GVS-12 attenuates non-alcoholic steatohepatitis by suppressing inflammatory responses via PPARγ/STAT3 signaling pathways

Non-alcoholic steatohepatitis (NASH), a type of fatty liver disease, is characterized by excessive inflammation and fat accumulation in the liver. Peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone has great potential in protecting against the development of NASH. However, long-term usage of rosiglitazone probably leads to many adverse reactions. In this research, GVS-12 was designed and synthesized as a PPARγ agonist with high selectivity, evidenced by increasing the activity of the PPARγ reporter gene and promoting the mRNA expression of the PPARγ responsive gene cluster of differentiation 36 (CD36). It was noteworthy that GVS-12 could ameliorate dysfunction and lipid accumulation by down-regulating the mRNA expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the liver of high fat diet (HFD)-induced rats and palmitic acid (PA)-stimulated hepatocellular carcinoma G2 (HepG2) cells. Moreover, PPARγ siRNA (siPPARγ) markedly diminished GVS-12 induced the down-regulation of mRNA expression of IL-1β, IL-6 and TNF-α in PA-stimulated HepG2 cells. Additionally, GVS-12 could reduce the phosphorylation level of STAT3 and up-regulate the protein expression of a suppressor of cytokine signaling 3 (SOCS3), which could be reversed by siPPARγ. In detail, SOCS3 siRNA (siSOCS3) diminished the inhibitory effect of GVS-12 on the mRNA expression of IL-1β, IL-6 and TNF-α. In conclusion, GVS-12 suppressed the development of NASH by down-regulating the mRNA expression of IL-1β, IL-6 and TNF-α via PPARγ/STAT3 signaling pathways.


Introduction
Nonalcoholic steatohepatitis (NASH), as the most common cause of chronic liver disease, is characterized by fatty inltration, fatty change, fat storage, necrosis and ballooning degeneration of hepatic cells accompanied by the inltration of inammatory cells. 1,2 It affects millions of people worldwide. Hypothesis shows that the proinammatory cytokines especially interleukin-1b (IL-1b), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) contribute to the occurrence and development of NASH, and consequently lead to liver cirrhosis and hepatocellular carcinoma. 3,4 Therefore, down-regulating the expression of the proinammatory cytokines might be a practical therapeutic strategy for NASH.
The key proinammatory cytokines such as IL-1b, IL-6 and TNF-a could be regulated by many signal pathways. For example, the activation of p38 mitogen-activated protein kinase (MAPKs) signal pathways, nuclear factor kappa-light-chainenhancer of activated B cells (NF-kB) signal pathways and signal transducer and activator of transcription 3 (STAT3) signal pathways could promote the production of the proinammatory cytokines. 5 In contrast, the inhibitors of these signal pathways could reduce the production of proinammatory cytokines aforementioned. Accumulative evidence suggested that STAT3 signal pathway played a vital role in the production of proin-ammatory cytokines in NASH. 6,7 Therefore, the abrogation of aberrant STAT3 signaling pathways might decrease the production of the proinammatory cytokines and inhibit the progress of NASH.
The thiazolidinediones, a class of heterocyclic compounds consisting of a ve-membered C3NS ring, usually refers to a family of drugs used in the treatment of diabetes mellitus type 2. 8 Rosiglitazone is an antidiabetic drug which activates peroxisome proliferator-activated receptor g (PPARg) in the thiazolidinedione class. 9 It works as an insulin sensitizer through binding to the PPARg in fat cells and making the cells more responsive to insulin. Increasing evidence showed that rosiglitazone could improve NASH through improving insulin resistance, lipid metabolism and reducing transaminase activity. 10,11 The effect of rosiglitazone on the promotion of NASH was accomplished through inhibiting the inammation responses via down-regulating the expression of proinammatory cytokines. 12 However, the long-term use of rosiglitazone could cause severe adverse reactions, such as increase of weight, retention of uid and increase of the risk of heart disease. 13,14 Therefore, to develop a new PPARg agonist with high selectivity might be a strong strategy for NASH treatment. 15,16 In combination of the advantage of structure features of rosiglitazone and SR1664, GVS-12 was designed and synthesized by our group. In the present study, we characterized the effects of GVS-12 on the inammatory responses in high fat diet (HFD)-induced NASH rats and palmitic acid (PA)-induced hepatocellular carcinoma G2 (HepG2) cells. In depth, we explored the potential molecular mechanisms and target protein of GVS-12 with a focus on the PPARg/STAT3 pathways.

Animals and experimental groups
Male 6 weeks-old SD rats were purchased from Cavens Laboratory Animals Co., Ltd. (Changzhou, China) and housed in 12 h light/12 h dark cycles in specic pathogen free laboratory animal rooms with unrestricted access to food and drinking water. This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH publication no. 85 -23 rev. 1985) and was approved by the Animal Ethics Committee of Guilin Medical University (Guilin, China). Aer an acclimatization period of 2 weeks, the rats were randomly divided into the following ve groups (n ¼ 6 per group), fed and treated as follows for 4 weeks: (1) normal group; (2) HFD group; (3) HFD + GVS-12 (2 mg kg À1 ) group; (4) HFD + GVS-12 (6 mg kg À1 ) group; (5) HFD + GVS-12 (18 mg kg À1 ) group. GVS-12 was orally administered seven times per week for the duration of the experiment. Thereaer, the rats were euthanized aer a 12 h fast, and the liver tissues and blood samples were collected for analyses. Blood samples were acquired from the heart for serum isolation by centrifugation at 2000 Â g for 10 min at 4 C. The liver sections xed with 4% paraformaldehyde were prepared for histological staining and the remained hepatic tissues were stored at À80 C for molecular biological assays.

Enzyme-linked immuno sorbent assay (ELISA) and biochemical analysis
The level of hepatic triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) was detected by enzymatic colorimetric methods using commercially available kits (Nanjing Jiancheng Bioengineering institute, Nanjing, China) according to the manufacturer's protocol. Serum level of alanine aminotransferase (ALT) and aspartate transaminase (AST) was assessed by the microplate method (Nanjing Jiancheng Bioengineering institute) according to the manufacturer's instructions. Serum level of IL-1b, IL-6 and TNF-a was assessed by the ELISA analyses (Dakewe Biotech Co., Ltd) according to the manufacturer's instructions.

Oil red O staining analysis
7 mm-thick frozen liver sections were xed in 4% paraformaldehyde quickly and washed in deionized water for 3 times, and then blocked with oil red O solution for 10 min at room temperature, then washed with deionized water and stained with hematoxylin. In the end, tissue sections were sealed with glycerin gelatin and analyzed by light microscopy (Olympus, Tokyo, Japan).

Cell culture
Hepatocellular carcinoma G2 (HepG2) cells was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in a humidied incubator at 37 C under 5% CO 2 atmospheric condition in corresponding medium supplemented with 10% FBS, 100 U mL À1 streptomycin and 100 U mL À1 penicillin.

Cell transfection
The PPARg siRNA (siPPARg) and SOCS3 siRNA (siSOCS3) were purchased from Jiman Biochemical Technology Co., Ltd (Shanghai, China). HepG2 cells were transfected with siPPARg and siSOCS3 by using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to manufacturer's instructions. Palmitic acid (PA, 1 mM)-stimulated HepG2 cells and liver specimens were treated with GVS-12, and the total RNA was isolated by using TRIzol reagent according to the supplier's instructions. The cDNA was transcribed from RNA by using HiScript RT Super Mix (Vazyme, Nanjing, China), then analyzed for the expression of IL-1b, IL-6, TNF-a, SOCS3 and PPARg by Ace Q-PCR SYBR Green Master Mix (Vazyme, Nanjing, China) with the help of MyiQ2 Detection System (Bio-Rad Laboratories, Hercules, USA).

Western blotting
PA-stimulated HepG2 cells and liver specimens were treated with GVS-12, and the total cell lysates were prepared by using nonidet P-40 (NP40) buffer (Beyotime, Nanjing, China). The equal concentration of protein lysate of all the samples was separated on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and further transferred to polyvinylidene uoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 2 h, and then incubated with specic primary antibodies overnight at 4 C. Aer being washed with tris buffered saline (TBS)-0.1% Tween, membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) secondary antibodies, and then visualized with electrochemiluminescence (ECL) reagent (Guge Biochemical Technology Co., Ltd., Wuhan, China).

Statistical analysis
The data was presented as the means AE standard error of measurement (SEM). Statistical analysis was performed by using one-way analysis of variance (ANOVA) followed by Tukey's test. P values less than 0.05 (P < 0.05) were accepted as a signicant difference.

The synthetic routes of GVS-12
As shown in Fig. 1A, (i) sodium hydride (NaH, 6.29 g, 12 mmol) was added in portions at 0 C to a stirred solution of methyl 4hydroxybenzoate 1 (1.52 g, 10 mmol) in dry N,N-dimethylformamide (DMF, 10 mL). Aer stirring for 30 min at 0 C, 1-(bromomethyl)-4-(triuoromethyl) benzene 2 (2.40 g, 10 mmol) was added and the mixture was then stirred at room temperature for 24 h. Aer this, the reaction mixture was cooled to ambient temperature, poured into H 2 O (100 mL) and extracted with ethyl acetate (EtOAc, 100 mL). The organic phase was dried over anhydrous sodium sulfate (Na 2 SO 4 ). When the evaporation of the solvents was under reduced pressure, the crude product was puried on a silica gel column using EtOAc/petroleumether (1 : 9) to obtain the pure 3 (2.48 g, 80%) as a white solid. LC-MS (ESI): m/z 309 [M À H] À . (ii) A suspension of 3 (1.55 g, 5 mmol) and 5 mL sodium hydroxide (NaOH, 4 mol) in tetrahydrofuran (THF, 20 mL) was stirred at 50 C until the consumption of the starting material. It was allowed to reach room temperature, hydrogen chloride (HCl, 2 mol) was added until the pH of the solution reached 3.0, and then extracted with EtOAc (50 mL) and washed by saturated solution of sodium chloride (NaCl). The combined organic phase was dried over anhydrous Na 2 SO 4 . Aer the evaporation of the solvents was under reduced pressure, the crude product was puried on silica gel column to afford the product 4 (1.33 g, 90%) as a white solid. LC-MS (ESI): m/z 295 [M À H] À . (iii) To a solution of product 4 (1.48 g, 5.0 mmol) in dry DMF (20 mL), dimethylaminopyridine (DMAP, 1.22 g, 10 mmol) and benzotriazol-1-yloxytris (dimethylamino) phosphonium hexauorophosphate (BOP, 4.42 g, 10 mmol) were added respectively at 0 C. Aer stirred for 0.5 h, furan-2ylmethanamine 5 (0.49 g, 5.0 mmol) were added, the reaction was allowed to stir until all starting material disappeared. Then, the mixture was poured into a 10% aqueous solution of HCl. The aqueous phase was extracted with EtOAc for twice, and the combined organic layers were dried over anhydrous Na 2 SO 4 and concentrated in vacuo. GVS-12 production (1.70 g, 98%) was puried on silica gel column as a white solid. 1

Effect of GVS-12 on the hepatic dysfunction and lipid accumulation in HFD-induced rats
As shown in Fig. 1B, aer 4 weeks of HFD-diet feeding, the liver sections of the HFD model group showed vacuolated hepatocytes and severe micro-and macro-vesicular steatosis with inltration of inammatory cells as evidenced by H&E staining, all these above indicated that a proper NASH animal model had been established. It was noteworthy that GVS-12 (6, 18 mg kg À1 ) improved HFD diet-induced hepatic dysfunction as evidenced by reducing hepatocyte ballooning, lobular inammation and steatosis in liver tissues. In addition, the HFD diet induced indicative damage of hepatocellular and increased the production of biochemical diagnostic markers such as ALT and AST in the serum of NASH rat, which were diminished by GVS-12 (2, 6, 18 mg kg À1 ) in a dose-dependent manner ( Fig. 1C and D).
In order to elucidate the improvement of GVS-12 on hepatic steatosis, which is mainly manifested by lipid deposition and accumulation of lipid droplets, the contents of LDL-C and TG in the liver were assayed. In comparison with the normal rats, the production of LDL-C and TG was elevated in the liver of HFDinduced rats. However, the elevated production of LDL-C and TG was sharpened by GVS-12 (18 mg kg À1 ) ( Fig. 1E and F).
An important pathological characteristic of NASH was hepatic inammation, and HFD-diet previously showed to be efficacious in causing obvious hepatic inammation. As shown in Fig. 1G, the production of IL-1b, IL-6 and TNF-a was increased in the liver of HFD rats compared with the normal group. GVS-12 (6, 18 mg kg À1 ) could reverse HFD-induced elevations of IL-1b, IL-6 and TNF-a in the liver. These results clearly showed the anti-steatosis effect of GVS-12 on HFD dietinduced NASH.

Effect of GVS-12 on the process of NASH in PA-induced HepG2 cells
MTT assay was used to determine the cell viability of the test compounds. The results showed that GVS-12 had little cytotoxicity on HepG2 cells at concentrations below 10 mM (Fig. 2A). Therefore, GVS-12 (1, 3, 10 mM) could be used for further experiments.
To disclose the protective mechanism of GVS-12 on NASH, we studied the effects of GVS-12 on the expression of lipid metabolism relative factors and proinammatory cytokines in PA-stimulated HepG2 cells. As shown in Fig. 2B-E, the contents This journal is © The Royal Society of Chemistry 2019 of ALT, AST, TC and TG were elevated in HepG2 cells treated with PA, and GVS-12 (3, 10 mM) reduced the contents of ALT, TC and TG with unobvious effect on the content of AST. Moreover, PA stimulation for 24 h resulted in a dramatical elevation of the mRNA expression of IL-1b, IL-6 and TNF-a in HepG2 cells, which were abrogated by GVS-12 (3, 10 mM) (Fig. 2F).

A role of PPARg in GVS-12-induced down-regulation of proinammatory cytokines
In order to identify the role of PPARg in the down-regulated expression of IL-1b, IL-6 and TNF-a induced by GVS-12, PPARg siRNA (siPPARg) was used in PA-stimulated HepG2 cells. As shown in the Fig. 4A, PPARg 1 showed stronger silence efficiency than PPARg 2-3. Therefore, PPARg 1 was used for further experiments. The results showed that siPPARg could reverse the down-regulation effect of GVS-12 on the expression of IL-1b, IL-6 and TNF-a in HepG2 cells (Fig. 4B), indicating that GVS-12 suppressed the expression of IL-1b, IL-6 and TNF-a in a PPARg-dependent manner.

A role of PPARg in GVS-12-induced inhibition of STAT3 pathway
To further explore the role of STAT3 in the inhibitory effect of GVS-12 on the expression of IL-1b, IL-6 and TNF-a, we observed the effect of GVS-12 on the activation of STAT3 in PA-stimulated HepG2 cells. Fig. 5A and B showed that the phosphorylation of STAT3 was inhibited by GVS-12 in PA-stimulated cells. Moreover, GVS-12 promoted the expression of SOCS3 (a suppressor of STAT3 signal transduction pathway) ( Fig. 5C and D). Moreover, GVS-12 could promote the expression of SOCS3 in the liver of NASH rats (Fig. 5E and F). Of note, siPPARg diminished the regulatory effect of GVS-12 on p-STAT3 and SOCS3, implying that GVS-12-induced the down-regulation of p-STAT3 level and

A role of SOCS3 in GVS-12-induced down-regulation of proinammatory cytokines
To ascertain the relationship between the up-regulation of SOCS3 and down-regulation of IL-1b, IL-6 and TNF-a expression induced by GVS-12, PA-stimulated HepG2 cells were treated with GVS-12 in combination with or without SOCS3 siRNA (siSOCS3). As shown in the Fig. 6A, SOCS3 1 showed stronger silence efficiency than SOCS3 2-3. Therefore, SOCS3 1 was used for further experiments. The results showed that siSOCS3 diminished the down-regulation effect of GVS-12 on the mRNA expression of IL-1b, IL-6 and TNF-a (Fig. 6B), suggesting that SOCS3 played an important role in GVS-12-induced downregulation of IL-1b, IL-6 and TNF-a expression.

Discussion
The primary hallmarks of NASH are fat storage, parenchyma hepatic cells fatty change, liver fatty inltration, hepatic cells necrosis, ballooning degeneration, and inammatory cells inltration. 17,18 Increasing evidence has shown that the inltration of inammatory cells in NASH mainly involves the secretion of inammatory factors in the accumulating macrophage cells. [19][20][21] It is worth noting that the secretion of inammatory factors mainly includes the production of proinammatory factors and the release of inammatory factors. GVS-12, a new type of PPARg, markedly down-regulated the mRNA expression of IL-1b, IL-6 and TNF-a in PA-induced HepG2 cells. Thus, we explored its potential mechanisms. The ndings would deepen the anti-inammatory effect of GVS-12 on the inammatory cells inltration of NASH, which could consecutively develop effective prevention or therapeutic strategies to combat NASH.
STAT3 is a member of the transcription and activation signal transduction family. Increasing evidence showed that the activation of STAT3 signaling pathway was critical in inammatory responses, which promoted the production of pro-inammatory factors such as IL-1b, IL-6 and TNF-a. 22,23 The activation of STAT3 was closely correlated with fat storage, parenchyma hepatic cells fatty change, liver fatty inltration, hepatic cells necrosis, ballooning degeneration and inammatory cells inltration in NASH. 24,25 Therefore, it is probably of great therapeutic interest to develop effective medicine to suppress the activation of STAT3 in NASH patients. STAT3 activation could be inhibited by SOCS3, which was considered as a suppressor of STAT3 signaling pathway activation. The low expression or deciency of SOCS3 results in the activation of STAT3 and secretion of inammatory factors in NASH rats. 26,27 Thus, upregulating the expression of SOCS3 is a good method for inhibiting the activation of STAT3. Our study showed that GVS-12 obviously inhibited the mRNA expression of IL-1b, IL-6 and TNF-a in the liver of HFD-diet rats and PA-treated HepG2 cells. Moreover, GVS-12 down-regulated the expression of p-STAT3 and up-regulated the expression of SOCS3, indicating that STAT3 signaling pathway played a pivotal role in the inhibition of NASH treated with GVS-12.
PPARg, a ligand-dependent transcription factor that can regulate fatty acid storage and glucose metabolism, plays a vital role in NASH. 28,29 Increasing evidence has indicated that PPARg activation might inhibit the inammatory responses in the liver of NASH rats. 30 There is growing evidence affirmed that PPARg agonist rosiglitazone could alleviate hepatocyte steatosis through inhibiting the activation of STAT3 pathway. 31 However, the long-term use of rosiglitazone might cause adverse reactions such as weight gain, uid retention, and an increase in the risk of heart disease. Therefore, developing a new type of PPARg agonist with high selectivity might be a better strategy for NASH treatment. Combining the superiority of rosiglitazone and SR1664, GVS-12 was designed and synthesized as a PPARg agonist by our team. We found that GVS-12 was a PPARg modulator with low toxicity (ESI †), and PPARg was involved in GVS-12-mediated inhibition of STAT3 phosphorylation and consequent inhibition of inammatory responses. The result showed that GVS-12 could promote the expression of CD36 in HepG2 cells. The TR-FRET assay further demonstrated that GVS-12 had the ability to bind PPAR with a binding constant of 0.32 mM. Moreover, GVS-12 was shown to increase the reporter gene activity in a PPAR-dependent manner. In summary, GVS-12 is a PPARg regulator, and it could activate PPARg. The effect and mechanism of GVS-12 on NASH might be as same as PPARg agonists. [32][33][34] We assumed that p-hydroxybenzoic acid might be The protein expression of STAT3, p-STAT3 and SOCS3 was detected by western blot; (E and F) rats were fed with HFD, and then treated with GVS-12 (2, 6, 18 mg kg À1 ). The expression of SOCS3 in the liver of HFD rats was assayed by western blot; (G-J) The protein expression of STAT3, p-STAT3 and SOCS3 in PA-induced HepG2 cells was detected by western blot. The data were expressed as the means AE SEM of three independent experiments. # p < 0.05, ## p < 0.01 vs. control, *p < 0.05, **p < 0.01 vs. PA (1 mM), $ p < 0.05, $$ p < 0.01 vs. GVS-12 (10 mM). a metabolic product of GVS-12. And it would be evaluated in a series of preclinical efficacy pharmacology, safety pharmacology, pharmacokinetic/toxicokinetic, and drug metabolism studies for further experiments.

Conclusion
In conclusion, GVS-12 alleviated the NASH process through inhibiting the expression of IL-1b, IL-6 and TNF-a both in vivo and in vitro. Moreover, it mainly functioned by regulating the SOSC3/STAT3 pathway via activating PPARg.

Conflicts of interest
The authors have no nancial conicts of interest.