Gypenosides improves nonalcoholic fatty liver disease induced by high-fat diet induced through regulating LPS/TLR4 signaling pathway

Abstract

Background The contents of lipopolysaccharide (LPS) and Toll-like receptor 4 (TLR4) are significantly increased during the progression of nonalcoholic fatty liver disease (NAFLD). The study investigated the role of the LPS/TLR4 signaling pathway in improving gypenosides (Gyp) on NAFLD. Methods NAFLD model were established in rats and treated by Gyp. Pathological changes of liver tissues were observed by Hematoxylin and Eosin (HE) staining. Lipid metabolism and insulin resistance were measured. Expressions of inflammatory factors and protein of LPS/TLR4 downstream pathway were detected by qRT-PCR and Western blotting. THLE-2 cells were treated by free-fatty acid (FFA), Gyp, and LPS, and then transfected with TLR4. Next, cell viability was detected by MTT. Lipid droplet deposition and Triglyceride (TG) content were determined by Oil Red O staining and ELISA. Results Gyp protected fatty liver tissues in NAFLD model, and significantly reversed cholesterol increased by high-fat diet. Moreover, Gyp increased SOD content and decreased the contents of AST, ALT, MDA, HSI, FBG, FINS, HOMA-IR, IL-1β, and TNF-α, and promoted the expressions of TLR4, LPS, MyD88, p-IκBα, and reduced the expressions of p-p65 and IκBα in the NAFLD model. Gyp treatment significantly reduced lipid droplet deposition, increased TG content and MyD88, p-IκBα, p-p65 in FFA-induced liver cells, but LPS and TLR4 greatly reversed improvement of FFA by Gyp. Conclusion Gypenosides could improve liver function, lipid metabolism, insulin resistance, and levels of inflammatory factors in NAFLD model by regulating LPS/TLR4 signaling pathway in vitro and in vivo.

Keywords: Gypenosides; LPS/TLR4 signaling pathway; insulin resistance; lipid metabolism; nonalcoholic fatty liver disease.

Figure 1.

Effect of Gypenosides on liver tissue damage and biochemical indicators in NAFLD rat model. (a) Pathological changes of liver tissue were observed by hematoxylin and eosin (HE) staining in NAFLD rat model that treated with Gyp. (b) The scores were counted according to HE staining. (c) Serum total cholesterol level was detected by serum total cholesterol kit. (d) Triglyceride level was measured by triglyceride kit. (e) Aminotransferase (AST) level was detected by AST kit. (f) Alanine transaminase (ALT) level was detected by ALT kit. (g) Superoxide dismutase (SOD) level was detected by SOD kit. (h) Malondialdehyde (MDA) level was detected by MDA kit. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, vs. Control; ^{^}P < 0.05, ^{^^}P < 0.01, ^{^^^}P < 0.001, vs. Model; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, vs. Control

Figure 2.

Effect of Gypenosides on biochemical indicators and inflammatory factor and LPS/TLR4 downstream pathway in NAFLD rat model (a) hepatic steatosis index (HSI) of rats was evaluated. (b) Fasting blood glucose (FBG) of rats was measured. (c) Fasting insulin (FINS) of rats was detected. (d) Insulin resistance (HOMA-IR) of rats was evaluated. (e) The expressions of IL-1β, TNF-α, and TLR4 were detected by qRT-PCR. (f) Serum LPS concentration was detected by ELISA kits. (G, H, I and J) The expressions of TLR4, MyD88, p-IκBα, κBα and p-p65 and p65 were detected by Western blotting. (k) QRT-PCR was used to detect the iNOS expression in liver tissue. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, vs. Control; ^{^}P < 0.05, ^{^^}P < 0.01, ^{^^^}P < 0.001, vs. Model; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, vs. Control

Figure 3.

The effect of Gypenosides on FFA-induced adipogenesis in THLE-2 cells and the effect on LPS/TLR4 downstream pathways in THLE-2 cells (a) MTT was used to detect the viability of THLE-2 cell treated with different concentrations of Gyp (10 μmol/L, 20 μmol/L, 40 μmol/L, 80 μmol/L, 160 μmol/L, and 320 μmol/L,). (b and c) Oil Red O staining was used to detect lipid droplet deposition of FFA-induced THLE-2 cell that treated with 40 μmol/L Gyp. (d) Kit used to detect triglyceride (TG) content in cells. (e) The iNOS expression was detected by qRT-PCR in cells. (F, G, H and I) The expression levels of TLR4, MyD88, p-IκBα, κBα, and p-p65 and p65 were detected by Western blotting. (J, K, and L) The p65 expression in cytoplasm and nucleus were detected by Western blotting. n = 3, ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, vs. 0; *P < 0.05, **P < 0.01, ***P < 0.001, vs. Control; ^{^}P < 0.05, ^{^^}P < 0.01, ^{^^^}P < 0.001, vs. FFA; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, vs. Gyp; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, vs. FFA+Gyp+NC

Figure 4.

Effects of LPS stimulation or upregulating TLR4 signaling pathway on Gyp’s inhibition of FFA-induced simple steatosis in THLE-2 cells. (a and b) Oil Red O staining was used to detect lipid droplet deposition. (c) Kit used to detect triglyceride (TG) content in cells. (D, E, F, and G) The expression levels of MyD88, p-IκBα, κBα, and p-p65 and p65 were detected by Western blotting. n = 3, ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, vs. 0; *P < 0.05, **P < 0.01, ***P < 0.001, vs. Control; ^{^}P < 0.05, ^{^^}P < 0.01, ^{^^^}P < 0.001, vs. FFA; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, vs. Gyp; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, vs. FFA+Gyp+NC