An integrated strategy for screening Q-markers and elucidating anti-fibrotic mechanisms of Ganoderma lucidum

Ganoderma lucidum (G. lucidum) is a medicinal mushroom known for its hepatoprotective effects against various liver diseases.

Addressing the variable quality of G. lucidum samples and the lack of efficacy-linked quality control criteria, an integrated strategy combining chemical analysis and biological validation was conducted to identify the Q-markers directly responsible for its anti-hepatic fibrosis (HF) activity and to preliminarily elucidate their action mechanism.

49 batches of G. lucidum samples were collected from 15 geographical origins across 7 provinces in China. Initially, UPLC fingerprinting integrated with chemometric analysis evaluated the quality and identified differential chemical components. The common compounds were further characterized by UPLC-Orbitrap-HRMS. Subsequently, the potential targets and binding affinity were predicted by network pharmacology and molecular docking. Finally, the anti-HF activity and underlying mechanism of the core Q-marker were verified in a TGF-β1-induced HSC-T6 model using CCK-8, flow cytometry, immunofluorescence, and Western blotting.

The UPLC chemical fingerprint of G. lucidum was successfully established, 49 common components were identified based on UPLC-Orbitrap-HRMS analysis, mostly were triterpenoids. Through chemometric analysis, 19 key differential markers were subsequently screened. Then, by integrating network pharmacology predictions with chemometric screening results, 7 triterpenoids were ultimately determined as potential Q-markers for the anti-HF activity of G. lucidum. Ganoderic acid J (GAJ) demonstrated robust binding specificity to pivotal targets within the PI3K/AKT signaling pathway through molecular docking. 5, 10, and 20 µg/mL at 24 h were confirmed as low, medium, and high doses of GAJ by CCK-8, respectively. Ultimately, HF was relieved by suppressing the PI3K/AKT pathway, promoting apoptosis in activated HSCs and normalizing the MMP1/MMP2 equilibrium.

The anti-HF effects of G. lucidum were mediated through multi-target synergistic actions, chiefly by suppressing the PI3K/AKT pathway and modulating extracellular matrix metabolism. The hepatoprotective mechanism of G. lucidum was elucidated at the molecular level, and a Q-marker-based theoretical framework was established for quality control of traditional medicines and innovative drug development.