基于MH7A细胞迁移、凋亡及NLRP3炎性小体探讨金铁锁总皂苷治疗类风湿关节炎的作用机制

Study on the Mechanism of the Total Saponins of Psammosilene tunicoides Root in the Treatment of Rheumatoid Arthritis Based on MH7A Cell Migration, Apoptosis and NLRP3 Inflammasome

目的:探讨金铁锁总皂苷对类风湿关节炎成纤维样滑膜细胞(MH7A)增殖、迁移、凋亡及相关炎性因子的影响及其机制。方法:培养MH7A细胞,给予不同浓度的金铁锁总皂苷(0.05、0.10、0.15μg/mL)干预后,采用Annexin V/PI染色后流式细胞仪检测MH7A细胞凋亡;细胞划痕法测定MH7A细胞迁移;接着将细胞分为空白对照组、模型组、mcc950(1μmol/L)阳性对照组及金铁锁总皂苷各浓度(0.05、0.10、0.15μg/mL)组,采用MTS比色法检测MH7A细胞增殖;蛋白免疫印迹法、免疫荧光、q-PCR检测各组细胞NLRP3、ASC、Caspase-1 mRNA及蛋白表达;ELISA试剂盒及q-PCR检测炎性因子IL-1β、IL-18、IL-6炎性因子的释放及mRNA表达。结果:与空白对照组比较,金铁锁总皂苷呈浓度依赖性显著升高MH7A细胞凋亡率(P<0.01);中、高浓度金铁锁能显著抑制MH7A细胞迁移(P<0.01)。与模型组比较,不同浓度金铁锁总皂苷能显著降低TNF-α导致的炎症因子IL-1β、IL-18、IL-6的释放及其mRNA表达,并能抑制TNF-α诱导的MH7A细胞内NLRP3、ASC mRNA及蛋白表达(P<0.01)。结论:金铁锁总皂苷能抑制NLRP3炎性小体激活,其机制可能与抑制MH7A细胞的增殖、迁移和诱导其凋亡有关。

Objective:To investigate the effects and the mechanism of total saponins of Psammosilene tunicoides root(TSPT)on the fibroblast-like synoviocytes(MH7A)proliferation,migration,apoptosis and related inflammatory factors in rheumatoid arthritis(RA).Methods:MH7A cells were cultured and treated with different concentrations of TSPT(0.05,0.10,0.15μg/mL).The apoptosis of MH7A cells was detected by flow cytometry after Annexin V/PI staining.The migration of MH7A cells was determined by cell scratch method.The cells were divided into normal control group,model group,positive control group of mcc950(1μmol/L)and TSPT(0.05,0.10,0.15μg/mL)groups.The proliferation of MH7A cells was detected by MTS colorimetry.Western blotting,immunofluorescence and q-PCR were used to detect the mRNA and protein expressions of NLRP3,ASC and Caspase-1 in cells of each group.ELISA kit and q-PCR were used to detect the releases and mRNA expressions of inflammatory factors IL-1β,IL-18 and IL-6.Results:Compared with the normal control group,the TSPT was significantly reduced the number of living cells,and significantly increased the apoptosis rate of MH7A cells in a concentration-dependent manner(P<0.01).The migration of MH7A cells could be significantly inhibited in medium and high concentrations of TSPT(P<0.01).Compared with model group,different concentrations of TSPT could significantly reduce the release of TNF-αinduced inflammatory cytokines IL-1β,IL-18,IL-6 and the mRNA expressions,and inhibit NLRP3,ASC mRNA and protein expressions in MH7A cells induced by TNF-α(P<0.01).Conclusion:The TSPT can inhibit the activation of NLRP3 inflammasomes,the mechanism may be related to the inhibition of proliferation,migration and induction of apoptosis of MH7A cells.