Blockade of the calpain-1/HSP90/TGF-β1 axis by Astragaloside IV ameliorates bleomycin-induced pulmonary fibrosis

Background Previous studies have confirmed the therapeutic potential of Astragaloside IV (AS-IV) in pulmonary fibrosis, yet the upstream regulatory mechanisms governing TGF-β1 stability, particularly involving proteolytic enzymes, are not fully understood. This study investigates the pivotal role of calpain-1 in modulating the HSP90/TGF-β1 signaling pathway, thereby elucidating the effects of AS-IV on bleomycin (BLM)-induced pulmonary fibrosis. Methods Calpain-1 knockout (CAPN1 −/− ) and wild-type (WT) mice were used to establish pulmonary fibrosis models via intratracheal instillation of BLM. Both animal and cell models were administered varying doses of AS-IV for intervention. Inflammatory, oxidative stress, fibrotic, and epithelial-mesenchymal transition (EMT) markers were subsequently evaluated. Additionally, molecular docking, molecular dynamics simulations, and surface plasmon resonance (SPR) experiments were employed to assess the binding interactions between AS-IV and the calpain-1/HSP90/TGF-β1 axis. Results The calpain-1/HSP90/TGF-β1 axis was significantly activated in BLM-induced pulmonary fibrosis mice. AS-IV ameliorated pulmonary fibrosis, EMT, oxidative stress, and inflammation while simultaneously suppressing this overactivated axis. Calpain-1 knockout inhibited HSP90 upregulation, attenuated TGF-β1/Smad2/3 activation, and reduced pulmonary fibrosis. In vivo experiments further confirmed the inhibitory effects of AS-IV and calpain-1 inhibition on lung epithelial cell injury and fibroblast proliferation. Notably, calpain-1 overexpression in MLE-12 cells abolished the protective effects of AS-IV against EMT, oxidative stress, and inflammation. Molecular docking, molecular dynamics simulations, and SPR experiments confirmed the direct binding between AS-IV and calpain-1, supporting its upstream regulatory role in this signaling axis. Conclusion AS-IV attenuates pulmonary fibrosis by targeting calpain-1 to disrupt the HSP90/TGF-β1 signaling axis. These findings identify calpain-1 as a novel therapeutic target and support AS-IV as a promising candidate for PF treatment.