Shugan Jianpi Formula attenuate liver fibrosis via regulation of miR-193a-3p/TGF-β2 in hepatic stellate cells: An in vivo and in vitro study.

Abstract

Ethnopharmacological relevance: The Chinese herbal medicine Shugan Jianpi Formula (SGJPF) has traditionally been used to treat various chronic liver disorders. Previous studies have indicated that SGJPF inhibits hepatic stellate cells (HSCs) activation in rats with liver fibrosis (LF) and that miR-193a-3p may be a crucial molecule in LF. However, the mechanisms by which SGJPF regulates HSCs activation through miR-193a-3p remain unclear.

Aim of the study: This study aimed to determine whether the effect of SGJPF on LF is related to its regulation of miR-193a-3p and TGF-β2, both in a carbon tetrachloride (CCl₄)-induced LF mouse model and in TGF-β1-induced JS-1 cells.

Materials and methods: A CCl₄-induced LF mouse model was established to evaluate the anti-fibrotic efficacy of SGJPF by examining liver histopathological changes, collagen deposition, and the expression of α-smooth muscle actin (α-SMA) and collagen-I. To investigate the role of miR-193a-3p in HSCs activation, miR-193a-3p mimics and inhibitors were transfected into TGF-β1-induced JS-1 cells. The potential targets of miR-193a-3p were identified using miRDB, TargetScan 8.0, RNA-seq, and dual-luciferase reporter assays. Finally, the effects of SGJPF on HSCs activation and the miR-193a-3p/TGF-β2 axis were assessed in TGF-β1-treated JS-1 cells using CCK-8, EDU, scratch, RT-qPCR, and Western blotting assays.

Results: SGJPF significantly reduced liver damage and fibrosis, inhibited HSCs activation, decreased TGF-β2 levels, and increased miR-193a-3p expression in CCl₄-induced LF tissue. Additionally, miR-193a-3p was upregulated in HSCs transfected with miR-193a-3p mimics and downregulated in those with miR-193a-3p inhibitors. High levels of miR-193a-3p, combined with miRNA mimics, inhibited HSCs activation, proliferation, and migration. TGF-β2, a target negatively regulated by miR-193a-3p, partially reversed the effects of miR-193a-3p on TGF-β1-induced HSCs activation. SGJPF also reduced HSCs activation, proliferation, and migration in TGF-β1-treated JS-1 cells. Moreover, treatment with SGJPF-containing serum and miR-193a-3p inhibition restored HSCs activation, proliferation, and migration in TGF-β1-induced JS-1 cells.

Conclusions: This study demonstrates that SGJPF ameliorates CCl₄-induced liver fibrosis, which is associated with the regulation of miR-193a-3p and TGF-β2 in HSCs. These findings provide a new pharmacological basis for SGJPF and suggest a novel strategy for treating LF through TCM by regulating miRNAs.