Tomatidine modulatesfibrosis-related markers by promoting autophagy in hepatic stellate cells via the ERK/MAPK-mTOR-ULK1 signaling pathway

Liver fibrosis is a progressive pathological process triggered by chronic liver disorders, which may progress to hepatocellular carcinoma (HCC) if left untreated. Currently, there are no specific therapeutic agents for liver fibrosis, highlighting an urgent need for novel pharmacological strategies. Tomatidine (TD), a major steroidal glycoal-kaloid abundant in immature tomato fruits, leaves, and stems, exhibits diverse biological activities including anti-inflammation, anti-tumor effects, and autophagy regulation. However, its role in liver fibrosis and the underlying molecular mechanisms remain incompletely understood. In this study, we combined network pharmacology, molecular docking, and experimental validation to investigate the potential effects of TD against liver fibrosis and its associated mechanism of action. In vitro experiments using the human hepatic stellate cell (HSC) line LX-2 demonstrated that TD inhibited HSC proliferation in a dose- and time-dependent manner, and downregulated the expression of fibrosis-related markers α-smooth muscle actin (α-SMA) and collagen type I α1 chain (COL1A1) at the gene, protein, and cellular levels. Network pharmacology analysis identified 18 common targets between TD and liver fibrosis, with core targets including MAPK3, RELA, and MAPK1 involved in intracellular signal transduction and stress-activated MAPK cascade. Although no autophagy-related targets were identified in the current database among these common targets, pharmacological evidence and experimental validation confirmed that TD promoted autophagy in LX-2 cells, as indicated by reduced P62 expression, increased LC3-II/LC3-I ratio and Beclin-1 levels, and enhanced autophagic flux. Further mechanism exploration revealed that TD exerted its autophagy-promoting effect by regulating the ERK/MAPK-mTOR-ULK1 signaling pathway: TD suppressed the phosphorylation of ERK and mTOR, while activating ULK1 phosphorylation. Molecular docking verified stable binding affinity between TD and key proteins in this pathway (ERK, MAPK, mTOR, ULK1) as well as autophagy-related proteins (P62, Beclin-1, LC3) and fibrosis-related protein COL1A1, with specific amino acid residues mediating hydrogen bond formation. Collectively, our findings demonstrate that TD modulates fibrosis-related markers in hepatic stellate cells by promoting autophagy in HSCs via the ERK/MAPK-mTOR-ULK1 pathway. This study enriches the biological function research of TD and provides a novel potential candidate and theoretical basis for the development of anti-liver fibrosis therapeutics.