新风胶囊含药血清抑制脂多糖诱导的类风湿关节炎大鼠的滑膜成纤维细胞焦亡

目的 观察新风胶囊（XFC）含药血清对脂多糖（LPS）诱导的类风湿关节炎滑膜成纤维细胞（RA-FLS）焦亡的影响及其机制。方法 将20只SD大鼠利用随机数字表法分为2组：空白组和XFC组，XFC组予0.324 mg/g灌胃7 d后制备含药血清。CCK-8法检测XFC含药血清作用于RA-FLS最佳干预浓度和时间。将RA-FLS分为空白组（RA-FLS）、模型组（RA-FLS+5 μg/mLLPS）、MCC950组（RA-FLS+LPS+MCC950）、XFC组（RA-FLS+LPS+XFC含药血清）。CCK-8法检测细胞活性，电镜观察RA-FLS焦亡形态，ELISA检测IL-1β、IL-18浓度，Western blotting和qRT-PCR检测NLRP3、caspase-1、GSDMD的蛋白和mRNA的表达。结果 XFC作用于RA-FLS最佳干预浓度和时间分别为20%和24 h。与空白组相比，模型组细胞活性显著上升（P<0.05），电镜下可见RA-FLS内形成大量小泡，膜上形成孔隙，胞膜破裂，细胞内容物流出，IL-1β、IL-18浓度和NLRP3、GSDMD、caspase-1mRNA及蛋白表达显著升高（P<0.05或P<0.01）；与模型组相比，XFC含药血清组、MCC950组细胞活性显著下降（P<0.01），细胞焦亡情况改善，IL-1β、IL-18浓度和NLRP3、GSDMD、caspase-1 蛋白及mRNA表达明显降低（P<0.05、P<0.01）。结论 XFC可能通过NLRP3/GSDMD通路抑制FLS细胞焦亡，下调炎症细胞因子的分泌，减轻关节局部炎症反应而发挥治疗作用。

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

Objective 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. Methods 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. Results 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). Conclusion 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.