**3.1 The role of nitric oxide in the Severe Cardiorenal Syndrome**

We developed a model of the SCRS based on CKD and depletion of NO availability. The rationale for these investigations was that the pathogenesis of CKD (in the presence of hypertension, diabetes or aging) is associated with low NO availability,76,77 while experimental SNX induces nephron number reduction in a healthy animal. Furthermore, in SNX, cardiac systolic function generally remains preserved, while in patients left ventricular dysfunction (LVSD) develops during the course of CKD progression.78

Reduced NO availability is considered a hallmark of CKD.46, 79 NO can function as an effector of the CRC by way of its vasodilatory action. It also modulates GFR and tubuloglomerular feedback.80 Reduced NO availability will result in tissue damage by oxidative stress. In extension to our proposal of the Cardiorenal Connection,44 we postulated that the balance between NO and ROS is a key modulator of the other cardiorenal connectors.81 Many effects of the other CRCs may be mediated by changes in the redox-balance and NO availability.82, 83

Also, it has been shown that constitutive NO production supports basal cardiac function.84 Apart from its role in endothelial dysfunction, NO availability also modulates cardiac contractility, as NO synthase (NOS) inhibition reduces cardiac output, and causes cardiac damage in high doses.85, 86

Origins of Cardiorenal Syndrome and the Cardiorenal Connection 117

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We thus hypothesized that a reduction in NO availability would accelerate the development of cardiac dysfunction. Indeed, treatment with an oral NO synthase (NOS) inhibitor (L-NNA), at a very low dose, induced NO depletion and severe cardiac dysfunction.87 Furthermore, proteinuria, severe glomerulosclerosis and cardiac interstitial fibrosis were worsened compared to rats with CKD without NOS inhibition. Another remarkable finding was that the effects on cardiorenal dysfunction but also on systemic NO production were irreversible after cessation of the NOS inhibitor, during a 7 week follow-up. A five times higher dose of NOS inhibition in control rats, which caused a similar level of hypertension and NO depletion, induced LVSD that was not as severe as in the CRS rats. Furthermore, all effects on blood pressure, cardiac function and NO availability were completely reversible, and had no effect on kidney structure and function. Combining NOS inhibition with SNX also, worsened kidney injury. The more severe hypertension and direct effects of NOS inhibition may have played a role in this.

We concluded that during CKD development the heart is very sensitive to depression of systemic NO availability. Compared to the normal kidney, the damaged kidney is more sensitive to alterations of NO availability as well, possibly because of a loss of autoregulation.88 Thus, maintaining adequate NO availability appears to be very important for progression of cardiorenal failure during progression of CKD, and the combination of CKD and NO depletion appears to produce a functional model of the SCRS in which cardiac function is further compromised.

That supplementation of NO is useful as a rescue therapy was shown in a subsequent study, where treatment with the oral tolerance-free NO donor molsidomine (MOLS) significantly improved cardiac diastolic and systolic function, abrogated mortality, and also slightly improved kidney function and injury.89 The cardiac effect of MOLS appeared to be a combination of reduced cardiac loading and improved contractility and relaxation. Systolic blood pressure was only mildly reduced and GFR was even slightly improved. Thus, MOLS appears to be an attractive and safe therapeutic option for CKD patients suffering from cardiac dysfunction of non-ischemic origin. The pathophysiology of the continuing low NO production in this model is likely very complex and may include low NOS expression or activity, substrate deficiency, high oxidative stress levels, and increased amounts of endogenous NOS inhibitors.79

In conclusion, the cardiorenal connection has intrigued scientists and physicians for centuries. The existence of a specific cardiorenal syndrome has been suggested since the start of the 20th century, but has recently gained widespread attention in the scientific literature. We proposed the Severe Cardiorenal Syndrome, in which CKD and HF induce derangements to cause a vicious cycle of cardiovascular damage and progression of failure of both organs. Understanding of pathophysiological mechanisms is expanding and animal models provide an invaluable tool to investigate the bidirectional nature of cardiorenal interactions.

#### **4. References**

[1] Parfrey PS, Harnett JD, Barre PE. The natural history of myocardial disease in dialysis patients. *J Am Soc Nephrol* 1991;2:2-12.

We thus hypothesized that a reduction in NO availability would accelerate the development of cardiac dysfunction. Indeed, treatment with an oral NO synthase (NOS) inhibitor (L-NNA), at a very low dose, induced NO depletion and severe cardiac dysfunction.87 Furthermore, proteinuria, severe glomerulosclerosis and cardiac interstitial fibrosis were worsened compared to rats with CKD without NOS inhibition. Another remarkable finding was that the effects on cardiorenal dysfunction but also on systemic NO production were irreversible after cessation of the NOS inhibitor, during a 7 week follow-up. A five times higher dose of NOS inhibition in control rats, which caused a similar level of hypertension and NO depletion, induced LVSD that was not as severe as in the CRS rats. Furthermore, all effects on blood pressure, cardiac function and NO availability were completely reversible, and had no effect on kidney structure and function. Combining NOS inhibition with SNX also, worsened kidney injury. The more severe hypertension and direct effects of NOS

We concluded that during CKD development the heart is very sensitive to depression of systemic NO availability. Compared to the normal kidney, the damaged kidney is more sensitive to alterations of NO availability as well, possibly because of a loss of autoregulation.88 Thus, maintaining adequate NO availability appears to be very important for progression of cardiorenal failure during progression of CKD, and the combination of CKD and NO depletion appears to produce a functional model of the SCRS in which cardiac

That supplementation of NO is useful as a rescue therapy was shown in a subsequent study, where treatment with the oral tolerance-free NO donor molsidomine (MOLS) significantly improved cardiac diastolic and systolic function, abrogated mortality, and also slightly improved kidney function and injury.89 The cardiac effect of MOLS appeared to be a combination of reduced cardiac loading and improved contractility and relaxation. Systolic blood pressure was only mildly reduced and GFR was even slightly improved. Thus, MOLS appears to be an attractive and safe therapeutic option for CKD patients suffering from cardiac dysfunction of non-ischemic origin. The pathophysiology of the continuing low NO production in this model is likely very complex and may include low NOS expression or activity, substrate deficiency, high oxidative stress levels, and increased amounts of

In conclusion, the cardiorenal connection has intrigued scientists and physicians for centuries. The existence of a specific cardiorenal syndrome has been suggested since the start of the 20th century, but has recently gained widespread attention in the scientific literature. We proposed the Severe Cardiorenal Syndrome, in which CKD and HF induce derangements to cause a vicious cycle of cardiovascular damage and progression of failure of both organs. Understanding of pathophysiological mechanisms is expanding and animal models provide an invaluable tool to investigate the bidirectional nature of cardiorenal

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endogenous NOS inhibitors.79

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**8**

*Canada*

**Sub-Types and Therapeutic**

Margot Davis and Sean A. Virani

*University of British Columbia*

**Management of the Cardiorenal Syndrome**

Cardiorenal syndrome (CRS) describes the inter-relationship and complex pathophysiological processes by which dysfunction of either the heart or the kidneys is related to dysfunction in the other organ system. Historical definitions may have been overly simplistic; newer definitions have tried to capture the complex interactions and feedback processes which exist between the two organs. These definitions classify the CRS into five discrete categories, based on both the organ system in which the primary

The CRS is more common than many clinicians realize. Over one third of patients in heart failure (HF) registries have evidence of renal dysfunction, and a similar proportion of dialysis patients have symptoms of congestive HF or clinical evidence of left ventricular dysfunction (Adams et al., 2005; Stack & Bloembergen, 2001). Importantly, the presence of the CRS is a strong adverse prognostic marker in patients with either primary cardiac

While originally thought to reflect renal hypoperfusion secondary to low cardiac output, it is now understood that the CRS is underpinned by far more complex processes. From a hemodynamic standpoint, it seems likely that venous congestion is at least as important to the pathophysiology of disease progression as is low forward flow. Other contributing factors include activation of neurohormonal axes, including the sympathetic nervous system and the renin-angiotensin-aldosterone system, as well as oxidative injury and endothelial dysfunction (Bock & Gottlieb, 2010). More recently, it has become recognized that anemia may also be intimately involved in the process, both as a consequence and as a causative agent of the CRS. Finally, it is well recognized that many common risk factors for cardiovascular disease and for chronic kidney disease (CKD) co-exist in these patient

Management of the CRS is challenging. Therapies for HF often cause worsening of renal function, while treatment of renal failure commonly involves fluid administration, which may precipitate disease decompensation among those with HF. Unfortunately, most large randomized trials in the HF population have excluded patients with elevated serum creatinine levels, and there is little evidence to guide therapy in this group of patients. Observational studies suggest that there may be a mortality benefit associated with the use

dysfunction occurs and the time course of disease development/progression.

**1. Introduction**

disease or primary renal disease.

cohorts.


**4. Discussion**

The purpose of this paper is to study an improved pedestrian navigation and location method based on the combination of indoor map assistance and adaptive particle filter. For the multi constraint integrated navigation method that only relies on the wearable inertial sensor node network for indoor pedestrians, there is an unavoidable problem of the accumulation and divergence of navigation errors. It is urgent to further improve the accuracy of the navigation system based on the available indoor auxiliary information. Considering that the indoor architectural plan is the most basic and accessible information source in the indoor rescue process, a navigation and positioning method based on the combination of indoor map assistance and particle filter is proposed. This method makes full use of the existing indoor map constraint information to assist in improving the performance of the pedestrian navigation and positioning system based on inertial sensor network. In this paper, the algorithm framework of indoor pedestrian navigation based on map assistance is designed. Combined with the characteristics that pedestrians cannot actually go through the walls and other obstacles when moving in buildings. By establishing the filtering algorithm under the property of particle "not going through the wall", the effective constraints on navigation error are realized; In view of the pedestrian's initial entry into an unfamiliar indoor environment, a map aided localization algorithm based on global search is proposed under the condition of unknown initial position and heading; In order to solve the problem that a large number of particles are required to complete the global search, which leads to low computational efficiency, a particle resampling method based

on adaptive particle number is proposed. While maintaining the accuracy of navigation and positioning, it also improves the computing efficiency and achieves accurate indoor positioning in unfamiliar environments. On this basis, based on indoor map constraints, the problem of inertial accumulation error divergence is well suppressed, which provides a strong support for pedestrian indoor navigation and positioning with high precision and reliability.

Through the verification and analysis of simulation data and measured data, the pedestrian navigation and positioning method based on the combination of improved indoor map assistance and adaptive particle filter proposed in this paper is suitable for the conditions of known and unknown initial position and heading. In the indoor environment of about 2600 m2, when the total distance exceeds 415.44 m, the mean error and the maximum error of the position relative to the reference point are both less than 2 m. It effectively suppresses the pedestrian navigation error based on inertial devices, and greatly improves the calculation efficiency, which can meet the needs of indoor pedestrians for a long time.

In fact, in the process of motion, both lateral and longitudinal errors are derived from step length error and heading errors. In a one-step correction process, if it is calculated that the coordinates of a particle in the lateral or longitudinal direction are in the inaccessible area, the particle is in the inaccessible area and needs to be resampled and given new navigation parameters. The pedestrian position is then calculated by the weighted average method.

The method proposed in this paper also has some limitations. It is based on the indoor building plan, and combines the characteristics of particles "not going through the wall" to constrain and modify the pedestrian trace. When the indoor structure is simple and the environment is open, then the distance between the walls on both sides of the walkway is very far, it is difficult to correct the pedestrian trace through this method.


*Article* **Monkfish Peptides Mitigate High Fat Diet-Induced Hepatic Steatosis in Mice**

**Jiena Ye 1,†, Xiaoxiao Tian 1,†, Qiongfen Wang 2, Jiawen Zheng 1, Yanzhuo Yang 1, Baogui Xu 1, Shuai Zhang 1, Falei Yuan 1,\* and Zuisu Yang 1,\***

**Abstract:** Non-alcoholic fatty liver disease (NAFLD) is a hepatic metabolic syndrome usually accompanied by fatty degeneration and functional impairment. The aim of the study was to determine whether monkfish peptides (LPs) could ameliorate high-fat diet (HFD)-induced NAFLD and its underlying mechanisms. NAFLD was induced in mice by giving them an HFD for eight weeks, after which LPs were administered in various dosages. In comparison to the HFD control group: body weight in the LP-treated groups decreased by 23–28%; triacylglycerol levels in the blood decreased by 16–35%; and low-density lipoproteins levels in the blood decreased by 23–51%. Additionally, we found that LPs elevated the activity of hepatic antioxidant enzymes and reduced the inflammatory reactions within fatty liver tissue. Investigating the effect on metabolic pathways, we found that in LP-treated mice: the levels of phospho-AMP-activated protein kinase (p-AMPK), and phospho-acetyl CoA carboxylase (p-ACC) in the AMP-activated protein kinase (AMPK) pathway were up-regulated and the levels of downstream sterol regulatory elementbinding transcription factor 1 (SREBP-1) were down-regulated; lipid oxidation increased and free fatty acid (FFA) accumulation decreased (revealed by the increased carnitine palmitoyltransferase-1 (CPT-1) and the decreased fatty acid synthase (FASN) expression, respectively); the nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant pathway was activated; and the levels of heme oxygenase-1 (HO-1) and nicotinamide quinone oxidoreductase 1 (NQO1) were increased. Overall, all these findings demonstrated that LPs can improve the antioxidant capacity of liver to alleviate NAFLD progression mainly through modulating the AMPK and Nrf2 pathways, and thus it could be considered as an effective candidate in the treatment of human NAFLD.

**Keywords:** monkfish peptides; NAFLD; the AMPK/Nrf2 pathway; lipid metabolism

#### **1. Introduction**

Non-alcoholic fatty liver disease (NAFLD) refers to a clinicopathological syndrome characterized by inflammation of the liver lobule and hepatic parenchymal steatosis. NAFLD can range from simple steatosis to more severe liver diseases, such as steatohepatitis, fibrosis, and even liver cancer [1]. The occurrence of NAFLD worldwide is between 14% and 24% of liver diseases, yet the underlying mechanism of the condition remains unclear [2]. HFD provides a high amount of saturated fatty acids, especially palmitic acid (C16:0). The excess of palmitic acid generates an increase in the hepatic inflammatory response [3]. With a lack of clinical intervention options, diet regulation remains the most effective means of managing NAFLD [4].

Recent research has focused largely on the effects of mitochondrial dysfunction, insulin resistance, obesity, and changes to the gut microbiome on the development of NAFLD. Mitochondrial dysfunction is involved in the progression of NAFLD, which changes the

**Citation:** Ye, J.; Tian, X.; Wang, Q.; Zheng, J.; Yang, Y.; Xu, B.; Zhang, S.; Yuan, F.; Yang, Z. Monkfish Peptides Mitigate High Fat Diet-Induced Hepatic Steatosis in Mice. *Mar. Drugs* **2022**, *20*, 312. https://doi.org/ 10.3390/md20050312

Academic Editors: Bin Wang and Chang-Feng Chi

Received: 9 April 2022 Accepted: 2 May 2022 Published: 5 May 2022

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**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/).

homeostasis of fatty liver, thereby producing higher levels of malondialdehyde (MDA) and reactive oxygen species (ROS) [5,6]. When oxidative stress occurs, nuclear factor erythroid-2-related factor 2 (Nrf2) combines with antioxidant redox elements to protect cells against stress. Together, they regulate the expression of downstream genes such as heme oxygenase-1 (HO-1) and nicotinamide quinone oxidoreductase 1 (NQO1) [7,8]. Both liraglutide and tetrahydrocurcumin ameliorated NAFLD by increasing the mRNA levels of Nrf2, HO-1, GCLM, and NQO1 [9,10]. The body's antioxidant defense enzymes include catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD). Decreasing these enzymes can also lead to oxidative stress.

Insulin resistance breaks down the adipose tissue, which in turn releases more free fatty acids (FFA), adipokines, and inflammatory factors to the liver and adipose tissue. Much attention has been paid to reducing the level of FFA in the treatment of NAFLD [11]. AMP-activated protein kinase (AMPK) is recognized as a major energy-related protein kinase that mediates the development of liver dysfunction [11]. Acetyl CoA carboxylase (ACC) is the target molecule of AMPK. ACC catalyzes the production of malonyl-CoA by prompting the synthesis and elongation of fatty acids as well as inhibiting the oxidation of fatty acids through the inhibition of carnitine palmitoyltransferase-1 (CPT-1) [12]. AMPK phosphorylation can induce ACC phosphorylation and inhibit ACC activity. ACC enters a negative feedback loop that increases the content of CPT-1 and oxidizes fatty acids, thus reducing fatty acid content in the liver [13]. The activation of AMPK weakens the proteolytic process of sterol regulatory element-binding proteins (SREBP)-1c and leads to accelerated fatty acid oxidation, thereby eliminating the abnormal accumulation of FFA. Liensinine significantly ameliorated HFD-triggered hepatic oxidative stress and dyslipidemia by mediating Nrf2/AMPK signaling, and dimethyl fumarate similarly mitigated NAFLD progression by mediating Nrf2, SREBP-1c, and nuclear factor-κB (NF-κB) signaling [14,15].  Obesity is closely related to NAFLD, because it is characterized by liverneutral lipid accumulation [18,19]. One of the morphological features of steatosis is the accumulation of lipid droplets in hepatic parenchymal cells.

Bioactive peptides, generally composed of three to twenty amino acid residues, are known for their antihypertensive and antilipemic properties [20]. Milk is recognized as a major source of bioactive peptides and fish proteins may be another important source [21]. Although it has been shown that protein hydrolysates from yellow catfish alleviate mouse NAFLD through inhibiting lipid metabolism, the underlying mechanisms have yet to be studied [22]. Herring milt protein hydrolysates attenuate insulin resistance from excessive fat consumption [23]. Fish protein hydrolysates can regulate lipid metabolism and improve dyslipidemia in Zucker rats [24,25]. Every year, approximately 12% of aquatic products worldwide are used for non-food purposes [24]. Monkfish meat has traditionally been considered a low-value food. Monkfish peptides (LPs) have been found to have strong antioxidant activities and fat absorption capabilities [26–28]. However, the regulatory effects of LPs on NAFLD, and the underlying molecular mechanisms are still unclear. Thus, in the current study, we tested the protective effects of LPs against high-fat diet-induced NAFLD in mice. We also sought to determine whether the protective effects of LPs involve the AMPK and Nrf2 signaling pathways.

#### **2. Results**

#### *2.1. Identification and Protein Analysis of LPs*

LPs with a molecular weight of less than 1 kDa were harvested, and 198 mg of LPs were prepared from 100 g of the fish meat. The total yield was 0.198%. Using database alignment, the corresponding sequences were found in the protein database of the Lophiiformes species in the National Center for Biotechnology Information (NCBI). As shown in Supplementary Table S1, database matching obtained 98 low molecular weight peptides that consisted of 17 heptapeptides, 31 octapeptides, 26 nonapeptides, 13 decapeptides, 9 undecapeptides, and 2 dodecapeptides. There were 96 peptides with two charges and 2 peptides with one

charge. A total of 3276 free peptides were identified by de novo sequencing, and the results did not overlap with the data obtained through the database comparison method. The shortest free peptide identified through de novo sequencing was a tetrapeptide and the longest was an octapeptide. Amino acid local confidence (ALC) is the confidence level of de novo sequencing data. Generally, ALC > 80% is adequately reliable. Accordingly, Supplementary Table S2 lists 66 free peptides with ALC > 80% identified by the de novo sequencing results. They had a distribution ranging from tetrapeptide to nonapeptide, including 13 tetrapeptides, 32 pentapeptides, 13 hexapeptides, 4 heptapeptides, and 4 nonapeptides. Additionally, 39 polypeptides had two charges and 27 polypeptides had one charge. Supplementary Table S3 lists the types of proteins. Each of these proteins plays a different role in organisms. For example, the protein nicotinamide adenine dinucleotide (NADH) dehydrogenase is an enzyme located in the inner membrane of mitochondria that catalyzes the transfer of electrons from NADH to coenzyme Q. This enzyme is an "entry enzyme" of oxidative phosphorylation in the mitochondria [29]. ATPase, also known as adenosine triphosphatase, catalyzes the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and a phosphate ion. Cytochrome c oxidase transfers the electron of a respiratory substrate directly to molecular oxygen (i.e., with automatic oxidation) through the cytochrome system. This shows that there is an abundance of low-molecular-weight peptides in LPs, of which tetrapeptides and pentapeptides account for most. These protein types also show that LPs are likely to have good antioxidant activity, which is consistent with the results of previous studies [27].

#### *2.2. LPs Lowered Body Weight and Liver Weight*

As shown in Figure 1A, after seven days of adaptive feeding, the mice in the experimental groups were fed with a high-fat diet (HFD) for eight weeks. The average body weight of mice in the HFD group increased 82% more than that of the normal diet (ND) group. From Week 9 onward, all groups were fed normal diets (Figure 1D) during which the average weight of all the experimental groups decreased (LP-50, 24%, LP-100, 23%, LP-200, 28%, and silibinin (SIL) 32%). After another four weeks of intervention, the average liver weight and liver weight index of the HFD group, calculated as the percentage of liver weight divided by body weight, was higher than that of the ND group by 51% and 34% (*p* < 0.01 Figure 1B,C). Meanwhile the average liver weight and liver weight indexes of the SIL, LP-50, LP-100, and LP-200 groups decreased by 32%, 17%, 29%, and 34%, and 19%, 13%, 22%, and 25%, respectively, as compared with the HFD group (*p* < 0.01 Figure 1B,C).

**Figure 1.** (**A**): Effects of LPs on the alterations of body weight. (**B**): Effects of LPs on the alterations of liver weight. (**C**): Effects of LPs on the liver index (Liver weight/Body weight × 100) of mice with non-alcoholic fatty liver disease (NAFLD). (**D**): Research schedule of NAFLD mice. Values are presented as means ± SD. Values with different labels are significantly different in the groups (\* *p* < 0.05, \*\* *p* < 0.01 vs. ND group, # *p* < 0.05, ## *p* < 0.01 vs. HFD group).

#### *2.3. Effects of LPs on Blood Lipids*

The results of the blood lipid indexes found in the serum are shown in Figure 2. Compared to the ND group serum, total cholesterol (TC) levels increased by a substantial 68% in the HFD group (*p* < 0.01, Figure 2A). Meanwhile the SIL and LPs-treated groups (LP-50, LP-100, and LP-200) decreased by 30%, 16%, 29%, and 35%, respectively, in contrast to the HFD group (*p* < 0.05, Figure 2A). There was no significant difference between the LP-200 group and the ND group, and the repair effect was the best between them. The changes in triacylglycerol (TG) and low-density lipoproteins (LDL)-c levels in the serum were the same. In comparison with the ND group, the TG and LDL-c concentrations in the HFD group increased by 91% and 137%, respectively (*p* < 0.01, Figure 2B,C). Compared with the HFD group, the concentration of TG decreased in the SIL, LP-50, LP-100, and LP-200 groups by 42%, 13%, 29%, and 45%, respectively. Furthermore, the concentration of LDL-c decreased in the SIL, LP-50, LP-100, and LP-200 groups by 47%, 23%, 41%, and 51%, respectively (*p* < 0.05, Figure 2B,C). The concentrations of TG and LDL-c in the SIL group and the LP-200 group were not significantly different from the ND group. Compared with the ND group, the concentration of high-density lipoproteins (HDL)-c in each experimental group decreased significantly (*p* < 0.05, Figure 2D). Compared with the HFD group, the level of HDL-c in the LP-50 group was not significantly different, and the level of HDL-c in the SIL, LP-100, and LP-200 groups increased by 46%, 9%, 23%, and 50%, respectively (*p* < 0.05, Figure 2D). The FFA levels in the serum of the HFD group were twice the amount of that in the ND group (*p* < 0.01, Figure 2E). The FFA levels in the serum decreased in the SIL, LP-50, LP-100, and LP-200 by 61%, 28%, 43%, and 62%, respectively, compared to the HFD group (*p* < 0.01, Figure 2E). There was no significant difference between the LP-200 group and the ND group, and the repair effect was the best in the LP-200 group. Thus, it can be concluded that the LP-200 group is most effective in regulating blood lipid levels.

**Figure 2.** *Cont*.

**Figure 2.** Effects of LPs on plasma total cholesterol (TC) (**A**), triacylglycerol (TG) (**B**), low-density lipoprotein (LDL-c) (**C**), high-density lipoprotein (HDL-c), (**D**) and free fatty acids (FFA) (**E**). Values are presented as means ± SD. Values with different labels are significantly different in the groups (\* *p* < 0.05, \*\* *p* < 0.01 vs. ND group, # *p* < 0.05, ## *p* < 0.01 vs. HFD group).

#### *2.4. Effects of LPs on Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT)*

An increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity in the blood is usually interpreted as a marker for lesions [30]. The ALT and AST levels in the HFD group increased by 105% and 93%, respectively, indicating that an HFD gave rise to liver injuries (*p* < 0.01, Figure 3). Compared with the HFD group, ALT contents in the serum of the SIL, LP - 50, LP-100, and LP-200 groups decreased by 52%, 20%, 37%, and 55%, respectively, and the concentration of AST in the SIL, LP-50, LP-100, and LP-200 decreased by 47%, 12%, 31%, and 44%, respectively (*p* < 0.01, Figure 3). Although no statistical significance was found among the SIL, LP-200, and ND groups, AST levels in the LP-50 and the LP-100 groups were significantly higher than baseline, a trend similarly followed by ALT levels. The ALT levels of LP-50 and LP-100 were significantly higher than those of the ND group (*p* < 0.01 Figure 3B), and so were the levels in the SIL group (*p* < 0.05 Figure 3B). The LP-200 group did not show any significant difference.

**Figure 3.** Effects of LPs on the levels of aspartate aminotransferase (AST) (**A**) and alanine aminotransferase (ALT) (**B**). Values with different labels are significantly different in the groups (\* *p* < 0.05, \*\* *p* < 0.01 vs. ND group, ## *p* < 0.01 vs. HFD group).

#### *2.5. Effects of LPs on Antioxidant Capacity*

The antioxidant capacity of liver tissues in different groups is shown in Table 1. The levels of total antioxidant capacity (T-AOC), CAT, SOD, and GSH-Px in the HFD group were significantly lower than those in the ND group (*p* < 0.01). The levels of these same indicators in the SIL and LPs-treated groups were significantly lower than those in the HFD group (*p* < 0.01). There was no significant difference between the SIL, LP-200, and ND groups. The MDA level in the HFD group was significantly higher than that in the ND group. Compared with the HFD group, the MDA content in the SIL, LP-100, and LP-200 groups significantly decreased (*p* < 0.05).

**Table 1.** Effects of LPs on Antioxidant Capacities in Liver.

The data are expressed as the mean ± SD (*n* = 8 per group). \*\* *p* < 0.01 vs. ND group. ## *p* < 0.01 vs. HFD group. \* *p* < 0.05 vs. ND group. # *p* < 0.05 vs. HFD group. Groups: ND = normal control; HFD = HFD control; SIL = HFD + silibinin; LP-50 = HFD + 50 LP mg/kg; LP-100 = HFD + 100 LP mg/kg; LP-200 = HFD + 200 LP mg/kg.

#### *2.6. Effects of LPs on the Level of Inflammatory Factors*

To determine the effects of LPs on inflammation, we examined the levels of IL-6, IL-1β, TNF-α, and IFN-γ in the blood. The concentration of TNF-α in the HFD group increased 94% in comparison with the ND group (*p* < 0.01, Figure 4A). The concentration of TNF-α in the SIL, LP-50, LP-100, and LP-200 groups decreased by 42%, 18%, 36%, and 45%, respectively, compared to the HFD group levels (*p* < 0.01, Figure 4A). There was no significant difference between the LP-200 group and the ND group. The concentration of IL-6 in each experimental group significantly increased in comparison to the ND group (*p* < 0.05, Figure 4B). The concentration of IL-6 in the SIL, LP-50, LP-100, and LP-200 groups decreased by 38%, 14%, 26%, and 39%, respectively, compared with the HFD group (*p* < 0.01, Figure 4B). The concentrations of IL-1β and IFN-γ in the HFD group were significantly higher than those in the ND group (*p* < 0.01, Figure 4C,D). Compared with the HFD group, IL-1β levels in the SIL, LP-50, LP-100, and LP-200 groups showed significant decreases of 46%, 16%, 40%, and 45%, respectively, and as did the levels of IFN-γ in these groups with decreases of 45%, 17%, 42%, and 51%, respectively (*p* < 0.01, Figure 4C,D). From these results, it can be concluded that the SIL group and the LP-200 group were the best at inhibiting the inflammatory response of mice with NAFLD.

**Figure 4.** Effects of LPs on the level of inflammatory factors: TNF-α (**A**), IL-6 (**B**), IL-1β (**C**), and IFN-γ (**D**). Data are presented as means ± SD. Values with different labels are significantly different among the groups (\* *p* < 0.05, \*\* *p* < 0.01 vs. ND group, ## *p* < 0.01 vs. HFD group).

#### *2.7. Effects of LPs on Hepatic Histopathology*

The results of the histopathological examination of the liver tissue are shown in Figure 5. The liver volume of the HFD group was enlarged, and white particles could be seen on the surface. In the SIL group and LPs groups, the size of the liver shrank, and the white particles of fat decreased (Figure 5A). Hematoxylin and eosin (H&E) staining revealed hypertrophy of the liver cells in the HFD group. The fat tissue was mainly composed of lipid droplets, and the hepatocyte cords were disordered. Compared with the HFD group, the LP-50 and LP-100 groups had fewer lipid droplets. The SIL and LP-200 groups greatly inhibited the accumulation of lipid droplets, attenuating the swelling and disorder of hepatocytes (Figure 5B). The oil red O staining and transmission electron microscopy (TEM) results were consistent with those of the H&E staining. In the ND group, there were no obvious lipid droplets in the hepatocytes. In contrast, there were many red lipid droplets in the model group. The numbers of these droplets in the LPs groups decreased, especially in the LP-200 group (Figure 5C). Hepatic steatosis is related to changes in the mitochondria of hepatocytes [31]. The results show that the electron density of lipid droplets was low, meaning the content of saturated fatty acids was high, and that the structure of hepatocytes in the model group was fuzzy with a blurry boundary. In the normal group, the amount of endoplasmic reticulum was reduced, and there were a large number of round lipid droplets of different sizes. After the intervention of SIL and LPs, the swelling degree of liposomes was reduced, the changes in the mitochondria and endoplasmic reticulum were significantly reduced, and the number of lipid droplets was significantly reduced. The electron density of liposomes was higher than that of the model group (Figure 5D). Livers also increased in size during HFD intervention and H&E staining showed macrovesicular steatosis, ballooning, and inflammation. According to the NAS scoring standard, the structural changes in the liver tissue in 10 visual fields were observed under a microscope, and the results in Figure 5E were obtained, which shows the NAS score: livers showed an average NAS score of 4.8. Quantitative analysis of oil red O staining showed that the fat area in the HFD group was 20%, while that in the LPs groups decreased to 14%, 11%, and 5%, respectively (Figure 5F).

**Figure 5.** *Cont*.

**Figure 5.** *Cont*.

**Figure 5.**  Data are presented as means ± SD. Values with different labels are significantly different among the groups (\* *p* < 0.05, \*\* *p* < 0.01 vs. ND group, # *p* < 0.05, ## *p* < 0.01 vs. HFD group).

#### *2.8. Effects of LPs on the Protein Expressions of AMPK Pathways*

To clarify the molecular mechanism of LPs inhibiting the abnormal development of FFA, we examined the expression level of the AMPK pathway-related proteins. The phosphorylation level of the AMPK and ACC proteins in the HFD group was much lower than that in the ND group (*p* < 0.01, Figure 6). The expression of the sterol regulatory element-binding transcription factor 1 (SREBP-1) protein downstream was significantly up-regulated, resulting in a significant increase in fatty acid synthase (FASN) expression compared with the ND group (*p* < 0.01, Figure 6), as well as a significant decrease in the CPT-1 protein (*p* < 0.01). After the intervention of SIL and LPs, the phosphorylation levels of AMPK and ACC were significantly enhanced compared with the HFD group (*p* < 0.01, Figure 6), and the expression of the SREBP-1 protein down-stream was downregulated. Thus, the content of the FASN protein was decreased and the protein content of CPT-1 was increased. The expression of peroxisome proliferator-activated receptor alpha (PPARα) in the HFD group was significantly decreased (*p* < 0.01, Figure 6), and its expression was significantly upregulated by SIL and LPs (*p* < 0.01, Figure 6). These results show that LPs can inhibit the abnormal accumulation of FFA by regulating the synthesis of lipid metabolism-related proteins, thus increasing lipid β oxidation and reducing fatty acid synthesis.

**Figure 6.** *Cont*.

**Figure 6.** Effects of LPs on the AMP-activated protein kinase (AMPK) pathway-related proteins: (**A**) Western blot analysis of AMPK, phospho-AMP-activated protein kinase (p-AMPK), acetyl CoA carboxylase (ACC), p-ACC, carnitine palmitoyltransferase-1 (CPT-1), sterol regulatory elementbinding transcription factor 1 (SREBP-1), fatty acid synthase (FASN), peroxisome proliferatoractivated receptor alpha (PPARα), and β-actin. The expressions of p-AMPK (**B**), p-ACC (**C**), CPT-1 (**D**), PPARα (**E**), SREBP-1 (**F**), and FASN (**G**). Values are presented as means ± SD. Values with different labels are significantly different among the groups (\* *p* < 0.05, \*\* *p* < 0.01 vs. ND group, ## *p* < 0.01 vs. HFD group).

#### *2.9. Effects of LPs on the Expressions of Hepatic Nrf2 Pathway Proteins*

Studies have shown that upregulation of Nrf2 expression in the liver can improve NAFLD [32–34]. Therefore, we can speculate that LPs mitigate hepatic oxidative stress through the Nrf2 pathway. The expression levels of Nrf2, downstream HO-1, and NQO1 in the HFD group were significantly lower than the levels of those indicators in the ND group (*p* < 0.01, Figure 7). Compared with the HFD group, Nrf2 expression was significantly increased after SIL and LPs intervention (*p* < 0.01, Figure 7), and the expression levels of HO-1 and NQO1 in the SIL and LP-200 groups significantly increased (*p* < 0.05, Figure 7).

**Figure 7.** Effects of LPs on the Nrf2 pathway-related proteins: (**A**) Western blot analysis of Nrf2, HO-1, NQO1, and β-actin. The expressions of Nrf2 (**B**), HO-1 (**C**), and NQO1 (**D**). Values are presented as means ± SD. Values with different labels are significantly different among the groups (\*\* *p* < 0.01 vs. ND group, # *p* < 0.05, ## *p* < 0.01 vs. HFD group).

#### **3. Discussion**

In this study, we investigated the effects of LPs on liver metabolism. We found that LPs were able to lower body weight in LP-50, LP-100, and LP-200 groups by 24, 23, and 28 percent; to lower TG levels by 13, 29, and 45 percent; to lower LDL-c levels by 23, 41, and 51 percent; and to lower FFA levels by 28, 43, and 62 percent. After the administration of LPs, the markers for liver lesions were decreased, antioxidant capacities were enhanced, and inflammation inhibited. Upon further investigation, we found the mechanism of action to be via AMPK and Nrf2 pathways.

The conventional hypothesis regarding the pathogenesis of NAFLD is the "second hit" theory proposed by Day and James in 1998 [35]. In this theory, the "first strike" is liver fat deposition and hepatocyte fatty change in response to various lipid metabolism disorders. Insulin resistance (IR) is considered to be the central link of the first strike. The accumulation of fat in the liver is also related to a high-calorie diet, sedentary lifestyle, and possible genetic predisposition. The "second hit" follows, which mainly includes the effects of oxidative stress, lipid peroxidation, mitochondrial dysfunction, and increased production of serum endotoxin and inflammatory factors. This "second hit" increases the liver's susceptibility to inflammatory necrosis and fibrosis, in turn accelerating the progression of NAFLD. Inflammation damage can also lead to non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and even liver cancer.

In recent years, marine active peptides have received growing attention due to their anti-hypertensive and anti-oxidative properties [36]. Mendisa et al. isolated bioactive peptides from squid and found them to inhibit lipid peroxidation by using a linoleic acid model [37]. In this study, functions of LPs were studied in a murine model of NAFLD, induced by an HFD. Excessive intake of fat leads to disorders of lipid metabolism in the liver, which can result in the clinical features of NAFLD such as fatty degeneration with increased serum FFA level and dyslipidemia [38,39]. As shown in Figures 1B and 2, the liver index of the LPs and SIL groups decreased significantly compared with the HFD control group (*p* < 0.05). The TG, TC, LDL-C, and FFA levels significantly increased in the HFD group, and decreased in the experimental groups. AST and ALT are markers generally used for identifying liver lesions and dysfunctions and also can be used as indicators of NAFLD [40]. The levels of AST and ALT in the HFD group significantly increased (*p* < 0.01), indicating that liver damage had occurred in the HFD mice. The AST and ALT levels in the LPs group, however, significantly decreased (*p* < 0.05), suggesting that the effect of NAFLD on the liver was somewhat mitigated by the peptides.

Oxidative stress plays a critical role in the progression of NAFLD as the excess of reactive oxygen species directly induces lipid peroxidation [41]. MDA accumulation is used as a marker for oxidative stress as it is one of the major products of lipid peroxidation, and the markers of the endogenous antioxidant system that counteract this stress are T-AOC, GSH, SOD, and CAT [42]. Table 1 shows a significantly higher level of MDA in the HFD group compared to the ND group (*p* < 0.01), while T-AOC, GSH, SOD, and CAT levels were significantly decreased (*p* < 0.05). The level of MDA in the LPs group was improved. Based on these data, we determined that the protective effects of LPs in NAFLD are due to partially enhancing the endogenous antioxidant system and reducing the MDA level in hepatocytes, thus protecting the liver from excessive reactive oxygen species.

Inflammatory cytokines, also important regulators, are often overexpressed in NAFLD [43]. For example, IL-6 inhibits fat decomposition and promotes fat storage in NAFLD, while TNF-α inhibits the transport of lipids and lipoproteins, leading to an accumulation of lipids in liver cells [44]. In addition, TNF-α also mediates superoxide formation and lipid peroxidation, triggering a cascade of cytokine responses [45]. By examining the levels of TNF-α, IL-1β, and IL-6 in mice during our study, we found LPs to have an inhibitory effect on the HFD-induced inflammation.

H&E staining is a more intuitive approach to analyzing the characteristics of NAFLD, and oil red O staining can be used to show the accumulation of fat in liver tissues. Additionally, in this study, the alterations the in morphology of the liver cell mitochondria were observed by transmission electron microscopy.  Compared with the HFD group, liver samples from the LPs group appeared ruddy and smooth, with neater hepatic cords, and a reduced amount of fat.

AMPK is a critical protein in energy regulation and *p*-AMPK reduces lipid synthesis through accelerating fatty acid β-oxidation. The AMPK-signaling pathway further affects liver lipid metabolism via downstream targets such as CPT-1 and FAS [46]. The FASN level is regulated by the inhibition of SREBP1 expression in response to AMPK phosphorylation, resulting in a decrease in lipid synthesis. AMPK also regulates lipid metabolism by adjusting the ACC and CPT-1 pathways, with previous studies having shown the role of PPAR-α in elevating fatty acid oxidation through the induction of CPT-1 expression [47]. The results of Figure 6 show that the HFD group had significantly reduced levels of p-AMPK, p-ACC, PPAR-α, and CPT-1, and significantly increased SREBP-1 and FASN content (*p* < 0.05). Notably, HFD generates mitochondrial dysfunction, and active PPAR-α negatively interferes with NF-κB activation [48]. Thus, liver PPAR-α downregulation in obesity also has a pro-inflammatory connotation. These outcomes indicate that in the HFD group, β-oxidation of fat in the liver was decreased and fatty acid synthesis was increased, leading to lipid accumulation in liver cells, eventually causing NAFLD. It can be seen that LPs altered the homeostasis of hepatic lipids and up-regulated the expression of the p-AMPK protein. A significant increase in PPAR-α and CPT-1 expression, and a significant decrease in the SREBP-1 and FASN content were seen in the LPs groups in a dose-dependent manner (*p* < 0.05).  Recent studies demonstrated that AMPK regulated Nrf2 nuclear translocation to induce HO-1 gene expression [49]. Nrf2 plays a pivotal role in cellular defenses against oxidative stress by regulating the gene expression of antioxidant and detoxication enzymes [50].

Next, we measured the expression of Nrf2 and its associated pathways. As shown in Figure 7, regardless of the dosage, LPs are able to significantly increase the level of Nrf2. As well as this, the levels of HO-1 and NQO1 in mouse liver are increased. This indicates that AMPK affects the expression of HO-1 by regulating Nrf2. Therefore, LPs may ameliorate high-fat diet-related oxidative damage by activating the AMPK pathway to modulate the Nrf2-mediated antioxidant pathway.

#### **4. Materials and Methods**

#### *4.1. Preparation of Monkfish Muscle Peptides*

Monkfish (*Lophius litulon*) with an approximate body length of 39.21 ± 1.71 cm was purchased from a local market in Zhoushan, Zhejiang Province. Fish muscle peptides were obtained following an already described procedure [22]. Briefly, tissue fat was removed by adding 95% ethanol extraction followed by centrifugation. The precipitate was subsequently washed with distilled water and centrifuged again. The defatted monkfish meat was digested with neutral protease (E/S, 2000 U/g) at 45 ◦C for five hours. The hydrolysate was separated using a 1000 Da ultrafiltration membrane, and a peptide solution with a molecular weight of 1000 Da or less was collected. Free amino acids were removed by a 150 Da ultrafiltration membrane and were vacuum freeze-dried to obtain LPs with a molecular weight less than 1000 Da. Ultra-high pressure liquid chromatography and mass spectrometry (UPLC-MS) were performed. PEAKS studio was used to analyze the spectrum information, and the NCBI database was used for comparison and de novo sequencing analysis to obtain the structural composition of <1 kDa peptide.

#### *4.2. Animals and Treatments*

Six weeks old male ICR mice were provided by the Experimental Animal Center of Zhejiang Province. The Experimental Animal Ethics Committee of Zhejiang Ocean University approved the procedures for the use of the laboratory animals (Certificate Number SCXK ZHE2014-0001). Mice were housed in an animal facility at a room temperature of 20–25 ◦C and had free access to water and food. After seven days of adaptive feeding, the mice were randomly divided into the following six groups (48 in total, *n* = 8 per group): ND, HFD, HFD with silibinin (SIL, 80 mg/kg/day), HFD with LP-50 (50 mg/kg/day), HFD

with LP-100 (100 mg/kg/day), and HFD with LP-200 (200 mg/kg/day). The HFD used was purchased from Research Diets, Inc. (New Brunswick, NJ, USA) (Product #D12492). NAFLD was induced by continuous feeding for eight weeks. Both LPs and silibinin were administered by intragastric needle feeding. After four weeks of intervention, the mice were fasted for sixteen hours before euthanization. Plasma was collected by centrifuging the blood at 4 ◦C and 4000 r/min for 10 min. The livers were acutely dissected and weighed.

#### *4.3. Determination of the Serum Lipid Index, AST, ALT, and Liver Oxidative Stress Index*

Serum was collected and analyzed for TC, FFA, LDL, TG, HDL, AST, ALT, MDA, CAT, T-AOC, GSH-Px, and SOD with the aid of detection kits according to the manufacturer's instructions (Nanjing Jiancheng Biotechnology Research Institute, Nanjing, China).

#### *4.4. Pro-Inflammatory Factors Analysis*

The levels of hepatic TNF-α, IL-6, IL-Iβ, and IFN-γ were determined by Quantikine ELISA kits (Elabscience Biotechnology, Inc., Wuhan, China). The 96-well plates were coated with primary antibodies before the reference standards and test samples were added. After rinsing the plates, a biotinylated antibody was applied. A peroxidase-conjugated streptavidin was then added, and the concentrations of the primary antibodies were detected using Tetramethylbenzidine through analyzing OD at λ max = 450 nm.

#### *4.5. Histopathological Examination of the Liver*

Liver tissues were harvested and fixed overnight in 4% paraformaldehyde and then embedded in paraffin. Four-millimeter paraffin sections were subsequently made and stained with H&E. Photomicrographs were taken under an optical microscope (Biological microscope CX31, Olympus, Tokyo, Japan). TEM was performed to examine the detailed information of the liver injuries. Oil Red O staining was carried out according to the method used by Shen et al. [51]. Fresh liver sections were then taken and stained with an analytical kit (Nanjing Jiancheng Biotech, Nanjing, China). TEM procedure was determined according to Huang et al. [52].

#### *4.6. Western Blotting*

The liver tissue was homogenized using ultrasound and then lysed in a RIPA buffer. The procedure of Western blotting as described by Tang et al. was used to differentiate the protein molecules [53]. The protein concentration was measured using the bicinchoninic acid (BCA) method (KeyGENbio, Nanjing, China). Protein extracts were loaded onto 12% SDS-polyacrylamide gel and subsequently transferred onto a PVDF membrane. After incubating with primary and secondary antibodies, protein bands were visualized with enhanced chemiluminescence (ECL). Western blotting bands were imaged and quantitated with the FluorChem FC3 software (<IP_ADDRESS>8, ProteinSimple, Waltham, MA, USA). All antibodies were purchased from Affinity (Affinity Biosciences, Cincinnati, OH, USA).

#### *4.7. Statistical Analysis*

Data were presented as mean ± standard deviation (SD) (*n* = 8). For comparing data in different groups, an ANOVA analysis was performed by the SPSS 19.0 software (IBM, Armonk, NY, USA). A probability value of less than 5% was considered statistically significant.

#### **5. Conclusions**

According to the results of our experiment, we hypothesize that LPs act by resisting the second attack of NAFLD pathogenesis (including oxidative stress, lipid peroxidation, and increased production of inflammatory factors), so as to assist in repairing liver function (Figure 8). We previously found that LPs had a high antioxidant activity and could effectively scavenge DPPH, hydroxyl, ABTS, and superoxide anion radicals, and protect RAW264.7 cells from H2O2-induced injury. LPs can be used as raw materials for natural

antioxidants [26]. In order to provide a theoretical basis for the stable development and application of LPs and related products, we systematically analyzed the composition of LPs and identified the corresponding proteins using both database search and de novo sequencing methods. In the field of drug discovery, these functional proteins can be used as the lead compound for further modification and screening of active peptide analogues.

**Figure 8.** Mechanisms of LPs on alleviating NAFLD.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20050312/s1, Table S1: The free peptide was obtained by database search, Table S2: Free peptides with ALC% over 80 in de novo sequencing, Table S3: Source proteins of the identified peptides in Low Molecular Weight Monkfish (*Lophius litulon*) Peptides.

**Author Contributions:** Conceptualization, J.Y., X.T. and Z.Y.; data curation, J.Y., X.T., Z.Y., S.Z., Q.W. and F.Y.; methodology, J.Y., X.T., J.Z. and B.X.; writing—original draft preparation, J.Y. and X.T.; writing—review and editing, J.Y., X.T., Y.Y. and F.Y.; funding acquisition, Z.Y. and F.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Zhejiang Provincial Public Welfare Technology Research Program (LGN21D060002), Fundamental Research Funds for the Provincial Universities of Zhejiang Province (2021JZ011), and National Science Foundation (81773629).

**Institutional Review Board Statement:** The Experimental Animal Ethics Committee of Zhejiang Ocean University approved the procedures for the use of the laboratory animals (Certificate Number SCXK ZHE2014-0001, approval date 1 March 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are indebted to Xiaoxia Jiang for her generous technical support. The authors have no conflict of interest to disclose.

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

#### **References**


**1. Introduction**

In medical imaging, different modalities replicate different details of human organs and tissues. For example, Magnetic Resosance Imaging (MRI) provides low density soft tissues such as blood vessels, whereas Computed Tomography(CT) provides clear detail about bone tissue and also provides the reference for location of the lesion [1]. As it is known, dose reduction lowers the radiation exposure risks, but at the same time decreases the image quality. By its nature, CT involves larger radiation doses than the more common, conventional x-ray imaging procedures [2]. We briefly discuss the nature of CT scanning and its main clinical applications, both in symptomatic patients and, in the screening of asymptomatic patients. We focus on the increasing number of CT scans being obtained, the associated radiation doses, and the consequent cancer risks in adults and particularly in children [3]. Although the risks for any one person are not large, the increasing exposure to radiation in the population may be a public health issue in the future. The use of CT has increased rapidly since 1980's, according to recent surveys, it is showing that

more than 62 million CT scans are currently obtained ever year in the United States, as compared with about 3 million in 1980's. The largest use of CT scan, however, are within the classes of pediatric identification and adult screening, and these trends are often expected to continue for ensuing few years [4]. The rise in use of CT scan in kids has been driven primarily by the decrease within the time required to scan, that is a smaller amount than a second, and additionally eliminating the necessity for physiological condition to forestall the kid from moving throughout image acquisition method. The foremost growth space in exploitation CT scan for youngsters has been presurgical identification of inflammation, that CT seems to be each correct and efficient.

**2.1 Histogram equalization**

from its neighbourhood region.

**2.2 Adaptive histogram equalization**

component with equal price.

**63**

a picture. Properties of Adaptive Histogram Equalisation:

Histogram equalization is a global processing technique used to spread the pixel

values over the dynamic range of image and the equalized histogram must be approximately uniformly distributed in the dynamic range [10]. It is a distribution

1.The frequency of the histogram reflects only the pixels in the image of a certain grey level values but not reflects the position of each pixel.

It is not sure that the contrast will always be increased by the histogram equalization. There may be some cases in which histogram equalization can be worse. In that cases the distinction could also be decreased. In general, normal bar graph exploit uses an equivalent transformation that comes from the image bar graph to rework all pixels. This works well once the distribution of pel values is comparable throughout the image [11]. However, once the image contains regions that square measure considerably lighter or darker than most of the image, the distinction in those regions won't be sufficiently increased. Adaptive bar graph exploit (AHE) improves during this side by remodeling every pel with a change perform derived

Adaptive bar graph feat (AHE) may be a pc image process technique wont to improve the distinction in pictures. It differs from normal bar graph feat within the respect that the adaptive technique computes many histograms, every admire a definite section of the image, and uses them to spread the brightness values of the image [12]. In its simplest type, every element is remodeled supported the bar graph of a sq. close that element. The transformation functions derived from the bar graphs is precisely constant as for normal histogram feat. The transformation perform is proportional to the accumulative distribution function (CDF) of element values within the neighbourhood. Pixels close to the image boundary have to be compelled to be treated specially, as a result of their neighbourhood wouldn't lie fully at intervals the image [13]. It is so appropriate for rising the native distinction and enhancing the definitions of edges in every region of a picture. However, AHE contains a tendency to over amplify noise in comparatively homogeneous regions of

• The size of the neighbourhood region is a parameter of the method. It improves the contrast at smaller scales and reduces the contrast at larger scales.

neighbourhood. This permits Associate in Nursing economical implementation of hardware that may compare the middle component with all different pixels within the neighbourhood [3]. Associate in Nursing unnormalized result price may be computed by adding two for every component with a smaller price than the middle component, and adding one for every

• Due to the character of bar chart feat, the resultant price of a component underneath AHE is proportional to its rank among the pixels in its

2.Histogram of an image doesn't overlap each sub section of an image.

function transformation method based on histogram modification.

Characteristics of Histogram of a digital image:

*A Hybrid Image Fusion Algorithm for Medical Applications*

*DOI: http://dx.doi.org/10.5772/intechopen.96974*

The radiation doses from CT scanning are considerably larger than those from corresponding conventional radiography. Michael F. McNitt-Gray [3] discussed that the radiation doses to a particular organ from any given CT scan depends on number of factors, such as range of scans, the tube current and scanning time in milliampseconds (mAs), the scale of the patient, the axial scan vary, the scan pitch (the degree of overlap between adjacent CT slices), the tube voltage within the potential unit peaks (kVp), and therefore the specific style of the scanner being employed. Patient dosimetry and evaluation of image quality are basic aspects of any quality control program in diagnostic radiology. Image quality must be adequate for diagnosis and obtained with reasonable patient doses [5]. As per the recommendations of International Commission on Radiological Protection, No dose limit applies to medical exposure to patients, but diagnostic reference levels or reference values have been proposed by the International Commission on Radiologic Protection [6]. Thomas Lehnert et al. said that it is always the relative noise in CT images will increase as the radiation dose decreases, which means that there will always be a tradeoff between the need for low-noise images and the desirability of using low doses of radiation [4]. The low dose CT scan image usually suffers from serious noise and artifacts by using analytical reconstruction methods. It is always preferable to have standard imaging techniques that diminish the patient dose with reasonable image quality [7]. As part of implementation efforts, an important clinical requirement has been addressed that low-dose CT (LDCT) images need to be improved in the Electronic Health Records (EHR). Khalid et al., proposed an enhanced dynamic quadrant equalization for image contrast enhancement, in which input image histogram is divided into 8 subhistograms by using median values. For individual subhistograms, clipping of histogram is done by the average pixels. New dynamic range is assigned to each subhistograms and HE is done separately. This approach preserves the mean brightness [8]. As there is no guarantee that the contrast will always be increased by the histogram equalization [1], Adaptive Histogram Equalisation has been applied on low dose CT scan image to improve the contrast.

This chapter gives a comparative study related to performance of the image fusion techniques. Organization of this paper is as follows; Section 2 explains the image enhancement techniques. The principle of PCA and DWT image fusion techniques are discussed in Section 3. In Section 4, fusion performance assessment techniques are explained. In Section 5, the results of fused images for two different data sets are compared with PCA and DWT applied to medical images by implementing in MATLAB.

### **2. Image enhancement**

The goal of an image enhancement is to improve the visual effects of the entire image or to enhance the certain information in accordance with specific needs [9].


2.6.2. Relative RMSE (rRMSE)

The relative RMSE is the ratio of the RMSE to the mean values of field measurements (yield (t ha<sup>−</sup>1)):

$$\text{rRMSE} = \frac{\text{RMSE}}{\frac{\sum\_{i=1}^{N} O i}{N}} \tag{2}$$

where *O* represents the observed value and *N* represents the number of samples.


**Di** ff**erential Transcription of Selected Cytokine and Neuroactive Ligand-receptor Genes in Peripheral Leukocytes from Calves in Response to Cautery Disbudding**

**Kavitha Kongara 1,\*, Venkata Sayoji Rao Dukkipati 1,2, Hui Min Tai 3, Axel Heiser 1,4, Alan Murray 1, James Webster 5 and Craig Brian Johnson 1**

Received: 13 May 2020; Accepted: 6 July 2020; Published: 14 July 2020

**Simple Summary:** Calf disbudding is a painful husbandry practice on dairy and beef cattle farms. Continuing e fforts to enhance the accuracy of pain assessment can aid in the application of e ffective anti-nociceptive (analgesic) agents in non-verbal animals. The aim of this study was to evaluate the changes in the expression of genes involved in inflammation and pain sensitisation in response to removal of horn buds in calves, using hot-iron cauterization. The e fficacy of an analgesic, meloxicam, was also tested in attenuating the changes in expression of the studied genes post-disbudding. It was revealed that cautery disbudding induces significant changes in the expression of genes involved in inflammation. Meloxicam was able to blunt the increased expression of some of the genes at 4 h and 24 h after disbudding, while it could not attenuate the increased expression of a few other genes associated with inflammation.

**Abstract:** Calf disbudding is a painful husbandry practice on dairy and beef cattle farms. An objective measurement of pain is useful to reliably evaluate the pain intensity and anti-nociceptive (analgesic) efficacy of therapeutic agents. The aim of this study was to investigate the changes in peripheral leucocyte inflammatory cytokine gene expression in calves after disbudding, and to assess whether the changes in cytokine gene expression could be an indicator of the e fficacy of analgesic drugs. In a randomised controlled study, 16 calves (aged 31 to 41 days and weighing 58 to 73 kg), undergoing routine disbudding, were randomly allocated into two groups (*n* = 8 in each group). Calves in the control group received no analgesic, while those in the treatment group received 0.5 mg kg−1 meloxicam subcutaneously prior to disbudding. Disbudding was performed using an electric debudder. Blood (10 mL) was sampled from the jugular vein just before and 4 and 24 h post-disbudding, RNA was extracted from leukocytes, and the transcription of 12 genes of interest was assessed using nCounter gene expression assay. The results showed significantly higher transcription (compared to baseline values) of the studied genes (except *CRH*, *IFN*γ, and *IL10*) in the control group calves at either 4 or 24 h post-disbudding. The administration of meloxicam one hour before disbudding significantly attenuated the upregulation of *IL6*, *PGHS2*, *TAC1*, *NOS1*, and *CRH* gene transcription post-disbudding, while it did not suppress the elevated transcription of acute and pro-inflammatory cytokines such as *IL1*β, *IFN*γ, *IL8*, and *TNF*α genes. In conclusion, nCounter gene expression assay seems to be a promising tool to study the expression of cytokine genes and thus could be used for the pre-clinical evaluation of novel analgesics.

**Keywords:** calves; disbudding; gene transcription; cytokines; pain; meloxicam; nCounter


<IP_ADDRESS>.2 The effect of the material of the metal fibers on SE

The metal fibers used in the fabric are different, and the different electrical conductivity of the metal may affect the electrical resistivity of the yarns and the

Figure 14. The shielding effectiveness of different types of yarns.

fabrics. For example, the electrical conductivity of copper fiber is 5.8 107 S/m, and the electrical conductivity of aluminum is 3.54 107 S/m. When the metal fiber content, linear density, and fabric specification parameters are the same, the SE of copper fiber fabrics are better than that of aluminum fiber fabrics.

The five grid samples with completely nonconducting period of 2 mm is shown in Figure 15, for which each grid sample of five different materials consisting of stainless steel bare wire (the diameter is 35 μm), core spun yarn and blended yarn of stainless steel/cotton (containing the stainless steel content of 30%), silver-plated nylon filament (the diameter is 50 μm), and bare copper wire (the diameter is 80 μm). The SE of the samples made of, respectively, stainless steel blended yarns, silver-plated filaments, and bare copper wires are substantially equal and higher that of the samples of core spun yarn and stainless steel bare wire.

#### <IP_ADDRESS>.3 The effect of metal fiber content on SE

The two blended yarns with the stainless steel content of 20 and 30% are woven in both warp and weft directions, and the SE of the obtained sample in the range of 1–18 GHz are shown in Figure 16. It can be seen that except for the frequency range of 10 and 12–14 GHz, the SE of the fabric with 30% stainless steel content is 5 dB higher than that of the fabric with 20% stainless steel content, and the difference of SE in other frequency bands is unobvious. The content of stainless steel fibers has a certain effect on the shielding effectiveness of the fabric, but after reaching a certain level, the difference is not significant.

3.3 Electromagnetic scattering fabrics

Electromagnetic Function Textiles

DOI: http://dx.doi.org/10.5772/intechopen.85586

invisible for certain direction Radar.

for incident electromagnetic waves.

structures

structure.

197

Figure 16.

3.3.1 The mechanism of electromagnetic scattering fabrics

The shielding effectiveness of the fabrics with different content of stainless steel.

When electromagnetic waves radiate into the macroscopic object, which will causing induced electric charges and currents of the object, then the electromagnetic wave radiated into the object will be scattered into various directions. This process is called electromagnetic scattering [11, 12]. The electromagnetic scattering fabric is an electromagnetic functional material with the specific design structure, which makes the electromagnetic waves incident on the target are no longer reflected back along the way of the reflection of mirror, but radiated out into different directions. Thereby, it can reduce the radiated electromagnetic waves in the direction of propagation, and can make the human body and military targets

The textile technology is relatively mature in the preparation of threedimensional structural fabrics. Thus, it is very feasible to design the threedimensional structure of metallized fabrics which have good scattering properties

<IP_ADDRESS> The electromagnetic wave scattering characteristics of linear column unit

When a simple harmonic uniform plane wave is incident on the threedimensional structure, the three-dimensional coordinate system XYZ shown in Figure 18 is selected. The XOY plane of the coordinate system coincides with the lower surface of the object, and the Z axis is perpendicular to the interface of the upper and lower surfaces. Electromagnetic waves are incident from the upper surface with the incident angle θ, and reflected waves and transmitted waves are generated at the interface of the upper surface. The transmitted waves enter the

As shown in Figure 17, the composite materials of the three-dimensional periodic structure can be simplified into two-phase dielectric materials when studying the transmission process of electromagnetic waves in the three-dimensional

It is assumed that the fibers are evenly distributed in the yarn. As the content of the metal fibers increases, the shielding effectiveness of the fabrics will increase, but when it is increased to a certain extent, the bending stiffness and flexural modulus of the yarns will increase, and the porosity among fibers during the fabric will increase. So the SE of the fabrics becomes slow down or even lower. Considering the cost, the content of stainless steel is generally 20–30% for fabrics containing stainless steel fibers.

Figure 15. Shielding effectiveness of different materials with grid period spacing of 2 mm in order.

fabrics. For example, the electrical conductivity of copper fiber is 5.8 107 S/m, and the electrical conductivity of aluminum is 3.54 107 S/m. When the metal fiber content, linear density, and fabric specification parameters are the same, the SE of

The five grid samples with completely nonconducting period of 2 mm is shown in Figure 15, for which each grid sample of five different materials consisting of stainless steel bare wire (the diameter is 35 μm), core spun yarn and blended yarn of stainless steel/cotton (containing the stainless steel content of 30%), silver-plated nylon filament (the diameter is 50 μm), and bare copper wire (the diameter is 80 μm). The SE of the samples made of, respectively, stainless steel blended yarns, silver-plated filaments, and bare copper wires are substantially equal and higher

The two blended yarns with the stainless steel content of 20 and 30% are woven in both warp and weft directions, and the SE of the obtained sample in the range of 1–18 GHz are shown in Figure 16. It can be seen that except for the frequency range of 10 and 12–14 GHz, the SE of the fabric with 30% stainless steel content is 5 dB higher than that of the fabric with 20% stainless steel content, and the difference of SE in other frequency bands is unobvious. The content of stainless steel fibers has a certain effect on the shielding effectiveness of the fabric, but after reaching a

It is assumed that the fibers are evenly distributed in the yarn. As the content of the metal fibers increases, the shielding effectiveness of the fabrics will increase, but when it is increased to a certain extent, the bending stiffness and flexural modulus of the yarns will increase, and the porosity among fibers during the fabric will increase. So the SE of the fabrics becomes slow down or even lower. Considering the cost, the content of stainless steel is generally 20–30% for fabrics containing

copper fiber fabrics are better than that of aluminum fiber fabrics.

that of the samples of core spun yarn and stainless steel bare wire.

Shielding effectiveness of different materials with grid period spacing of 2 mm in order.

<IP_ADDRESS>.3 The effect of metal fiber content on SE

Electromagnetic Materials and Devices

certain level, the difference is not significant.

stainless steel fibers.

Figure 15.

196

Figure 16. The shielding effectiveness of the fabrics with different content of stainless steel.

### 3.3 Electromagnetic scattering fabrics

### 3.3.1 The mechanism of electromagnetic scattering fabrics

When electromagnetic waves radiate into the macroscopic object, which will causing induced electric charges and currents of the object, then the electromagnetic wave radiated into the object will be scattered into various directions. This process is called electromagnetic scattering [11, 12]. The electromagnetic scattering fabric is an electromagnetic functional material with the specific design structure, which makes the electromagnetic waves incident on the target are no longer reflected back along the way of the reflection of mirror, but radiated out into different directions. Thereby, it can reduce the radiated electromagnetic waves in the direction of propagation, and can make the human body and military targets invisible for certain direction Radar.

The textile technology is relatively mature in the preparation of threedimensional structural fabrics. Thus, it is very feasible to design the threedimensional structure of metallized fabrics which have good scattering properties for incident electromagnetic waves.

## <IP_ADDRESS> The electromagnetic wave scattering characteristics of linear column unit structures

As shown in Figure 17, the composite materials of the three-dimensional periodic structure can be simplified into two-phase dielectric materials when studying the transmission process of electromagnetic waves in the three-dimensional structure.

When a simple harmonic uniform plane wave is incident on the threedimensional structure, the three-dimensional coordinate system XYZ shown in Figure 18 is selected. The XOY plane of the coordinate system coincides with the lower surface of the object, and the Z axis is perpendicular to the interface of the upper and lower surfaces. Electromagnetic waves are incident from the upper surface with the incident angle θ, and reflected waves and transmitted waves are generated at the interface of the upper surface. The transmitted waves enter the

Figure 17. Dielectric column periodic three-dimensional structure.

Figure 18. The schematic diagram of electromagnetic wave incident three-dimensional structure.

periodic three-dimensional structure, and after being attenuated in the threedimensional structure, the reflected waves and the transmitted waves are again generated at the lower surface interface.