Silibinin alleviates lung fibrosis by targeting Annexin A6 to suppress endoplasmic reticulum stress and oxidative stress.

INTRODUCTION

Endoplasmic reticulum (ER) stress and oxidative stress are closely linked in driving the TGF-β1-mediated proliferation and myofibroblast differentiation of lung fibroblasts during pulmonary fibrosis. Silibinin (SIL), a bioactive compound derived from Silybum marianum l., has been proven to suppress inflammatory responses in a bleomycin (BLM) mouse model, but the protective mechanisms and specific molecular target through which SIL acts in pulmonary fibrosis remain unclear.

OBJECTIVES

This study aimed to identify the direct molecular target of SIL, and investigate the mechanisms by which SIL exerts its anti-fibrotic effect.

METHODS

A mouse model of BLM-induced pulmonary fibrosis and a cellular model of TGF-β1-induced fibroblast activation were utilized to explore the mechanism by which SIL inhibits fibroblast functions. Limited proteolysis-mass spectrometry (LiP-MS) technology, molecular docking and surface plasmon resonance (SPR) were used to identify the molecular target of SIL.

RESULTS

SIL treatment significantly attenuated BLM-induced pulmonary fibrosis in mice by suppressing both the proliferation and differentiation of lung fibroblasts. SIL also reduced BLM-induced ER stress and oxidative stress in mouse lung fibroblasts. In vitro, SIL markedly inhibited TGF-β1-induced fibroblast proliferation, myofibroblast differentiation, and extracellular matrix (ECM) deposition by attenuating ER stress and oxidative stress in primary normal human lung fibroblasts (NHLFs), IPF-derived lung fibroblasts (IPF-HLFs), and the MRC-5 cell line. Using an integrated approach combining LIP-MS, molecular docking, and SPR, we identified Annexin A6 (ANXA6) as the direct molecular target of SIL. ANXA6 knockdown in lung fibroblasts reduced TGF-β1-induced ROS accumulation, ER stress, and subsequent fibroblast activation, whereas ANXA6 overexpression exacerbated these effects. Mechanistically, SIL bound to ANXA6, leading to inactivation of the PI3K/AKT/mTOR signaling and thereby alleviating fibroblast activation.

CONCLUSION

Our finding identifies ANXA6 as a promising therapeutic target for pulmonary fibrosis and proposes SIL as its natural inhibitor, thereby providing crucial mechanistic insights for developing ANXA6-targeted therapies against IPF.