[Effect of Fuzi Lizhong decoction in reducing liver injury of rats with non-alcoholic fatty liver via activating AMPK and suppressing NF-κBp65 pathway].

To investigate the protective effect and relevant mechanism of Fuzi Lizhong decoction (FZLZD) on liver of rats with non-alcoholic fatty liver disease (NAFLD), totally 32 male SPF Wistar rats were randomly divided into 4 groups: control group, model group, Yishanfu (YSF) group (200 mg·kg⁻¹·d⁻¹) and FZLZD group (10 g·kg⁻¹·d⁻¹), with 8 rats in each group. Rat model of NAFLD was prepared through the intragastric administration with fat emulsion for 4 weeks. After the successful modeling, rats in each administration group were continuously administered for 4 weeks. After 8 weeks, the rats in each group were put to death, and the pathological changes in liver tissue were detected by HE staining. Automatic biochemical analyzer was used to detect fasting serum lipid levels (T-Chol, TG, LDL-C, HDL-C) and liver functions (ALT, TP, ALB) of rats in each group. The rat liver index was calculated by weighing method. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of inflammatory cytokines TNF-α and IL-6 in liver tissue. Real-time quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of fat metabolism-related factors SREBP-1c and FASN in liver tissue. Western blot was used to detect the p-AMPK and p-NF-κBp65 protein expressions in liver tissue. The results of HE staining showed that compared with the control group, the pathological changes in liver tissue in the model group rats were obvious; specifically, the outline of hepatic lobule was unclear, the hepatic cells showed diffuse steatosis of adipose tissue, and were accompanied by inflammatory infiltration, nuclear condensation, coloring deep; compared with the model group, liver lesions of all of the treatment groups were significantly alleviated; especially, the FZLZD group showed the most significant degree of remission. The results of serum test showed that the levels of serum lipids (T-Chol, TG, LDL-C, HDL-C), liver functions (ALT, TP, ALB) and liver index in model group were significantly higher than those in control group (P<0.01); compared with the model group, the indexes of serum lipid and liver function of rats in each treatment group were significantly decreased (P<0.01), and those in FZLZD group were significantly decreased (P<0.05), while those in YSF group were not significantly changed. The results of ELISA and qRT-PCR showed that compared with the control group, the secretion levels of TNF-α, IL-6 and the mRNA levels of SREBP-1c and FASN in the liver tissue of model group rats were significantly increased (P<0.01); compared with model group, the secretion levels of TNF-α, IL-6 and the mRNA levels of SREBP-1c, FASN in liver tissue of rats in each treatment group were significantly decreased (P<0.01); compared with YSF group, the secretion levels of TNF-α and IL-6 and the mRNA levels of SREBP-1c and FASN in FZLZD group were significantly different (P<0.01). Western blotting showed that compared with the model group, the protein expression of p-AMPK in liver tissue of rats in FZLZD group was significantly increased (P<0.01), while the protein expression of p-NF-κBp65 was significantly decreased (P<0.01). FZLZD can significantly improve hepatic pathological changes, reduce serum lipid levels, promote liver function and liver index in NAFLD rats, which may be associated with the activation of the AMPK pathway and thereby the inhibition of the expressions of SREBP-1c and FASN, and the inhibition of the NF-κBp65 pathway and thereby the reduction of the release of inflammatory factors.

[Effects of three Wenyang Jianpi Tang on cell proliferation and apoptosis of nonalcoholic fatty liver cells].

To investigate the effects of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang on the cell proliferation and apoptosis of nonalcoholic fatty liver cells through the nonalcoholic fatty liver cell model established by inducing L02 cells with oleic acid. Different concentrations of oleic acid were added into L02 cells to induce the nonalcoholic fatty liver cell model. Oil red O staining was used to observe fatty droplets of fatty liver cells. Automatic biochemical analyzer was used to detect the levels of aspartic transaminase(AST), alanine aminotransferase(ALT), total cholesterol(TC), and triglyceride(TG) in the cell supernatants. There were five groups, namely normal group, model group, model and Sijunzi Tang group, model and Lizhong Tang group, and model and Fuzi Lizhong Tang group. The cell proliferation and apoptosis of the five groups were detected by MTT colorimetry test and flow cytometer. The expressions of PCNA, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax and Bcl-2 proteins of the five groups were detected by Western blot. The oil red O staining results showed that the optimum concentration of oleic acid that was used to induce nonalcoholic fatty liver cell models was 80 mg•L-1. The levels of AST, ALT, TC and TG in the nonalcoholic fatty liver cell supernatants were higher than that in normal liver cell supernatants(P<0.01). MTT colorimetry test and flow cytometer results showed that all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells(P<0.01). And Fuzi Lizhong Tang showed the best effect. Western blot results showed that Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could down-regulate the expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax proteins, and up-regulate the expressions of PCNA and Bcl-2 proteins of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. In conclusion, all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. The pharmacodynamic mechanism may be related to the expressions of key factors in pathways related with proliferation and apoptosis mediated by the three decoctions.