those in the H0 group. Intestinal total antioxidant capacity, peroxidase, catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase activities in the H480 group were significantly lower than those in the H0 group, whereas intestinal malondialdehyde content exhibited the opposite trend. Intestinal complement 3, complement 4, immunoglobulin M, and Recombinant Mucin 2 in the H480 group were significantly lower than those in the H0 group, in contrast to intestinal lipopolysaccharide content. Intestinal *IL-10* gene expression in the H480 group was significantly lower than that in the H0 group, whereas the *TNF-α*, *IL-1*, *IL-6*, and *IL-8* gene expression exhibited opposite results. Scanning and transmission electron microscopic observation of the gastrointestinal tract revealed severe damage to the gastric mucosa and intestinal epithelium in the H480 group. The abundance of *Treponema* in the histamine groups was significantly higher than that in the H0 group. These results indicated that high dietary histamine decreases intestinal immunity and antioxidant capacity, inducing digestive tract oxidative damage and ultimately decreasing the growth of striped catfish.

**Keywords:** histamine; striped catfish; digestive tract; mucosal barrier; oxidative damage

#### **1. Introduction**

The striped catfish (*Pangasianodon hypophthalmus*) is native to the Mekong and Chao Phraya River basins and is widely farmed in many Asian countries because of its fast growth, strong adaptability, and high disease resistance [1]. In Vietnam, exports of striped catfish already accounted for 23.2% of the national aquatic product exports in 2019 [2]. Due to its delicious meat quality, no intermuscular spines, and low price, striped fish is popular among consumers [3]. As a result, the culture scale of this species is still expanding. It is well known that reducing farming costs is one of the main strategies to improve farming economic efficiency [4]. In modern aquaculture, feed costs usually account for

**Citation:** Liu, Y.; Fu, X.; Huang, H.; Fan, J.; Zhou, H.; Deng, J.; Tan, B. High Dietary Histamine Induces Digestive Tract Oxidative Damage in Juvenile Striped Catfish (*Pangasianodon hypophthalmus*). *Antioxidants* **2022**, *11*, 2276. https:// doi.org/10.3390/antiox11112276

Academic Editors: Bo Liu, Changyou Song and Cunxin Sun

Received: 12 October 2022 Accepted: 15 November 2022 Published: 17 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

more than 50% of total costs, since fish meal is increasingly expensive [5–7]. Currently, fish meal remains an integral part of commercial feed for striped catfish, remaining at 20–60% [8]. In Vietnam, stale fishmeal has been routinely used in catfish farming because of its local availability and it facilitates the control of feed costs [9]. However, fishmeal stored for long periods usually contains high doses of histamine due to the decarboxylation of histidine [10–12]. Moreover, striped catfish are cultivated commercially in subtropical and tropical regions where unfavorable storage conditions (humidity and heat) often lead to feed spoilage and produce large amounts of histamine [13]. Therefore, striped catfish may face dietary challenges with high doses of histamine.

Histamine is a low molecular weight organic nitrogen compound with the chemical formula of C5H9N3 [14], which extensively participated in numerous metabolic activities [15–17]. Excessive intake of histamine may cause human health disorders, poisoning, headaches, and even anaphylaxis [18–20]. Therefore, some regions and countries legislate to limit the histamine content in food; for the European Union, Australia, and South Africa, histamine content should be less than 100 mg/kg [21–23], and for the United States of America, below 50 mg/kg [24]. Recently, the effects of histamine on the growth, metabolism, and health of aquatic animals have attracted widespread attention [12,25–29], with results showing that dietary histamine interferes with normal metabolism, causes inflammatory responses, and reduces antioxidant capacity and growth in aquatic animals. Moreover, the toxic effects of dietary histamine can directly damage the morphology, physiology, and health of the digestive tract of aquatic animals [29–32] and disrupt the normal gut microbiota structure [28]. Thus, striped fish may suffer from the toxic effects of dietary histamine; however, limited information is available about this.

The digestive tract is the main nutrient intake place of fish, and its physiological function and health maintenance are closely related to antioxidant and immune status. Specifically, the antioxidant status usually depends on the activity of antioxidant enzyme systems, including peroxidase, catalase superoxide dismutase, glutathione peroxidase, and glutathione reductase; the immune status is determined by both the innate and acquired immune systems, with the innate immune system being the dominant type in fish [29], which consists of complement and immunoglobulins. To date, no studies have reported the effects of dietary histamine on the intestinal antioxidant and immune system of striped catfish. Therefore, this study evaluated the effects of dietary histamine on the antioxidant capacity, gastric and intestinal barrier functions, and growth performance of striped catfish. Our data will contribute to the healthy farming of striped fish.

#### **2. Materials and Methods**

#### *2.1. Experimental Diets Preparation*

Before making the test diet, we tested the histamine content of 22 commercial feeds for striped catfish (Table 1), and the results showed that the histamine contents ranged from 33.70–177.00 mg/kg. Among them, the histamine content exceeded 80 mg/kg in eight feeds, accounting for 36.36%. Subsequently, the histamine content of the experimental diet was designed based on these data.

**Table 1.** Histamine content of commercial feeds.

**Table 1.** *Cont.*

Seven isonitrogenous (34.0% crude protein) and isolipidic (10.5% crude lipid) diets were formulated with supplemental 0, 15, 30, 60, 120, 240, and 480 mg/kg of histamine, named H0, H15, H30, H60, H120, H240, and H480 groups, respectively. To reduce the histamine content in the basal diet, we used fresh white fish meal (PILENGA 2, histamine ≤ 40 mg/kg, purchased from fishing vessel IMO: 9120310). All ingredients were ground into meals and sifted out with a 60 μm sieve, and then accurately weighed and mixed with a V-mixer (M-256, South China University of Technology, Guangzhou, China). Subsequently, the soybean oil and pure water were supplemented to prepare a dough and then extruded with an extruder (School of Chemical Engineering, South China University of Technology, Guangzhou, China) to produce 2.0 mm diameter pellets. The moist pellets were dried in an air-conditioned room at 25 ◦C (dehumidification mode) and then collected in self-sealing bags and stored at −20 ◦C until use. Diet formulation and proximate composition are shown in Table 2.

**Table 2.** Ingredients and nutritional compositions of the experimental diets.

 Animal and Feeding Trial*

A total of 630 striped catfish juveniles (body weight = 31.38 ± 0.09 g) were randomly assigned to 21 cages with 30 fish per cage (0.7 m × 0.7 m × 1 m). The feeding trial lasted 8 weeks, and the fish were fed twice daily at 7:00 and 17:30 h to apparent satiation during this period. The amount of feed fed to each net box was recorded daily and checked for fish mortality. This trial was conducted in the Freshwater Base of Guangdong Ocean University with the following water parameters: temperature keep at 29–32 ◦C, dissolved oxygen > 4.0 mg/L, ammonia nitrogen < 0.04 mg/L.

#### *2.3. Sample Collection*

At the end of the feeding trial, the fish were starved for 1 day, then weighed and counted. Subsequently, the fish were anesthetized with eugenol solution (1:10,000 dilution, Macklin, Shanghai, China) before starting the sampling. Four fish per cage were individually measured in terms of body weight and length, visceral weight, liver weight, intestinal length, and intestinal weight to calculate condition factors (CF), viscerosomatic index (VSI), hepatosomatic index (HSI), relative intestinal length (RIL) and relative intestinal weight (RIW). Four fish from each cage were selected for dissection, and the hindgut tissues were harvested and placed in an Eppendorf (EP) tube, then stored at −80 ◦C for enzyme activities analysis. Another four fish per cage were selected for dissection, and the hindgut was collected in an RNAlater-added EP tube for intestinal gene quantitative analysis, and the digesta in the hindgut was collected for intestinal flora analysis.

#### *2.4. Gastrointestinal Tract Histomorphological Observation*

One fish per cage from the H0, H60, and H480 groups was selected for dissection, and the stomach and hindgut were harvested and placed in an EP tube containing glutaraldehyde fixative (Wuhan Servicebio Technology Co., Ltd., Wuhan, China). Subsequently, the stomach tissue blocks were washed with 0.1 mol/L phosphoric acid buffer (PSB) solution 3 times, 15 min each, and then the tissue was transferred to PBS containing 1% OsO4 and left for 1–2 h at room temperature. Afterward, the tissue blocks were washed three times with PBS for 15 min each, followed by dehydration with an alcohol gradient. The tissues were placed in an isoamyl acetate solution and then dried with a critical point dryer (K850, Quorum, Nottingham, United Kingdom). Finally, the specimens were attached to metal stakes for 30 s using a carbon sticker and sputtering apparatus (MC1000, Hitachi, Tokyo, Japan) and then observed using a scanning electron microscope (SEM, SU8100, Hitachi, Japan).

Hindgut tissue was fixed and dehydrated according to the procedure of gastric tissue, and the treated hindgut tissue was placed in resin for permeabilization and embedding, followed by cutting into ultrathin sections (Leica UC7, Leica, Wetzlar, Germany). Subsequently, ultrathin sections were stained with 2% uranyl acetate and 2.6% lead citrate and observed using a transmission electron microscope (TEM, HT7800, HITACHI, Japan).

#### *2.5. Intestinal Biochemical Parameters Analysis*

Wet intestine samples were first accurately weighted, and then a ninefold volume (v/m) of phosphate buffer (pH 7.4) was added to prepare crude enzyme extract solution with the following method: homogenized evenly using a homogenizer (IKA Works Asia, Bhd, Kuala Lumpur, Malaysia) and then centrifuged at 875 g for 10 min at 4 ◦C; the supernatant was then removed for subsequent analysis. The total protein concentration of each intestine sample was measured by the bicinchoninic acid method (BCA) using the commercially available kit (No. A045-3-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Intestinal total antioxidant capacity (T-AOC, ABTS method, No. A015- 2-1), peroxidase (POD, colorimetric method, No. A084-1-1), catalase (CAT, ultraviolet method, No. A007-2-1), superoxide dismutase (SOD, WST-1 method, No. A001-3-2), glutathione peroxidase (GPX, colorimetric method, No. A005-1-2), glutathione reductase (GR, ultraviolet method, No. A062-1-1), and lysozyme (LZM, turbidimetric method, A050-1-1) activities and intestinal malondialdehyde (MDA, TBA method, No. A003-1-2) content were determined by using commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Intestinal recombinant lipopolysaccharide (LPS, No. ml505648), complement 3 (C3, ml003460), complement 4 (C4, ml003461), and immunoglobulin M (IgM, No. ml326413) contents were determined by the ELISA method using commercial kits purchased from Shanghai Enzyme Link Biotechnology Co., Ltd. (Shanghai, China). The determination procedures of each parameter were performed in strict accordance with the corresponding instructions, and unit conversion was performed by dividing by the protein concentration of the corresponding sample.

#### *2.6. Real-Time Quantitative PCR*

Total intestinal RNA was extracted using the TRIzol™ Reagent kit (TransGenBiotech, Beijing, China), followed by concentration and integrity detection using a spectrophotometer (NanoDrop® ND-2000, Thermo, Waltham, MA, USA) and a 1.2% denatured agarose, respectively. Subsequently, the qualified samples (1 μg RNA) were used as reverse transcription templates to synthesize cDNA using commercial kits (Accurate Biology, Changsha, China). Real-Time Quantitative PCR analysis was performed under a 10 μL SYBR® Green Premix Pro Taq HS qPCR Kit II reaction system (Accurate Biology, China) using a quantitative PCR instrument (Roche LightCycler® 480) following the method described in a previous study [33]. Briefly, the 10 μL reaction system contains a 10 ng cDNA template, 0.4 <sup>μ</sup>M forward primers, 0.4 <sup>μ</sup>M reverse primers, 5 <sup>μ</sup>L 2 × SYBR® Green Pro Taq HS Premix II, and the rest is RNase-free water. The PCR instrument was set up with a program of denaturation at 95 ◦C for 30 s, followed by 40 amplification cycles with denaturation at 95 ◦C for 5 s and annealing at 60 ◦C for 30 s. The primers used in this trial are shown in Table 3. The expression level or target genes were calculated by the 2−ΔΔCT method [34] and normalized with the expression level of β-actin in the H0 group.

**Table 3.** Primers pair sequences used for real-time PCR.

TNF-α, tumor necrosis factor a; IL-10, interleukin-10; IL-6, interleukin-6; IL-8, interleukin-8; IL-1β, interleukin 1-beta.

#### *2.7. Intestinal Microbiome Analysis*

Intestinal microbiota total DNA was extracted by using the HiPure Soil DNA Extraction Kit (Magen, Guangzhou, China) according to the instructions, followed by a quality test with a UV spectrophotometer (Thermo, Waltham, MA, USA). Subsequently, the V3- V4 region of the 16SrRNA gene was amplified using a universal primer pair (341F/806R, CCTACGGGNGGCWGCAG/GGACTACHVGGGTATCTAAT) and a commercially available kit (New England Biolabs, MA, USA). The amplification conditions were set as follows: pre-denaturation at 95 ◦C for 5 min, followed by denaturation at 95 ◦C for 1 min, annealing at 60 ◦C for 1 min, extension at 72 ◦C for 1 min, and incubation at 72 ◦C for 7 min after performing 30 cycles. PCR amplification was performed using a 50 μL reaction system containing 10 μL of 5 × Q5@ Reaction Buffer, 10 μL 5 × Q5@ High GC Enhancer, 1.5 μL of 2.5 mM dNTPs, 1.5 μL of forward and reverse primers (10 μM), 0.2 μL of Q5@ High-Fidelity DNA Polymerase, and 50 ng of template DNA. Afterward, the amplification products are purified and quantified, and then a library is constructed before initiating the sequencing work (Illumina platform, Illumina, San Diego, CA, USA). Intestinal microbiome analysis was entrusted to Guangzhou Genedenovo Biotechnology Co., Ltd. (Guangzhou, China).

#### *2.8. Statistical Analysis*

The obtained data were subjected to one-way analysis of variance (ANOVA) by SPSS software (version 22, IBM, Chicago, IL, USA), followed by Duncan's multiple comparison test when *p*-value < 0.05. All data were expressed as mean ± standard error of measurement (SEM).

#### **3. Results**

#### *3.1. Growth Performance and Morphologic Indexes*

As shown in Table 4, the SR, VSI, and RIW were not significantly affected by experimental diets (*p* > 0.05). The FMW, WGR, SGR, and RIL in the H240 and H480 groups and the CF in the H480 group were significantly lower than those in the H0 group, whereas the FCR in the H240 and H480 groups exhibited an opposite result (*p* < 0.05). The HIS in the H0 group was significantly lower than that in the H60, H120, H240, and H480 groups (*p* < 0.05).

$$WGR(\%) = 100 \times (W\_{\rm F} - W\_{\rm I}) / W\_{\rm I}$$

$$SGR(\%/\text{day}) = 100 \times (\text{LnW}\_{\rm F} - \text{LnW}\_{\rm I}) / 56$$

$$FCR(\%) = 100 \times W\_{\rm F1} / (W\_{\rm F} - W\_{\rm I})$$

$$SR(\%) = 100 \times N\_{\rm F} / N\_{\rm I}$$

$$VSI(\%) = 100 \times W\_{\rm V} / W$$

$$HSI(\%) = 100 \times W\_{\rm L} / W$$

$$CF\left(\%/cm^{3}\right) = W / L^{3}$$

$$RIL(\%) = 100 \times L\_{\rm I} / L$$

$$RIV(\%) = 100 \times W\_{\rm I} / W$$

where *W*I, *W*F, *W*FI, *N*I, and *N*<sup>F</sup> are the initial body weight (g), final body weight (g), feed intake weight (g), initial fish numbers, and final fish numbers, respectively; *W*, *W*V, *W*L, *W*I, *L*, and *L*<sup>I</sup> are the body weight (g), visceral weight (g), liver weight (g), intestinal weight (g), body length (cm), and intestinal length (cm) of the same sampled fish.

**Table 4.** Effect of dietary histamine on growth performance and morphologic indexes of striped catfish.

Data in the same row with different superscript letters indicate significant differences between groups (*n* = 3; *p* < 0.05).

#### *3.2. Intestinal Antioxidant Capacity*

As shown in Table 5, intestinal POD and SOD activities in the H480 groups, intestinal GR activity in the H240 and H480 groups, and intestinal T-AOC and GPX activities in the H120, H240, and H480 groups were significantly lower than those in the H0 group, whereas the intestinal MDA content in the H480 group exhibited an opposite result (*p* < 0.05). The intestinal CAT activity in the H30, H60, H120, H240, and H480 groups was significantly lower than that in the H0 group (*p* < 0.05).

**Table 5.** Effect of dietary histamine on the intestinal antioxidant ability of striped catfish.

Data in the same row with different superscript letters indicate significant differences between groups (*n* = 3; *p* < 0.05).

#### *3.3. Intestinal Immune Status*

As shown in Table 6, the intestinal LZM activity was not significantly affected by experimental diets (*p* > 0.05). The intestinal C3 content in the H240 and H480 groups and the IgM content in the H60, H120, H240, and H480 groups were significantly lower than those in the H0 group (*p* < 0.05). The intestinal C4 content in the H30, H60, H120, H240, and H480 groups was significantly lower than that in the H0 group, whereas the LPS content exhibited an opposite result (*p* < 0.05).

**Table 6.** Effect of dietary histamine on intestinal immunity of striped catfish.

Data in the same row with different superscript letters indicate significant differences between groups (*n* = 3; *p* < 0.05).

#### *3.4. Intestinal Inflammatory Response*

The intestinal IL-6 and IL-8 expressions in the H240 and H480 groups and the intestinal TNF-α and IL-1 expressions in the H120, H240, and H480 groups were significantly higher than those in the H0 group, whereas the IL-10 expression in the H60, H120, H240, and H480 groups was significantly lower than that in the H0 group (*p* < 0.05; Figure 1).

**Figure 1.** Intestinal inflammation-related gene expression of striped catfish fed with experimental diets. Values in each column with different superscripts represent significant differences (*p* < 0.05).

#### *3.5. Gastric SEM and Intestinal TEM Observations*

The gastric SEM and intestinal TEM observations are presented in Figure 2A,B, respectively. As shown in Figure 2A, the degree of gastric mucosal damage increased with increasing dietary histamine levels. Furthermore, as shown in Figure 2B, dietary histamine damaged intestinal epithelial cells, and organelle lysis was observed in the H480 group.

**Figure 2.** Gastric SEM and intestinal TEM observations of striped catfish fed with experimental diets. (**A**), gastric SEM observations; (**B**), intestinal TEM observations. M, mitochondria; TJ, tight junction; MV, microvillus; L, lysosome. Mitochondrial dissolution, lysosome volume increase, and microvilli damage were clearly observed in the H480 group.

#### *3.6. Microbiota Structure*

As shown in Table 7, the Goods coverage of all the groups was above 99%, indicating that the sequencing depth in this study was adequate. The Shannon, Simpson, Chao1, and ACE indices were not significantly affected by the experimental diets (*p* > 0.05). The

structural composition analysis of microbiota exhibited that Bacteroidetes, Firmicutes, Fusobacteria, and Spirochaetes were the dominant phyla of all groups (Figure 3A). The top 10 genera of all groups are shown in Figure 3B, and the *Bacteroides*, *Clostridium sensu stricto*, and *Cetobacterium* were the three dominant genera in all groups. As shown in Figure 3C, the abundance of *Bacteroides* in the H60 group, the abundance of Treponema2 in the H240 group, and the abundance of *Parabacteroides* in the H120 group were significantly higher than those in the H0 group, whereas the abundance of *Catobacterium* in the H480 group and the abundance of *Terrisporobacter* in the H240 group were significantly lower than those in the H0 group (*p* < 0.05).

**Table 7.** Effect of dietary histamine on microbiota α-diversity of striped catfish.

**Figure 3.** Intestinal flora composition of striped catfish fed with different experimental diets. (**A**), phylum level; (**B**), general level; (**C**), genera with significant differences. Values in each column with different superscripts represent significant differences (*p* < 0.05).

#### **4. Discussion**

This study first investigated the effects of dietary histamine on the antioxidant capacity, gastric and intestinal barrier functions, and growth performance of striped catfish. As a toxic dietary component, histamine has attracted much attention from aquatic animal nutritionists in recent years. Previous studies have shown that dietary histamine significantly decreased the growth performance of the juvenile group (*Epinephelus coioides*) [35], mysis (*Neomysis japonica Nakazawa*) [36,37], American eels (*Anguilla rostrata*) [38,39], yellow catfish (*Pelteobagrus fulvidraco*) [29], and rainbow trout (*Oncorhynchus mykiss*) [40]. In this study, our data exhibited that dietary histamine below 60 mg/kg has limited effects on the growth of striped catfish, whereas dietary histamine above 120 mg/kg significantly decreased the growth, suggesting that the growth-inhibiting effect of dietary histamine was

dose-dependent. Of note, the striped catfish in the H15 group showed better growth than in the H0 group. Similarly, He et al. (2018) [40] found that diets containing 18.0 mg/kg of histamine had a growth-promoting effect on yellow catfish. This evidence suggests that low-dose histamine may benefit the growth of fish. Conversely, diets containing 4000 mg/kg of histamine showed no detrimental effects on the performance of Chinese mitten crab (*Eriocheir sinensis*) [32], and high-dose dietary histamine (2400 mg/kg) even showed a growth-promoting effect on blue shrimp (*Litopenaeus stylirostris*) [41]. The difference in these results may be related to differences in the digestive physiology of different aquatic animals.

A healthy digestive tract is important for maintaining proper immunity and growth in fish, and the health of the digestive tract is regulated by dietary ingredients [42,43]. Oxidative damage is an unavoidable part of the physiological activity of aerobic organisms, due to the continuous production of oxygen radicals during their physiological metabolism [44]. The scavenging of oxygen radicals is accomplished by the antioxidant system, including antioxidant enzymes and antioxidant active substances [45]. Therefore, the antioxidant capacity is commonly used to evaluate the health status of fish. In this study, fish in the H480 group exhibited the lowest intestinal T-AOC, POD, CAT, SOD, GPX, and GR activities and the highest MDA content, suggesting that high dietary histamine is detrimental to the intestinal antioxidant capacity of striped catfish. Similarly, high-dose dietary histamine significantly decreased the antioxidant capability of grouper [35], American eel [38], and Pacific white shrimp (*Litopenaeus vannamei*) [12]. Liu et al. [35] concluded that the long-term intake of histamine disrupts the ability of fish to respond to reactive oxygen intermediates, thereby disrupting the antioxidant system and causing oxidative damage. However, considering that the intestinal antioxidant capacity of striped catfish fed low-dose dietary histamine (below 60 mg/kg) was not significantly reduced, we hypothesized that dietary histamine disrupts the intestinal antioxidant system in a dose-dependent manner.

Intestinal innate immunity plays an essential role in maintaining intestinal health, which constitutes the first line of defense against colonization by disease-causing microorganisms [46]. Intestinal immune activity is accomplished by the synergistic action of multiple immune enzymes and immunologically active substances, including immunoglobulins M (IgM), lysozyme, and the complement (C3, C4) [47,48]. In this study, dietary histamine (above 120 mg/kg) significantly decreased intestinal C3, C4, and IgM contents, suggesting that high-dose dietary histamine is detrimental to the innate immunity of striped catfish, thereby disrupting intestinal health. Besides, the complement system also plays an equally important role in regulating inflammation in fish [49]. The inflammatory response is usually regulated by a dynamic balance of pro- and anti-inflammatory factors [26,50]. In this study, dietary histamine (above 120 mg/kg) caused the downregulation of anti-inflammatory factors (e.g., IL-10 and NF-κB) and the upregulation of pro-inflammatory factors (e.g., IL-1, IL-6, IL-8, and TNF-α), suggesting that high doses of histamine may cause intestinal inflammation in striped catfish. Similarly, dietary histamine also induced inflammation in Pacific white shrimp and Perciformes [12,51]. Galindo-Villegas et al. [51] suggested that dietary histamine regulates the inflammatory response by acting directly with macrophages. Therefore, this evidence suggests that high doses of dietary histamine may trigger an inflammatory response mediated by macrophages, thereby impairing intestinal health.

Dong et al. [52] suggested that inflammation usually occurs when immune cells are infected or tissues are damaged. In this study, the intestinal TEM observation exhibited that high-dose dietary histamine (480 mg/kg) severely damaged the structure and morphology of intestinal epithelial cells, including an increase in the number of lysosomes and swelling of mitochondria and endoplasmic reticulum. Moreover, the gastric SEM observation also showed that high-dose dietary histamine severely damaged the mucosal layer. Combined with the poor intestinal antioxidant capacity and upregulated inflammation levels, these results sufficiently suggest that high-dose dietary histamine disrupts the intestinal antioxidant system, causing oxidative damage to intestinal tissues and inducing inflammation in striped catfish. More importantly, the morphology of the digestive tract is closely related

to its physiological function [53]. Thus, the poor digestive tract morphology also plausibly explains the reduced intestinal immune function and growth performance of striped catfish [54]. Similar results were observed in grouper [35] and yellow catfish [29]. This evidence suggests that high-dose dietary histamine may reduce fish growth by disrupting digestive tract health and function.

As a component of the intestinal mucosal layer, the intestinal flora is also critical to intestinal health and function [55,56]. Meanwhile, the structure of the intestinal flora is easily affected by dietary ingredients [57,58]. In this study, Firmicutes, Fusobacteria, Bacteroidetes, and Spirochaetes were the four dominant phyla of all groups, which was highly consistent with a previous study reported by Hieu et al. [59]. These results indicate that these phyla may constitute the core flora of striped catfish and are essential for maintaining intestinal health and function [60]. In this study, dietary histamine altered the relative abundance of individual phyla, although it did not change the species of the core flora, suggesting that dietary histamine may induce the migration of intestinal function.

In this study, Fusobacteria, mainly *Cetobacterium* genera, have been reported to produce vitamin B12, acetate, and propionate through their fermentation process [61,62]. These metabolites extensively participate in the regulation of energy metabolism, gut health, and gut microecology [62,63]. Therefore, a decrease in the abundance of *Cetobacterium* in the H480 group suggests that high-dose dietary histamine is detrimental to the intestinal physiological function and health of striped catfish. *Bacteroidetes* mainly consist of Bacteroides and Parabacteroides in this trial. *Bacteroidetes* are considered to be the main catabolite of polysaccharides in the intestine, producing large amounts of short-chain fatty acids that contribute to improved intestinal health [64]. *Parabacteroides* are a butyrate producer with equally positive effects on improving intestinal health [65,66]. Thus, an increase in the abundance of *Bacteroides* and *Parabacteroides* may suggest that low-dose dietary histamine has an ameliorative effect on intestinal health. Nevertheless, this degree of ameliorative effect did not effectively mitigate the toxic effects of dietary histamine, and therefore, dietary histamine ultimately impaired the intestinal health of striped catfish. Moreover, considering that dietary histamine caused a significant decrease in intestinal maltase activity in striped catfish (Table S1) and that both *Bacteroides* and *Parabacteroides* usually use carbohydrates as the carbon source, we speculate that the increase in the abundance of these two genera caused by dietary histamine may be related to poor carbohydrate utilization efficiency. Firmicutes mainly consist of *Terrisporobacter* genera in this study. *Terrisporobacter* is an acetate-producing bacterium that is positively correlated with host health [67,68]. *Treponema\_2* genera are the dominant genera of Spirochaetes in this study, which has been reported as pathogenic bacteria [69]. Therefore, a decrease in the abundance of *Terrisporobacter* and a decrease in the abundance of *Treponema\_2* in the H240 group suggests that high-dose dietary histamine is detrimental to the intestinal health of striped catfish. Moreover, the colonization of pathogenic microorganisms indicates a decrease in the immune viability of the intestinal mucosa and an imbalance in the balance of the microbial community. Combined with the poor intestinal immunity, decreased intestinal antioxidant capacity, and increased oxidative stress in the H240 group, the increased abundance of pathogenic microorganisms in the H240 group suggests that a high dose of dietary histamine disrupts intestinal antioxidant and immune defenses, leading to colonization by pathogenic microorganisms.

#### **5. Conclusions**

In conclusion, high-dose dietary histamine decreased intestinal antioxidant capacity, thereby inducing intestinal oxidative damage and decreasing the growth of striped catfish. In addition, high-dose dietary histamine reduces intestinal immunity, induces the colonization of pathogenic microorganisms and intestinal inflammation, and impairs intestinal health. High-dose dietary histamine severely damaged the mucosal layer of the digestive tract in striped catfish. More importantly, our data confirmed that histamine is a toxic

dietary component for aquatic animals and that increases in histamine levels should be prevented during aquafeed production and storage.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox11112276/s1, Table S1: Intestinal digestive enzyme activities of striped catfish fed with different experimental diets.

**Author Contributions:** Y.L. and X.F.: Formal analysis, Data curation, Writing—original draft, Writing—review and editing; H.H., J.F. and H.Z.: Formal analysis, Data curation; J.D. and B.T.: Conceptualization, Methodology, Writing—original draft, Writing—review and editing, Supervision, and Funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the National Key R&D Program of China (2019YF-D0900200), the Program for Scientific Research Startup Funds of Guangdong Ocean University<PHONE_NUMBER>07), the Foundation of Tongwei Co., Ltd. (TA2019A003), National Natural Science Foundation of China (31760761), and the Natural Science Foundation of Yunnan Province (2018FA018).

**Institutional Review Board Statement:** The striped catfish juveniles used in this study were provided by Guangdong Evergreen Feed Industry Co., Ltd. The procurement of test fish was approved by the Animal Research and Ethics Committee of Guangdong Ocean University (Zhanjiang, China; approval ID: GDOU-IACUC-2021-A2220, approval date: 8 September 2021), and all experimental procedures were carried out in strict accordance with its guidelines.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and in the Supplementary Material.

**Acknowledgments:** The authors would like to thank Yixiong Cao, Yumeng Zhang, and Xiaomei Dou for their help during the experiments.

**Conflicts of Interest:** The authors declare that no conflict of interest could be perceived as prejudicing the impartiality of the research reported.

#### **References**

### *Article* **Effects of Different Dietary** β**-Glucan Levels on Antioxidant Capacity and Immunity, Gut Microbiota and Transcriptome Responses of White Shrimp (***Litopenaeus vannamei***) under Low Salinity**

**Yanbing Qiao, Li Zhou, Yayu Qu, Kunyu Lu, Fenglu Han \* and Erchao Li \***

Key Laboratory of Tropical Hydrobiology and Biotechnology of Hainan Province, Hainan Aquaculture Breeding Engineering Research Center, College of Marine Sciences, Hainan University, Haikou 570228, China

**\*** Correspondence<EMAIL_ADDRESS>(F.H<EMAIL_ADDRESS>(E.L.)

**Abstract:** β-Glucan could significantly improve the antioxidant capacity of aquatic animals. The effects of different dietary levels (0 (control), 0.05, 0.1, 0.2 or 0.4%) of β-glucan on the growth, survival, antioxidant capacity, immunity, intestinal microbiota and transcriptional responses of *Litopenaeus vannamei* under low salinity (≤3) were investigated. The dietary growth trial lasted 35 days (initial shrimp 0.26 ± 0.01 g). The results indicated that the growth performance of the 0.1% and 0.2% groups was significantly better than that of the control group. A second-order polynomial regression analysis of growth performance against dietary β-glucan indicated that the optimal dietary β-glucan level was 0.2% of dry matter. The digestive enzyme activity of the hepatopancreas was enhanced with increasing β-glucan levels. The antioxidant and nonspecific immunity capacities of the hepatopancreas were also enhanced in the 0.1% group. The α-diversity index analysis of the intestinal microbiota showed that the intestinal microbial richness of *L. vannamei* increased in the 0.1% group. The relative abundance of Proteobacteria decreased in the 0.1% group compared with the control group. The transcriptome results indicate that the prebiotic mechanisms of β-glucan include upregulating the expression of nonspecific immune genes and osmoregulation genes and activating KEGG pathways associated with carbohydrate metabolism under low-salinity stress. These results suggested that dietary supplementation with β-glucan markedly increased growth performance and alleviated the negative effects of low-salinity stress by contributing to the activity of biochemical enzymes and enriching carbohydrate metabolism in *L. vannamei*.

**Keywords:** *Litopenaeus vannamei*; aquaculture; low salinity; β-glucan; transcriptome

#### **1. Introduction**

White shrimp (*Litopenaeus vannamei*) can tolerate salinities ranging from 0.5 to 50 due to its strong ability to maintain osmosis and ion regulation, making it a widely farmed shrimp worldwide [1,2]. In 2021, freshwater aquatic animal production in China reached 665,202 t, with the freshwater production of *L. vannamei* accounting for 35% of the total production [3]. Although *L. vannamei* can be cultured on a large scale in freshwater, this factor does not mean that white shrimp can produce an optimal physiological response. Shrimp reared at low salinity can experience passive effects, including low survival (53.3%), which was observed at a salinity of 5, and increases in oxygen consumption and feed coefficients were reported [4,5]. Some research also documented that low salinity could reduce innate immune parameters [6,7], increase the toxicity of ammonia (NH3) and nitrite (NO2 −) [8,9], decrease resistance against pathogens [7,10] and even destroy the structure of the intestinal microbiota during aquatic animal culture [11,12]. These negative effects ultimately lead to the poor aquaculture production of *L. vannamei*. Therefore, the

**Citation:** Qiao, Y.; Zhou, L.; Qu, Y.; Lu, K.; Han, F.; Li, E. Effects of Different Dietary β-Glucan Levels on Antioxidant Capacity and Immunity, Gut Microbiota and Transcriptome Responses of White Shrimp (*Litopenaeus vannamei*) under Low Salinity. *Antioxidants* **2022**, *11*, 2282. https://doi.org/10.3390/ antiox11112282

Academic Editors: Bo Liu, Changyou Song and Cunxin Sun

Received: 22 October 2022 Accepted: 16 November 2022 Published: 18 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

exploitation of novel strategies to overcome the above problems is particularly important for the rapid development of the *L. vannamei* aquaculture industry. Nutritive regulation is an effective strategy to alleviate the adverse effects of low-salt stress. Previous studies have documented that dietary mineral supplements, proteins and prebiotics can improve growth performance [12–16] and enhance the antistress ability and immune defense ability of *L. vannamei* to alleviate the unfavorable effects of low salinity.

Prebiotics are commonly added to feed to improve the immune activity of white shrimp to environmental stress [13,17–19]. Among the many prebiotics, β-glucan is commonly used as an immunostimulant in the aquaculture industry [20]. β-Glucan is a complex polysaccharide present in grains, seaweeds, mushrooms, yeast and certain bacteria, distinguished by the presence of pathogen-associated molecular patterns (PAMPs) in molecules with immunomodulatory activity [21]. β-Glucan can improve the immunity and disease resistance of crustaceans, including tiger shrimp (*Penaeus monodon*) [22] and banana shrimp (*Penaeus merguiensis*) [23]. Feeding *L. vannamei* 0.2% β-glucan has positive effects on growth performance [24]. Dietary 0.05–0.20% β-glucan can improve the tolerance of Golden Pompano (*Trachinotus ovatus*) to low-salt stress [25]. Dietary β-glucans could alter the dominance of the intestinal microbiota structure in *L. vannamei* [26,27] and turbot (*Scophthalmus maximus*) [28]. Although studies have confirmed that β-glucan could promote nonspecific immune defense and improve low-salt adaptation [13], previous research involving the optimal dosage of β-glucan has not presented consistent results for *L. vannamei* under environmental stress. In addition, the effects of prebiotics on aquaculture are primarily focused on their growth and physiological and biochemical functions, but little is known about the prebiotic mechanism of prebiotics at the molecular level in shrimp under low salinity.

This study aimed to explore the optimal supplementation level of β-glucan as a prebiotic under low salinity. This study examined the effects on the growth, antioxidant capacity, immunity and intestinal microflora of *L. vannamei* under low salinity (≤3). The results of transcriptome and microbiology techniques further reveal the prebiotic mechanism of β-glucan in alleviating the unfavorable effects of low salinity.

#### **2. Materials and Methods**

#### *2.1. Experimental Diets*

The basic feed formula and nutrients are displayed in Table 1. The basic diet was formulated according to the nutritional requirements of *L. vannamei* and previous research [13]. The diet was supplemented with isonitrogenous and isolipids containing different levels (0, 0.05, 0.1, 0.2 or 0.4%) of β-glucan. Yeast (1,3)-(1,6)-β-glucan with a purity of 88% was the experimental β-glucan source. The diet material was ground with a grinder and filtered with a 60-mesh screen. All single raw materials were thoroughly mixed, and distilled water was added to make a dough, which was extruded into 2 mm diameter pellets by an extruder (CD4-1TS extruder, Guangzhou Huagongguang Electromechanical Technology Co., Ltd., Guangzhou, China). The prepared pellets were air-dried. After drying, the pellets were classified into different sizes with the most suitable sieve (12, 14, 16 and 20 mm) to ensure effective feeding and stored at −20 ◦C.

#### *2.2. Experimental Design and Breeding*

Larvae of *L. vannamei* (P5) were purchased from a commercial shrimp farm (Wenchang, China). During the acclimation period, larvae were fed a commercial diet (Alpha feed Co., Ltd., Protein 48%, Shenzhen, China) at 4% of their body weight per day. All shrimp were acclimated to a salinity of 3 at a rate of 2% reduction per day by adding fresh water. Fresh water was adequately aerated prior to adjusting the salinity of the seawater.

After finishing acclimation, shrimp (0.26 ± 0.01 g) were randomly separated into five groups: 0% β-glucan (control), 0.05% β-glucan (0.05%), 0.1% β-glucan (0.1%), 0.2% β-glucan (0.2%) and 0.4% β-glucan (0.4%), divided into five groups in 20 tanks (60 × 30 × 36 cm) with 20 shrimp, each with four replicates (October to November 2020). During the five-week experiment, daily water exchange was carried out at a rate of 1/2 the culture water volume

using low-salinity (≤3) water, and residual food and feces were removed. The temperature (27 ± 2 ◦C), pH (7.5–8.0), total nitrogen concentration (<0.03 mg/L) and dissolved oxygen (≥7 mg/L) were maintained and checked twice a week throughout the experimental session. Shrimp were fed three times a day (7:00, 12:00 and 18:00).

**Table 1.** Dietary formulations and proximate composition of the experimental diets (% dry matter).

<sup>a</sup> Vitamin premix (g/kg premix): vitamin A acetate (500,000 IU/g), 0.960; L-ascorbyl-2-polyphosphate 35% Active C, 71.420; folic acid, 0.360; biotin, 5.000; riboflavin, 6.000; DL Ca-pantothenate, 10.000; pyridoxine HCl, 2.000; 1% vitamin B12, 0.400; thiamin HCl, 1.000; Menadione, 4.000; DL-alpha-tocopheryl acetate (250 IU/g), 16.000; nicotinic acid, 10.000; vitamin D (500,000 IU/g), 1.600; defatted rice bran, 871.260. <sup>b</sup> Mineral premix (g/kg premix): zinc sulfate monohydrate, 20.585; calcium iodate, 0.117; cupric sulfate pentahydrate, 0.625; manganous sulfate monohydrate, 1.625; magnesium sulfate monohydrate, 39.860; cobalt chloride, 0.010; ferrous sulfate monohydrate, 11.179; sodium selenite, 0.025; calcium hydrogen phosphate dihydrate, 166.442; defatted rice bran, 759.532. <sup>c</sup> β-Glucan was purchased from Xi'an Ruilin Biotechnology Co., Ltd., Xi'an, China.

#### *2.3. Sampling and Growth Performance*

After completing the 35-day dietary trial, all shrimp fasted for 24 h before sampling. Before sampling began, the shrimp were counted, and then they were anesthetized in an ice-water bath. The body length and body weight were randomly measured. The middle intestines and whole hepatopancreas were aseptically dissected and placed into a 1.5 mL sterile centrifuge tube, and the two tissues were prepared for the biochemical and multiomics analyses. All tube samples were immediately stored in liquid nitrogen and transferred to −80 ◦C for storage until further analyses. Three intestine samples were pooled into one sample per tank for microbiota analysis. Hepatopancreas samples were examined for digestive enzyme, antioxidant and immune enzyme activities. Growth-related indicators were evaluated using the following formulas:

Survival (%) = (final shrimp number/initial shrimp number) × 100

Weight gain (WG, %) = (final weight − initial weight)/initial weight × 100

Specific growth rate (SGR, % day) = 100 (Ln final weight − Ln initial weight)/number of days

#### *2.4. Biochemical Assay*

Eight samples of the hepatopancreas (1 g) from two shrimp per tank per treatment were accurately weighed, transferred to sterile centrifuge tubes and homogenized in a cold 0.86 saline solution (1:9, *w*/*v*) using a tissue grinder (Shanghai Jingxin, Shanghai, China) (60 Hz, 30 s) and centrifuged at 1500× *g* (4 ◦C/15 min, 3–18 KS, Sigma, Osterode, Germany). After centrifugation, the supernatant was collected to determine the total protein concentration, digestive enzymes (protease, amylase and lipase), total antioxidant capacity (T-AOC), catalase (CAT), malondialdehyde content (MDA), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). These biochemical enzyme activities in the hepatopancreas were measured according to procedures described in [29,30]. All hepatopancreatic biochemical indices were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the operating procedures were used according to the manufacturer's instructions.

#### *2.5. Intestinal Microbiota Analysis*

Total genomic DNA was extracted from intestinal samples using the TAB/SDS method. DNA quality was measured using a NanoDrop spectrophotometer (Thermo, Wilmington, DE, USA). Generation sequencing was performed on an Illumina HiSeq system according to the protocol of Majorbio Biopharm Technology Co., Ltd. (Shanghai, China). The hypervariable V3–V4 region of the 16S rRNA gene was amplified by using primers 338 (forward 5-ACTCCTACGGGAGGCAGCA-3) and 806 (reverse 5-GGACTACHVGGGTWTCTAAT-3). A TruSeq® DNA PCR-free Sample Preparation Kit (Illumina, CA, USA) was used to construct the library, and index codes were added. Finally, the libraries were carried out on an Illumina HiSeq 2500 platform. All statistical analyses of the results were performed using the R package software (version 2.11 http://sourceforge.net/projects/rdp-classifier/; accessed on 10 January 2021). Alpha-diversity indices (ACE, Chao1, Simpson, Shannon and Observed\_species) were determined using QIIME software (http://www.mothur.org/ wiki/Calculators; accessed on 10 January 2021), and statistical analyses were performed using one-way analysis of variance (ANOVA) (SPSS 23.0). The raw sequences of intestinal microbiota in this study were submitted to Sequence ReadArchive (SRA) under GenBank database number PRJNA842883.

#### *2.6. Transcriptomic Analysis*

Total RNA was extracted from the hepatopancreas of individual shrimp (three samples per treatment) using TRIzol Reagent (Ambion, CA, USA). The quality and concentration of the RNA were determined using a 1% agarose gel on a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and ND-2000 (Thermo, Wilmington, DE, USA). One microgram of total RNA per sample was used to construct a sequencing library using a TruSeq RNA sample preparation kit from Illumina (San Diego, CA, USA). The prepared cDNA libraries were sequenced on the Illumina HiSeq™ 4000 platform at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Raw paired-end reads were quality-checked with default parameters. Clean reads were individually screened using Trimmomatic software (http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic; accessed on 13 January 2021). Further, total clean reads were separately aligned to the reference *L. vannamei* genome in orientation mode using TopHat (http://tophat.cbcb.umd.edu; accessed on 13 January 2021). The expression levels of unigenes were calculated according to the fragments per kilobase of transcripts per million reads (TPM) to identify differentially expressed genes (DEGs) between different samples. RSEM was used to estimate gene abundances (http://deweylab.biostat.wisc.edu/rsem/; accessed on 10 January 2021). Essentially, differential expression analysis was performed using DESeq2/DEGseq/EdgeR with a Q value of 0.05. DEGs with |log2FC| > 1 and Q value ≤ 0.05 (DESeq2 or EdgeR)/Q value ≤ 0.001 (DEGseq) were considered DEGs. Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway analysis of the DEGs was performed using KOBAS software (http://kobas.cbi.pku.edu.cn/home.do; accessed on 10 January 2021). In addition, KEGG

enrichment analyses were conducted with Bonferroni-corrected *p* values < 0.05. A false discovery rate < 0.01 and FC > 2 were considered for the significance analysis. The RNA-seq data were deposited in the NCBI SRA database (PRJNA846923).

#### *2.7. Statistical Analysis*

To clarify the mechanism of dietary β-glucan on growth, biochemical and omics analyses were performed. SPSS 23 software (IBM, New York, NY, USA) was used to carry out all statistical analyses. The data are expressed as the mean ± standard error (mean ± SE). One-way ANOVA was chosen to determine the significant effects among different groups, followed by multiple comparisons by Duncan's multiple comparison test. The statistical significance level was set at *p* < 0.05. In addition, to determine whether the effect was quadratic, a follow-up trend analysis using the method of orthogonal polynomial contrasts was performed using SPSS 23. The assumptions of normality and homoscedasticity were confirmed before conducting any statistical analyses. The relationship between WG, SGR and dietary β-glucan was estimated through second-order polynomial regression [31], and the optimal dietary β-glucan requirement was also evaluated.

#### **3. Results**

#### *3.1. Growth Performance*

The growth performance results of the 35-day trial are shown in Table 2. There was no significant difference in survival among the groups (*p* > 0.05). The growth performance of shrimp was the highest in the 0.2% group, which was significantly higher than that of the other groups (*p* < 0.05) but not significantly different from that of the 0.1% group (*p* > 0.05). Furthermore, a second-order polynomial regression analysis of WG and SGR against dietary β-glucan indicated that the ideal dietary β-glucan level was 0.2–0.23% of dry matter (Figure 1).

**Table 2.** Regression analysis of the growth performance of Juvenile *L. vannamei* fed different dietary β-glucan levels for 5 weeks.

Note: <sup>a</sup> Values represent the means of four replicate tanks (*n* = 4). Different superscript lowercase letters within a column indicate significant differences (*p* < 0.05). IW, initial weight; FW, final weight; WG, weight gain; SGR, specific growth rate; SOP, second-order polynomial; Adj. R2, adjusted R-squared.

#### *3.2. Hepatopancreatic Digestive Enzyme Activity*

The hepatopancreatic digestive enzyme activities are shown in Figure 2 and Table 3. Hepatopancreas protease and lipase activities were significantly higher in the 0.1%, 0.2% and 0.4% groups than in the control group (*p* < 0.05) (Figure 2A,B). The hepatopancreas amylase activity was also significantly increased with the addition of β-glucan compared with the control group (*p* < 0.05) (Figure 2C).

**Figure 1.** Relationships of (**A**) specific growth rate and (**B**) weight gain with dietary β-glucan levels based on second−order polynomial regression analysis of Juvenile *L. vannamei*, where Xopt represents the dietary β-glucan level (*n* = 4).

**Figure 2.** Effects of different doses of β-glucan on the (**A**) protease, (**B**) lipase and (**C**) amylase enzyme activities in Juvenile *L. vannamei*. Different letters indicate significant differences (*p* < 0.05) among groups. All data are expressed as the mean ± SE (*n* = 4).

**Table 3.** Regression analysis of biochemical assays in the hepatopancreas of Juvenile *L. vannamei* fed different dietary β-glucan levels.

Note: <sup>a</sup> T-AOC, total antioxidant capacity; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde content; ACP, acid phosphatase; AKP, alkaline phosphatase. <sup>b</sup> SOP, second-order polynomial; Adj. R2, adjusted R-squared.

#### *3.3. Antioxidant Capacity and Immunity*

The hepatopancreatic antioxidant performance and nonspecific immune response results are shown in Figure 3 and Table 3. The T-AOC enzyme activity in the hepatopancreas of shrimp in the 0.1%, 0.2% and 0.4% groups was significantly higher than that in the control group (*p* < 0.05) (Figure 3A). The SOD enzyme activity in the hepatopancreas of shrimp in the 0.1% group was significantly higher than that in the other groups (*p* < 0.05) (Figure 3B). The CAT enzyme activity in the hepatopancreas of shrimp in the 0.1% group was significantly lower than that in the other groups (*p* < 0.05) but not significantly different from that in the 0.4% group (*p* > 0.05) (Figure 3C). The MDA content of the hepatopancreas in shrimp in the 0.2% group was significantly increased compared with that in shrimp in the other groups (*p* < 0.05) but was not significantly different from that in shrimp in the 0.4% group (*p* > 0.05) (Figure 3D). The ACP enzyme activity in the hepatopancreas of shrimp in the 0.1% and 0.2% groups was significantly higher than that in the other groups

(*p* < 0.05) (Figure 3E). The AKP enzyme activity in the hepatopancreas of shrimp in the 0.05% and 0.1% groups was significantly higher than that in the other groups (*p* < 0.05) (Figure 3F).

**Figure 3.** Effects of different doses of β-glucan on the (**A**) total antioxidant capacity, (**B**) superoxide dismutase, (**C**) catalase, (**D**) malondialdehyde content, (**E**) acid phosphatase and (**F**) alkaline phosphatase of Juvenile *L. vannamei*. Different letters indicate significant differences (*p* < 0.05) among groups. All data are expressed as the mean ± SE (*n* = 4).

#### *3.4. Intestinal Microbiota Analysis*

In this study, the analysis of the intestinal microflora showed that a total of 2,384,213 high-quality sequences were acquired in all samples. Each sample had an average of 99,342 sequences. The observed species ranged from 338.00 to 828.25. Supplementation with 0.1% β-glucan significantly increased the alpha-diversity indices (Shannon and Observed\_species) compared with those of the other groups (*p* < 0.05), as shown in Figure 4 and Table S1. At the phylum level, the most dominant phylum was Proteobacteria, followed by Firmicutes, Actinobacteriota and Bacteroidota in all samples (Figure 5A,B), and Firmicutes and Actinobacteriota were dominant in the 0.1% group (Figure 5C and Table S1). Supplementation with 0.1% significantly induced an increase in the relative abundance of intestinal microbial species compared with those of the other groups of *L. vannamei* (*p* < 0.05). At the genus level, the relative abundances of Vibrio, Rheinheimera and Demequina in the 0.1% group were significantly lower than those in the other groups, but the relative abundance of Lacrobacillus in the 0.1% group was significantly higher than those in the other groups (Figure 5D–F and Table S1). In addition, the principal coordinate analysis (PCoA) cluster analysis indicated that the overall structure of the intestinal microflora in the 0.1% group was significantly different compared with the control group (Figure 6).

**Figure 4.** Effects of different doses of β-glucan on the alpha-diversity using (**A**) Chao1 estimator, (**B**) ACE estimator, (**C**) Shannon estimator and (**D**) observed species in gut microbiota in Juvenile *L. vannamei*. Different letters indicate significant differences (*p* < 0.05) among groups. All data are expressed as the mean ± SE (*n* = 4).

**Figure 5.** Effects of different doses of β-glucan on the gut microbiota composition of Juvenile *L. vannamei* at the phylum and genus levels. (**A**) Microbiota composition at the phylum level with the relative abundance of the top four. (**B**) Relative abundance of gut microbiota at the phylum level. (**C**) Differences in the relative abundance of phylum taxa among groups. (**D**) Microbiota composition at the genus level with the relative abundance of the top thirteen. (**E**) Relative abundance of gut microbiota at the genus level. (**F**) Differences in the relative abundance of genus taxa among groups. Different letters indicate significant differences (*p* < 0.05) among groups. All data are expressed as the mean ± SE (*n* = 4).

**Figure 6.** Effects of different doses of β-glucan on the β−diversity of gut microbiota in Juvenile *L. vannamei*. PCoA of the microbiota at the OTU level based on Jaccard distances. Analysis of similarity (ANOSIM) was performed to evaluate the overall differences in bacterial community structure based on Jaccard distance (*n* = 4).

#### *3.5. Transcriptome Analysis*

Among the five treatment groups, 2354 differentially expressed genes (DEGs) were identified. As shown in Figure 7, a total of 27 DEGs were shared among the treatment groups, while 32, 23, 300 and 1114 DEGs were significantly expressed only in the control vs. 0.05% group, control vs. 0.1% group, control vs. 0.2% group and control vs. 0.4% group, respectively (Figure 7A,B). A total of 183 DEGs were found in the control vs. 0.05% group, including 94 upregulated and 89 downregulated genes; 134 DEGs were found in the control vs. 0.1% group, including 68 upregulated and 66 downregulated genes; 902 DEGs were found in the control vs. 0.2% group, including 467 upregulated and 435 downregulated genes; and 1801 DEGs were found in the control vs. 0.4% group, including 808 upregulated and 993 downregulated genes (Figure 7C). Compared with the control group, there were

27 differentially expressed genes treated with different doses of β-glucan. Specific information on these 27 common DEGs is shown in Table S2. Significant DEGs were studied using KEGG pathway enrichment statistics to study the important functional pathways of DEGs (Table S3). The phenoloxidase gene, antimicrobial peptide gene and Na+/K+/2Cl<sup>−</sup> cotransporter gene expression levels were significantly upregulated in the β-glucan addition group. The KEGG analyses of DEGs showed that dietary β-glucan under low salinity also regulated signal transduction pathways (RIG-I-like receptor, PPAR, JAK-STAT, prolactin, neurotrophic factor, AMPK, Toll-like receptor, TNF and sphingolipid) and activated KEGG pathways associated with carbohydrate metabolism (starch and sucrose metabolism, carbohydrate digestion and absorption and galactose metabolism).

**Figure 7.** Changes in transcriptome levels of differentially expressed genes with different doses of β-glucan in Juvenile *L. vannamei*. (**A**,**B**) The Venn diagram shows the number of common or unique differential genes among groups (0.05%, 0.1%, 0.2% and 0.4%) compared with the control group. (**C**) Volcano map showing the number of upregulated and downregulated genes in the control vs. 0.05%, control vs. 0.1%, control vs. 0.2% and control vs. 0.4% groups; the *X*-axis is represented by log2 (fold change), and the larger the variation, the wider the distribution; the *Y*-axis is represented by −log10 (Padj), a negative logarithm of the adjusted *p*-value; the green dots represent downregulated genes, the red dots represent upregulated genes, and the gray dots represent genes that do not differ significantly (*n* = 4).

#### **4. Discussion**

*L. vannamei* can adapt to extensive environmental salinity fluctuations; its optimal growth rate occurs at ≥20 salinity [11], and it faces hyposaline stress under low salinity [32]. Various prebiotics have been proven to alleviate hyposaline stress; for instance, inulin can cope with low-salinity stress in shrimp [19]. Dietary β-glucan significantly increased the WG, SGR and PO activity of Pacific white shrimp [27] and post-larval *P. monodon* [33]. This study revealed that 0.1% and 0.2% β-glucan increased the growth performance, protease activity, lipase activity and amylase activity of *L. vannamei* under low-salinity stress. β-Glucan could improve the growth of *L. vannamei*, which could be due to the increased digestive capacity and the absorption and accumulation of nutrients. As with almost all functional substances, β-glucan is often underutilized in practical farming due to a lack of scientific strategies for its use, but this study showed that β-glucan improved growth performance at low salinity, suggesting that β-glucan has superior production applications. Similar results showed that the addition of 0.02% β-glucan (0.04%) increased the weight gain of *L. vannamei* [13]. Prebiotic xylooligosaccharides fed to crucian carp (*Carassius auratus*) caused an increase in digestive enzymes [34], while mannanoligosaccharides significantly enhanced digestive enzymes in *Pangasianodon hypophthalmus* [35]. Multiple stressors, such as low salinity, nitrite and ammonia, can produce excess reactive oxygen species (ROS) that damage the organism [36,37]. SOD and CAT enzymes, as antioxidant enzymes, can remove excess ROS, and GPx also has an efficient antioxidant capacity [38]. This study demonstrated that the antioxidant enzyme activity was increased due to supplementation with β-glucan compared with the control. Similarly, this research showed that 0.02% and 0.04% β-glucan could have a positive effect on SOD and GPX enzyme activities in *L. vannamei* [13]. Dietary β-glucan enhanced the SOD activity in *Penaeus monodon* [27,39], and similar results were found with the addition of inulin in Pacific white shrimp [19]. These results suggest that dietary β-glucan could reduce oxidative damage caused by low-salinity stress by increasing the activities of antioxidant enzymes. In addition, this experiment showed that 0.1% β-glucan helped to increase the ACP and AKP activities in *L. vannamei*. ACP and AKP are capable of assisting, modulating and accelerating phagocytosis [40]. A previous study also showed that the oral administration of β-glucan enhanced ACP activity [41]. In this study, the significant activation of SOD, ACP and AKP activities clearly illustrates that β-glucan can be used as an immunostimulatory agent in Pacific white shrimp, which is beneficial for improving the immunity of shrimp in a low-salinity environment.

The effects of prebiotics are closely related to the dosage in aquatic animals [17–19]. In this study, the growth performance of *L. vannamei* increased with the additive dose (0–0.2%) but started to decrease when β-glucan was 0.4%. This indicated that there was no linear relationship between the growth-promoting effect of prebiotics on *L. vannamei* and the additive dose. In addition to the growth performance indicators, other indicators, such as digestive enzyme activity, total antioxidant capacity, malondialdehyde content, AKP, ACP activity and so on, all showed the same phenomenon. A previous study found that the addition of 0.064% β-glucan improved the weight gain of *L. vannamei* [42]. Moreover, 0.02% dietary β-glucan showed higher levels of immune gene expression in *L. vannamei* [13]. In this study, based on the growth performance, oxidation resistance and immune indices, it was speculated that the optimal β-glucan additive level in the diet of *L. vannamei* in low-salinity culture conditions was 0.1%. It should be noted that the most appropriate prebiotic dose may vary depending on the species of aquatic animal, the type of prebiotic, the structure and dietary conditions, so there is no simple linear relationship.

The intestine not only plays a vital role in nutrient absorption and metabolic activity but also improves environmental stress and provides a defense against pathogens [43]. Intestinal microorganisms and their metabolites can directly or indirectly affect the physiological functions of animals, including metabolism and immunity [44]. Recently, most research on Pacific white shrimp has paid more attention to the relationships between shrimp health and intestinal microbiota [45,46]. Dietary prebiotics can modulate the microbial population in the intestine of the host [47]. This study showed that adding 0.1%

β-glucan significantly increased the intestinal microflora values of α-diversity indices. The effect of prebiotics on the α-diversity of intestinal flora in aquatic animals is still controversial. Some studies have shown that inulin and β-glucan have no effect on the α-diversity of intestinal flora in white shrimp [13,19]. However, some studies have documented that prebiotics increase the α-diversity of intestinal flora [48]. Some results suggested that prebiotics enhanced immunity in the host by stimulating the growth of probiotics such as *Lactobacillus* and *Bacillus* [49,50]. This study showed that the dominant phylum and genus in the intestine were significantly changed by the addition of β-glucan. The addition of 0.1% β-glucan could affect the bacterial community in the intestine of *L. vannamei* by increasing the relative abundance of *Lactobacillus*, thereby maintaining a more stable intestinal environment and resisting the invasion of pathogens [45]. The vast majority of *Lactobacillus* species are considered probiotics for aquatic animals [51]. Therefore, dietary β-glucan could have beneficial effects by promoting the occupancy of these intestinal probiotics in the host. Furthermore, we hypothesize that the practical value of β-glucan in aquaculture is mainly through intestinal microbiota and physiological changes that synergistically improve the physiological health of farmed shrimp.

Transcriptome sequencing technology is a significant approach for quantifying transcriptional expression in nonmodel species [52,53]. This strategy has been used in the environmental stress response, such as different salinities [54] and temperatures in *L. vannamei* [55]. In this study, differential gene expression analysis revealed that there were 27 common differential gene changes between the β-glucan groups at different doses and the control group, and these differential gene changes ranged from 1 to 3. The number of upregulated DEGs was much larger than that of downregulated DEGs. This result indicated that β-glucan alleviated low-salinity stress mainly by activating gene expression levels in *L. vannamei*. The expression levels of phenoloxidase genes and antimicrobial peptide genes were significantly upregulated with β-glucan addition. Phenol oxidase activity has a positive correlation with disease [56]. Shrimp encode and produce antimicrobial peptides (AMPs) as part of their innate immune response [57]. These results suggest that dietary β-glucan could enhance the nonspecific immune function of white shrimp under low salinity by enhancing the expression of immune-related genes, thus producing a probiotic effect on the host. In addition, compared with the control group, the expression level of the Na+/K+/2Cl<sup>−</sup> cotransporter gene was significantly upregulated in the β-glucan group at different doses and was closely related to osmotic pressure regulation [58]. Similar results were found in a previous study, where prebiotic inulin significantly upregulated metabolic pathways related to osmotic pressure regulation (aldosterone-mediated sodium reuptake metabolic pathway) in *L. vannamei* under low-salinity stress [19]. These results suggest that the prebiotic effect of prebiotics on the host may be mediated by regulating osmotic pressure at low salinity. However, there is limited information on the effect of prebiotics on osmotic regulation, and more research is needed. In the present study, transcriptome analyses were performed in *L. vannamei* receiving dietary β-glucan to investigate how β-glucan plays an active role. KEGG analyses of DEGs showed that β-glucan has significant effects on pathways related to energy metabolism and immune defense. Low-salinity stress may affect the metabolism and immune system of *L. vannamei*. It was speculated that β-glucan could exert its probiotic effect on the host by regulating these signaling pathways. Osmotic regulation requires a certain amount of energy in crustaceans to ensure normal metabolism [2]. Many enzymes and transporters are relevant to iono- and osmoregulatory processes, which consume large amounts of energy [59]. In this study, KEGG pathways related to carbohydrate metabolism, such as starch and sucrose metabolism, carbohydrate digestion and absorption, and galactose metabolism, were significantly enriched under low salinity by adding β-glucan. To date, dietary supplementation with xylooligosaccharides (XOS) has been demonstrated to be effective in improving blood glucose [60]. A previous study showed that the prebiotic inulin enriches different metabolic pathways, including glycolysis and gluconeogenesis [61], consistent with this study. These results indicate that dietary β-glucan addition can improve immune defense and energy metabolism disorders under low-salinity stress. Furthermore, the prebiotic β-glucan could meet energy requirements under low salinity by activating KEGG pathways related to carbohydrate metabolism, thus producing a probiotic effect on the host.

#### **5. Conclusions**

The addition of β-glucan could improve the antioxidant capacity of *L. vannamei* by enhancing the activities of T-AOC, SOD and CAT to relieve low-salinity stress (≤3). In addition, dietary β-glucan addition could enhance growth performance and effectively alleviate the physiological effects by enhancing biochemical indicators (digestive and nonspecific immune activity) and improving intestinal microbiota and carbohydrate metabolism (starch and sucrose metabolism, carbohydrate digestion and absorption, galactose metabolism, etc.). In addition, it is recommended that the β-glucan dose added to the diet be 0.1–0.2% to improve growth performance and relieve low-salinity stress (≤3). These results can provide a reference for the practical application value and farming costs of β-glucan in aquaculture, which will set a model for subsequent research on functional substances in *L. vannamei* and contribute to the green, healthy and sustainable development of the global aquaculture industry.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/antiox11112282/s1. Table S1: Regression analysis of the gut microbiota composition in *L. vannamei* fed different dietary β-glucan levels; Table S2: Shared differentially expressed genes between the control, 0.05%, 0.1%, 0.2% and 0.4% groups; Table S3: KEGG pathway enrichment of the differentially expressed genes between control, 0.05%, 0.1%, 0.2% and 0.4%.

**Author Contributions:** Y.Q. (Yanbing Qiao) was responsible for feeding management, data analysis and the writing of the original manuscript. Y.Q. (Yayu Qu) and K.L. were responsible for feeding management and validation of the data. Y.Q. (Yanbing Qiao), F.H., L.Z. and E.L. were responsible for the research investigation and experimental design. F.H. and E.L. were responsible for reviewing and revising the article. E.L. was responsible for the overall planning of funds and review of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was co-sponsored by the National Natural Science Foundation of China (grant number 32060832), the Research and Development Program Projects in Key Areas of Guangdong Province (grant number 2020B0202010001) and the initial fund from Hainan University for R & D (grant number KYQD(ZR)21091).

**Institutional Review Board Statement:** The animal experiments in this study were reviewed and approved by the Committee on the Ethics of Animal Experiments of Hainan University (HNUAUCC-2021-00119).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data provided in this study have been uploaded to the NCBI database. The accession numbers are PRJNA842883 and PRJNA846923, and the links are https://www.ncbi.nlm. nih.gov/sra/PRJNA842883 (accessed on 23 May 2022) and https://submit.ncbi.nlm.nih.gov/subs/ PRJN-A846923 (accessed on 23 May 2022).

**Acknowledgments:** The authors would acknowledge Hainan Blue Ocean Biotechnology Co., Ltd., for the contribution of experimental sites and experimental materials, the Beijing Novogene Co., Ltd., for assistance in microbial 16S bioinformatics analysis, and Shanghai Majorbio Co., Ltd., for assistance in the transcriptome analysis of hepatopancreas.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

#### *Article*

## **Berberine Regulation of Cellular Oxidative Stress, Apoptosis and Autophagy by Modulation of m6A mRNA Methylation through Targeting the** *Camk1db***/ERK Pathway in Zebrafish-Hepatocytes**

**Meijuan Zhang 1, Jin Liu 1, Chengbing Yu 1, Shangshang Tang 1, Guangzhen Jiang 2, Jing Zhang 1, Hongcai Zhang 1, Jianxiong Xu <sup>1</sup> and Weina Xu 1,\***

**Abstract:** Berberine (BBR) ameliorates cellular oxidative stress, apoptosis and autophagy induced by lipid metabolism disorder, however, the molecular mechanism associated with it is not well known. To study the mechanism, we started with m6A methylation modification to investigate its role in lipid deposition zebrafish hepatocytes (ZFL). The results showed that BBR could change the cellular m6A RNA methylation level, increase m6A levels of *Camk1db* gene transcript and alter *Camk1db* gene mRNA expression. Via knockdown of the *Camk1db* gene, *Camk1db* could promote cellular ERK phosphorylation levels. Berberine regulated the expression level of *Camk1db* mRNA by altering the M6A RNA methylation of the Camk1db gene, which further affected the synthesis of calmodulin-dependent protein kinase and activated ERK signaling pathway resulting in changes in downstream physiological indicators including ROS production, cell proliferation, apoptosis and autophagy. In conclusion, berberine could regulate cellular oxidative stress, apoptosis and autophagy by mediating *Camk1db* m6A methylation through the targeting of the *Camk1db*/ERK pathway in zebrafish-hepatocyte.

**Keywords:** berberine; m6A methylation; *Camk1db*; ERK; zebrafish hepatocytes (ZFL)

#### **1. Introduction**

The long-term excessive exposure to a high-fat diet results in lipid metabolism disorders and over-production of reactive oxygen species (ROS), which triggers oxidative stress and cell damage at the cellular and molecular levels [1]. Studies have demonstrated cell damage biochemical mechanisms involved in the cellular signaling pathways [2] and the interaction between genes and protein expression changes at the transcriptional level [3,4]. Gene RNA is a key linking DNA to protein in the transmission of genetic information, but the level of synthesized protein does not necessarily correlate positively with the level of mRNA, suggesting the importance of post-transcriptional RNA modifications. There are more than 100 known post-transcriptional modifications of RNA. Among these, m6A methylation is the most common type [5], and also the most widely studied dynamic modification method of RNA. M6A RNA methylation, the sixth methylation modification of adenine on nitrogen atoms, is an evolutionarily-conserved RNA modification that is present in most organisms from bacteria to mammals [6,7]. M6A RNA methylation is also a reversible epigenetic RNA modification that exists at the transcriptional level of the nitrogen or oxygen atom of S-adenosylmethionine, and regulates mRNA stability, splicing, export, localization, and translation [8–10]. M6A RNA methylation can affect most aspects of gene

**Citation:** Zhang, M.; Liu, J.; Yu, C.; Tang, S.; Jiang, G.; Zhang, J.; Zhang, H.; Xu, J.; Xu, W. Berberine Regulation of Cellular Oxidative Stress, Apoptosis and Autophagy by Modulation of m6A mRNA Methylation through Targeting the *Camk1db*/ERK Pathway in Zebrafish-Hepatocytes. *Antioxidants* **2022**, *11*, 2370. https://doi.org/ 10.3390/antiox11122370

Academic Editors: Zhihui Feng and Mark G. Clemens

Received: 30 September 2022 Accepted: 28 November 2022 Published: 30 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

expression. Recent studies have identified m6A RNA methylation modifications plays an important role in regulating cell biological function, controlling cell proliferation and differentiation [11], cellular oxidative stress response [12,13], DNA damage response [14], and lipid metabolism [15–18]. Now, biological functions for this m6A modification are emerging through more mechanistic analyses.

The Chinese medicine berberine (BBR, chemical formula C20H18NO4) is an isoquinoline quaternary alkaloid isolated from several medicinal plants, including *Berberis aristata* and *Coptis chinensis* [19,20]. Berberine can inactivate highly active molecules such as O2 •−, HO•, NO and OONO−, so as to facilitate the ability to remove reactive oxygen species clusters (ROS) directly [21]. In addition, modern pharmacological studies have shown that berberine exhibits hepatoprotective, anti-inflammatory, antioxidant, hypoglycemic, hypolipidemic properties [22,23], and reduces obesity in mammals [24]. Our previous studies showed berberine-supplemented diets could attenuate oxidative stress, improve the function of the mitochondrial respiratory chain, reduce apoptosis and inhibit the inflammation response as well as modulate the intestinal microflora profile caused by high-fat and high-carbohydrate diets in fish in vitro and vivo [25–28]. However, the molecular mechanism of berberine's role in oxidative stress, apoptosis, and autophagy is still limited. In this paper, we investigated whether berberine could regulate cellular oxidative stress, apoptosis and autophagy by changing the methylation level of cell gene m6A RNA.

Zebrafish (*Danio rerio*, Cyprinidae) is an important vertebrate model organism for development, genetics, and reproduction [29]. The liver is the main organ for fat synthesis, and about 90% of the fat in fish is synthesized in the liver [30], which plays an important role in maintaining the metabolic balance of carbohydrates, amino acids, and fatty acids [31]. There were many studies using Zebrafish hepatocytes (ZFLs) to study fat deposition in vitro. Therefore, in this study, we selected ZFL as model system for the study of metabolic syndrome combined with oxidative stress to investigated the roles of m6A RNA methylation modification in lipid-deposition ZFL treated with berberine in vitro. The results may have implications for a molecular theoretical basis for the research and development of hepatoprotective herbal treatments in fish farming.

#### **2. Materials and Methods**

#### *2.1. Materials*

Zebrafish hepatocytes (ZFL, CRL-2643) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). Other materials and reagents purchased included the following: DMEM-F12 medium, DMEM medium, L-15 medium, fetal bovine serum (Gibco, Waltham, MA, USA); penicillin, murine epithelial growth factor, insulin, DPBS and 0.25% trypsin (Thermo Fisher, Shanghai, China); total RNA extraction kit (OMEGA, Cambridge, MA, USA); reverse transcription kit, and fluorescent quantitative PCR kit (Takara, Beijing, China); the targeted antibody (ERK, P-ERK, P62, LC3-B) and the internal antibody tubulin (because the size of the antibody protein of interest is between 10 and 70 kDa, in order to ensure that the target internal reference is on a membrane, and our sample is a whole cell protein, the loading amount is 30 micrograms, using ripa lysate, so for comprehensive consideration, tubulin was selected as the internal reference protein) are purchased from CST (Parsons, KS, USA). Instrumentation included the following: high-speed frozen centrifuge (Eppendorf, Hamburg, Germany, 5804 R, Shanghai, China); real-time fluorescence quantitative PCR instrument (Applied Biosystems, StepOnePlusTM, Waltham, MA, USA); inverted microscope (Nikon Corporation, Hong Kong, China); multifunctional enzyme labeling instrument (SYNERGY 2 BioTek, Waltham, MA, USA). Oil Red O staining solution, sodium palmitate, and berberine, were purchased from Sigma-Aldrich (Shanghai, China) Trading Co. CCK-8 cell activity assay kit, BCA protein assay kit, triglyceride assay kit, ROS, MDA, and T-AOC assay kits, were purchased from Nanjing Jiancheng Institute of Biological Engineering. Total RNA extraction kit was purchased from OMEGA (Cambridge, MA, USA). Primers were synthesized by Thermo Fisher Scientific China Ltd (Shanghai, China). The reverse transcription kit PrimeScript RT reagent Kit (Perfect Real

Time) and its matching fluorescent quantitative PCR kit TB GreenTM Premix Ex TaqTM (Tli RNaseH Plus) were purchased from Takara Corporation (Beijing, China).

#### *2.2. Lipid Accumulation Injury Treatment and Berberine Repair Treatment in ZFL* 2.2.1. Lipid Accumulation Injury Treatment in ZFL

Mixed media was composed of 50% L-15, 30% DMEM/F12, and 20% DMEM (containing 5% fetal bovine serum, 1% penicillin, 1% insulin and 50 ng/mL murine epithelial growth factor). ZFL cells were cultured to 70–80% fusion with a mixed culture base at 28◦C and 5% CO2. Cells were collected after treatment with sodium palmitate (SP) culture solution at a concentration of 0.25 mmol/L for 24 h, thus establishing a validated model of hepatocyte fat deposition [32].

#### 2.2.2. Berberine Repair Treatment in ZFL

ZFL cells were treated with SP culture solution at a concentration of 0.25 mmol/L for 24 h and then collected. The cells were then treated with BBR working solution (solvent 0.1% DMSO) at a concentration of 25 μmol/L for 6 h and then collected, thus establishing an effective model of hepatocyte fat deposition repair [32].

#### *2.3. Oxidative Stress Indicator Test*

#### 2.3.1. ROS Content Detection

The intracellular ROS (reactive oxygen species) content was detected using a DCFH-DA probe. After each group of cells was treated, cells were rinsed with DPBS three times, incubated with DCFH-DA probe for 30 min, and rinsed with DPBS three times, and ROS fluorescence photographs were taken with fluorescence microscopy. Cells received the same treatment as above, with a microplate reader to determine the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The calculation formula is as follows:

ROS content = Fluorescence intensity of the treatment group − Fluorescence intensity of blank group Fluorescence intensity of the control group <sup>−</sup> Fluorescence intensity of blank group ÷ Cell density × 100%

#### 2.3.2. MDA and T-AOC Contents Measurement

After the cell culture treatment, cells were placed on ice. The cell culture medium was discarded, rinsed 3 times with DPBS, and then 200 μL of cell lysate was added to each well for lysis. The cell lysate was collected, and the intracellular MDA and T-AOC contents were measured separately according to the method of the kit (Nanjing Jiancheng Institute of Biological Engineering).

#### *2.4. Cell Apoptosis*

Apoptotic cells were detected by Annexin V/FITC as described previously [33]. Each group of cells was collected in a centrifuge tube and centrifuged at 1000× *g* for 5 min. Supernatant was discarded and 195 μL of membrane linked protein V-FITC binding solution was added to gently suspend the cells. Next, 5 μL of Annexin V-FITC were added and mixed well before further adding 10 μL of propyl iodide solution. The mixture was incubated at room temperature for 10–20 min on ice, protected from light. Apoptosis was detected using flow cytometry counting, where the apoptosis rate (%) = number of apoptotic cells/total number of cells × 100%.

#### *2.5. Western Blot*

Cells were collected by centrifugation, lysed on ice for 30 min with RIPA cell lysis solution, and the total protein concentration was determined by BCA method. Each well was sampled with 30 μg of protein and separated by 10% polyacrylamide gel electrophoresis. The separated proteins were electro transferred (200 mA current for 2 h) to PVDF membrane. ERK (cst, 9102), P-ERK (cst, 9101), P62 (cst, 5114), LC3-B (cst, 2775) (1:1000 volume dilution) were taken from the primary antibody rabbit, and the internal reference primary antibody tubulin (cst, 2146) (1:2000 volume dilution) was taken, and incubated

overnight at 4 ◦C. The next day, the membranes were washed three times with TBST for 10 min each time, followed by the addition of secondary antibodies against rabbit anti-IgG (1:2000 *v*/*v*) for 2–4 h at room temperature, and then washed three times with TBST for 10 min each time, and developed according to the instructions of the ECL kit. The grayscale values of the protein bands were analyzed using the LK5100 electrochemiluminescence analysis system.

#### *2.6. Cellular Microstructure Detection*

A sufficient number of cells were cultured in a 10 cm dish. Cells grown in the petri dish were scraped off directly and the cell suspension was transferred to the centrifuge tube. Cells were centrifuged at 1000–3000 rpm, the supernatant was discarded, and PBS was added to transfer the cells to a 1.5 mL centrifuge tube. After centrifugation, supernatant was discarded. PBS was added to repeat the above steps, and 2.5% glutaraldehyde fixative for tissue and cell electron microscopy was added at 4 ◦C overnight. Samples were then sent to Wuhan Max Bio-Technology Co., Ltd. (Wuhan, China) the next day for subsequent electron microscopy detection.

#### *2.7. m6A Methylation and Transcriptome Merip-Seq Sequencing*

The RNA of the total samples was isolated and purified using TRizol, and the amount and purity of the total RNA was tested for quality control and integrity, while the protocol was verified by agar electrophoresis. Magnetic beads with Poly adenylic acid were used to specifically capture the mRNA. Fragmentation was performed with a magnesium ion interruption kit, and the fragmented RNA was pre-mixed with immunomagnetic beads with m6A antibody, and subjected to IP. The IP product was subjected to duplex synthesis, converting the DNA and RNA duplexes into DNA duplexes, and complementing the ends. Base A was added at each end, ligated to the end with base T, and purified by screening with magnetic beads. After digestion of the duplexes, PCR was pre-denatured at 95 ◦C for 3 min, denatured at 98 ◦C for 15 s, cycled 8 times, annealed at 60 ◦C for 15 s, extended at 72 ◦C for 30 s, and held at 72 ◦C for 5 min to obtain fragments of approximately 300 bp size. Finally, it was sequenced using Illumina NovaSeq TM 6000 according to the sequencing mode PE150.

#### *2.8. Methylation Level Detection*

RNA samples were fragmented and immunoprecipitated magnetic beads were prepared, immunoprecipitated reaction was performed. Then, the obtained RNA samples were eluted and purified. The MeRIP RNA was analyzed together with the corresponding Input RNA by quantitative RT-PCR, or constructed using standard library building kits for RNA-seq library construction and identification of RNA methylation regions within the transcriptome by deep sequencing.

#### *2.9. SiRNA Transfection*

For the study, 1 × 106 cells were inoculated into 6-well plates so that the cell density during transfection could reach 30~50%. After 24 h, samples were washed twice with DPBS, then 2 mL of the configured mixed reagent were added to each well. After 6 h, this was replaced with normal culture medium until the end of the corresponding transfection time when cells were collected for subsequent assay tests. To configure the mixed reagent, 10 μL of lipo2000 were added to 190 μL of serum-free culture medium, which was then gently mixed for 5 min. Next, 5 nmol of siRNA (sequence shown in Table 1) was added to 250 μL of DEPC water, i.e., dry powdered siRNA was configured into a 20 nmol/mL solution. Then, 5 μL of the prepared solution was combined with 195 μL of serum-free culture medium, and gently mixed for 5 min. The two liquid solutions described above were then mixed 1:1 and incubated for 15 min at room temperature to prepare the transfection complex. The complete culture medium was supplemented to 2 mL, which is the required transfection mixture for one well.

**Table 1.** SiRNA sequence of target genes.

#### *2.10. Cell Activity Assay*

The CCK-8 colorimetric assay was used to determine cell activity [34]. Cells were inoculated on 96-well plates at an inoculation density of 1 × <sup>10</sup>4/mL. After incubation with SP or BBR, 10 μL of CCK-8 solution was added to each well in a humidified incubator with 5% CO2 at 28 ◦C for 2 h. The absorbance value (OD) of each well was measured using ELISA (single wavelength, 450 nm).

#### *2.11. Cellular TG Content Assay*

Cells were inoculated into 96-well plates. The original cell culture medium was discarded after each group of cells was treated, followed by rinsing three times with DPBS. Next, 100 μL of cell lysis solution was added to each well and oscillated to fully lyse the cells. Intracellular TG level was measured with GPO-POD method using Triglyceride Colorimetric Assay Kit (Nanjing, Jiangsu, China). The content of protein in each sample was determined by BCA Protein Assay Kit (Nanjing, Jiangsu, China).

#### *2.12. Total RNA Extraction, Reverse Transcription and Real-Time PCR*

Total cellular RNA was extracted and reverse transcribed, and the expression level of *Camk1db* in each group of cells was detected by real-time fluorescence quantitative PCR. Real-time quantitative primer sequences for the genes to be tested were designed by Primer Premier 5.0 software and synthesized by Inweigeki (Table 2). The reaction solution was prepared on ice, mixed thoroughly, and the configured mixture was divided into reaction tubes of 18 μL each. Next, cDNA was added to the samples, which were then centrifuged thoroughly for the amplification reaction. The specific reaction conditions were as follows: 95 ◦C for 30 s; 95 ◦C for 5 s, 60 ◦C for 34 s, 40 cycles and 95 ◦C for 15 s; 60 ◦C for 60 s, 95 ◦C for 15 s. The relative expression of the genes to be tested in the samples was calculated using the 2−ΔΔCT method [35].

**Table 2.** Nucleotide sequences of primers of target genes.

#### *2.13. Statistical Analysis*

All results are shown as mean ± standard error (X ± SEM) and one-way ANOVA, and Duncan's multiple range test were performed using SPSS 20.0. The significance level was *p* < 0.05, which was considered statistically significant.

#### **3. Results**

#### *3.1. Modulation of Oxidative Stress, Apoptosis and Autophagy in Palmitic Acid-Induced Fat-Deposited Zebrafish Hepatocytes by Berberine*

3.1.1. Berberine on Sodium Palmitate-Induced Oxidative Stress in Zebrafish Hepatocytes

To detect the oxidative stress status of cells, the intracellular ROS (reactive oxygen species) content, MDA (malondialdehyde) content, and T-AOC (total antioxidant capacity), were measured. Excess ROS causes cellular damage, and MDA is an important marker of ROS-induced oxidative damage [36]. High levels of T-AOC can effectively protect cells from reactive oxygen species [37]. Four groups were created for the experiment: a control group (Control), a lipid accumulation group (SP), a berberine group (BBR), and a lipid accumulation + berberine group (SP + BBR), respectively. As seen in Figure 1A, the intracellular MDA content was significantly higher in the lipid accumulation group compared with the control group, but there was no significant change in the MDA content in the berberine group or the lipid accumulation + berberine group, and the MDA content in the lipid accumulation + berberine group was significantly reduced compared with the lipid accumulation group (*p* < 0.05). As seen in Figure 1B, the intracellular T-AOC (total antioxidant capacity) was significantly lower in the lipid accumulation group compared with the control group; there was no significant change in the T-AOC in the berberine group and the lipid accumulation + berberine group; and the T-AOC in the lipid accumulation + berberine group was significantly higher compared with the lipid accumulation group (*p* < 0.05). As seen in Figure 1C, the intracellular ROS (reactive oxygen species) content was significantly higher in the lipid accumulation group compared with the control group; there was no significant change in the berberine group; and the ROS content was significantly higher in the lipid accumulation + berberine group (*p* < 0.05). In addition, we also examined the ROS fluorescence graph, in which the brightness of the green spot represented the level of intracellular ROS content, and the brighter fluorescence indicated higher intracellular ROS concentration (red arrows). This can be seen from the luminescence trend compared with the control group, as the fluorescence intensity of all other groups was significantly higher, with the lipid accumulation + berberine group having the highest fluorescence intensity, which was consistent with the results in Figure 1D. These data indicated that lipid accumulation treatment caused oxidative stress in cells, and the total antioxidant capacity was reduced. Conversely, the addition of appropriate amounts of berberine were found to effectively alleviate the level of oxidative stress in cells, and improve the lipotoxicity caused by lipid accumulation.

**Figure 1.** Indicators of oxidative stress in different treatment groups within ZFL and transmission electron microscopy pictures of ZFL. (**A**) is MDA (malondialdehyde). (**B**) is T-AOC (total antioxidant capacity). (**C**) is the relative expression of ROS. (**D**) is the ROS fluorescence microscope picture. The red arrow is the fluorescence of ROS in zebrafish liver cells. The higher the ROS content, the stronger the fluorescence intensity. Dates were presented as mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\*\* *p* < 0.0001. (**E**) shows transmission electron microscopy pictures of ZFL. The red arrow is the nucleus, the blue arrow is the lipid drop, and the yellow arrow is the mitochondria, the green arrow is bubbles.

3.1.2. Berberine on Sodium Palmitate-Induced Hepatocyte Apoptosis and Autophagy

To investigate changes of the apoptosis rates in each treatment group, flow cytometry was applied (Figure 2A), and the data were counted in Figure 2B plots (*p* < 0.05). Results indicated that the apoptosis rate in the lipid accumulation group was significantly higher than the control group, and the apoptosis rate in the berberine group was significantly lower, while the apoptosis rate in the lipid accumulation + berberine group was significantly lower than the lipid accumulation group. Among all groups, the apoptosis rate was lowest in the berberine group, followed by the control group and the lipid accumulation + berberine group, while the apoptosis rate was highest in the lipid accumulation group. To detect changes in the level of cellular autophagy in each group, Western blotting was applied to detect the expression of proteins associated with cellular autophagy in each group (Figure 2C). The relative content of P62 protein, which was calculated as shown in gray in Figure 2D, was significantly lower in the lipid accumulation group compared with the control group, significantly higher in the berberine group, and significantly higher in the lipid accumulation + berberine group, compared with the lipid accumulation group (*p* < 0.05). The relative content of LC3-B protein is presented in Figure 2E, indicating that LC3-B protein content was significantly higher in the lipid accumulation group compared with the control group, significantly lower in the berberine group, and significantly lower in the lipid accumulation + berberine group, compared with the lipid accumulation group (*p* < 0.05). The expression of P62 protein was negatively correlated with the level of cellular autophagy, and the expression of LC3-B protein was positively correlated with the level of cellular autophagy. The P62 and LC3-B protein expression levels indicated that the autophagy levels in the lipid accumulation group was significantly higher than in the control group, and that the autophagy level in the lipid accumulation + berberine group was significantly lower than in the lipid accumulation group, while the autophagy level in the berberine group was the lowest, and the autophagy level in the lipid accumulation group was the highest.

**Figure 2.** Berberine on sodium palmitate-induced hepatocyte apoptosis and autophagy. (**A**) is flow cytometry to detect ZFL apoptosis, each group has a cell count of 10,000. (**B**) is apoptosis and apoptosis rate of ZFL. (**C**) is WB diagram of P62 protein and LC3-B protein. (**D**) is relative expression level of P62 protein. (**E**) is relative expression level of LC3-B protein. Dates were presented as mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001.

3.1.3. Berberine on the Morphology of Zebrafish Hepatocytes Induced by Sodium Palmitate

As seen in Figure 1E, transmission electron micrographs of the cells were taken. The cells in the control group were morphologically intact, with round and regular shaped nuclei (red arrow), clear nuclear membranes, and normal endoplasmic reticulum and mitochondrial morphology. The morphology of the cells in the lipid accumulation group was significantly changed. The cells were severely vacuolated (green arrow); the nucleoli were extruded and deformed; the intracellular mitochondria were swollen (yellow arrow); and a large number of obvious lipid droplets appeared (blue arrow), indicating that the lipid accumulation treatment induced by sodium palmitate had an adverse effect on the cell morphology. In contrast, after the addition of an appropriate amount of berberine, there was a significant trend of cell morphology recovery compared with the lipid accumulation group, but there were still small amounts of swollen mitochondria and lipid droplets compared with the control group.

#### *3.2. Effect of Berberine on Palmitic Acid-Induced m6A Methylation and Transcription Levels in Zebrafish Hepatocytes*

#### 3.2.1. m6A Methylation Analysis

Cells were collected from four experimental groups: control group (Control), lipid accumulation group (SP), berberine group (BBR), and lipid accumulation + berberine group (SP + BBR), and methylation analysis was conducted by Merip-seq high-throughput sequencing to investigate the effect of berberine on lipid accumulation-induced m6A methylation in zebrafish cells. As seen in Table 3, in terms of the number of methylation sites, the highest number was detected in the berberine group, followed by the lipid accumulation + berberine group and the control group, with the least number of methylation sites detected in the lipid accumulation group. Results indicated that the addition of either lipid accumulation or berberine affected the number of m6A methylation sites in zebrafish hepatocytes.

**Table 3.** Number of methylation sites.

To study the distribution of m6A sites on mRNA, m6A peaks were then divided into four parts according to their position on the transcript: 5 untranslated region (5 UTR); 3 untranslated region (3 UTR), 1st Exon (1st Exon), and Other Exon (Other Exon). Table S1 and Figure 3 summarize the distribution of m6A sites in each group, with most peaks located in the 3 untranslated region, followed by the 5 untranslated region and other exon regions. Compared with the control group, there was a significant increase in the proportion of m6A peaks in the 3 and 5 untranslated regions in the lipid accumulation group, while the proportion within the 3 untranslated region was significantly reduced and the proportion within 5 untranslated region was further increased after the addition of berberine. M6A peaks possessed a higher enrichment ploidy. This indicated that lipid accumulationinduced zebrafish hepatocytes possessed different m6A methylation patterns, and the addition of appropriate amounts of berberine changed these m6A methylation patterns.

The methylation and demethylation process of RNA requires binding proteins to the motifs of methylation sites. The analysis software HOMER was used to look for motifs with high feasibility in the peak region, as seen in Figure 4A, for the four groups of samples with motif prediction results. Motifs with multiple occurrences and high similarity in the four groups revealed potential motifs of zebrafish methylation sites. Results indicated that all four different treatment groups contained the "RRACH" motif, which indicated that zebrafish also conformed to the general pattern of other species, and confirmed that the results of this experiment were reliable and could be used for subsequent analysis.

**Figure 3.** Distribution of m6A peaks of each group within ZFL.

**Figure 4.** M6A methylation analysis. (**A**) is top ten motif analysis for different treatment groups within ZFL. (**B**) is top six motif analysis of each comparison group. \*—possible false positive.

Table S2 illustrates the significant changes in methylation levels in each comparison group. The number of genes with significant methylation differences was 231 in the lipid accumulation group compared with the control group; 488 in the berberine group compared with the control group; 107 in the lipid accumulation + berberine group compared with the lipid accumulation group; and 441 in the lipid accumulation + berberine group compared with the berberine group. This indicated that the effect of the treatment with berberine on methylation levels was more significant compared to the lipid accumulation treatment. The distribution of the screened differentially methylated gene peaks was further counted, and data in Table S3 demonstrates that most of the differentially m6A methylated loci in each group was located in the 3 untranslated region, followed by distribution in the 5 untranslated region, then other exons, and finally, the first exon region. From the table, it can be seen that the proportion of m6A peaks in the 3 and 5 untranslated regions was significantly increased in the lipid accumulation versus control group, while the proportion of the 3 untranslated region was significantly decreased and the proportion

of the 5 untranslated region was significantly increased in the berberine-repaired lipid accumulation group versus the control group. The addition of an appropriate amount of berberine was observed to change the m6A methylation pattern of differential genes in lipid accumulation-induced zebrafish hepatocytes, with a decrease in the proportion of the 3 untranslated region and an increase in the proportion of the 5 untranslated region. The differentially methylated peaks screened for each comparison group were subjected to motif analysis, and the top six ranked differentially methylated motifs of lipid accumulation versus control are listed in Figure 4B. From the figure, we can find that lipid accumulation treatment affected m6A methylation at these loci, and the m6A methylation occurring at these loci was also associated with berberine treatment. Similarly, these motifs contained multiple "RRACH" sequences, which verified the reliability of the results.

#### 3.2.2. Transcriptome Analysis

Cells were collected for transcriptomic analysis from four experimental setup groups: control (Control), lipid accumulation group (SP), berberine group (BBR), and lipid accumulation + berberine group (SP + BBR). Significant changes in transcript levels in each comparison group are summarized in Table S4. In terms of up- and down-regulation of transcript levels, the number of genes up-regulated by methylation was greater than the number of genes down-regulated by methylation in all comparison groups, except for the lipid accumulation + berberine group, where the number of genes up-regulated by methylation was smaller than the number of genes down-regulated by methylation compared with the lipid accumulation group.

The results of KEGG pathway enrichment analysis with significant differences in transcript levels are shown in Figure 5. Compared with the control group, the significantly differentially expressed genes in the lipid accumulation group were mainly involved in PPAR signaling pathway, phenylalanine, tyrosine and tryptophan biosynthesis, steroid biosynthesis, necroptosis, herpes simplex virus 1 infection, fructose and mannose metabolism, amino sugar and nucleotide sugar metabolism. Compared with the control group, the significantly differentially expressed genes in the berberine group were mainly involved in the TGF−beta signaling pathway, sphingolipid metabolism, selenocompound metabolism, primary bile acid biosynthesis, PPAR signaling pathway, FoxO signaling pathway, focal adhesion, arginine and proline metabolism, arachidonic acid metabolism, and the AGE−RAGE signaling pathway in diabetic complications. Compared with the control group, the significantly differentially expressed genes in the lipid accumulation + berberine group were mainly involved in the TGF−beta signaling pathway, terpenoid backbone biosynthesis, steroid biosynthesis, fatty acid elongation, cytokine−cytokine receptor interaction, biosynthesis of unsaturated fatty acids, and the AGE−RAGE signaling pathway in diabetic complications. Compared with the lipid accumulation + berberine group, the significantly differentially expressed genes in the lipid accumulation group were mainly involved in the TGF−beta signaling pathway, steroid biosynthesis, notch signaling pathway, MAPK signaling pathway, fatty acid elongation, biosynthesis of unsaturated fatty acids, and the AGE−RAGE signaling pathway in diabetic complications. Compared with the berberine group, the significantly differentially expressed genes in the lipid accumulation group were mainly involved in the sphingolipid metabolism, PPAR signaling pathway, FoxO signaling pathway, fatty acid degradation, cytokine−cytokine receptor interaction, biosynthesis of unsaturated fatty acids, and the AGE−RAGE signaling pathway in diabetic complications. Compared with the lipid accumulation + berberine group, the differentially expressed genes in the berberine group were mainly involved in the TGF−beta signaling pathway, steroid biosynthesis, taurine and hypotaurine metabolism, MAPK signaling pathway, linoleic acid metabolism, glycerolipid metabolism, cytokine−cytokine receptor interaction, and arachidonic acid metabolism. From the above results of the analysis of KEGG, the addition of high-fat and berberine significantly differentiates the transcriptional expression of genes in zebrafish liver cells, which in turn regulates related signaling pathways, mainly manifested as pathways related to lipid metabolism (PPAR signaling pathway,

MAPK signaling pathway, glyceril lipid metabolism, etc.), and pathways related to oxidative stress (FoxO signaling pathway, TGF-beta signaling pathway, etc.). This indicates that lipid deposition and berberine treatment have obvious effects on lipid metabolism and oxidative stress in zebrafish hepatocytes.

**Figure 5.** KEGG enrichment analysis of transcriptional differential genes in each comparison group. (**A**) is the comparison group of SP and Control. (**B**) is the comparison group of BBR and Control. (**C**) is the comparison group of SP + BBR and Control. (**D**) is the comparison group of SP + BBR and SP. (**E**) is the comparison group of BBR and SP. (**F**) is the comparison group of SP + BBR and BBR comparison group.

### 3.2.3. Analysis of m6A Methylation and Transcriptome Association

For the correlation analysis of transcription levels and methylation levels, we counted the number of genes with changes in methylation and transcription levels, respectively, and the number of genes with significant differences in methylation and transcription levels at the same time in each comparison group, as seen in Table S5. We also analyzed the KEGG pathway for genes with significant differences in methylation and transcription together (Figure 6). Compared with the control group, the significantly differentially expressed genes in the lipid accumulation group were mainly involved in adrenergic signaling in cardiomyocytes. Compared with the control group, the significantly differentially expressed genes in the berberine group were mainly involved in PPAR signaling pathway, DNA replication, and arachidonic acid metabolism. Compared with the control group, the significantly differentially expressed genes in the lipid accumulation + berberine group were mainly involved in terpenoid backbone biosynthesis, synthesis and degradation of ketone bodies, ABC transporters, glycosaminoglycan biosynthesis − chondroitin sulfate/dermatan sulfate. Compared with the lipid accumulation + berberine group, the significantly differentially expressed genes in the lipid accumulation group were mainly involved in glycosaminoglycan biosynthesis − heparan sulfate/heparin, glycosaminoglycan biosynthesis − chondroitin sulfate/dermatan sulfate, beta−alanine metabolism, arginine biosynthesis, arginine and proline metabolism, and ABC transporters. Compared with the berberine group, the significantly differentially expressed genes in the lipid accumulation group were mainly involved in purine metabolism, PPAR signaling pathway, p53 signaling pathway, fatty acid degradation, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, and N−Glycan biosynthesis. Compared with the lipid accumulation + berberine group, the differentially expressed genes in the berberine group were mainly involved in VEGF signaling pathway, tyrosine metabolism, sphingolipid metabolism, regulation of actin cytoskeleton, PPAR signaling pathway, MAPK signaling pathway, glycosaminoglycan biosynthesis − keratan sulfate, focal adhesion, adherens junction, and autophagy—other. From the above results of KEGG analysis of differentially methylated coding genes, it can be speculated that some genes related to lipid metabolism and translation regulation in zebrafish hepatocytes were differentially modified by m6A methylation under lipid accumulation and berberine treatment, and thus their expression was regulated, which in turn affected the phenotype.

#### 3.2.4. Screening of Target Genes

The screening process for methylation-transcription level association analysis focused on genes that showed significant changes in methylation levels and also showed significant differences in transcript levels (*p* < 0.05) (Fc ≥ 2). From these, the *Camk1db* gene of zebrafish was screened as the key gene for the next study. Trends are summarized in Table S6. The M6A RNA methylation of the *Camk1db* gene was significantly lower in the lipid accumulation group compared with the control group, and the transcript level was reduced but not significantly different. The methylation levels and transcript levels were significantly higher in the berberine group compared to the lipid accumulation group. Methylation levels and transcript levels were also significantly increased in the lipid accumulation + berberine group compared to the lipid accumulation group.

#### *3.3. Validation of Camk1db Gene Methylation and Its Regulation of Cellular Homeostasis in ZFL*

#### 3.3.1. Validation of m6A Methylation Level and Transcription Level of Camk1db Gene

Changes in m6A methylation levels and gene expression levels occurring in the *Camk1db* gene in each treatment group were also evaluated at a low throughput level. Merip PCR was used to detect the m6A methylation level of the *Camk1db* gene (Figure 7A), and q-PCR was applied to detect the mRNA expression of the *Camk1db* gene (Figure 7B). Compared with the control group, the methylation and mRNA expression levels of *Camk1db* gene were significantly lower in the lipid accumulation group, and significantly higher in the berberine and lipid accumulation + berberine groups. Compared with the lipid accumulation group, the *Camk1db* gene methylation and mRNA expression levels were

significantly higher in the lipid accumulation + berberine group. The trends in each set are basically consistent with the previous Merip-seq high-throughput sequencing results.