Jinshui Chenfei formula alleviates SiO2-induced pulmonary fibrosis by inhibiting macrophage M2 polarization via Grb2/STAT6 in rats

Background Jinshui Chenfei Formula (JCF), a proprietary Chinese medicinal prescription, has demonstrated remarkable therapeutic efficacy in treating pneumoconiosis patients. However, the precise mechanisms remain to be elucidated. This study aimed to investigate how JCF counteracts M2 macrophage polarization and thereby attenuates SiO 2 -triggered silicosis progression. Methods This study aimed to observe the effects of JCF on collagen deposition and macrophage M2 polarization in silicotic lungs induced by silica at 14 days, 28 days, and 42 days in rats. The active fraction of JCF was isolated and extracted using macroporous resin. Subsequently, RNA-seq was performed to predict the potential mechanism involved in the improvement of IL4-induced bone marrow-derived macrophages (BMDMs) by JCF. Finally, the targets of the active fraction of JCF were identified by the DARTS experiment. The underlying mechanisms were elucidated by combining siRNA and plasmid overexpression techniques. Results JCF significantly improved pulmonary function and pathological remodeling in silicosis rats across different time points, reduced collagen deposition in lung tissues, and downregulated the expression of M2 macrophage markers. JCF5, the active fraction of JCF, potently inhibited IL4-induced M2 polarization of bone marrow-derived macrophages (BMDMs). RNA-seq identified 96 differential expressed genes (DEGs) associated with M2 polarization and JCF5 treatment, which enriched signaling pathways such as JAK/STAT. In vitro experiment confirmed that JCF5 markedly reduced STAT6 phosphorylation. Drug affinity responsive target stability (DARTS) assays revealed that JCF5 suppress STAT6 activation by down-regulating Grb2, siRNA-mediated Grb2 knockdown potently reduced IL-4-induced M2 macrophages, whereas Grb2 overexpression significantly alleviated the inhibitory effects of JCF5 on STAT6 activation and M2 polarization. Further, Hesperidin is the main active ingredient verified by molecular docking and cell function assays. Conclusion JCF downregulate STAT6 activation by regulating Grb2, thereby inhibit M2 polarization of macrophage and improve pulmonary fibrosis in silicotic rats.