Integrated analysis of single-cell RNA sequencing, transcriptomics, and thermal proteome profiling identifies PLCG1 as the therapeutic target of isopimpinellin in treating rheumatoid arthritis

BACKGROUND: Isopimpinellin (ISOP), derived from Toddalia asiatica (L.) Lam, is thought to possess anti-inflammatory potential. However, the effects of ISOP on rheumatoid arthritis (RA) and corresponding mechanisms remain unclear. METHODS: We evaluated the therapeutic effect of ISOP on RA using the collagen-induced arthritis (CIA) rat model. Subsequently, we determined the potential mechanisms of ISOP by combining single-cell RNA sequencing of rat synovial tissue in vivo with transcriptome analysis of macrophages in vitro. Molecular docking, thermal proteome profiling, cellular thermal shift assays, and drug affinity responsive target stability were then employed to identify the molecular targets of ISOP. Finally, we validated this target and explored the interaction between macrophages and RA-fibroblast-like synoviocytes (FLS) in vitro. RESULTS: We found that ISOP improved pathological changes in CIA rats. Mechanistically, ISOP inhibited macrophage migration and M1 macrophage polarization, and downregulated Spp1 expression. Furthermore, ISOP restrained the RAS/ERK pathway in M1 macrophages. Importantly, PLCG1 is a direct target of ISOP. Silencing PLCG1 reduced the related inhibitory effects of ISOP on M1 macrophage polarization. In addition, the supernatant of macrophages treated with ISOP reduced the proliferation and activation of RA-FLS. Silencing PLCG1 eliminated the regulatory effects of ISOP in the macrophage-RA-FLS co-culture system. CONCLUSIONS: ISOP exerts its anti-RA effects by targeting PLCG1 to inhibit the production of SPP1 in M1 macrophages.