Exploration of potential mechanism of Rougan formula against hepatic fibrosis by network analysis and experimental assessment

Rougan Formula (RG) has long been clinically applied to treat hepatic fibrosis in patients with different chronic liver diseases. However, the core active substances and the potential pharmacological mechanisms of RG remain unclear.The purpose of this study is to explore bioactive components, key targets, and potential mechanisms of RG by performing network pharmacological analyses and experimental model validation.All chemical components in RG extract were identified using ultraperformance liquid chromatography-quadrupole/time-of-flight tandem mass technology. The candidate components and drug targets of RG, as well as disease-related genes, were extracted from TCMSP and GeneCards databases. The potential pathways related to genes were predicted by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The core bioactive components, key targets, and signaling pathways were ultimately obtained by analyzing protein-protein interaction (PPI) and component-target-pathway (C-T-P) networks. Subsequently, the efficacy and underlying mechanisms of RG on hepatic fibrosis were experimentally validated in transforming growth factor-beta 1 (TGF-β1)-induced hepatic stellate cell activation model and CCL4-induced hepatic fibrosis mouse model.A total of 52 components in RG extract were obtained, and 22 of them were selected as the core bioactive components. Five hundred and thirty-nine overlapped targets were determined by matching drug targets with disease-related targets. The results of PPI and C-T-P network analyses revealed 100 key targets and 19 signaling pathways associated with RG efficacy. In vitro and in vivo studies further verified that RG exerted a significant anti-hepatic fibrotic effect by suppressing the activation of hepatic stellate cells by downregulating the TGF-β1/Smads signaling pathway.These results may provide some evidence for further clinical research and development of RG formula as an effective and safe drug for hepatic fibrosis treatment.