Homogenates (50µg of protein) were subjected to SDS-PAGE on a 12% polyacrylamide gel (12% acrylamide, 0.1% *w/v* SDS, 0.1% (NH4) 2S2O8, Temed, 0.39 M Tris pH 8.8); 10% of the elution volume of each extract was mixed with 4× buffer (100 mM Tris-HCl pH 6.8, 10% *v/v* 2-mercaptoethanol, 4% *w/v* SDS, 0.02% *w/v* bromophenol blue, and 20% glycerol). Prior to being added to the gel, the samples were denatured by heating at 100 ◦C for 5 min. The potential difference applied was 200 V, and the current intensity was 150 mA. The running buffer contained 25 mM Tris-base, 250 mM glycine and 0.1% SDS. After electrophoresis, the proteins were transferred to a nitrocellulose membrane in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS and 20% methanol) and subjected to a potential difference of 60 V and a current intensity of 150 mA. The transfer was performed at 4 ◦C for 3 h. Nonspecific binding was then blocked by incubating the membrane with 5% skimmed milk powder in PBS-T (phosphate-buffered saline, 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4, and 0.1% Tween) for 1 h at room temperature under constant stirring. After blocking, the membrane was washed (3×) in PBS-T solution for 10 min and cut based on the size of the proteins to be evaluated. For BDNF detection, the membrane was incubated with a BDNF primary antibody (dilution, 1:700) (700 rabbit Abcam®, Inc., Cambridge, MA, USA) for 16 h at 4 ◦C under constant stirring. An anti-α-tubulin antibody) (dilution, 1: 10.000) (Sigma-Aldrich, St Louis, MO, USA) was used as the loading control. After incubation, the membrane was incubated with a conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1: 10.000 in 5% skimmed milk powder in PBS-T for 1 h at room temperature under constant agitation.

Finally, the membrane was washed (3×) times in PBS-T for 10 min. Immunoreactivity was evaluated with the Immobilon Western Chemiluminescent HRP Substrate kit (Millipore, Billerica, MA, USA). The bands were quantified using ImageQuant LAS 4000 (GE, Buckinghamshire, UK). The data were analysed using repeated-measures ANOVA followed by Dunnett's multiple comparisons.

#### **5. Conclusions**

Based on the findings from the present study, the persistence of memory is a process associated with mechanisms initiated during consolidation that persist over time. Furthermore, we reveal for the first time that EGb treatment before the acquisition of ORM increases BDNF expression in the hippocampus in a dose-dependent manner, which is associated with the persistence of object recognition memory. This finding was substantiated by the different levels of BDNF expression observed during the T1 and T2 sessions following treatment with 250 or 1000 mg/kg EGb. Conversely, rats treated with 500 mg/kg EGb showed upregulation during the T2 session, which was not sufficient to maintain the storage of recognition memory traces over time.

**Supplementary Materials:** The following are available online. Table S1: Recognition Index (RI) values in the retention and persistence tests. Table S2: The levels of rearing and grooming during analysis of long-term memory for objects sessions.

**Author Contributions:** Conceptualization, S.M.C. and J.M.C.; methodology, B.G.M., C.R.Z., T.B.M. and B.H.N.N.; formal analysis, B.G.M., C.R.Z., T.B.M. and B.H.N.N.; investigation, B.G.M., S.M.C. and J.M.C.; resources, B.G.M., C.R.Z., J.M.C. and S.M.C.; data curation, B.G.M., C.R.Z., T.B.M., B.H.N.N., J.M.C. and S.M.C.; writing—original draft preparation, B.G.M., J.M.C. and S.M.C.; writing—review and editing, B.G.M., J.M.C. and S.M.C.; supervision, S.M.C. and J.M.C.; project administration, S.M.C.; funding acquisition, S.M.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the São Paulo State Research Foundation (FAPESP) (grant **2016/18039-9** to SMC). BGM is a scholar from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)—Finance Code 001. CRZ is a Postdoctoral fellow from CNPq.

**Institutional Review Board Statement:** All procedures were approved by the Local Committee Governing the Ethics on the Use of Animal Experimentation of the Federal University of Sao Paulo (CEUA UNIFESP<PHONE_NUMBER>) and were conducted in accordance with the Brazilian Law for the use of animals in scientific research (Nº11.794) as suggested by the APA Guidelines for Ethical Conduct in the Care.

**Informed Consent Statement:** Not applicable for studies not involving humans.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

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

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**

## *Article* **Astragaloside IV Suppresses Hepatic Proliferation in Regenerating Rat Liver after 70% Partial Hepatectomy via Down-Regulation of Cell Cycle Pathway and DNA Replication**

**Gyeong-Seok Lee 1 , Hee-Yeon Jeong 1 , Hyeon-Gung Yang 2 , Young-Ran Seo 1 , Eui-Gil Jung 3 , Yong-Seok Lee 1 , Kung-Woo Nam 1 and Wan-Jong Kim 1,\***

**Abstract:** Astragaloside IV (AS-IV) is one of the major bio-active ingredients of huang qi which is the dried root of *Astragalus membranaceus* (a traditional Chinese medicinal plant). The pharmacological effects of AS-IV, including anti-oxidative, anti-cancer, and anti-diabetic effects have been actively studied, however, the effects of AS-IV on liver regeneration have not yet been fully described. Thus, the aim of this study was to explore the effects of AS-IV on regenerating liver after 70% partial hepatectomy (PHx) in rats. Differentially expressed mRNAs, proliferative marker and growth factors were analyzed. AS-IV (10 mg/kg) was administrated orally 2 h before surgery. We found 20 core genes showed effects of AS-IV, many of which were involved with functions related to DNA replication during cell division. AS-IV down-regulates MAPK signaling, PI3/Akt signaling, and cell cycle pathway. Hepatocyte growth factor (HGF) and cyclin D1 expression were also decreased by AS-IV administration. Transforming growth factor β1 (TGFβ1, growth regulation signal) was slightly increased. In short, AS-IV down-regulated proliferative signals and genes related to DNA replication. In conclusion, AS-IV showed anti-proliferative activity in regenerating liver tissue after 70% PHx.

**Keywords:** astragaloside IV; *Astragalus membranaceus*; huang qi; Astragali Radix; liver; liver regeneration; 70% partial hepatectomy; proliferation; rat

#### **1. Introduction**

For a long time, medicinal plants have played a key role in pharmacological research studies and drug development. *Astragalus membranaceus* is medicinal plant that has been used in traditional Chinese medicine throughout history; it is also called Astragali Radix. Huang qi (黃芪, in Chinese) is the name for the dried root of *Astragalus membranaceus* and it is mainly produced in China, Mongolia, and Korea [1]. The main components of huang qi are saponins, polysaccharides and flavonoids [2–4]. It has been used in traditional Chinese medicine for over 2000 years to treat various diseases including anemia, cardiovascular disorder, weakness, fatigue and fever [4,5]. In recent years, it has been reported that huang qi has anti-oxidative, anti-aging, anti-inflammatory, anti-diabetic, and anti-cancer properties [1–4].

Astragaloside IV (AS-IV) is a representative bio-active ingredient of huang qi [4,6]. AS-IV is classified as a tetra cyclic triterpenenoid, otherwise known as a protostane which means prototype of steroid (Figure 1) [7]. AS-IV has a similar carbon-tetra-cyclic structure to steroids which have a bio-activities as cholesterol. Recently, it was reported that long-term dietary cholesterol overload negatively affects liver regeneration after PHx. Cholesterol overload reduced hepatic DNA synthesis [8]. While, other steroid, estradiol accelerates

**Citation:** Lee, G.-S.; Jeong, H.-Y.; Yang, H.-G.; Seo, Y.-R.; Jung, E.-G.; Lee, Y.-S.; Nam, K.-W.; Kim, W.-J. Astragaloside IV Suppresses Hepatic Proliferation in Regenerating Rat Liver after 70% Partial Hepatectomy via Down-Regulation of Cell Cycle Pathway and DNA Replication. *Molecules* **2021**, *26*, 2895. https:// doi.org/10.3390/molecules26102895

Academic Editors: Raffaele Pezzani and Sara Vitalini

Received: 19 April 2021 Accepted: 11 May 2021 Published: 13 May 2021

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

**Copyright:** © 2021 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/).

liver regeneration [9]. Thus, it was expected that the AS-IV directly has an effects on liver regeneration after PHx. Many reports have shown that AS-IV has anti-oxidative properties and protective effects against hypoxic injury and ischemia-reperfusion injury [10–15]. The anti-cancer activities of AS-IV via inhibition of migration and invasion of cancer cells have also been demonstrated [16,17]. Moreover, AS-IV improves diabetic nephropathy, retinopathy, gastropathy, and wound healing [18–21] Hepato-protective effects against hepatic fibrosis, oxidative stress, and hepatitis B virus were also reported [10,22–25].

**Figure 1.** Chemical structure of astragaloside IV. (C41H68O14, molecular weight: 794.97 g/mol).

The liver has ability to recover lost functional capacity after chemical or physical injury. After injury, the liver quickly regenerates to meet metabolic demand via the proliferation of hepatic cells. Liver regeneration involves a complex network of growth factors, signaling pathways, and transcriptional factors [26]. Uniquely, liver regeneration occurs without functional loss and through repopulation of mature cells rather than progenitor or stem cells [27].

The 70% partial hepatectomy (PHx) model is a commonly used model to investigate new aspects of liver regeneration [28]. It was first described by Higgins and Anderson in 1931. PHx is a surgical procedure to resect the median and left lateral lobes of the liver, which constitute about 70% of the liver mass [29–31]. Liver regeneration is especially rapid in small rodents; full-size restorations have been reported within 7 days in most rodents [27,32]. After PHx, remnant liver tissues undergo three phases. First is priming phase, which is characterized by the stimulation of hepatic mitogen. Hypoxic conditions after PHx and hemo-dynamic factors such as blood pressure are thought to be major stimulators of liver regeneration [33–36]. The liver is not alone in promoting its own regeneration, and cooperative signals for priming also come from the pancreas, spleen, duodenum, and adrenal glands [37,38]. Second is proliferating phase; DNA replication in hepatocytes started. The last step is growth termination. Transforming growth factor β1 (TGFβ1) is secreted by non-parenchymal cells including hepatic stellate cells, Kupffer cells, and platelets. TGFβ1 plays an important role in ending regeneration through suppression of hepatic proliferation [33,39,40].

The pharmacological effects of AS-IV have been actively studied in recent years and hepato-protective effects of AS-IV have been extensively reported. However, the effects of AS-IV on liver regeneration are not yet fully elucidated. In this study, the effects of AS-IV were investigated in a 70% PHx rat model through measurement of gene expression, the expression of hepatocyte growth factor (HGF, primary hepatic mitogen), and the expression of hepatic proliferation marker protein.

#### **2. Results**

#### *2.1. mRNA Sequencing Analysis*

To determine the effects of AS-IV on the gene expression of regenerating liver tissues after PHx, changes of gene expression were measured 12 h after PHx by mRNA sequencing analysis. The count of differentially expressed genes (DEGs) was 17,048; DEGs exhibiting changes more than 2-fold were considered significant. DEGs exhibiting changes more than 2-fold and 5-fold were 975 and 103 genes, respectively (Figure 2a,b). In the results of the two-fold DEGs, 370 DEGs were up-regulated and 605 DEGs were down-regulated by AS-IV (Figure 2c). Results from the 5-fold DEGs showed that 31 DEGs were up-regulated and 72 DEGs were down-regulated by AS-IV (Figure 2d).

**Figure 2.** DEGs and gene network. (**a**) heatmap with hierarchical clustering of 2-fold changed DEGs, (**b**) expression pattern for 2-fold changed DEGs, (**c**) heatmap with hierarchical clustering of 5-fold changed DEGs, (**d**) expression pattern for 5-fold changed DEGs, (**e**) Gene network of 5-fold changed DEGs. 20 DEGs (in blue dotted line) and 12 DEGs (in red dotted line) were clustered. Markedly, DEGs within the blue dotted line showed strong relation. 14 other DEGs (not marked) were linked with only one or two genes. The green color in the figure indicates decreased expression; the red color in the figure indicates increased expression.

#### 2.1.1. Key Gene Screening and Functional Annotation

To determine the pharmacological effects of AS-IV, we listed key genes from the result and investigated their function through corresponding protein of genes. We listed highly changed DEGs (more than 5-fold) and generated gene networks for the DEGs using a multiple protein search tool within the STRING database; two big cluster networks of DEGs were generated (Figure 2e). One network, which is marked by a blue dotted line in Figure 2e, consisted of 20 DEGs including *Mcm5*, *Cdc45*, *Cdc7*, *Cenpm*, *Asf1b*, *Donson*, *Tfdp2*, *Ccdc64*, *Cenpn*, *Brca2*, *Dbf4*, *Enahm*, *Mcm10*, *Ttk*, *Spdl1*, *Cdc6*, *Ndc80*, *Kif20b*, *Rad9b* and *Bora*. Another big network, marked by the red dotted line of Figure 2e, consisted of 12 genes including *Mmp9*, *Atf3*, *Cyr61*, *Fos*, *Il1b*, *Tlr1*, *Egr2*, *Spp1*, *Il15*, *Prlr*, *Hoxa2*, and *Hand2*. Functional annotation of DEGs in cluster network was conducted using the database for annotation, visualization and integrated discovery (DAVID) bioinformatic tool in three GO (gene ontology) categories including biological process, cellular component, molecular function (Table 1). The 20 DEGs in blue dotted line of Figure 2e were matched with

DNA replication initiation, cell division, double-strand break repair via break-induced replication, and DNA duplex unwinding, all of which fall within the biological process category. In the cellular component category, nucleoplasm was matched. In the molecular function category, DNA replication origin binding and chromatin binding were matched. Results from the functional annotation of the DEGs in this cluster (blue dotted line) showed that these genes were related to the DNA replication process of cell division. DEGs in the other clustered network within the red dotted line of Figure 2e were matched with positive regulation of angiogenesis, positive regulation of protein phosphorylation, and positive regulation of apoptotic process. They were matched only the biological process category (Table 1). From the changes in gene expression, strong interaction and function, we considered DEGs within the blue dotted line to be the key DEGs showing the effects of AS-IV.

**Table 1.** Functional annotation of clustered DEGs by DAVID in Figure 2e.

#### 2.1.2. Pathway Mapping of DEGs

DEGs exhibiting 2-fold changes were further analyzed using the KEGG pathway database to evaluate the effect of AS-IV on signaling in regenerating liver tissues. Remarkably, DEGs were highly matched with three pathways including MAPK signaling, PI3K-Akt signaling, and cell cycle pathways (Table 2). The DEGs in these three pathways showed similar down-regulated patterns; overall, most matched DEGs were downregulated (Figure 3).

**Table 2.** Results of pathway mapping for 2-fold changed DEGs by AS-IV.

**Figure 3.** Results of KEGG pathway mapping. 2-fold changed DEGs were analyzed using the KEGG pathway database. 24, 22, and 22 DEGs were included in cell cycle, MAPK signaling, and PI3K-Akt signaling pathways, respectively. AS-IV showed down-regulatory effects on these pathways. Only a few DEGs were up-regulated in these three pathways. MAPK signaling and PI3K-Akt signaling pathways were upstream of the cell cycle pathway. The red and blue color in the figure indicate up-regulation and down-regulation of genes, respectively. Intensity of color is proportional to fold change.

#### *2.2. Hepatic Proliferation of Regenerating Liver Tissue*

Hepatic proliferation of regenerating liver tissue after AS-IV administration was evaluated via immunohistochemical staining and Western blot analysis for hepatocyte growth factor (HGF) (a mitogen for hepatocytes), cyclin D1 (a marker protein for proliferation), and transforming growth factor β1 (TGF β1) (a terminator for proliferation).

As HGF is a major stimulator of proliferation of hepatocyte during liver regeneration, the expression of HGF was evaluated to determine the hepato-proliferative signal after 70% PHx. Thus, the ratio of HGF positive cells increased immediately after 70% PHx and peaked 12 h after PHx. The ratio of immuno-positive cells in the AS-IV group showed a decrease when compared to controls (Figure 4). Remarkably, immediately after PHx, HGF expression decreased in half of the control group. Cyclin D1 was evaluated to determine the level of proliferation of hepatocyte after proliferative signal. It showed different expression. Cyclin D1 increased since 12 h after PHx with progression for cell cycle. In the AS-IV group, cyclin D1 expression was markedly decreased at 12 and 24 h after PHx (Figure 5). Results of Western blot analysis for cyclin D1 showed a decrease similar to that of the immunohistochemical results in Figure 5 (Figure 6). Cyclin D1 expression was dramatically decreased by AS-IV 24 h after PHx. TGF β1 was also determined by Western blot analysis to be a signal of growth regulation (Figure 6). The expression of TGFβ1 showed no difference 12 h after PHx, however, it was slightly increased with AS-IV treatment 24 h after PHx.

**Figure 4.** Immunostaining for HGF. HGF-immuno-positive reactions were a brown color after immunostaining. HGF peaked at 12 h after PHx. The AS-IV group showed a low expression of HGF. Hematoxylin was used as a counter stain. (Scale bar indicates 100 µm, CV: central vein, *n* = 3, mean ± standard deviation, \*: *p* < 0.05).

**Figure 5.** Immunostaining for cyclin D1. Cyclin D1 expression in regenerating liver tissues rapidly increased 12 h after PHx, peaking at 12 h after PHx then decreasing by 24 h. The AS-IV group showed a low expression of cyclin D1. Hematoxylin was used as a counter stain. (Scale bar indicates 100 µm, CV: central vein, *n* = 3, mean ± standard deviation, \*\*: *p* < 0.01).

**Figure 6.** Relative expressions of cyclin D1 and TGF β1. Expression of cyclin D1 and TGFβ1 were analyzed by Western blot analysis. 12 h after PHx, cyclin D1 expression decreased in the experimental group and decreased further by 24 h while showing increased TGFβ1 expression. Relative protein expressions are presented as a ratio of the β-actin loading control. (mean ± standard deviation, *n* = 3, \*\*: *p* < 0.01).

#### **3. Discussion**

*Aastragalus membranaceus* is one of the oldest known medicinal plants in traditional Chinese medicine. The dried root of this plant is called huang qi and an astragaloside IV (AS-IV) is the core bio-active ingredient of huang qi. Recently, it was reported that AS-IV showed anti-oxidative, anti-cancer, and hepato-protective activity in a variety of experiments [41–43]. Looking at recent research, it seems likely that AS-IV has many pharmacological potential. In 2019 Wei et al. reported that AS-IV could improve liver cirrhosis [44]. However, the effects of AS-IV on liver regeneration have not yet been elucidated. The 70% partial hepatectomy (PHx) model has been used in numerous studies of liver regeneration. In this study, we investigated the effects of AS-IV on liver regeneration in a 70% PHx model using mRNA sequencing, immunohistochemistry, and Western blot analysis. Our results suggest that AS-IV could suppress liver regeneration after 70% PHx. After oral administration of AS-IV, many genes changed their expression significantly (Figure 2). To determine the effects of AS-IV, we focused on the functions that are carried out by a group of differently expressed genes (DEGs) rather than the increases or decreases in expression of each individual gene. From the fold change, corresponding protein interactions, and function annotation of DEGs, we found 20 key DEGs including *Mcm5*, *Cdc45*, *Cdc7*, *Cenpm*, *Asf1b*, *Donson*, *Tfdp2*, *Ccdc64*, *Cenpn*, *Brca2*, *Dbf4*, *Enahm*, *Mcm10*, *Ttk*, *Spdl1*, *Cdc6*, *Ndc80*, *Kif20b*, *Rad9b*, and *Bora*, as shown in Figure 2e and Table 1. These genes showed strong functional relationship with cell division and our research suggests that these are the core genes responsible for the function of AS-IV in the regenerating liver. These results provide a potential mechanism for the therapeutic effects of AS-IV. Thus, as it relates to gene expression levels, by suppressing the genes required for molecular binding during DNA replication in the nucleus, AS-IV could inhibit DNA replication during cell division. These results suggest that AS-IV has a potent anti-proliferative effect in the regenerating liver.

DEGs were further analyzed via the KEGG pathway database to investigate the effects of AS-IV on proliferative signaling. Three signaling pathways (MAPK signaling pathway, PI3K-Akt signaling pathway, and cell cycle pathway) were down-regulated by AS-IV (Table 2, Figure 3). Other researchers have also reported similar down-regulation of MAPK signaling by AS-IV [45–47]. Decreased hepatocyte growth factor (HGF, hepato-proliferative signal molecule) and cyclin D1 (proliferation marker protein) expression also suggest the same anti-proliferative effects (Figures 4 and 5). Furthermore, AS-IV could affect the growth termination signal as well as could affect proliferative signal. Our extended study results show increased level of transforming growth factor β1 (TGF β1, growth termination signal) in the liver 24 h after PHx (Figure 6). In summary, AS-IV was revealed to have

anti-proliferative effects in the regenerating liver via changes in gene expressions and protein expressions related to cell division and proliferative signals.

And So, AS-IV showed anti-proliferative effects in regenerating liver tissues. Thus, for the purpose of tissue regeneration through encouraging normal cell division, it is likely that AS-IV is unsuitable for liver regeneration. However, AS-IV could be applied for other purposes, such as for reducing oxidative injury after PHx surgery. After PHx, the liver is rapidly regenerated. In parallel, oxidative stress also rapidly increased. Some reports have shown that AS-IV has anti-oxidative and hepato-protective activities [48,49]. Additionally, our results could be used as basic study data for the anti-cancer activities of AS-IV with other studies. In recent, similar anti-cancer effects of AS-IV were reported including blocking of MAPK signal, decreased PCNA and Ki67 expression, and trigging G1 arrest in tumor cell [45,50].

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

#### *4.1. Animals and Experimental Design*

Male rats (SD strain, 8 weeks) were obtained from DBL Co., Ltd. (Eumseong, Korea). They were housed in an environmentally controlled room at 25 ± 1 ◦C, 12/12-light/dark cycle, and relative humidity 60 ± 5% with free access to standard food pellets and water (ad libitum). All animal experiments and procedures were performed in accordance with the guidelines for the care and use of laboratory animals of the national institutes of health (NIH) and after approval by the institutional animal care and use committee (IACUC) at the Sooonchunhyang University (permission No.: SCH20-0002).

Rats were randomly divided into control and experimental groups depending on treatment and sacrifice time. Both groups consisted of six rats. Three rats of each group were sacrificed 12 h after PHx and the other three rats were sacrificed 24 h after PHx. The rats in the experimental group received intragastric administration of astragaloside IV (AS-IV, 10 mg/kg) [4,13,19,22,51]. AS-IV was diluted in 1.5 mL of D.W. [52]. AS-IV (product No. #A3305, purity > 98.0%, HPLC) was purchased from the Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The rats in the control group received the same volume of D.W. by intragastric administration. AS-IV and D.W. were administrated 2 h before surgery (Figure 7) [53–58].

To establish the liver regeneration model, a 70% partial hepatectomy (PHx) involving resection of the median and left lateral lobes was performed under anesthesia as previously described by Higgins and Anderson [29]. Animals were fasted for 12 h before surgery. Rats were sacrificed at 12 or 24 h after 70% PHx [39]. Regenerated remnant liver tissue was collected for analyses.

**Figure 7.** Administration of AS-IV and 70% PHx. Rats were received 10mg/kg of AS-IV diluted in D.W. (experimental group) or D.W. (control group) 2 h before 70% PHx and were sacrificed at 12 h or 24 h after PHx.

#### *4.2. RNA Sequencing Analysis*

TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) was used for isolation of total RNA from liver tissue [59]. cDNA libraries were generated and purified using QuantSeq 30 mRNA-Seq Library Prep Kit for Illumina (LEXOGEN, Vienna, Austria) according to manu-

facturer's instructions. High-throughput sequencing was performed as single-end 75 base pair sequencing using a NextSeq 500 (Illumina, Inc., San Diego, CA, USA). Raw reads were processed by BBDuk and aligned to the reference genome (rat, rn6, UCSC) using Bowtie2 [60]. The alignment file was assembled and estimated their abundances. Differentially expressed genes (DEGs) were determined based on counts from unique and multiple alignments with coverage in the Bedtools. The read count data were processed based on a quantile normalization method using EdgeR within R [61]. Genes were classified based on the database for annotation, visualization and integrated discovery (DAVID) and Medline databases (http://david.abcc.ncifcrf.gov/, accessed on 8 November 2020). DEGs exhibiting changes more than 2-fold were considered significant [59]. DEGs were also analyzed via the Kyoto Encyclopedia of Genes and Genomes (KEGG) mapper (address: https://www.genome.jp/kegg/tool/map\_pathway2.html, accessed on 8 November 2020). DEGs were also analyzed based on protein-protein interactions via multiple protein searching tool within STRING database (ver. 11.0, address: http://string-db.org/db.org/, accessed on 8 November 2020).

#### *4.3. Immunohistochemistry*

Liver tissue was removed and immediately fixed in 10% neutral buffered formalin. It was then embedded in paraffin according to the routine process for light microscopy. The paraffin block was then cut into 4 µm thicknesses by rotary microtome (RM2235, Leica Biosystems, Germany). Antigen retrieval was conducted with sodium citrate buffer (10 mM, pH 6.0) at 95 ◦C. Heated sections were cooled for 30 min at room temperature. Sections were then incubated in 3% H2O2 (#1146, DUCSAN PURE CHEMICALS, Korea) and incubated in 5% bovine serum albumin (BSA, A7906, Merck KGaA, Darmstadt, Germany). Primary antibodies against Cyclin D1 (ab134175, Abcam plc., UK) and HGF (ab83760, Abcam plc., UK) were added. An HRP-conjugated secondary antibody (Thermo fisher scientific, Waltham, MA, USA) was then added. Immuno-detection was conducted with 3,30 -diaminobenzidine (SIGMAFAST™, Merck KGaA, Darmstadt, Germany). Counter staining was conducted using hematoxylin. All procedures were carried out in a humidified chamber to prevent the drying out of tissues. Tissues were observed using a microscope (CKX53, Olympus, Tokyo, Japan). Counts of positive reacted cells and total cells were measured by the color deconvolution tool within TMARKER (Ver. 2.146, open-source software) [62,63].

#### *4.4. Western Blot Analysis*

Total protein was extracted from liver tissues using protein extraction solution (PRO-PREP™, iNtRON BIOTECHNOLOGY, Seongnam, Korea) on ice and was determined by bicinchoninate (BCA) calorimetric assay kit (#23227, Thermo fisher scientific, USA) according to manufacturer's instruction. The protein was separated by 12% SDS-PAGE and transferred to PVDF (IPVH00010, Merck KGaA, Germany). The PVDF was then blocked with 5% skim milk (#232100, BD, Franklin Lakes, NJ, USA) and incubated with a primary antibody diluted in 0.5% skim milk overnight at 4 ◦C. The primary antibody against β-actin (A5316, Merck KGaA, Darmstadt, Germany) was diluted to 1:6000. Primary antibodies against cyclin D1 (ab134175) and TGF β1 (ab92486) were purchased from Abcam plc. (Cambridge, UK) and diluted to 1:3000. The membrane was further incubated with an HRP-conjugated secondary antibody (Thermo fisher scientific, USA) at room temperature for 1 h, followed by washing with PBS. Protein expression was detected by enhanced chemiluminescence (ECL) solution (K-12049-D50, Advansta Inc., San Jose, CA, USA) and chemiluminescence imaging system (GBox ichemi XL, Syngene, UK). β-actin was used as loading control. The relative expression level of target protein was expressed as ratio of β-actin [64].

#### *4.5. Statistical Analysis*

All quantitative data are presented as mean ± standard deviation (SD) from three independent experiments. Statistical analyses were performed using IBM SPSS statistics for windows (ver. 25, IBM, New York, NY, USA). Student's *t*-tests were used to analyze differences between control and the experimental group. Data with a *p*-value less than 0.05 were considered statistically significant. (\*: *p*-value < 0.05, \*\*: *p*-value < 0.01)

#### **5. Conclusions**

Astragaloside IV (AS-IV) is the major bio-active component of huang qi (the dried root of *Astragalus membranaceus*, a traditional Chinese medicinal plante). In present study, we demonstrated the pharmacological effects of AS-IV on regenerating rat liver tissue after 70% partial hepatectomy. AS-IV down-regulated proliferative signals, genes related to DNA replication, and cyclin D1 expression (Figure 8). In conclusion, AS-IV showed anti-proliferative activities in regenerating liver tissue.

**Figure 8.** Graphical abstract of study. AS-IV suppressed hepatic proliferation of regenerating liver tissues after 70% PHx.

**Author Contributions:** Conceptualization: G.-S.L. and W.-J.K.; methodology: G.-S.L. and E.-G.J.; software: G.-S.L. and Y.-S.L.; validation: G.-S.L. and K.-W.N.; formal analysis: G.-S.L.; investigation: G.-S.L., H.-Y.J., H.-G.Y. and Y.-R.S.; resources: H.-Y.J., H.-G.Y. and Y.-R.S.; data curation: H.-Y.J. and H.-G.Y.; writing—original draft preparation: G.-S.L.; writing—review and editing: E.-G.J., Y.- S.L., K.-W.N. and W.-J.K.; visualization: G.-S.L., H.-Y.J. and H.-G.Y.; supervision: W.-J.K.; project administration: G.-S.L.; funding acquisition: E.-G.J., Y.-S.L., K.-W.N. and W.-J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Soonchunhyang University Research Fund.

**Institutional Review Board Statement:** The study was conducted according to the guidelines for the care and use of laboratory animal of the national institutes of health (NIH) and approved by the institutional animal care and use committee (IACUC) at the Soonchunhyang University (protocol No.: SCH20-0002, approved at February 2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** We want to exclude this statement. Our study did not report public dataset.

**Acknowledgments:** This work was supported by the Soonchunhyang University Research Fund.

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

**Sample Availability:** Astragaloside IV is available from the authors or Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Product No. #3305.

#### **References**

## *Article* **Protective Effects of Swertiamarin against Methylglyoxal-Induced Epithelial-Mesenchymal Transition by Improving Oxidative Stress in Rat Kidney Epithelial (NRK-52E) Cells**

**Kirti Parwani † , Farhin Patel † , Dhara Patel and Palash Mandal \***

> Department of Biological Sciences, P. D. Patel Institute of Applied Sciences, Charotar University of Science and Technology, Changa 388421, India<EMAIL_ADDRESS>(K.P<EMAIL_ADDRESS>(F.P<EMAIL_ADDRESS>(D.P.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +91-9666164654

† These authors contributed equally to this work.

**Abstract:** Increased blood glucose in diabetic individuals results in the formation of advanced glycation end products (AGEs), causing various adverse effects on kidney cells, thereby leading to diabetic nephropathy (DN). In this study, the antiglycative potential of Swertiamarin (SM) isolated from the methanolic extract of *E. littorale* was explored. The effect of SM on protein glycation was studied by incubating bovine serum albumin with fructose at 60 ◦C in the presence and absence of different concentrations of swertiamarin for 24 h. For comparative analysis, metformin was also used at similar concentrations as SM. Further, to understand the role of SM in preventing DN, in vitro studies using NRK-52E cells were done by treating cells with methylglyoxal (MG) in the presence and absence of SM. SM showed better antiglycative potential as compared to metformin. In addition, SM could prevent the MG mediated pathogenesis in DN by reducing levels of argpyrimidine, oxidative stress and epithelial mesenchymal transition in kidney cells. SM also downregulated the expression of interleukin-6, tumor necrosis factor-α and interleukin-1β. This study, for the first time, reports the antiglycative potential of SM and also provides novel insights into the molecular mechanisms by which SM prevents toxicity of MG on rat kidney cells.

**Keywords:** advanced glycation end product (AGE); oxidative stress; epithelial to mesenchymal transition; AGE-inhibitor; swertiamarin; diabetic nephropathy

#### **1. Introduction**

Type 2 Diabetes (T2D) is a metabolic syndrome, which results due to peripheral insulin resistance, affecting both metabolism and the disposal of glucose. The occurrence of diabetes is accelerating worldwide, with a consequent increase in diabetes associated complications like diabetic nephropathy (DN), neuropathy, retinopathy, and cardiovascular complications. Hyperglycaemia is one of the main reasons for causing DN, and various clinical trials have established that the progression to DN can be slowed and also reversed by good and strict glycemic controls [1–3]. Elevated blood glucose, which induces reactive oxygen species (ROS) formation, is understood to be one of the main reasons for the formation of advanced glycation end products (AGEs) in the intracellular and extracellular environment [4]. AGEs are the products of nonenzymatic glycation between free amino acids and reducing sugar via the Maillard reaction resulting into yellowish-brown fluorescent and insoluble adducts.

The formation of AGEs is divided into two stages. In the earlier stage, Amadori product, a stable compound is formed from an unstable Schiff base [5]. Amadori products can either form reactive dicarbonyls like glyoxal and methylglyoxal (MG) or undergo various chemical reactions like condensation, dehydration and oxidation to form AGEs.

**Citation:** Parwani, K.; Patel, F.; Patel, D.; Mandal, P. Protective Effects of Swertiamarin against Methylglyoxal-Induced Epithelial-Mesenchymal Transition by Improving Oxidative Stress in Rat Kidney Epithelial (NRK-52E) Cells. *Molecules* **2021**, *26*, 2748. https:// doi.org/10.3390/molecules26092748

Academic Editor: Raffaele Pezzani

Received: 11 March 2021 Accepted: 20 April 2021 Published: 7 May 2021

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

**Copyright:** © 2021 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/).

The mechanism and the different stages involved in the formation of AGEs are explained in Supplementary Materials Figure S1. These adducts can alter the normal physiological functions of a protein upon glycation [6]. This can alter the half-life of the proteins and affect their physiological clearance. Patients with persistent T2D are established to have vastly higher levels of AGEs [7]. The role of AGEs in DN has been documented by reports suggesting the negative correlation between the accumulation of AGEs in plasma and renal function [8]. AGEs bind to their receptor called receptor for advanced glycation end products (RAGE) and primarily lead to the generation of ROS and oxidative stress which further leads to the damage of the renal tubular cells and mesangial cells leading to DN. Further, AGEs lead to an imbalance between synthesis and degradation of extracellular matrix components like collagen and cause their accumulation in the mesangium, tubular interstitial cells and glomerulus basement membrane leading to their hardening, which is a hallmark feature of DN [9].

Reduced antioxidant defenses have also been reported in diabetic patients thereby providing acceleration to the development of chronic complications [10]. It is reported that AGEs could alter the physiological functions of an antioxidant enzyme like superoxide dismutase (SOD) and could inactivate it [11]. Glycation has been found to induce aggregation and structural modifications in catalase by targeting lysine residues [12]. This reduced antioxidant machinery along with increased ROS due to hyperglycaemia can be detrimental for a cell. Many synthetic inhibitors that can restrain the formation of AGEs have been identified and studied but they have been withdrawn back due to their lower potency and side effects. Therefore, there arises a rationale to identify some inhibitor, which along with being potent and effective, should have low toxicity.

Traditional and herbal therapies are gaining importance slowly over conventional medicines due to their advantage of having lower or no side-effects. The molecules present in the plants are shown to be hypoglycaemic, hypolipidemic, and antioxidant in nature. Compounds in the plants like phenolics [13], polysaccharides [14] and carotenoids [15], etc. are shown to possess antiglycative properties. In addition, the regular consumption of edible products rich in antioxidants and polyphenolic compounds could be beneficial in the prevention of diabetes and its related complications [16]. *Enicostemma littorale* commonly called Mamejava has been shown to possess a hypoglycaemic effect in T2D rats [17]. *E. littorale* contains important phytoconstituents like gentianine, enicoflavine, gentiocrucine, swertiamarin (SM), etc., to name a few. SM has been shown to possess anticholinergic [18], antihyperlipidaemic [19], hypoglycaemic [20] and antioxidant effects [21]. SM in combination with quercetin, ameliorated T2D and oxidative stress in streptozotocin-treated Wistar rats [22].

Since glycation is a process associated with increased free radical formation, compounds with antioxidant activities could also act as an inhibitor for the formation of AGEs [23]. The antiglycation potential of SM has not been explored yet, although there exists a report where *Swertia Chirayata* extract, which contains various active molecules along with SM, showed inhibition of fructose mediated glycation. Given the important role of SM in diabetes mellitus and its antioxidant potential, the present study was executed to understand and assess its antiglycation potential. To our knowledge, this is the first report that shows inhibition of fructose mediated glycation by SM and also unravels the molecular mechanism involved in its protective role in MG-induced damage on kidney cells, thereby preventing DN.

#### **2. Results**

*2.1. Isolation of SM from E. littorale and Characterization Using High-Performance Liquid Chromatography (HPLC), Fourier-Transform Infrared Spectroscopy (FTIR) and Liquid Chromatography-Mass Spectra(LC-MS)*

Using the solvent chromatography on silica, SM was successfully isolated from *E. littorale* as confirmed by TLC. The presence of SM was confirmed with reference to the standard SM using the chloroform: methanol (8:2 *v/v*) as a mobile phase with Rf of 0.58. The visualization was done at 254 nm as shown in Figure 1a. By our isolation method, we

report 7% yield of SM from the methanolic extract of *E. littorale*, which to our knowledge is the highest yield obtained until now. To analyze the purity of the isolated sample, the HPLC of the isolated compound was done and was compared with the standard SM. The HPLC profiles of the isolated SM (a) and the standard (b) are shown in Figure 1b. Both the standard and the lab isolated SM gave a characteristic peak at 238 nm, which is the absorbance maxima of SM as reported earlier. The retention time of the lab isolated SM was found to be 3.801 which fairly matched to 3.805 of that of the standard with the acetonitrile: water (10:90 *v/v*) as the mobile phase. The validation of the molecular weight of the isolated SM was done using LC-MS. The LC-MS spectrum of SM reported earlier showed a molecular ion peak at *m/z* of 374, and LC-MS spectrum for SM isolated in our lab as shown in Figure 1d, showed *m/z* of 375.1 in positive ion mode, indicating the addition of H+ to the compound, thereby increasing molecular ion peak by 1, as shown in Merck Index. Together the characterization data using different methods confirmed the isolated compound to be SM and by our isolation method, we report 7% yield of SM from the methanolic extract of *E. littorale*, which to our knowledge is the highest yield obtained until now (Figure 1).

standard SM using the chloroform: methanol (8:2 *v/v*) as a mobile phase with Rf of 0.58. The visualization was done at 254 nm as shown in Figure 1a. By our isolation method, we report 7% yield of SM from the methanolic extract of *E. littorale*, which to our knowledge is the highest yield obtained until now. To analyze the purity of the isolated sample, the HPLC of the isolated compound was done and was compared with the standard SM. The HPLC profiles of the isolated SM (a) and the standard (b) are shown in Figure 1b. Both the standard and the lab isolated SM gave a characteristic peak at 238 nm, which is the absorbance maxima of SM as reported earlier. The retention time of the lab isolated SM was found to be 3.801 which fairly matched to 3.805 of that of the standard with the acetoni-

The analysis of the different functional groups present in the isolated sample was done using FTIR and was also compared with the standard SM. The lab isolated SM fairly matched the FTIR spectrum of the standard SM as shown in Figure 1c. FTIR spectra of both, isolated and standard swertiamarin showed definite peaks between wavenumbers

, C=C stretch at 1617 cm−1

.This validates the purity of the isolated compound as the wave-

, C-H stretch at

, C-O-C at 1411 cm−1, and

. The characteristic O-H stretch peak was found at 3381 cm−1

numbers for both compounds (lab isolated SM and standard) match with each other.

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trile: water (10:90 *v/v*) as the mobile phase.

, C=O stretch at 1694 cm−1

4000–500 cm−1

C=CH2 stretch at 844 cm−1

2292 cm−1

**Figure 1.** (**a**) TLC profile: Lane 1, 2: Fractions containing SM along with impurities. Lane 3: Standard Lane 4: Methanolic extract of *E. littorale* Lane 5: Fraction containing SM in the TLC mobile phase (Chloroform: methanol in 8:2 *v/v* proportion) (**b**) The HPLC chromatogram of SM isolated in the lab (**b1**) and of the standard SM (**b2**) at 1 mg/mL using acetonitrile: water (10:90) as the mobile phase. (**c**) An overlay of FT-IR spectrum of standard SM (red) and SM isolated in the lab (blue). (**d**) The mass spectrum (LC-MS) of isolated swertiamarin showing characteristic *m/z* of 375.1. **Figure 1.** (**a**) TLC profile: Lane 1, 2: Fractions containing SM along with impurities. Lane 3: Standard Lane 4: Methanolic extract of *E. littorale* Lane 5: Fraction containing SM in the TLC mobile phase (Chloroform: methanol in 8:2 *v/v* proportion) (**b**) The HPLC chromatogram of SM isolated in the lab (**b1**) and of the standard SM (**b2**) at 1 mg/mL using acetonitrile: water (10:90) as the mobile phase. (**c**) An overlay of FT-IR spectrum of standard SM (red) and SM isolated in the lab (blue). (**d**) The mass spectrum (LC-MS) of isolated swertiamarin showing characteristic *m/z* of 375.1.

> The analysis of the different functional groups present in the isolated sample was done using FTIR and was also compared with the standard SM. The lab isolated SM fairly matched the FTIR spectrum of the standard SM as shown in Figure 1c. FTIR spectra of both, isolated and standard swertiamarin showed definite peaks between wavenumbers 4000–500 cm−1 . The characteristic O-H stretch peak was found at 3381 cm−1 , C-H stretch at 2292 cm−1 , C=O stretch at 1694 cm−1 , C=C stretch at 1617 cm−1 , C-O-C at 1411 cm−1, and C=CH2 stretch at 844 cm−1 .This validates the purity of the isolated compound as the wavenumbers for both compounds (lab isolated SM and standard) match with each other.

> The validation of the molecular weight of the isolated SM was done using LC-MS. The LC-MS spectrum of SM reported earlier showed a molecular ion peak at *m/z* of 374, and LC-MS spectrum for SM isolated in our lab as shown in Figure 1d, showed *m/z* of 375.1 in positive ion mode, indicating the addition of H+ to the compound, thereby increasing molecular ion peak by 1, as shown in Merck Index. Together the characterization data using different methods confirmed the isolated compound to be SM and by our isolation method, we report 7% yield of SM from the methanolic extract of *E. littorale*, which to our knowledge is the highest yield obtained until now (Figure 1).

#### *2.2. SM Inhibits the Formation of AGEs* possess characteristic property of fluorescence after glycation. The formation of AGEs was

*2.2. SM Inhibits the Formation of AGEs*

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The nonenzymatic glycation of the proteins leads to the formation of AGEs. AGEs possess characteristic property of fluorescence after glycation. The formation of AGEs was analyzed by checking the fluorescence at excitation and emission wavelengths of 370 nm and 440 nm, respectively. The fluorescence intensity was used as a measure of glycation, i.e., higher the intensity of fluorescence, the higher is the glycation of proteins. The intensity of fluorescence was significantly decreased (*p* ≤ 0.0001) in SM treated samples as compared to untreated samples that contained neither SM nor metformin. In addition, SM could inhibit the formation of AGEs better than metformin (*p* < 0.0001) at similar doses (Figure 2). This indicates that SM could be a better glycation inhibitor and therefore can prevent the formation of endogenous AGEs due to persistent hyperglycaemia in diabetic subjects. analyzed by checking the fluorescence at excitation and emission wavelengths of 370 nm and 440 nm, respectively. The fluorescence intensity was used as a measure of glycation, i.e., higher the intensity of fluorescence, the higher is the glycation of proteins. The intensity of fluorescence was significantly decreased (*p* ≤ 0.0001) in SM treated samples as compared to untreated samples that contained neither SM nor metformin. In addition, SM could inhibit the formation of AGEs better than metformin (*p* < 0.0001) at similar doses (Figure 2). This indicates that SM could be a better glycation inhibitor and therefore can prevent the formation of endogenous AGEs due to persistent hyperglycaemia in diabetic subjects.

The nonenzymatic glycation of the proteins leads to the formation of AGEs. AGEs

**Figure 2.** The intensity of fluorescence of AGEs in the presence and absence of swertiamarin (SM) and its comparison with metformin (M) at different concentrations measured using an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The results are Mean ± SD of 3 individual experiments. #### *p* < 0.0001 represents comparison between bovine serum albumin (BSA) negative and BSA + Fructose (Fru) group. \*\*\*\* *p* < 0.0001 represents the statistical significance w.r.t the BSA + Fru group. aaaa *p* < 0.0001 represents the comparison between SM and M. **Figure 2.** The intensity of fluorescence of AGEs in the presence and absence of swertiamarin (SM) and its comparison with metformin (M) at different concentrations measured using an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The results are Mean ± SD of 3 individual experiments. #### *p* < 0.0001 represents comparison between bovine serum albumin (BSA) negative and BSA + Fructose (Fru) group. \*\*\*\* *p* < 0.0001 represents the statistical significance w.r.t the BSA + Fru group. aaaa *p* < 0.0001 represents the comparison between SM and M.

#### *2.3. SM Reduces Fructose Mediated Hyperchromicity 2.3. SM Reduces Fructose Mediated Hyperchromicity*

Glycation of the proteins results in the structural changes of the proteins, which modifies their characteristic UV absorption spectrum. The changes in the structure of BSA and its absorbance were additionally analyzed by spectrophotometer (Figure 3a). As shown in Figure 3b, compared to normal BSA (Black line), glycated BSA (Blue line) shown increased absorbance and hyperchromicity (89%) due to fructose mediated changes in the structure of bovine serum albumin (BSA). SM could prevent the fructose mediated changes in the BSA (Green line) as depicted by reduced hyperchromicity (57.9%) indicating the inhibition of glycation (*p* < 0.0054) (Figure 3c). Glycation of the proteins results in the structural changes of the proteins, which modifies their characteristic UV absorption spectrum. The changes in the structure of BSA and its absorbance were additionally analyzed by spectrophotometer (Figure 3a). As shown in Figure 3b, compared to normal BSA (Black line), glycated BSA (Blue line) shown increased absorbance and hyperchromicity (89%) due to fructose mediated changes in the structure of bovine serum albumin (BSA). SM could prevent the fructose mediated changes in the BSA (Green line) as depicted by reduced hyperchromicity (57.9%) indicating the inhibition of glycation (*p* < 0.0054) (Figure 3c).

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**Figure 3.** Antiglycative effect of swertiamarin at 100 µg/mL (SM 100) as shown by absorbance at 280 nm using UV-spectrophotometer (**a**), the structural changes as seen by the shift in the peaks due to glycation (**b**), suggesting hyperchromicity (**c**). The results are Mean ± SD of 3 individual experiments. \*\*\*\* *p* < 0.0001, \*\* *p* < 0.054241. **Figure 3.** Antiglycative effect of swertiamarin at 100 µg/mL (SM 100) as shown by absorbance at 280 nm using UVspectrophotometer (**a**), the structural changes as seen by the shift in the peaks due to glycation (**b**), suggesting hyperchromicity (**c**). The results are Mean ± SD of 3 individual experiments. \*\*\*\* *p* < 0.0001, \*\* *p* < 0.054241. trophotometer (**a**), the structural changes as seen by the shift in the peaks due to glycation (**b**), suggesting hyperchromicity (**c**). The results are Mean ± SD of 3 individual experiments. \*\*\*\* *p* < 0.0001, \*\* *p* < 0.054241. *2.4. SM Reduces Carbonyl Content in the Glycated BSA*

#### *2.4. SM Reduces Carbonyl Content in the Glycated BSA 2.4. SM Reduces Carbonyl Content in the Glycated BSA* The carbonyl content was analysed as a marker for oxidation of protein during gly-

The carbonyl content was analysed as a marker for oxidation of protein during glycation. As shown in Figure 4, the glycated BSA showed remarkably higher carbonyl content upon the reaction with fructose, indicating the formation of radicals and dicarbonyls via Maillard reaction (\*\*\* *p* < 0.001). The treatment with SM could significantly limit the formation of carbonyls, which is due to its ability to scavenge free radicals as an antioxidant (\*\* *p* < 0.01). The carbonyl content was analysed as a marker for oxidation of protein during glycation. As shown in Figure 4, the glycated BSA showed remarkably higher carbonyl content upon the reaction with fructose, indicating the formation of radicals and dicarbonyls via Maillard reaction (\*\*\* *p* < 0.001). The treatment with SM could significantly limit the formation of carbonyls, which is due to its ability to scavenge free radicals as an antioxidant (\*\* *p* < 0.01). cation. As shown in Figure 4, the glycated BSA showed remarkably higher carbonyl content upon the reaction with fructose, indicating the formation of radicals and dicarbonyls via Maillard reaction (\*\*\* *p* < 0.001). The treatment with SM could significantly limit the formation of carbonyls, which is due to its ability to scavenge free radicals as an antioxidant (\*\* *p* < 0.01).

**Figure 4.** Estimation of carbonyl content. As compared to the native BSA, glycated BSA showed remarkably higher level of carbonyl content. The treatment with SM showed reduction in the carbonyl content, as compared to the glycated BSA. (\*\*\* *p* < 0.001 and \*\* *p* < 0.01). Results are mean ± **Figure 4.** Estimation of carbonyl content. As compared to the native BSA, glycated BSA showed remarkably higher level of carbonyl content. The treatment with SM showed reduction in the carbonyl content, as compared to the glycated BSA. (\*\*\* *p* < 0.001 and \*\* *p* < 0.01). Results are mean ± SD of 3 individual experiments. **Figure 4.** Estimation of carbonyl content. As compared to the native BSA, glycated BSA showed remarkably higher level of carbonyl content. The treatment with SM showed reduction in the carbonyl content, as compared to the glycated BSA. (\*\*\* *p* < 0.001 and \*\* *p* < 0.01). Results are mean ± SD of 3 individual experiments.

#### SD of 3 individual experiments. *2.5. SM Prevents Fructose Mediated Side-Chain Modifications in BSA 2.5. SM Prevents Fructose Mediated Side-Chain Modifications in BSA*

*2.5. SM Prevents Fructose Mediated Side-Chain Modifications in BSA* The modifications in the functional group in the side chains of the BSA was analysed The modifications in the functional group in the side chains of the BSA was analysed using FTIR. As shown in Figure 5, FTIR spectrum of the native BSA showed a peak at 1655 The modifications in the functional group in the side chains of the BSA was analysed using FTIR. As shown in Figure 5, FTIR spectrum of the native BSA showed a peak at 1655 cm−1 corresponding to amide I, while red-shift was observed in case of glycated

using FTIR. As shown in Figure 5, FTIR spectrum of the native BSA showed a peak at 1655

samples, where the amide I shifted to 1651 cm−1 . The treatment with SM showed a minor shift, indicating the relatively stable BSA molecule, as the amide I shift indicates the presence of α-helical structure in the protein. The amide III band shifted 9 cm−1 , from 1245 cm−1 to lower wavenumber 1236 cm−1 in the glycated BSA as compared to native BSA. However, the treatment of glycated BSA with SM did not show major shift in the wavenumber compared to native BSA with wavenumber 1242 cm−1 . Together these results indicate that glycated BSA exhibited various side-chain modifications, affecting the structure of BSA upon glycation. Table 1 shows the different bands and wavenumbers obtained in all the samples. **Table 1.** Comparative analysis of bands and wavenumbers in all samples. **Samples Bands and Wavenumber (cm−1 ) Amide I Amide III** Native BSA 1655 1245 Glycated BSA 1651 1236 Glycated BSA + SM 1654 1242

cm−1 corresponding to amide I, while red-shift was observed in case of glycated samples,

dicating the relatively stable BSA molecule, as the amide I shift indicates the presence of

lower wavenumber 1236 cm−1 in the glycated BSA as compared to native BSA. However, the treatment of glycated BSA with SM did not show major shift in the wavenumber com-

cated BSA exhibited various side-chain modifications, affecting the structure of BSA upon glycation. Table 1 shows the different bands and wavenumbers obtained in all the sam-

. The treatment with SM showed a minor shift, in-

. Together these results indicate that gly-

, from 1245 cm−1 to

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α-helical structure in the protein. The amide III band shifted 9 cm−1

where the amide I shifted to 1651 cm−1

ples.

pared to native BSA with wavenumber 1242 cm−1

**Figure 5.** FTIR spectra of BSA (black line), glycated BSA (red line) and the glycated BSA treated with SM (blue line). **Figure 5.** FTIR spectra of BSA (black line), glycated BSA (red line) and the glycated BSA treated with SM (blue line).

*2.6. Effect of MG and SM on the Viability of NRK-52E Cells* **Table 1.** Comparative analysis of bands and wavenumbers in all samples.

#### *2.6. Effect of MG and SM on the Viability of NRK-52E Cells*

The toxicity of MG on NRK-52E cells was analyzed for fixing the dose for further experiments on NRK-52E. The treatment with MG induced cell death of NRK-52E and the dose-dependent decrease in cell viability was observed with an increase in the concentration of MG (*p* < 0.0001) (Figure 6a). To determine the dose of MG to be used for further experiments with NRK-52E, we determined the IC50 value for MG, which was found to be 200 µM and therefore the cells were challenged with 200 µM MG for testing the protective effects of SM in presence of MG. However, SM did not show any toxicity on NRK-52E cells at all the different concentrations used for the experiment (Figure 6b).

under 10× objective.

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**Figure 6.** (**a**) The effect MG on the cell viability in NRK-52E cells. NRK-52E cells when treated with different concentrations of MG caused cell death in a dose-dependent manner as compared to control. (**b**) The cytotoxicity of SM was checked on NRK-52E cells. The treatment with SM did not show any toxicity on the NRK-52E cells at various concentrations. Results are Mean ± SD of 3 individual experiments. **Figure 6.** (**a**) The effect MG on the cell viability in NRK-52E cells. NRK-52E cells when treated with different concentrations of MG caused cell death in a dose-dependent manner as compared to control. (**b**) The cytotoxicity of SM was checked on NRK-52E cells. The treatment with SM did not show any toxicity on the NRK-52E cells at various concentrations. Results are Mean ± SD of 3 individual experiments. of MG caused cell death in a dose-dependent manner as compared to control. (**b**) The cytotoxicity of SM was checked on NRK-52E cells. The treatment with SM did not show any toxicity on the NRK-52E cells at various concentrations. Results are Mean ± SD of 3 individual experiments. *2.7. SM Improves the Morphology of the NRK-52E Cells and Ameliorates against MG-Induced*

#### *2.7. SM Improves the Morphology of the NRK-52E Cells and Ameliorates against MG-Induced Damage 2.7. SM Improves the Morphology of the NRK-52E Cells and Ameliorates against MG-Induced Damage Damage* The effect of MG on the morphology of NRK-52E cells can be seen in Figure 7. MG

The effect of MG on the morphology of NRK-52E cells can be seen in Figure 7. MG treatment on the cells changed the cobblestone morphology of NRK-52E cells to a round and elongated fibroblast-like shape, indicating the cellular damage due to MG, which was prevented when the cells were treated with MG along with SM. The morphological changes are indicative of various inflammatory changes occurred due to the presence of MG, which was ameliorated by SM, as seen by the intact epithelial morphology of NRK-The effect of MG on the morphology of NRK-52E cells can be seen in Figure 7. MG treatment on the cells changed the cobblestone morphology of NRK-52E cells to a round and elongated fibroblast-like shape, indicating the cellular damage due to MG, which was prevented when the cells were treated with MG along with SM. The morphological changes are indicative of various inflammatory changes occurred due to the presence of MG, which was ameliorated by SM, as seen by the intact epithelial morphology of NRK-52E cells. treatment on the cells changed the cobblestone morphology of NRK-52E cells to a round and elongated fibroblast-like shape, indicating the cellular damage due to MG, which was prevented when the cells were treated with MG along with SM. The morphological changes are indicative of various inflammatory changes occurred due to the presence of MG, which was ameliorated by SM, as seen by the intact epithelial morphology of NRK-52E cells.

**Figure 7.** Morphology of NRK-52E cells in each treatment group at 24 h. The untreated NRK-52E cells showed its characteristic epithelial morphology (**a**). The treatment with 200 µM MG changed the epithelial morphology of NRK-52E cells to elongated fibroblast like morphology, indicated with arrows (**b**). Cotreatment with 100 µg/mL SM prevented MG—in-**Figure 7.** Morphology of NRK-52E cells in each treatment group at 24 h. The untreated NRK-52E cells showed its characteristic epithelial morphology (**a**). The treatment with 200 µM MG changed the epithelial morphology of NRK-52E cells to elongated fibroblast like morphology, indicated with arrows (**b**). Cotreatment with 100 µg/mL SM prevented MG—induced morphological changes in NRK-52E cells (**c**). Above images are representative microscopy images of each group under 10× objective. **Figure 7.** Morphology of NRK-52E cells in each treatment group at 24 h. The untreated NRK-52E cells showed its characteristic epithelial morphology (**a**). The treatment with 200 µM MG changed the epithelial morphology of NRK-52E cells to elongated fibroblast like morphology, indicated with arrows (**b**). Cotreatment with 100 µg/mL SM prevented MG—induced morphological changes in NRK-52E cells (**c**). Above images are representative microscopy images of eachgroup under 10× objective.

duced morphological changes in NRK-52E cells (**c**). Above images are representative microscopy images of each group

*2.8. SM Alleviates the Formation of Argpyrimidine Like AGEs in NRK-52E Cells*

*2.8. SM Alleviates the Formation of Argpyrimidine Like AGEs in NRK-52E Cells*

#### *2.8. SM Alleviates the Formation of Argpyrimidine Like AGEs in NRK-52E Cells* Dicarbonyls like MG react with arginine in the proteins to form AGEs. Argpyrimi-

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Dicarbonyls like MG react with arginine in the proteins to form AGEs. Argpyrimidine is a major modification of amino acid arginine by MG. As shown in Figure 8 NRK-52E cells when treated with MG showed higher levels of argpyrimidine as compared to the untreated cells. The treatment of SM along with MG alleviated the levels of argpyrimidine which could be due to the inhibition of modification of arginine by MG, thereby reducing the glycation induced damage to the cells. dine is a major modification of amino acid arginine by MG. As shown in Figure 8 NRK-52E cells when treated with MG showed higher levels of argpyrimidine as compared to the untreated cells. The treatment of SM along with MG alleviated the levels of argpyrimidine which could be due to the inhibition of modification of arginine by MG, thereby reducing the glycation induced damage to the cells.

**Figure 8.** Detection of argpyrimidine levels. As compared to the control group, treatment with 200 µM MG in the NRK-52E cells after 24 h resulted in the formation of argpyrimidine by MG-induced modifications of arginine which could be prevented in MG and 100 µg/mL SM cotreatment group **Figure 8.** Detection of argpyrimidine levels. As compared to the control group, treatment with 200 µM MG in the NRK-52E cells after 24 h resulted in the formation of argpyrimidine by MG-induced modifications of arginine which could be prevented in MG and 100 µg/mL SM cotreatment group (\* *p* < 0.05). Results are Mean ± SD of 3 individual experiments.

#### (\* *p* < 0.05). Results are Mean ± SD of 3 individual experiments. *2.9. SM Alleviates the Formation of MG—Induced ROS and Mitigates the Oxidative Stress in NRK-52E Cells*

*2.9. SM Alleviates the Formation of MG—Induced ROS and Mitigates the Oxidative Stress in NRK-52E Cells* The treatment with MG on NRK-52E cells resulted in the elevated oxidative stress, which could be due to the increased levels of AGEs formation in the presence of MG. Increased oxidative stress leads to the peroxidation of lipids resulting in the formation of malondialdehyde (MDA), a marker for oxidative stress in the cells. MG treatment in the NRK-52E cells exacerbated the levels of MDA as measured by HPLC (Figure 9a). This was ameliorated by the treatment of SM as seen by the reduced MDA levels. Oxidative stress further increased the formation of ROS in the cells, measured through fluorescence spec-The treatment with MG on NRK-52E cells resulted in the elevated oxidative stress, which could be due to the increased levels of AGEs formation in the presence of MG. Increased oxidative stress leads to the peroxidation of lipids resulting in the formation of malondialdehyde (MDA), a marker for oxidative stress in the cells. MG treatment in the NRK-52E cells exacerbated the levels of MDA as measured by HPLC (Figure 9a). This was ameliorated by the treatment of SM as seen by the reduced MDA levels. Oxidative stress further increased the formation of ROS in the cells, measured through fluorescence spectroscopy (Figure 9b). Treatment with SM in the presence of MG, prevented the formation of ROS, thereby unraveling the role of SM in preventing the MG—induced oxidative stress.

troscopy (Figure 9b). Treatment with SM in the presence of MG, prevented the formation of ROS, thereby unraveling the role of SM in preventing the MG—induced oxidative

stress.

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**Figure 9.** (**a**) HPLC chromatograms of MDA measured from NRK-52E cells after DNPH derivatization. The treatment with 200 µM MG in NRK-52E cells increased the levels of MDA measured using ODS2 reverse phase column in the presence of acetonitrile and milliQ water containing 0.2% acetic acid, with a ratio 38:62 respectively as the mobile phase. Treatment with 100 µg/mL SM in the presence of MG attenuated the production of MDA. (**b**) The levels of ROS were also measured using fluorescence spectroscopy with excitation and emission wavelengths 495 nm and 529 nm, respectively. Treatment with SM in the presence of MG could inhibit the elevation of ROS significantly (\*\* *p* < 0.01) as shown in MG treated group (\*\* *p* < 0.01), proving the antioxidative characteristic of SM. Results are Mean ± SD of 3 individual experiments. **Figure 9.** (**a**) HPLC chromatograms of MDA measured from NRK-52E cells after DNPH derivatization. The treatment with 200 µM MG in NRK-52E cells increased the levels of MDA measured using ODS2 reverse phase column in the presence of acetonitrile and milliQ water containing 0.2% acetic acid, with a ratio 38:62 respectively as the mobile phase. Treatment with 100 µg/mL SM in the presence of MG attenuated the production of MDA. (**b**) The levels of ROS were also measured using fluorescence spectroscopy with excitation and emission wavelengths 495 nm and 529 nm, respectively. Treatment with SM in the presence of MG could inhibit the elevation of ROS significantly (\*\* *p* < 0.01) as shown in MG treated group (\*\* *p* < 0.01), proving the antioxidative characteristic of SM. Results are Mean ± SD of 3 individual experiments. 200 µM MG in NRK-52E cells increased the levels of MDA measured using ODS2 reverse phase column in the presence of acetonitrile and milliQ water containing 0.2% acetic acid, with a ratio 38:62 respectively as the mobile phase. Treatment with 100 µg/mL SM in the presence of MG attenuated the production of MDA. (**b**) The levels of ROS were also measured using fluorescence spectroscopy with excitation and emission wavelengths 495 nm and 529 nm, respectively. Treatment with SM in the presence of MG could inhibit the elevation of ROS significantly (\*\* *p* < 0.01) as shown in MG treated group (\*\* *p* < 0.01), proving the antioxidative characteristic of SM. Results are Mean ± SD of 3 individual experiments. *2.10. SM Alleviates MG—Induced ER Stress in NRK-52E Cells*

#### *2.10. SM Alleviates MG—Induced ER Stress in NRK-52E Cells 2.10. SM Alleviates MG—Induced ER Stress in NRK-52E Cells* further results in the upregulation of ER stress by activating the unfolded protein response

The treatment of the NRK-52E cells with MG leads to the production of AGEs which further results in the upregulation of ER stress by activating the unfolded protein response (UPR). The mRNA expression of ER stress markers like CCAAT-enhancer-binding protein homologous protein (CHOP) and glucose regulatory protein 78 (Grp78) were upregulated in the MG-treated NRK-52E cells. The treatment with SM along with MG could alleviate the upregulation of CHOP and Grp78, indicating the protection against ER stress (Figure The treatment of the NRK-52E cells with MG leads to the production of AGEs which further results in the upregulation of ER stress by activating the unfolded protein response (UPR). The mRNA expression of ER stress markers like CCAAT-enhancer-binding protein homologous protein (CHOP) and glucose regulatory protein 78 (Grp78) were upregulated in the MG-treated NRK-52E cells. The treatment with SM along with MG could alleviate the upregulation of CHOP and Grp78, indicating the protection against ER stress (Figure 10). (UPR). The mRNA expression of ER stress markers like CCAAT-enhancer-binding protein homologous protein (CHOP) and glucose regulatory protein 78 (Grp78) were upregulated in the MG-treated NRK-52E cells. The treatment with SM along with MG could alleviate the upregulation of CHOP and Grp78, indicating the protection against ER stress (Figure 10).

The treatment of the NRK-52E cells with MG leads to the production of AGEs which

**Figure 10.** mRNA expression of ER stress genes (**a**) CHOP and (**b**) Grp-78 using qRT-PCR. The expression of CHOP and Grp78 was significantly upregulated in MG-exposed cells as compared to the untreated control NRK-52E cells after 24 h. The treatment with 100 µg/mL SM along with 200 µM MG could alleviate the upregulation of CHOP and Grp78, indicating the protection against ER stress. Results are Mean ± SD of 3 individual experiments analysed by ANOVA. The symbol \*\*\* *p* < 0.001 indicates the comparison of MG group w.r.t control and # *p* < 0.05, ### *p* < 0.001 indicates the comparison of SM group w.r.t. MG group.

group w.r.t. MG group.

#### *2.11. Treatment with SM Improves the Inflammatory Response in MG Treated NRK-52E Cells 2.11. Treatment with SM Improves the Inflammatory Response in MG Treated NRK-52E Cells*

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**Figure 10.** mRNA expression of ER stress genes (**a**) CHOP and (**b**) Grp-78 using qRT-PCR. The expression of CHOP and Grp78 was significantly upregulated in MG-exposed cells as compared to the untreated control NRK-52E cells after 24 h. The treatment with 100 µg/mL SM along with 200 µM MG could alleviate the upregulation of CHOP and Grp78, indicating

*p* < 0.001 indicates the comparison of MG group w.r.t control and # *p* < 0.05, ### *p* < 0.001 indicates the comparison of SM

Cells when treated with 200 µM MG caused the upregulation of the mRNA levels of RAGE, that could be due to elevated levels of AGEs in MG-treated NRK-52E cells. The interaction of RAGE with AGEs and MG alone induced oxidative stress which resulted in the upregulation of ROS via increased NADPH oxidase levels in NRK-52E cells. The oxidative stress and the upregulated expression of RAGE induced the expression of various proinflammatory cytokines like interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) in the cells treated with MG as compared to untreated control NRK-52E cells. This further exacerbated the expression of cell adhesion molecules like intracellular cell adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) in NRK-52E cells, leading to inflammation and cellular injury to kidney cells. Nonetheless, SM treatment at a concentration of 100 µg/mL to MG exposed cells (Figure 11) could prevent the upregulation of all the inflammatory cytokines under investigation (#### *p* < 0.0001; ### *p <* 0.001; ## *p* < 0.01), therefore explaining the preventive role of SM in inflammation of kidney cells. Cells when treated with 200 µM MG caused the upregulation of the mRNA levels of RAGE, that could be due to elevated levels of AGEs in MG-treated NRK-52E cells. The interaction of RAGE with AGEs and MG alone induced oxidative stress which resulted in the upregulation of ROS via increased NADPH oxidase levels in NRK-52E cells. The oxidative stress and the upregulated expression of RAGE induced the expression of various proinflammatory cytokines like interleukin-6 (IL-6), tumor necrosis factor-α (TNF- α), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) in the cells treated with MG as compared to untreated control NRK-52E cells. This further exacerbated the expression of cell adhesion molecules like intracellular cell adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) in NRK-52E cells, leading to inflammation and cellular injury to kidney cells. Nonetheless, SM treatment at a concentration of 100 µg/mL to MG exposed cells (Figure 11) could prevent the upregulation of all the inflammatory cytokines under investigation (#### *p* < 0.0001; ### *p <* 0.001; ## *p* < 0.01), therefore explaining the preventive role of SM in inflammation of kidney cells.

**Figure 11.** qRT-PCR analysis results of (**a**) RAGE (**b**) NADPH oxidase (**c**) iNOS (**d**) IL-6 (**e**) TNF-α (**f**) IL-1β (**g**) ICAM-1, and (**h**) MCP-1 in MG-treated NRK-52E cells alone and SM for 24 h. The gene expression levels were calculated after normalizing against housekeeping 18S rRNA and are presented as relative mRNA expression units. Values represent mean ± SD of 3 individual experiments. The symbol \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001 indicate the comparison of MG group w.r.t control and ## *p* < 0.01, #### *p* < 0.0001 indicate the comparison between MG and SM group. **Figure 11.** qRT-PCR analysis results of (**a**) RAGE (**b**) NADPH oxidase (**c**) iNOS (**d**) IL-6 (**e**) TNF-α (**f**) IL-1β (**g**) ICAM-1, and (**h**) MCP-1 in MG-treated NRK-52E cells alone and SM for 24 h. The gene expression levels were calculated after normalizing against housekeeping 18S rRNA and are presented as relative mRNA expression units. Values represent mean ± SD of 3 individual experiments. The symbol \* *p* < 0.05, \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001 indicate the comparison of MG group w.r.t control and # *p* < 0.05, ## *p* < 0.01, #### *p* < 0.0001 indicate the comparison between MG and SM group.

#### *2.12. SM Improves MG-Induced Epithelial Mesenchymal Transition (EMT) in NRK-52E Cells 2.12. SM Improves MG-Induced Epithelial Mesenchymal Transition (EMT) in NRK-52E Cells*

NRK-52E cells when treated with MG, showed morphological changes and the epithelial nature of cells changed to elongated, and fibroblast-like phenotype (Figure 10), possibly due to epithelial-mesenchymal transformation (EMT). SM treated group, did not show any change in the epithelial morphology of NRK-52E cells. MG induces the upregulation of p38 MAPK and also the downstream signaling by activating the transcription factors leading to various molecular changes in the kidney cells. It is previously reported that transforming growth factor- β (TGF-β) plays a very crucial role in the pathogenesis of DN. TGF-β induces the upregulation of fibroblast markers like alpha-smooth muscle actin (α-SMA), and fibronectin-1, along with downregulation of epithelial marker E-cadherin. Treatment with MG showed upregulated expression of these markers in NRK-52E cells which was ameliorated in the cells cotreated with MG and SM. These results indicate that MG induces the EMT changes in epithelial NRK-52E cells (Figure 12). As oxidative stress plays a very important role in the development of renal fibrosis via EMT, we also analyzed the levels of key intracellular antioxidant machinery like nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) and heme oxygenase-1 (HO-1) expression levels. The treatment with MG in NRK-52E cells significantly attenuated the levels of Nrf-2 and HO-1, which were induced by the treatment with SM. Together these results indicate that SM ameliorated EMT in NRK-52E cells by inhibiting the expression of TGF-β via upregulation of HO-1 and Nrf-2 (Figure 12). As shown in Figure 13, MG treatment upregulated the protein levels of TGF-β and decreased the levels of HO-1, which was alleviated by SM treatment. NRK-52E cells when treated with MG, showed morphological changes and the epithelial nature of cells changed to elongated, and fibroblast-like phenotype (Figure 10), possibly due to epithelial-mesenchymal transformation (EMT). SM treated group, did not show any change in the epithelial morphology of NRK-52E cells. MG induces the upregulation of p38 MAPK and also the downstream signaling by activating the transcription factors leading to various molecular changes in the kidney cells. It is previously reported that transforming growth factor- β (TGF-β) plays a very crucial role in the pathogenesis of DN. TGF-β induces the upregulation of fibroblast markers like alpha-smooth muscle actin (α-SMA), and fibronectin-1, along with downregulation of epithelial marker E-cadherin. Treatment with MG showed upregulated expression of these markers in NRK-52E cells which was ameliorated in the cells cotreated with MG and SM. These results indicate that MG induces the EMT changes in epithelial NRK-52E cells (Figure 12). As oxidative stress plays a very important role in the development of renal fibrosis via EMT, we also analyzed the levels of key intracellular antioxidant machinery like nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) and heme oxygenase-1 (HO-1) expression levels. The treatment with MG in NRK-52E cells significantly attenuated the levels of Nrf-2 and HO-1, which were induced by the treatment with SM. Together these results indicate that SM ameliorated EMT in NRK-52E cells by inhibiting the expression of TGF-β via upregulation of HO-1 and Nrf-2 (Figure 12). As shown in Figure 13, MG treatment upregulated the protein levels of TGF-β and decreased the levels of HO-1, which was alleviated by SM treatment.

**Figure 12.** qRT-PCR analysis results of (**a**) p38 MAPK (**b**) TGF-β (**c**) α-SMA (**d**) Fibronectin-1 (**e**) E-cadherin (**f**) HO-1 and (**g**) Nrf-2 in MG-treated NRK-52E cells alone and SM for 24 h. The gene expression levels were calculated after normalizing against housekeeping 18S rRNA and are presented as relative mRNA expression units. Values represent mean ± SD of 3 individual experiments. The symbol \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 indicate the comparison of MG group w.r.t control and ## *p* < 0.01, ### *p* < 0.001, #### *p* < 0.0001, indicate the comparison between MG and SM group. **Figure 12.** qRT-PCR analysis results of (**a**) p38 MAPK (**b**) TGF-β (**c**) α-SMA (**d**) Fibronectin-1 (**e**) E-cadherin (**f**) HO-1 and (**g**) Nrf-2 in MG-treated NRK-52E cells alone and SM for 24 h. The gene expression levels were calculated after normalizing against housekeeping 18S rRNA and are presented as relative mRNA expression units. Values represent mean ± SD of 3 individual experiments. The symbol \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 indicate the comparison of MG group w.r.t control and ## *p* < 0.01, ### *p* < 0.001, #### *p* < 0.0001, indicate the comparison between MG and SM group.

group.

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**Figure 13.** Protein levels of TGF-β and HO-1 in NRK-52E measured using ELISA. The treatment with 200 μM MG for 24h, up-regulated the levels of TGF-β proteins which was prevented by the co-treatment with 100 μg/mL SM. The levels of HO-1 were declined in the MG treated group after 24 h which was alleviated by SM, proving the role of SM in inhibiting TGF-β expression by upregulating HO-1. Values represent mean ± SD of 3 individual experiments. \*\*\*\* *p* < 0.0001 indicate **Figure 13.** Protein levels of TGF-β and HO-1 in NRK-52E measured using ELISA. The treatment with 200 µM MG for 24 h, up-regulated the levels of TGF-β proteins which was prevented by the co-treatment with 100 µg/mL SM. The levels of HO-1 were declined in the MG treated group after 24 h which was alleviated by SM, proving the role of SM in inhibiting TGF-β expression by upregulating HO-1. Values represent mean ± SD of 3 individual experiments. \*\*\*\* *p* < 0.0001 indicate the comparison of MG group w.r.t control and ### *p* < 0.001, #### *p* < 0.0001, indicate the comparison between MG and SM group. **Figure 13.** Protein levels of TGF-β and HO-1 in NRK-52E measured using ELISA. The treatment with 200 μM MG for 24h, up-regulated the levels of TGF-β proteins which was prevented by the co-treatment with 100 μg/mL SM. The levels of HO-1 were declined in the MG treated group after 24 h which was alleviated by SM, proving the role of SM in inhibiting TGF-β expression by upregulating HO-1. Values represent mean ± SD of 3 individual experiments. \*\*\*\* *p* < 0.0001 indicate the comparison of MG group w.r.t control and ### *p* < 0.001, #### *p* < 0.0001, indicate the comparison between MG and SM group.

the comparison of MG group w.r.t control and ### *p* < 0.001, #### *p* < 0.0001, indicate the comparison between MG and SM

#### *2.13. SM Shows Better Affinity and Binding with RAGE as Compared to Argpyrimidine 2.13. SM Shows Better Affinity and Binding with RAGE as Compared to Argpyrimidine*

*2.13. SM Shows Better Affinity and Binding with RAGE as Compared to Argpyrimidine* AGEs interact with their cellular receptor RAGE and initiate the inflammatory pathways by production of proinflammatory cytokines which further results in the oxidative stress. The selective inhibition of AGE-RAGE axis by preventing the binding of AGEs with RAGE can inhibit the deleterious effects observed due to their interaction. Therapeutic molecules that can bind with RAGE, thereby blocking the binding of AGEs with RAGE can be used as an approach to attenuate the progression of DN. In our study we have shown that SM not only reduces the expression of RAGE but also reduces the levels of AGEs like Argpyrimidine which is formed in the presence of MG. Therefore, to understand the possible role and mechanism of how SM prevents the MG-induced damage on NRK-52E cells, we adopted an in silico approach, where we checked if SM could interact with RAGE so as to prevent the binding of AGEs and therefore inhibit the activation of AGE-RAGE axis. Therefore, we docked both SM and Argpyrimidine with RAGE to check the affinity of both the ligands with RAGE. A total of 13 different molecules that encompassed different states of SM and Argpyrimidine were generated by LigPrep module. All the ligands and their various states were docked in the upstream C-Domain Type 1 using the Glide XP module. Further, the molecules were quantitatively evaluated based on their AGEs interact with their cellular receptor RAGE and initiate the inflammatory pathways by production of proinflammatory cytokines which further results in the oxidative stress. The selective inhibition of AGE-RAGE axis by preventing the binding of AGEs with RAGE can inhibit the deleterious effects observed due to their interaction. Therapeutic molecules that can bind with RAGE, thereby blocking the binding of AGEs with RAGE can be used as an approach to attenuate the progression of DN. In our study we have shown that SM not only reduces the expression of RAGE but also reduces the levels of AGEs like Argpyrimidine which is formed in the presence of MG. Therefore, to understand the possible role and mechanism of how SM prevents the MG-induced damage on NRK-52E cells, we adopted an in silico approach, where we checked if SM could interact with RAGE so as to prevent the binding of AGEs and therefore inhibit the activation of AGE-RAGE axis. Therefore, we docked both SM and Argpyrimidine with RAGE to check the affinity of both the ligands with RAGE. A total of 13 different molecules that encompassed different states of SM and Argpyrimidine were generated by LigPrep module. All the ligands and their various states were docked in the upstream C-Domain Type 1 using the Glide XP module. Further, the molecules were quantitatively evaluated based on their scores and qualitatively based on the type of interactions made with the residues of the amino acids. The chemical structures of the ligands are shown is Figure 14. AGEs interact with their cellular receptor RAGE and initiate the inflammatory pathways by production of proinflammatory cytokines which further results in the oxidative stress. The selective inhibition of AGE-RAGE axis by preventing the binding of AGEs with RAGE can inhibit the deleterious effects observed due to their interaction. Therapeutic molecules that can bind with RAGE, thereby blocking the binding of AGEs with RAGE can be used as an approach to attenuate the progression of DN. In our study we have shown that SM not only reduces the expression of RAGE but also reduces the levels of AGEs like Argpyrimidine which is formed in the presence of MG. Therefore, to understand the possible role and mechanism of how SM prevents the MG-induced damage on NRK-52E cells, we adopted an in silico approach, where we checked if SM could interact with RAGE so as to prevent the binding of AGEs and therefore inhibit the activation of AGE-RAGE axis. Therefore, we docked both SM and Argpyrimidine with RAGE to check the affinity of both the ligands with RAGE. A total of 13 different molecules that encompassed different states of SM and Argpyrimidine were generated by LigPrep module. All the ligands and their various states were docked in the upstream C-Domain Type 1 using the Glide XP module. Further, the molecules were quantitatively evaluated based on their scores and qualitatively based on the type of interactions made with the residues of the amino acids. The chemical structures of the ligands are shown is Figure 14.

scores and qualitatively based on the type of interactions made with the residues of the

**Figure 14. Figure 14.**  Chemical structure of swertiamarin and Argpyrimidine. Chemical structure of swertiamarin and Argpyrimidine.

**Figure 14.** Chemical structure of swertiamarin and Argpyrimidine.

SM showed a G score of −4.7 kcal/mol while the docking score was −4.7 kcal/mol. The interactions formed by this ligand are as follows: OH acts as hydrogen bond acceptor for Leu 163 and Glu 174, and as a hydrogen bond donor to the amino acid Trp 156 (Figure 15a). Argpyrimidine on the other hand showed G score of −2.75 kcal/mol while the docking score is −2.73 kcal/mol. The interactions formed by this ligand are as follows: O− acts as hydrogen bond acceptor for TRP 156, and hydrogen from amino group as a hydrogen bond donor to the amino acids Leu 154 and Glu 174. Further, the protonated nitrogen of argpyrimidine forms a salt bridge with GLU 174 (Figure 15b). Table 2 summarizes different interactions between SM-RAGE and Argpyrimidine-RAGE docking. SM showed a G score of −4.7 kcal/mol while the docking score was −4.7 kcal/mol. The interactions formed by this ligand are as follows: OH acts as hydrogen bond acceptor for Leu 163 and Glu 174, and as a hydrogen bond donor to the amino acid Trp 156 (Figure 15a). Argpyrimidine on the other hand showed G score of −2.75 kcal/mol while the docking score is −2.73 kcal/mol. The interactions formed by this ligand are as follows: O− acts as hydrogen bond acceptor for TRP 156, and hydrogen from amino group as a hydrogen bond donor to the amino acids Leu 154 and Glu 174. Further, the protonated nitrogen of argpyrimidine forms a salt bridge with GLU 174 (Figure 15b). Table 2 summarizes different interactions between SM-RAGE and Argpyrimidine-RAGE docking.

**Figure 15.** (**a**) Binding of SM with RAGE (left) and the amino acids involved in the interaction (right panel). (**b**) Binding of Argpyrimidine with RAGE (left) and the amino acid involved in the interaction (right panel). **Figure 15.** (**a**) Binding of SM with RAGE (left) and the amino acids involved in the interaction (right panel). (**b**) Binding of Argpyrimidine with RAGE (left) and the amino acid involved in the interaction (right panel).

**Table 2.** Interaction of ligands, type of interaction and total binding energies of RAGE with SM and Argpyrimidine.

#### **3. Discussion**

In the present study, we report isolation of SM and its role in inhibiting the formation of AGEs. The SM was isolated in the lab from *E. littorale* with better yield (7%) by column chromatography, which to our knowledge is the highest yield using silica of 60–120 mesh size as compared to the 5% yield obtained using silica based column chromatography [24], 0.6% using sephadex LH-20 column [25] and 2% using centrifugal partition chromatography [26]. The absorbance maxima of isolated SM matched with the absorbance maxima of the SM isolated by Rana et al. in the HPLC [26] and with the UV spectral analysis by Vishwakarma et al. to be at 238 nm [24,27]. The molecular ion peak of the isolated SM was 375.1 which matched with the results obtained in the literature along with their FT-IR fingerprint, proving the isolated compound to be SM [28]. Owing to its various properties like anticholinergic [18] antihyperlipidaemic [19] and given the important role of SM in diabetes mellitus [20] and its antioxidant potential [29], the present study was executed to understand and assess its antiglycation potential.

The persistent hyperglycaemia in the blood leads to the production of ROS which results in the oxidative stress. Oxidative stress results not only due to excessive ROS but may also result due to the defects or impairment in the antioxidant enzymes which otherwise can clear the ROS [30]. Increased blood glucose exacerbates the formation of ROS and is one of the prime reasons for AGEs formation, which further leads to various complications of T2D like DN [31–33]. Free radicals formed due to higher glucose levels can induce the formation of AGEs from Amadori products [34]. This is explained by the study which shows the increased levels of MG in the blood of diabetics as compared to nondiabetic individuals [35] and also supported by a study proving the accumulation of AGEs in most DN patients [36]. Under diabetic conditions, the excess of glucose leads to the activation of polyol pathway, resulting in the formation of fructose [37]. Fructose leads to the formation of AGEs and ROS by promoting the glycation of proteins and lipids, also aided by MG which is a dicarbonyl formed during glycolytic pathway for glucose metabolism [38]. Hence, preventing the glycation induced by fructose and MG could be one of the prime strategies to inhibit the formation of AGEs and prevent the diabetes related complications [39]. We, therefore assessed the potential of SM in inhibiting the fructose induced glycation. Fructose mediated glycation of proteins increases their fluorescence intensity at 440 nm which is due to formation of AGEs [40]. In the extant study, BSA glycated with fructose showed increased fluorescence intensity as compared to BSA treated with fructose in the presence of SM, which explains the role of SM in being able to prevent the formation of AGEs. To understand the potency of SM in being able to prevent the formation of AGEs, we compared it with metformin which is already known to prevent MG mediated glycation of albumin and also prevent renal tubular injury [41,42]. SM at similar dose to that of metformin could significantly inhibit the formation of AGEs much better as compared to metformin, which proves it to a better glycation inhibitor than metformin. The glycation of the BSA results in the increase in the carbonyl content, indicative of oxidative changes in the BSA [43]. In the present study, in accordance with the literature, we found increased carbonyl content in the glycated BSA. The treatment of SM in presence of glycated BSA, could reduce the carbonyl content, which possibly can be attributed to the antioxidative potential of SM.

The glycation induced by fructose leads to the modifications of various proteins, which renders the loss of function of proteins. This can be understood by hyperchromicity that can be attributed to the loss or fragmentation of the proteins, which causes the exposure of the aromatic amino acids responsible for its higher absorption at 280 nm [44]. It is of prime importance to understand the changes in the proteins caused due to their frutosylation, as the concentration of fructose increases up to as high as 5 mM in the kidneys and peripheral nerves due to polyol pathway [45] The structural changes as understood by hyperchromicity could be due to side chain modifications in the proteins due to glycation. Such modifications lead towards the structural as well as functional group modifications in the proteins, which can be analysed by FT-IR. We found the changes in the peaks

corresponding to amide I from 1655 cm−1 of the native BSA to 1651 cm−1 in the glycated BSA. The treatment with SM showed a minor shift, indicating the relatively stable BSA molecule. The amide I band is a characteristic feature attributed to α-helix in the protein, and the decrease in the wavenumber indicates loss of α-helix in the glycated BSA, and therefore the structural change in the BSA due to glycation. Similarly, the amide III band of native BSA shifted from 1245 cm−1 to lower wavenumber 1236 cm−1 in the glycated BSA as compared to native BSA and the treatment of glycated BSA with SM did not show major shift in the wavenumber compared to native BSA with wavenumber 1242 cm−1 . The pronounced effect on shifting of amide III wavenumbers could be due to increase in the β-sheet and β-turns in the glycated BSA sample [46]. The treatment with SM however prevented the structural changes in the BSA as indicated by minor shift, suggesting the preventive role of SM in inhibiting the glycation induced structural changes in the protein. Our results show that the fructose mediated glycation was prevented when the glycation of BSA by fructose was carried out in presence of SM, as seen by reduced hyperchromicity which indicates the role of SM in preventing the fructose or AGEs mediated damage to kidney cells.

As discussed earlier, AGEs lead to the formation of ROS, which results in the oxidative stress. The formation of ROS and the repair of the damage caused due to ROS and oxidative stress is normally rescued in the cells by its antioxidant enzyme machinery comprising of antioxidant enzymes like catalase and SOD. However, glycation of enzymes like catalase and SOD lead to the loss of their activity, rendering an imbalance in homeostasis of oxidants and antioxidants in the cell [47]. ROS being toxic to kidney cells promotes cell death by inflammatory and fibrogenic reactions in the kidney cells [48]. Therefore, to understand the cellular effects of AGEs and how SM could prevent AGEs mediated progression of DN, we used an in vitro MG-induced model of DN. The concentration of MG to be used in the study was determined using MTT based cell viability assay. The dose-dependent decrease in the cell viability was observed, possibly because MG with increasing concentration may have induced apoptosis in the cells. Since nearly 50% cell death was observed at 199.5 µM concentration of MG, we chose 200 µM MG concentration for our further experiments, which was supported by the studies on rat mesangial cells, where 200 µM MG resulted in apoptosis of mesangial cells after 8 h of incubation with MG [49].

MG in particular can act as a source to the formation of a specific fluorescent AGE called Argpyrimidine, which is formed by a reaction of MG with the guanidine group of arginine [38]. Argpyrimidine was also found to be accumulated in the intima and media of small arteries of the kidneys of diabetic patients, which suggested its role in the progression of DN [50]. Approximately two to three-fold higher levels of argpyrimidine are found in diabetic patients as compared to nondiabetic individuals. The argpyrimidine levels also are found to positively correlate with glycosylated hemoglobin [51]. Higher levels of argpyrimidine were found to be present in the mesangial cells of rat kidney cultured in high glucose-containing medium [52]. Our study shows that SM can prevent the formation of AGEs and specifically could also inhibit the formation of MG mediated argpyrimidine formation in NRK-52 E cells. We also found increased levels of ROS in MG treated cells which as discussed earlier could be possibly due to the formation of AGEs [34]. However, the cells treated with SM along with MG showed reduced ROS, suggesting the antiglycative property of SM.

It is well established that oxidative stress and increased free radical can induce lipid peroxidation. This is because lipids are most vulnerable to the attack by ROS and reactive nitrogen species (RNS) [53]. This can generate increased levels of MDA, which is one of the major mutagenic and toxic products formed during the process of lipid peroxidation [54]. The present study shows that cotreatment of NRK-52E cells with MG and SM reduced the levels of ROS which led to reduced oxidative stress as evidenced by decreased production of MDA, which otherwise was higher due to lipid peroxidation in MG treated NRK-52E cells. Chronic hyperglycaemia, if unmanaged, not only culminates into various complications

but also leads to pancreatic β-cell death. In a recent study, it was shown that MG leads to β-cell death by the formation of AGEs [55].

The MG mediated AGEs could readily interact with their membrane-bound receptor RAGE and result in the kidney dysfunction by inducing chronic inflammation. In a study on OVE26 mouse, it was shown that RAGE deletion could prevent renal function in diabetic mice, thereby explaining the crucial role played by RAGE in DN [56]. Therefore, elucidating the role of SM in MG-mediated RAGE expression became crucial, and we found that SM could decrease the mRNA expression of RAGE as compared to that of MG-treated cells. The activation of RAGE due to AGEs can lead to various inflammatory reactions as discussed earlier, by inducing the expressions of inflammatory markers like NADPH oxidase, TNF-α, IL-1β, IL-6, which gets accelerated due to the expression of cellular adhesion molecules like ICAM-1 [57]. In the current study, we found that treatment of MG not only induced the up regulated expression of proinflammatory cytokines like TNF-α, IL-1β, IL-6 but also of NADPH oxidase and ICAM-1, which was ameliorated in the cells treated with SM along with MG. The interaction of RAGE with AGEs can lead to the generation of ROS via NADPH oxidase which can promote the expression of TGF-β via NFκB, mitogen-activated protein kinase (MAPK), or PKC pathways in mesangial and renal tubulointerstitial cells in the kidneys [58]. We in the current study showed the MG mediated upregulation of p38 MAPK, which by further activation of inflammatory cascades led to the increase in the pro-inflammatory markers, causing the progression towards DN.

TGF-β being a pro-fibrotic cytokine plays a very crucial role in the renal fibrosis via EMT. EMT remains a very important phenomenon in the pathogenesis of DN, as it induces the expression of pro-fibrotic markers and mesenchymal markers like fibronectin 1 and α-SMA, inducing the tubulointerstitial fibrosis [59,60]. In addition, inhibition of particular stages involved in EMT, reduced the formation of fibrotic lesions in the kidney and therefore EMT is being considered to be significant in the development of DN [61–63]. We found that MG exposure changed the epithelial morphology of NRK-52E to more extended, fibroblast-like morphology, possibly due to higher mRNA expression of TGF-β as compared to lower expression of TGF-β in the cells co-treated with SM and MG. In the current study, MG induced the expression of fibronectin 1 and α-SMA along decreasing the epithelial marker e-cadherin, confirming the MG mediated EMT. These changes were prevented by SM. Our results are in agreement with a previous study, where TGF-β led to fibrosis of renal proximal tubules cells, leading to the death of the cells [64].

AGEs are known to induce pro-inflammatory signaling culminating in oxidative stress, further inducing apoptosis in the nephrons leading to renal fibrosis and therefore DN [65]. It is well established that ROS and oxidative stress plays a very important role in development of renal complications including EMT [66]. Therefore, the cellular antioxidants could be chosen as one of the prime targets for inhibiting EMT. The role of cellular antioxidant machinery like Nrf-2 and HO-1 has been demonstrated by investigations which showed that Nrf2−/− mice were highly sensitive to high glucose induced oxidative damage, and the HO-1 deficiency is associated with increased fibrosis and increased tubular expression of TGF-β [67,68]. In our study, we found that MG induced downregulation of HO-1 and Nrf-2 was upregulated in SM treated cells.

To understand the possible mechanism by which SM inhibited the expression of RAGE, we explored the in silico approach adopted by researchers [69] to analyze the role of SM in binding with RAGE, thereby blocking the binding of AGEs with RAGE. As MG leads to the production of Argpyrimidine, we docked both Argpyrimidine and SM with RAGE to check for their binding energies. Argpyrimidine is known to bind in the C-Domain type 1 present in the RAGE; therefore, we chose the region ranging from 123 to 219 amino acids for performing the docking [69]. SM showed better binding energy and docking score as compared to Argpyrimidine, which suggests that RAGE binds with more preference with SM than Argpyrimidine, thereby blocking the binding of Argpyrimidine with RAGE. The difference in binding energy is −1.95 kcal/mol. This docking result is complementary to the experimental results obtained in vivo, which also shows that binding of Swertiamarin results in reduction of the AGEs and the expression of its receptor RAGE.

In conclusion, we report that SM inhibits binding of AGEs with RAGE and hence prevents the activation of RAGE signaling and p38 MAPK, thereby inhibiting the downregulation of HO-1 and Nrf-2 to prevent the EMT in rat kidney cells.

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

Bovine serum albumin (BSA, fraction V) was acquired from Himedia (Mumbai, India). Potassium bromide (FTIR grade) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (HPLC grade), HPLC grade water for HPLC; hexane (AR grade), ethyl acetate (AR grade) and methanol (AR grade) were acquired from Merck (Darmstadt, Germany) for the isolation of SM using column chromatography. All the solvents were used in their pure form without any preprocessing.

#### *4.1. Isolation of SM Using Silica Chromatography*

The dry plant *E.littorale* was acquired from Saurashtra region, Gujarat, India. The plant was press dried and then powdered in a crusher. The powder was immersed overnight in double the volume of 70% methanol (SRL, Mumbai, India) and 30% water. The process was repeated three times for a single batch and then filtered. The filtrate was vacuum evaporated using a Heidolph rotary evaporator (Schwabach, Germany), to get a dry hydroalcoholic extract.

To obtain SM from the hydroalcoholic extract, silica based column chromatography was done. Furthermore, 1 g of hydroalcoholic extract was coated with 3 g silica. The extract was chromatographed using silica gel (60–120 mesh size, Merck, Germany) with hexane and then with hexane containing ethyl acetate (60–97%), followed by ethyl acetate and then with ethyl acetate mixed with methanol in the order of increasing polarity (0.5–1.5%). Fractions with different polarities were assessed for the presence of SM by thin-layer chromatography (TLC) using the solvent system of chloroform: methanol (8:2). The presence of SM was established by co-chromatography of the standard SM (TCI, Japan) along with different fractions. Fractions with SM were pooled and dried. They were further purified by precipitating the methanol dissolved residue with nonpolar solvents like diethyl ether (yield: 7%).

#### *4.2. Characterization of Isolated SM for Its Purity*

The characterization as well as the purity of the isolated compound was confirmed by various methods which included TLC, HPLC, LC-MS and FTIR.

#### 4.2.1. Characterization Using High Performance Liquid Chromatography (HPLC)

For HPLC analysis, the solution of SM isolated in the lab was prepared to achieve a final concentration of 1 mg/mL in methanol. The samples and solvents to be used as mobile phases were filtered through 0.2 µm syringe filters (Axiva, India). HPLC was carried out using the previously described mobile phase for SM with slight modifications by using acetonitrile: water (10:90) as the mobile phase [70]. The flow rate was 1 mL/min and the column temperature was maintained at 25 ◦C. The detection wavelength and mode for SM was 238 nm and photodiode detector (PDA).

#### 4.2.2. Characterization Using Fourier-Transform Infrared Spectroscopy (FTIR) and Mass Spectrometer (MS)

The isolated compound was analyzed by FTIR for the identification of functional groups present in the compound. The standard was also used as a reference for the isolated compound. A homogenous solution of both standard and the compound was individually prepared in potassium bromide (KBr) and subjected to FTIR spectrophotometer (Thermofisher Scientific, Waltham, MA, USA). The compound isolated was also subjected to

positive-ion Electrospray Ionization, i.e., ESI using Perkin-Elmer Applied Biosystem Sciex API 2000 (Waltham, MA, USA) for identification of its characteristic molecular ion peak.

#### *4.3. Analysis of Antiglycative Potential of SM Using In Vitro Glycation Assay*

Antiglycative studies were done as suggested by McPherson et al. with slight modifications [71]. Bovine Serum Albumin (BSA, Himedia, Mumbai, India) and Fructose (Merck, Darmstadt, Germany) were used to induce glycation. In brief, in a 1.5 mL eppendorf, 100 µL of 60 mg/mL BSA (20 mg/mL final concentration) was incubated with 100 µL of 1.5 M fructose (0.5 M final concentration) and 100 µL of potassium phosphate buffer (pH 7.4). Negative control was incorporated as well, which consisted of 100 µL of 60 mg/mL BSA (20mg/mL final concentration) and 200 µL of potassium phosphate buffer (pH 7.4). To understand the antiglycative potential of the compound, the same reaction was carried out in the presence of varying concentrations of SM (1 µg/mL to 10 µg/mL). For the comparative studies, metformin was used as a positive antiglycative control at the same concentrations as of SM. The reaction was incubated at 60 ◦C for 24 h and samples were analyzed for fluorescence due to AGEs on a Perkin Elmer LS-55 spectrofluorometer (Waltham, MA, USA) by using an excitation wavelength of 370 nm and an emission wavelength of 440 nm.

#### *4.4. Analysis of Protein Modifications Due to Glycation by UV Absorbance Spectroscopy*

The native, glycated BSA and samples in which BSA was glycated in presence of SM were analyzed for their absorption spectra on Shimadzu UV–1700 spectrophotometer (Kyoto, Japan) in 200 to 800 nm wavelength range using a quartz cuvette. The samples were analyzed for the hyperchromic shift due to glycation by fructose as suggested by Allarakha et al. [45].

#### *4.5. Estimation of Protein Carbonyl Content*

The analysis of the carbonyl content as a marker for oxidative damage to protein due to glycation was done according to the protocol suggested by Meeprom et al. [43]. From each group, 100 µL of the sample was taken and mixed with 400 µL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) prepared in 2.5 M HCl solution, followed by the incubation for 1 h in the dark. After the incubation, the protein was precipitated on ice using 500 µL of 20% *w/v* tri-choloroacetic acid (TCA) solution and subjected to centrifugation at 10,000× *g* for 10 min at 4 ◦C. The protein in the pellet was washed thrice with 500 µL of 1:1 solution of ethanol and ethyl acetate followed by resuspension in 250 µL of 6 M guanidium hydrochloride solution. The absorbance was recorded at 370 nm and the carbonyl content was measured using the molar extinction coefficient of DNPH, i.e., ε = 22,000 M−1 cm−1 . The results were expressed as nM carbonyl content/mg of protein.

#### *4.6. Fourier Transform Infrared Spectroscopy of the Protein Samples*

To check and analyze the functional and conformational changes in the BSA due to fructose mediated glycation, FTIR spectroscopy was done as suggested by Liu et al. [46]. The samples were lyophilized and then mixed with FTIR grade KBr in 1: 100 ratio (sample: KBr) to make a homogenous mixture and pressed to prepare a transparent pellet. The pellet was subjected to analysis in the transmission mode in the range of 400–4000 cm−1 on FTIR spectrophotometer (Thermofisher Scientific, Waltham, MA, USA).

#### *4.7. Culturing of NRK-52 E Cells*

Normal Rat Kidney (NRK-52E) cell line was acquired from National Centre for Cell Sciences (NCCS), Pune, India. The cells were cultured in Dulbecco's Modified Eagle Medium Low Glucose (5.5 mM/L) medium with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin (Thermofisher Scientific, Waltham, MA, USA) at 37 ◦C in a 5% CO2 incubator. The cells were grown till 80–90% confluency, after which they were used for experiments with MG in the presence and absence of SM.

#### Cell Viability and Dose Determination of MG and SM Using MTT Assay

The concentration of MG (Sigma-Aldrich, St. Louis, MO, USA) used during the experiments was determined by checking MG toxicity on NRK-52E cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay described by Riss et al. [72]. In brief, 1 × 104 cells were seeded in a 96-well plate. After 24 h, cells were exposed to different concentrations MG (1 mM, 500 µM, 250 µM, 125 µM, 62.5 µM, 31.25 µM, 15.625 µM, 7.812 µM, and 3.906 µM), along with an untreated control well, for 24 h. Similarly, the toxicity of SM was checked on NRK-52E. NRK-52E cells were treated with different concentrations of SM (50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL and 250 µg/mL) along with a group left untreated with SM for 24 h. After treating them for 24 h, 10 µL of MTT solution was added to each well at a final concentration of 0.5 µg/mL and the cells were incubated at 37 ◦C in a 5% CO2 atmosphere for 3 h in the dark. Following the incubation period, formazan crystals formed in each well were solubilized using 100 µL dimethyl sulfoxide (DMSO) solution. The absorbance was then measured at 570 nm to determine the viability of cells.

#### *4.8. MG Stimulation and Different Treatment Groups*

To check the effect of MG on NRK-52E cells, 8 × 105 cells were seeded in a 60 mm culture plates followed by incubation for 24 h. After this, cells received different treatments based on the respective groups like control (only growth medium), MG (medium containing 200 µM MG) and MG + SM 100 µg/mL (medium containing 200 µM MG in the presence of 100 µg/mL SM). The effect of MG-induced stress on kidney cells was assayed by checking the ROS production, lipid peroxidation, argpyrimidine levels and transcript levels of various genes involved in the progression of DN by qRT PCR.

#### *4.9. Estimation of Argpyrimidine*

To estimate the levels of Argpyrimidine, NRK-52E cells (8 × 106 cells/well) were seeded onto 60mm plates, and the cells were incubated for 24 h. After 24 h, cells were treated with MG, in the presence and absence of SM for 24 h. After the incubation, the cells were lysed using the lysis buffer followed by centrifugation at 16,000 rpm for 10 min at 4 ◦C. The supernatant was then analyzed using a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with an excitation wavelength of 330 nm and an emission wavelength of 380 nm for the presence of Argpyrimidine [51].

#### *4.10. Estimation of Reactive Oxygen Species (ROS)*

In order to estimate oxidative stress caused by MG in the presence or absence of SM, the cells were treated with 200 µM MG alone or along with SM and incubated for 24 h. After treatment, cells were incubated with 5(6)-carboxy-20,70-dichlorofluorescein Diacetate (Carboxy-H2-DCFDA) (Sigma-Aldrich, St. Louis, MO, USA) in the dark with a final concentration of 30 µM at 37 ◦C for 1 h. The cells were then harvested, washed with PBS, and resuspended in PBS. ROS production in the cells was measured by measuring the fluorescence of the sample at an excitation wavelength—485 nm and an emission wavelength—530 nm, using a fluorescence spectrophotometer (Perkin Elmer LS-55, Waltham, MA, USA [73].

#### *4.11. Estimation of MDA as a Measure of Lipid Peroxidation by HPLC*

Total malondialdehyde (MDA) in the cultured cells was estimated using HPLC by following the method described by Tukozkan et al. with some modifications [74]. Summarily, the medium was removed from the plates and the cells were rinsed with PBS. Cells were homogenized in cold 1.15% KCl to make 10% homogenate. 500 µl of the homogenate was then mixed with 100 µl of 6 M NaOH, and the samples were incubated in a water bath at 60 ◦C for 45 min. The hydrolyzed sample was then acidified with 250 µL of 35% perchloric acid. The samples were subjected to centrifugation at 15,000× *g* for 10 min. After centrifugation, 250 µL of the supernatant was collected and mixed with 25 µL of DNPH solution, followed by 10 min incubation in the dark. The samples were then analyzed by

HPLC in an ODS2 reverse column using acetonitrile: water (38:62) containing 0.2% acetic acid as a mobile phase. Isocratic conditions were maintained during HPLC with a flow rate of 1 mL/min and the MDA was detected in the samples at 310 nm with the UV detector with a retention time of about 10 min. The concentration of MDA was detected in the sample by comparing it with the standard curve prepared using 1,1,3,3 tetraethoxypropane.

#### *4.12. Isolation of RNA, cDNA Synthesis and Analysis of Gene Expressions of Various Markers by qRT-PCR*

Total RNA was isolated from the control and treated cells using TRIzol reagent. The purity and concentration of RNA in the sample were measured at 260 nm and A260/A280 ratio of the sample was determined using NanoDrop 2000 (Thermofisher Scientific, Waltham, MA, USA). A total of 1 µg RNA was used to synthesize cDNA using first strand cDNA synthesis kit. For qPCR, maxima SYBR Green/ROX qPCR master mix was used to quantify the mRNA expression levels for all genes under investigation on the Agilent Strategene Mx3005P system. The qRT-PCR system comprised of 1 µL cDNA, 0.5 µL of 10 µM FP (forward primers), 0.5 µL of 10 µM RP (reverse primers), 10 µL SYBR Green Mastermix, and 8.0 µL milli-Q water in a 20 µL reaction system. qRT-PCR cycling conditions were set according to Kema, V.H. et al. 2017 [75]. The 2-∆∆Ct method (fold change over basal) was applied to evaluate mRNA expression levels in both control and treated cells. 18S rRNA was presented as an internal reference gene control. The list of primer sequences sets used are tabulated in Table 3.

**Table 3.** The list of *Rattus norvegicus* primers used for quantification of mRNA using qRT-PCR.

#### *4.13. Estimation of Protein Levels of TGF-β and HO-1 by ELISA*

The protein levels of TGF-β and HO-1 were analysed from the cultured NRK-52E cells after the treatment with 200 µM MG in the absence and presence of 100 µg/mL SM. Briefly, the cultured cells from different groups were removed using cell scraper after washing thrice with 1X PBS. The cells were then centrifuged at 1500 rpm for 10 min under cooling conditions. The collected cell free supernatant was analyzed as per the manufacturer's protocol (Abcam, Cambridge, UK). The levels of TGF-β and HO-1 were measured in pg/mL.

#### *4.14. Molecular Docking Studies*

#### 4.14.1. Homology Modelling

Receptor for Advanced Glycation End Products (RAGE) from *Rattus norvegicus* consists of 402 amino acids [76]. The FASTA sequence for building up the 3D model was obtained from UniProt database (UniProt ID: Q63495) [77]. The 3D model of RAGE was constructed using RAGE (*Mus musculus*) (PDB ID: 4IM8) as the template as it showed a sequence identity of 92.04% [76,78]. The 3D model was created using SWISS-MODEL server which uses homology modelling method, wherein it aligns the amino acid sequence of the desired protein with the protein that has closest identity to our desired protein and whose structure has been solved, thus developing a model for the unknown desired protein whose sequence is known. The generated model was further used for docking studies. The molecular docking was performed using Glide module (XP) of Maestro software (Schrodinger) version 12.7.156 (New York, NY, USA) [79].

#### 4.14.2. Protein Preparation Protocol

The protein structure of Receptor for Advanced Glycation End Products (RAGE) from *Rattus norvegicus* was constructed using SWISS-MODEL server. The structure so generated needs to be prepared using protein preparation wizard, which helps in pre-processing, optimizing hydrogen bonds and minimizing the protein, from its raw state to a better state which can be useful for the further calculations. The protein was preprocessed by assigning bond order, adding the missing hydrogen atoms, and converting the selenomethionine to methionine. The missing side chains and loops were filled in using Prime module, which is the protein refinement application. The termini were capped with N-acyl and N-methyl groups and finally, the water molecules which were more than 5 Å away from the het groups were deleted. A pH of 7.0 ± 2.0 was set in Epik to account for the possible tautomeric state of amino acid residues. The hydrogen bonds were optimized with PROPKA at a pH of 7. Lastly, the RAGE protein model was minimized using optimized potentials for liquid simulations 4 (OPLS4) force field [80–82].

#### 4.14.3. Receptor Grid Generation

Receptor grid generation forms the first step of molecular docking protocol. A volume of the active site is represented by the generated grid file. The ligands of interest are then docked in this grid. Initially, the minimized protein was selected to generate the grid. It has been reported in the literature that Argpyrimidine binds to the upstream C-Domain Type 1 in RAGE [69]. Hence, the amino acids from 123 to 219, which represent the upstream C-Domain Type 1, were selected for generating the grid for docking SM and Argpyrimidine. The centroid was generated around these amino acids, and further criteria were set to default, to generate the grid for docking [80–82].

#### 4.14.4. Glide Molecular Docking and Analysis of Docking

Glide is the molecular docking module in Maestro. It has a widespread use from knowing the ideal interactions between a particular ligand and the protein to screening large libraries of molecules for high throughput screening. The molecular docking using Glide starts with procuring the grid file and the Ligprep output file. Further, XP mode was selected for better accuracy and further criteria were set as default. The docking module was run and results were obtained and analysed using XP visualizer in Schrodinger [80–82].

#### 4.14.5. Statistical Analysis

All respective experiments were performed at least thrice. Data of the replicates were calculated as mean ± SD. GraphPad Prism 7 software (GraphPad Software Inc., California Corporation, San Diego, CA, USA) was used to analyze the results. The differences between all groups were analyzed using one way analysis of variance (ANOVA). Variations between groups were considered to be significant at *p* values less than or equal to 0.05.

#### **5. Conclusions**

The present study depicted the protective effect of SM in inhibiting the formation of fructose induced AGEs and its role in ameliorating the progression of DN. SM could prevent the fructose mediated conformational changes in the BSA. The formation of AGEs like argpyrimidine from MG upon interaction with RAGE led to the increased production of ROS and therefore resulted in the generation of oxidative stress. Moreover, treatment with SM in the presence of MG-treated NRK-52E cells prevented the oxidative stress and inflammation, by upregulating the antioxidants level of Nrf-2 and HO-1, thereby reducing the activation of TGF-β and prevented against the MG-induced EMT changes in NRK-52E. We for the first time report here that the inhibition of the of RAGE/ MAPK/ TGF-β pathway could be a possible mechanism contributing towards protecting effects of SM in preventing the DN induced changes in NRK-52E cells in presence of MG. In addition, the current study for the first time demonstrates that the molecular interactions between RAGE and SM can inhibit the binding of Argpyrimidine and block the AGE-RAGE axis, which also is supported by the cellular studies. Additionally, this may provide strong evidence for an in vivo study as a future prospect to unravel the mechanism of SM in inhibiting AGEs induced DN.

**Supplementary Materials:** The following are available online, Figure S1: The stages involved in the formation of advanced glycation end products.

**Author Contributions:** K.P. and F.P. contributed equally to this manuscript. The design and conception of the study was done by K.P., F.P. and P.M., K.P. did the acquisition of the data. Analysis and/or interpretation of data was done by K.P., F.P and P.M. The drafting of the manuscript was done by K.P. and F.P. while revision of the manuscript critically for important intellectual content was done by K.P., F.P., D.P. and P.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analyzed during the current study are included in the manuscript.

**Acknowledgments:** The author would like to acknowledge Charotar University of Science and Technology for providing the author with CHARUSAT Ph.D. Scholar Fellowship (CPSF). Authors acknowledge Vaibhav Patel, Assistant Professor at P. D. Patel Institute of Applied Sciences, CHARUSAT for his help in the HPLC analysis. Authors would like to thank Mitesh Patel, Assistant Professor at P. D. Patel Institute of Applied Sciences, CHARUSAT for his help in doing FTIR of BSA. Authors are grateful to Vijay Thiruvenkatam, Pranav Bhagwat and Haritha D. for helping in the molecular docking studies and providing the facility at Indian Institute of Technology, Gandhinagar for the in silico analysis.

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

#### **References**

## *Article* **Constituents of** *Aquilaria sinensis* **Leaves Upregulate the Expression of Matrix Metalloproteases 2 and 9**

**Sui-Wen Hsiao 1,†, Yu-Chin Wu 2,†, Hui-Ching Mei 3 , Yu-Hsin Chen 4 , George Hsiao 5,\* and Ching-Kuo Lee 1,2,6,7,\***

**Abstract:** In this novel study, we isolated 28 compounds from the leaves of *Aquilaria sinensis* (Lour.) Gilg based on a bioassay-guided procedure and also discovered the possible matrix metalloprotease 2 (MMP-2) and 9 (MMP-9) modulatory effect of pheophorbide A (PA). To evaluate the regulatory activity on MMP-2 and MMP-9, the HT-1080 human fibrosarcoma cells were treated with various concentrations of extracted materials and isolated compounds. PA was extracted by methanol from the leaves of *A. sinensis* and separated from the fraction of the partitioned ethyl acetate layer. PA is believed to be an active component for MMP expression since it exhibited significant stimulation on MMP-2 and proMMP-9 activity. When treating with 50 µM of PA, the expression of MMP-2 and MMP-9 were increased 1.9-fold and 2.3-fold, respectively. PA also exhibited no cytotoxicity against HT-1080 cells when the cell viability was monitored. Furthermore, no significant MMP activity was observed when five PA analogues were evaluated. This study is the first to demonstrate that C-17 of PA is the deciding factor in determining the bioactivity of the compound. The MMP-2 and proMMP-9 modulatory activity of PA indicate its potential applications for reducing scar formation and comparative medical purposes.

**Keywords:** *Aquilaria sinensis*; pheophorbide A; MMP-2; MMP-9; HT-1080

#### **1. Introduction**

*Aquilaria sinensis* (Lour.) Gilg is an evergreen woody plant with high economic value that is widely distributed in the tropical area of China. Possessing analgesic, sedative, and antiemetic effects, the resinous heartwood of *A. sinensis*, which is the major source of agarwood in China, has been used as traditional medicine for centuries [1]. Apart from the agarwood, the non-medical parts of leaves and flowers are consumed as a healthy herbal tea as well. The bioactivity of agarwood has been highly valued for years, while studies on the leaves have still been limited. The major metabolic components of *A. sinensis* are polysaccharides, amino acids, flavonoids and their glycosides, phenols, and xanthones [2–5]. Previous studies have reported anti-inflammatory and analgesic activity [6,7], laxative activity [8],

**Citation:** Hsiao, S.-W.; Wu, Y.-C.; Mei, H.-C.; Chen, Y.-H.; Hsiao, G.; Lee, C.-K. Constituents of *Aquilaria sinensis* Leaves Upregulate the Expression of Matrix Metalloproteases 2 and 9. *Molecules* **2021**, *26*, 2537. https://doi.org/ 10.3390/molecules26092537

Academic Editor: Raffaele Pezzani

Received: 20 March 2021 Accepted: 24 April 2021 Published: 26 April 2021

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