Non-Targeted Metabolomics and Network Pharmacology of Taohong Siwu Decoction in Hepatic Fibrosis Mouse Model Using High Resolution Mass Spectrometry

Shengsheng Li¹Shenglan Qi^2,3Rongsheng Li^2,3Fangming Yang⁴Zhenyi Niu⁵Wei Liu^2,3*Suping Ma^4*Zhun Xiao^1,4*

• ¹The First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou, China
• ²Key Laboratory of Liver and Kidney Diseases, Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
• ³Shanghai Key Laboratory of Traditional Chinese Medicine, Shanghai, China;, Shanghai, China
• ⁴Department of Digestive Diseases, The First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou, China;, Zhengzhou, China
• ⁵Department of Pathology, The First Affiliated Hospital of Henan University of traditional Chinese Medicine, Zheng Zhou, China

Taohong Siwu Decoction (THSW Decoction), a classic formula for treating blood stasis, has demonstrated significant clinical efficacy in the treatment of hepatic fibrosis. However, its primary active components and mechanisms of action remain unclear. In this study, a CCl₄-induced hepatic fibrosis mouse model was used to evaluate the therapeutic effects of THSW Decoction. Key blood-borne components were identified using UHPLC-Q-Exactive Orbitrap HRMS, and non-targeted metabolomics was conducted with a Synapt G2-Si QTOF HRMS system. Integrated network pharmacology and metabolomics analyses revealed potential targets, differential metabolites, and related pathways. Molecular docking, binding affinity prediction, and molecular dynamics simulations were employed to assess interactions between core compounds and targets. In vivo validation of key targets was also performed. THSW Decoction effectively reduced CCl₄-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, alleviating liver inflammation and collagen deposition. Forty-five blood components were detected, with 616 corresponding drug targets identified, including 419 overlapping targets for anti-hepatic fibrosis.The core protein-protein interaction (PPI) network comprised 59 nodes and 570 edges.Enrichment analysis revealed that THSW Decoction's blood components primarily modulated biological processes such as positive regulation of response to external stimuli and oxygen content. Key signaling pathways included PI3K-Akt, estrogen, relaxin, and MAPK. Non-targeted metabolomics identified 148 differential metabolites between the model and normal groups, and 156 between the THSW Decoction and model groups. Thirty-five overlapping metabolites were enriched in cAMP, phospholipase D, and GnRH signaling pathways. Twenty intersection targets linked blood components, metabolites, and hepatic fibrosis. PPI analysis ranked JUN, PTGS2, BCL2, ESR1, and PPARG as the top five targets. A "drug-component-target-metabolite" network highlighted ferulic acid 、 p-hydroxycinnamic acid 、 3-hydroxy-4-methoxycinnamic acid 、 ferulaldehyde、vanillic acid as the top five blood components.Molecular docking and molecular dynamics simulations revealed that 3-hydroxy-4-methoxycinnamic acid binds stably to the core target PPARG, exhibiting a binding free energy of -93.68 kJ/mol. In vivo validation showed that THSW Decoction upregulated JUN and downregulated ESR1 expression in the liver. This study elucidates THSW Decoction's key blood components, potential targets, and mechanisms in the treatment of hepatic fibrosis treatment, providing a foundation for further research.