Eupalinolide B ameliorates liver fibrosis by targeting PKCα to suppress the AKT/mTOR axis in hepatic stellate cells

Ethnopharmacological relevance: Eupatorium lindleyanum DC. (Asteraceae), traditionally known as "Yemazhui," is a renowned herbal medicine historically used for heat-clearing, detoxification, and resolving phlegm-properties closely associated with potent anti-inflammatory activities. Eupalinolide B (EB), a major bioactive sesquiterpene lactone isolated from this plant, has been reported to alleviate inflammation and inhibit hepatocellular carcinoma. However, the pharmacological effects and potential mechanisms of EB in the context of liver fibrosis remain to be elucidated.

Aim of the study: This study aimed to evaluate the anti-fibrotic efficacy of EB in preventing liver fibrosis and to systematically identify its direct molecular targets and underlying signaling mechanisms, with a specific focus on hepatic stellate cell (HSC) activation.

Materials and methods: The therapeutic effects of EB were evaluated in vivo using carbon tetrachloride (CCl₄)-induced and bile duct ligation (BDL)-induced liver fibrosis mouse models, and in vitro using TGF-β1-activated LX-2 cells. To identify the underlying mechanisms and direct targets, a comprehensive approach was employed, integrating RNA Sequencing (RNA-seq), network pharmacology, molecular docking, molecular dynamics (MD) simulations, and chemical biology assays (cellular thermal shift assay [CETSA], drug affinity responsive target stability [DARTS], and cyanogen bromide [CNBr]-activated Sepharose pull-down). Functional validation was performed using gain-of-function rescue experiments.

Results: EB treatment significantly attenuated hepatic injury and Collagen deposition in both CCl₄-and BDL-induced fibrotic mice and markedly inhibited the activation and proliferation of HSCs in vitro. RNA-seq analysis revealed that EB primarily exerts its effects by suppressing the PI3K-AKT signaling pathway. Through chemical biology and biophysical assays, Protein Kinase C alpha (PKCα) was identified as a high-affinity, direct binding target of EB. Mechanistically, EB binds to PKCα to inhibit its kinase activity, thereby blocking the downstream Akt/mTOR signaling cascade. Furthermore, overexpression of PKCα in HSCs abolished the anti-fibrotic effects of EB, confirming PKCα as the functional target.

Conclusion: This study demonstrates that EB, a natural compound from E. lindleyanum, serves as a potent anti-fibrotic agent by directly targeting PKCα and inhibiting the PKCα/Akt/mTOR axis. These findings provide a novel therapeutic lead for liver fibrosis and offer scientific validation for the traditional use of E. lindleyanum in treating inflammation-related disorders, highlighting the value of exploring traditional herbal constituents for modern molecular targets.

Akt/mTOR pathway; Eupalinolide B; HSC activation; Liver fibrosis; PKCα.