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

Abstract

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.