Astragaloside IV Improves High-Fat Diet-Induced Hepatic Steatosis in Nonalcoholic Fatty Liver Disease Rats by Regulating Inflammatory Factors Level via TLR4/NF-κB Signaling Pathway

Abstract

Objective: Astragaloside IV (AS-IV) is the primary bioactive component purified from Astragalus membranaceus which is one of the traditional Chinese medicines. Research studies found that AS-IV has significant pharmacological effects on focal cerebral ischemia/reperfusion, cardiovascular disease, pulmonary disease, liver cirrhosis, and diabetic nephropathy, but little is known about the effects of AS-IV on nonalcoholic fatty liver disease (NAFLD). In this study, we investigated whether AS-IV has beneficial effects on NAFLD in rats and its potential mechanisms. Methods: Male SD rats were fed with high-fat diet (HFD) for 12 weeks to establish NAFLD rat model, and then, the rats were divided into five groups. The control group rats were fed with normal diet for 12 weeks and then were given normal saline (1.0 ml kg^-1 day^-1) by intragastric administration for 4 weeks. The model group rats were fed with HFD for 12 weeks and then were given normal saline (1.0 ml kg^-1 day^-1) by intragastric administration for 4 weeks. The AS-IV-L, AS-IV-M, and AS-IV-H groups were treated with 20, 40, and 80 mg kg^-1 day^-1 of AS-IV by intragastric administration for 4 weeks and given HFD diet. Then, we detected serum transaminase (ALT, AST), blood lipid (TG, TC), inflammatory cytokines (IL-6, IL-8 and TNF-α), liver histology(NAFLD activity score), TLR4/MyD88 signaling pathway in liver tissue. Results: We found AS-IV significantly reduced serum levels of AST, ALT, TG, TNF-α, IL-6, and IL-8 in NAFLD rats and downregulate the expression of TLR4 mRNA, MyD88 mRNA, NF-κB mRNA, and proteins in liver tissue. Moreover, AS-IV could significantly reduce the NAFLD activity score of NAFLD rat liver. Conclusion: In this study, we demonstrated that AS-IV have a protective effect on NAFLD by inhibiting TNF-α, IL-6 and IL-8 levels and down-regulating TLR4, MyD88 and NF-κB expression in rat liver tissues.

Keywords: astragaloside IV; myeloid differentiation factor 88; non-alcoholic fatty liver disease; nuclear factor-kappa B; toll like receptor 4.

FIGURE 1

Chemical structure of astragaloside IV.

FIGURE 2

Body weight and daily food intake of rats. (A) The body weight of rats. (B) The body weight gain after 12 weeks. (C) The average daily food intake of rats in different groups. Data present the mean ± SD. Comparisons between two groups use t-tests, and data comparison among multiple groups use one-way ANOVA. *p < 0.05 and **p < 0.01 compared with the control group; ^# p < 0.05 and ^## p < 0.01 compared with the model group. CON, control group; MOD, model group; AS-IV-L, astragaloside IV low-dose group; AS-IV-M, astragaloside IV middle-dose group; AS-IV-H, astragaloside IV high-dose group.

FIGURE 3

Effects of astragaloside IV on biochemical parameters in HFD-fed rats. CON: 10 SD rats fed with normal diet for 12 weeks and then treated with intragastric administration of saline (1.0 ml/kg/day) and normal diet for 4 weeks. MOD: 10 SD rats fed with HFD for 12 weeks and then treated with intragastric administration of saline (1.0 ml/kg/day) and HFD diet for 4 weeks. AS-IV-L: 10 SD rats fed with HFD for 12 weeks and then treated with intragastric administration of 20 mg kg⁻¹ day⁻¹ and HFD for 4 weeks. AS-IV-M: 10 SD rats fed with HFD for 12 weeks and then treated with intragastric administration of 40 mg kg⁻¹ day⁻¹ and HFD for 4 weeks. AS-IV-H: 10 SD rats fed with HFD for 12 weeks and then treated with intragastric administration of 80 mg kg⁻¹ day⁻¹ and HFD for 4 weeks. (A) Serum ALT concentration of each group. (B) Serum AST concentration of each group. (C) Serum TC concentration of each group. (D) Serum TG concentration of each group. Data present the mean±SD. Comparisons between two groups use t-tests and data comparison among multiple groups use one-way ANOVA. *p < 0.05 and **p < 0.01 compared with the control group; ^# p < 0.05 and ^## p < 0.01 compared with the model group. HFD, high-fat diet; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TC, total cholesterol; TG, triglyceride; CON, control group; MOD, model group; AS-IV-L, astragaloside IV low-dose group; AS-IV-M, astragaloside IV middle-dose group; AS-IV-H, astragaloside IV high-dose group.

FIGURE 4

Effects of astragaloside IV on liver histology in HFD-fed rats. (A) Steatosis score of rat liver. (B) Lobular inflammation score of rat liver. (C) Ballooning score of rat liver. (D) NAFLD activity score of rat liver. (E) Representative histological change of steatosis in liver sections stained with H&E (magnification 400×). Data present the mean±SD. Comparisons between two groups use t-tests, and data comparison among multiple groups use one-way ANOVA. *p < 0.05 and **p < 0.01 compared with the control group; ^# p < 0.05 and ^## p < 0.01 compared with the model group. CON, control group; MOD, model group; AS-IV-L, astragaloside IV low-dose group; AS-IV-M, astragaloside IV middle-dose group; AS-IV-H, astragaloside IV high- dose group.

FIGURE 5

Effects of astragaloside IV on expression of hepatic TLR4, MyD88, and NF-κB p65 in NAFLD rats. (A) TLR4 mRNA expression. (B) MyD88 mRNA expression. (C) NF-κB p65 mRNA expression. (D) TLR4 protein expression. (E) MyD88 protein expression. (F) NF-κB p65 protein expression. (G) Representative protein expression bands of hepatic TLR4, MyD88, and NF-κB p65 were analyzed by Western blotting. Data present the mean ± SD. Comparisons between two groups use t-tests, and data comparison among multiple groups use one-way ANOVA.*p < 0.05 and **p < 0.01 compared with the control group; ^# p < 0.05 and ^## p < 0.01 compared with the model group. CON, control group; MOD, model group; AS-IV-L, astragaloside IV low-dose group; AS-IV-M, astragaloside IV middle-dose group; AS-IV-H, astragaloside IV high-dose group.

FIGURE 6

Effects of astragaloside IV on inflammatory factors in NAFLD rats. (A) Serum IL-6 concentration. (B) Serum IL-8 concentration. (C) Serum TNF-α concentration. Data present the mean ±SD. Comparisons between two groups use t-tests, and data comparison among multiple groups use one-way ANOVA. *p < 0.05 and **p < 0.01 compared with the control group; ^# p < 0.05 and ^## p < 0.01 compared with the model group. CON, control group; MOD, model group; AS-IV-L, astragaloside IV low-dose group; AS-IV-M, astragaloside IV middle-dose group; AS-IV-H, astragaloside IV high-dose group.