Sini San ameliorates CCl4-induced liver fibrosis in mice by inhibiting AKT-mediated hepatocyte apoptosis

Ethnopharmacological relevance: Sini San (SNS) is recorded in Zhang Zhongjing's "Treatise on Typhoids" and is used in the treatment of non-alcoholic fatty liver disease, hepatitis, and other liver diseases, with good efficacy in liver fibrosis. However, its anti-liver fibrosis mechanism remains unclear.Aim of the study: This study aimed to evaluate the ameliorative effect of SNS on carbon tetrachloride (CCl4)-induced liver fibrosis in mice and the underlying mechanisms.Materials and methods: The active ingredients in the water extract of SNS were determined using high-performance liquid chromatography (HPLC). CCl4-induced liver fibrosis mice were subsequently treated with different doses of SNS for 3 weeks, and AST, ALT, and T-BIL were detected in the serum. The pathological characteristics of the liver were observed using hematoxylin and eosin (H&E) and Masson's staining. Hepatocyte apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The proteins expression of PI3K, p-PI3K, AKT, p-AKT, FXR, caspase-8, Bax, and Bcl-2 was analyzed using western blotting and immunofluorescence. FXR mRNA expression was measured using quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR). Using network pharmacology and bioinformatics to search for active ingredients that regulate PI3K/AKT signaling in the SNS. The material basis for regulating PI3K/AKT signaling in SNS was searched using network pharmacology and bioinformatics. Based on the network phar-macology results, isorhamnetin or SNS-containing serum was added to HepG2 cells stimulated with TNF-alpha. The Cell Counting Kit (CCK)-8 assay was used to analyze cell viability and apoptosis of HepG2 cells was detected using flow cytometry.Results: SNS reduced serum levels of AST, ALT and T-BIL, down-regulated caspase-8 protein expression and the ratio of Bcl-2/Bax protein expression, and improved apoptosis in liver fibrosis mice. In addition, SNS down -regulated the ratio of p-PI3K/PI3K and p-AKT/AKT protein expression and increased FXR expression. Network pharmacology studies showed that quercetin, kaempferol and isorhamnetin in SNS can bind to AKT. In vitro experiments showed that isorhamnetin inhibited HepG2 cell apoptosis, upregulated FXR expression and sup-pressed AKT activity, whereas AKT inhibitors blocked the effects of isorhamnetin. The effect of the SNS-containing serum was similar to that of isorhamnetin.Conclusion: SNS ameliorated the progression of fibrosis and improved hepatocyte apoptosis in liver fibrosis mice. The anti-apoptotic mechanism was related to the inhibition of AKT-mediated down-regulation of FXR expression by its active ingredient, isorhamnetin.