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

Abstract

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.

Fig. 1. The synthesis route of GVS-12 and effect of GVS-12 on high fat diet (HFD)-induced rats. (A) The synthesis of GVS-12; (B) the pathological changes of the liver sections were detected by H&E staining, and the arrows indicated pathological changes of liver; (C–F) the serum level of liver function (ALT, AST, LDL-C and TG) was detected by biochemical quantitation kits; (G) the protein level of IL-1β, IL-6 and TNF-α in hepatic tissue was analyzed by ELISA. The data were expressed as the means ± SEM ^#p < 0.05, ^##p < 0.01 compared with the normal group; *p < 0.05, **p < 0.01, compared with the HFD group.

Fig. 2. Effect of GVS-12 on NASH biomarkers in PA-induced HepG2 cells. (A) The effect of GVS-12 on HepG2 cell viability; HepG2 cells were induced by PA (1 mM) with or without GVS-12 (1, 3, 10 μM) and rosiglitazone (Ros, 1 μM). (B–E) The contents of ALT, AST, TC and TG were detected by biochemical quantitation kits; (F) the mRNA expression of IL-1β, IL-6 and TNF-α in PA-induced HepG2 cells was analyzed. The data were expressed as the means ± SEM of three independent experiments. ^#p < 0.05, ^##p < 0.01 vs. normal; *p < 0.05, **p < 0.01 vs. PA (1 mM).

Fig. 3. Effect of GVS-12 on the activation of PPARγ. (A) Effect of GVS-12 on the mRNA expression of CD36 in HepG2 cells. The cells were treated with GVS-12 (1, 3, 10 μM) and rosiglitazone (Ros, 1 μM) for 24 h. The mRNA expression of CD36 was detected using qPCR analysis. (B) Effect of GVS-12 on PPARγ reporter gene activity. HepG2 cells were transiently transfected with REPO™ PPARγ and then subjected to indicated treatments for 24 h. Cells were then harvested and assayed for luciferase activity. (C) Binding of GVS-12 to PPARγ-LBD in a competitive TR-FRET assay. Data were expressed as means ± SEM of three independent experiments. *p < 0.05 vs. control, **p < 0.01 vs. control.

Fig. 4. Effects of PPARγ on GVS-12 induced the down-regulation expression of IL-1β, IL-6 and TNF-α in PA-induced HepG2 cells. (A and B) Cells were transduced with siPPARγ, and then treated with GVS-12 (10 μM) or rosiglitazone (Ros, 1 μM) for 24 h before assay. The mRNA expression of IL-1β, IL-6 and TNF-α was analyzed by qPCR. The data were expressed as the means ± 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 μM).

Fig. 5. Effect of PPARγ on GVS-12 inhibited the activation of STAT3 in HFD-induced rats and PA-induced HepG2 cells. (A–D), HepG2 cells were incubated with PA (1 mM) in the accordance with GVS-12 (1, 3, 10 μM) or rosiglitazone (Ros, 1 μM) for the indicated internals. (A–D) 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⁻¹). 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 ± 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 μM).

Fig. 6. Effect of SOCS3 on GVS-12 induced the down-regulation expression of IL-1β, IL-6 and TNF-α in PA-induced HepG2 cells. (A and B) Cells were transduced with siSOCS3, and then treated with GVS-12 (10 μM) or rosiglitazone (Ros, 1 μM) for 24 h before assay. The mRNA expression of IL-1β, IL-6 and TNF-α was analyzed by qPCR. The data were expressed as the means ± 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 μM).