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.
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/mL LPS）、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RAFLS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-1 mRNA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。
Keywords: NLRP3/GSDMD; Xinfeng Capsule; pyroptosis; synovial fibroblasts.