Effects of serum from Xinfeng capsule-treated rats on lipopolysaccharide-induced pyroptosis in rheumatoid arthritis synovial fibroblasts

Abstract

To observe the effect of serum from rats treated with Xinfeng Capsule (XFC) on lipopolysaccharide (LPS)-induced pyroptosis of rheumatoid arthritis synovial fibroblasts (RA-FLS) and explore the possible mechanism. Twenty SD rats were divided into blank control group and XFC group. The rats in XFC group was given 0.324 mg/g XFC by gavage for 7 days to prepare the drug-containing serum. CCK-8 assay was used to determine the optimal concentration and duration of the serum for cell treatment. The effect of the drug-containing serum or MCC950 on viability of RA-FLS stimulated with 5 μg/mL LPS was assessed with CCK-8 assay, and pyroptosis of the cells was observed using electron microscope; the levels of IL-1β and IL-18 in the cell culture supernatant were detected by ELISA, and the protein and mRNA expressions of NLRP3, caspase-1 and GSDMD were detected using Western blotting and qRT-PCR. The optimal concentration and duration of XFC for RA-FLS treatment were 20% and 24 h, respectively. Compared with the blank control cells, the cells with LPS stimulation showed significantly increased cell viability (P<0.05) and electron microscopy revealed a large number of vesicles in the cells with formation of membrane pores, cell membrane rupture, and leakage of cell contents. LPS stimulation significantly increased IL-1β and IL-18 levels and expressions of NLRP3, GSDMD, and caspase-1 in the cells (P<0.05 or 0.01). Treatment with the drug-containing serum or MCC950 significantly decreased the viability of LPS-stimulated RA-FLS (P<0.01), reduced cell pyroptosis, and lowered the concentrations of IL-1β and IL-18 and expressions of NLRP3, GSDMD, and caspase-1 (P<0.05 or 0.01). XFC alleviates local inflammatory response of joints in RA possibly by inhibiting pyroptosis of the FLS through inhibition of the NLRP3/GSDMD pathway, which results in reduced secretion of inflammatory cytokines.