Study on the mechanism of anti-pulmonary fibrosis action of Jingfang granules based on the Keap1-Nrf2-GPX4 pathway

Jingfang granules (JF) is derived from the original formula of Jingfang Baidu Powder. Modern pharmacological studies have shown its antipyretic, analgesic, and anti-inflammatory effects. It has been clinically used to treat viral respiratory infections and human mycoplasma pneumonia, with proven efficacy and significant improvement in patients' clinical symptoms. However, the mechanism by which JF treats pulmonary fibrosis (PF) remains unclear. This study aims to investigate the therapeutic efficacy of JF in PF and elucidate its potential therapeutic mechanisms. To explore the therapeutic potential of JF in PF, we constructed a mouse model of PF induced by bleomycin (BLM) and an integrated in vitro co-culture system consisting of MLE-12 cells and primary mouse pulmonary fibroblasts, combined with serum pharmacology of traditional Chinese medicine. The study evaluated the effects of JF on pathological alterations in vivo and its regulatory influence on cell proliferation and fibrotic processes in vitro . In addition, the activation of the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-glutathione peroxidase 4 (GPX4) signaling pathway was examined, and the direct binding interaction between JF and Keap1 was verified. JF significantly alleviated BLM-induced PF in mice. In murine lung epithelial cells (MLE-12) and primary pulmonary fibroblasts, JF demonstrated the ability to directly bind to Keap1, thereby promoting Keap1 autophagic degradation. This activation of Keap1-Nrf2-GPX4 effectively restrained cell proliferation and migration, thereby mitigating fibrotic progression. In summary, JF exerts its anti-fibrotic effects by targeting Keap1 and competitively disrupting the Keap1-Nrf2 interaction, thereby promoting Nrf2 protein translocation into the nucleus. This mechanism provides experimental evidence supporting its clinical application and new drug development. JF targets Keap1, promoting Keap1 clearance via the p62-mediated autophagy degradation pathway. This releases Nrf2 from Keap1 repression, allowing it to enter the nucleus and bind to transcription factors such as Maf. Consequently, Nrf2 significantly upregulates antioxidant genes including NQO1 and SOD2, restores GPX4 activity to inhibit lipid ROS production, and ultimately alleviates pathological damage in PF. • This study is the first to demonstrate that Jingfang Granules (JF) directly target Keap1, promoting its binding with the autophagy receptor p62 and facilitating its degradation through the autophagic pathway, thereby activating Nrf2. • Unlike the traditional mechanism of Nrf2 activation via competitive dissociation from Keap1, JF achieves sustained and steady-state Nrf2 activation by enhancing the autophagic degradation of Keap1, providing a new theoretical model for redox homeostasis regulation and antioxidant therapy. • The use of the Nrf2 inhibitor ML385 partially reversed the effects of JF on GPX4 expression and its cytoprotective activity, further confirming that the antifibrotic effects of JF are dependent on the Keap1–Nrf2 signaling pathway. • This study elucidates, at the mechanistic level, that JF exerts preventive and therapeutic effects against pulmonary fibrosis in its early stages by regulating oxidative stress and ferroptosis, providing experimental evidence supporting its potential as a candidate antifibrotic agent.