Butin regulates the NLRP3/CASP-1/GSDMD pyroptosis axis to inhibit proliferation and pyroptosis in human fibroblast-like synoviocytes of rheumatoid arthritis

To investigate the mechanism by which Butin inhibits proliferation and Pyroptosis in human fibroblast-like synoviocytes of rheumatoid arthritis (HFLS-RA) via the NLRP3/Caspase-1/ GSDMD Pyroptosis axis. Flavonoid compounds were extracted from Hei Gu Teng, and Butin was analyzed for its interaction with RA target proteins and signaling pathways using network pharmacology. An in vitro HFLS-RA model was established, and cells were induced with IL-1β. Groups included control, IL-1β-stimulated, IL-1β + Leflunomide, and IL-1β + Butin at low, medium, and high concentrations. Cell inhibition rates were measured using the MTT assay to determine the optimal Butin concentration, while scratch and Transwell assays assessed cell proliferation, migration, and invasion. LDH assays measured cell membrane damage; ELISA, RT-qPCR, and Western blot analyzed the expression of inflammation-related proteins NLRP3, GSDMD, CASP1, CASP3, IL1β, and IL18; Hoechst33342/PI staining and flow cytometry examined the cell cycle and Pyroptosis. Hei Gu Teng yielded Butin (> 96% purity). Network pharmacology identified 48 target genes, predominantly involved in pathways like IL-1 signaling, HIF-1 signaling, sphingolipid signaling, and osteoclast differentiation. Cell assays demonstrated significantly reduced proliferation in Butin-treated groups compared to control (P < 0.05). Compared to the IL-1β + Leflunomide group, the Butin group showed decreased LDH release and lower levels of NLRP3, GSDMD, IL-1β, IL-18, CASP1, and CASP3 proteins and mRNAs (P < 0.05). Cell cycle analysis indicated increased DNA content in G0/G1 phases and decreased in G2/S phases for medium and high Butin concentrations (P < 0.05). Butin suppresses the expression of multiple inflammatory factors induced by IL-1β within the NLRP3/CASP-1/GSDMD Pyroptosis axis, reducing proliferation and Pyroptosis of HFLS-RA, thus alleviating synovial inflammation and representing a potential mechanism for RA treatment.

Butin; Cell proliferation; Human rheumatoid arthritis synovial fibroblasts; Inflammatory factors; NLRP3/Caspase-1/GSDMD pyroptosis axis; Pyroptosis mechanism; Rheumatoid arthritis.