[经胶囊处理的大鼠血清对类风湿性关节炎滑膜成纤维细胞中脂多糖诱导的细胞焦亡的影响]
[Effects of serum from capsule-treated rats on lipopolysaccharide-induced pyroptosis in rheumatoid arthritis synovial fibroblasts].
作者信息
Wang L, Wang Y, Liu J, Huang C, Zhang W, Wan L, Sun Y, Huang D
机构信息
Anhui University of Traditional Chinese Medicine, Hefei 230038, China.
Department of Rheumatology, First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230031, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Dec 20;42(12):1846-1851. doi: 10.12122/j.issn.1673-4254.2022.12.13.
OBJECTIVE
To observe the effect of serum from rats treated with 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 (<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 (<0.05 or 0.01). Treatment with the drug-containing serum or MCC950 significantly decreased the viability of LPS-stimulated RA-FLS (<0.01), reduced cell pyroptosis, and lowered the concentrations of IL-1β and IL-18 and expressions of NLRP3, GSDMD, and caspase-1 (<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
观察消风胶囊（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，并探讨其可能机制。
方法
将20只SD大鼠分为空白对照组和XFC组。XFC组大鼠按0.324mg/g的剂量灌胃给予XFC，连续7天，制备含药血清。采用CCK-8法测定血清处理细胞的最佳浓度和作用时间。用CCK-8法评估含药血清或MCC950对5μg/mL LPS刺激的RA-FLS细胞活力的影响，用电子显微镜观察细胞焦亡情况；采用ELISA法检测细胞培养上清液中IL-1β和IL-18水平，采用蛋白质免疫印迹法和qRT-PCR法检测NLRP3、caspase-1和GSDMD的蛋白及mRNA表达。
结果
XFC处理RA-FLS的最佳浓度和作用时间分别为20%和24h。与空白对照细胞相比，LPS刺激的细胞活力显著增加（P<0.05），电子显微镜观察显示细胞内有大量囊泡形成，细胞膜出现孔洞、破裂，细胞内容物泄漏。LPS刺激显著增加细胞中IL-1β和IL-18水平以及NLRP3、GSDMD和caspase-1的表达（P<0.05或P<0.01）。含药血清或MCC950处理显著降低LPS刺激的RA-FLS细胞活力（P<0.01），减少细胞焦亡，降低IL-1β和IL-18浓度以及NLRP3、GSDMD和caspase-1的表达（P<0.05或P<0.01）。
结论
XFC可能通过抑制NLRP3/GSDMD途径抑制FLS焦亡，从而减轻RA关节局部炎症反应，减少炎性细胞因子分泌。
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