Network Pharmacology Analysis and Animal Experiment Validation of Penthorum chinense Pursh in Treating Cholestatic Liver Disease

INTRODUCTION: Cholestatic Liver Disease (CLD) remains a major clinical challenge because currently available therapies show limited efficacy in current therapies in a subset of patients. Penthorum chinense Pursh. (PCP), A traditional Miao ethnomedicine has reported hepatoprotective activity; however, its bioactive constituents and mechanisms in CLD remain unexplored. METHODS: An integrated strategy combining network pharmacology, molecular docking, 100 ns Molecular Dynamics (MD) simulations, and in vivo validation was applied. Active compounds and targets of PCP were identified through literature mining and database screening. Binding propensities between selected compounds and core targets were evaluated by molecular docking and MD. A mouse model of CLD induced by 0.1% 3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) was used for multi-dimensional validation through serum biochemistry, histopathology, proteomics, and protein expression analyses. RESULTS: A total of 32 bioactive compounds of PCP and 74 overlapping CLD-associated targets were identified. On the basis of the results of the protein-protein interaction and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, five potential active flavonoid components (kaempferol, luteolin, pinocembrin-7-O- β-D-glucoside (PCBG), pinocembrin chalcone, and quercetin) and core targets were selected to explore the mechanism of PCP in CLD. The molecular docking results indicated that there were good interactions between serine/threonine kinase proteins (AKT) and all five flavonoids, and PCBG showed potent or strong binding activity with Peroxisome Proliferator-Activated Receptor Gamma (PPARG), matrix metalloproteinase 9 (MMP9), interleukin-6 (IL6), Hypoxia-Inducible Factor 1 alpha (HIF), apoptosis regulator Bcl-2 (BCL2), and AKT1. In DDC-fed mice, PCP significantly improved biochemical and histopathological indices of cholestatic injury, altered proteomic signatures related to cellular processes/signaling and metabolism, and significantly decreased the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), AKT, and mammalian Target Of Rapamycin (mTOR). DISCUSSION: Integrating computational analyses with experimental validation suggests that PCP may exert anti-cholestatic effects primarily via flavonoid constituents (notably PCBG) through suppression of the PI3K/ AKT pathway. These findings suggest that PCP represents a promising novel treatment strategy for CLD. Further clinical studies are warranted to evaluate the efficacy of PCP-derived compounds, especially PCBG, in patients with this disease. CONCLUSION: This study integrates network pharmacology with experimental validation to implicate the PI3K/ AKT signaling as a key pathway targeted by PCP in CLD, providing a scientific basis for the clinical application of Gansukeli in CLD.