INTRODUCTION/OBJECTIVE: Sanghuang, a traditional Chinese medicinal fungus, exhibits well-documented anti-inflammatory, antioxidant, and antitumor activities. Emerging preclinical evidence indicates its efficacy in ameliorating bleomycin-induced Pulmonary Fibrosis (PF) in murine models. However, the molecular mechanisms mediating its anti-fibrotic effects remain poorly elucidated. This study was designed to systematically identify the bioactive constituents, pharmacological targets, and regulatory signaling networks that mediate the anti-PF effects of Sanghuang. METHODS: A bleomycin-induced mouse PF model was established to evaluate the in vivo therapeutic efficacy of Sanghuang. Integrative network pharmacology was applied to predict Sanghuang-derived bioactive compounds and their putative targets, followed by PF-related target screening and machine learning-based prioritization of high-confidence anti-fibrotic targets. Protein-Protein Interaction (PPI) network construction and GO/KEGG enrichment analyses were performed to identify key anti-fibrotic components and signaling pathways. Molecular docking and molecular dynamics simulations were conducted to evaluate the binding affinity and stability of prioritized constituents with core targets. In vitro functional validation was performed in TGF-β-stimulated A549 cells to assess the effects of stigmastane-3,5,6-triol on Epithelial-Mesenchymal Transition (EMT) and cell migration. RESULTS: Sanghuang treatment significantly alleviated bleomycin-induced histopathological damage and collagen deposition in lung tissues. Integrated network pharmacology and machine learning analyses identified 61 high-probability anti-PF targets and 20 candidate bioactive compounds, among which stigmastane‑3,5,6‑triol and genkwanin exhibited prominent multi‑target binding potential. ARG1, BCHE, MAOA, and MMP8 were recognized as core hub targets. Stigmastane‑3,5,6‑triol displayed strong predicted binding affinity and stable complex formation with these four core targets. KEGG pathway enrichment analysis revealed significant enrichment in AGE/RAGE, PI3K/AKT, and MAPK signaling cascades. Functionally, stigmastane‑3,5,6‑triol dose‑dependently reversed TGF‑β‑induced EMT by restoring E‑cadherin expression, suppressing the upregulation of N‑cadherin and vimentin, and inhibiting cell migration, with favorable in silico ADMET properties. DISCUSSION: Stigmastane-3,5,6-triol may be a key anti-fibrotic constituent of Sanghuang, exerting its therapeutic effects through high-affinity multi-target interactions with ARG1, MAOA, MMP8, and BCHE, as well as by inhibiting EMT. Among these targets, ARG1, MAOA, and MMP8 are critically implicated in PF pathogenesis, whereas BCHE represents a novel, under-investigated target warranting further experimental validation. Mechanistically, the anti-fibrotic activity of Sanghuang is achieved through the coordinated regulation of inflammation responses, oxidative stress, EMT, and Extracellular Matrix (ECM) homeostasis, accompanied by crosstalk between PI3K/AKT and MAPK signaling pathways, which aligns with the multi-target pharmacological characteristics of traditional Chinese medicines. CONCLUSION: These findings demonstrate that Sanghuang exerts anti-fibrotic effects via a multi-component, multi-target, and multi-pathway regulatory mode, orchestrating inflammatory and oxidative stress responses, ECM homeostasis, and EMT through crosstalk between PI3K/AKT and MAPK signaling pathways. Stigmastane- 3,5,6-triol may represent a potential key active constituent targeting ARG1, BCHE, MAOA, and MMP8. Despite some limitations, this work provides a mechanistic framework to support the preclinical and clinical development of Sanghuang and stigmastane-3,5,6-triol as promising therapeutic interventions for PF.
Lin et al. (Sun,) studied this question.