金乌健骨胶囊对类风湿关节炎滑膜细胞增殖、迁移及凋亡的影响

Effects of Jinwu Jiangu Capsule on proliferation,migration and apoptosis of RA-FLS cells

目的:探讨金乌健骨胶囊对类风湿关节炎成纤维样滑膜(RA-FLS)细胞增殖、迁移及凋亡的影响。方法:采用组织块贴壁法分离培养RA-FLS细胞,HE染色和免疫化学法鉴定细胞。根据含药血清比例将细胞分为空白对照组,兔血清对照组,金乌健骨胶囊含药血清高、中、低剂量组和来氟米特对照组。CCK-8法检测细胞增殖,划痕实验检测细胞迁移,Western Blot检测Caspase8和Caspase3蛋白表达水平,ELISA检测TNF-α、TNFR1的分泌。结果:组织块法分离培养的细胞为RA-FLS。与空白组比较,不同浓度金乌健骨胶囊24、48 h能抑制RA-FLS增殖(P<0.05)。与兔血清对照组比较,金乌健骨胶囊各剂量组抑制RA-FLS伤口愈合(P<0.05),下调Caspase8、Caspase3蛋白表达(P<0.05),下调细胞培养上清中TNF-α浓度(P<0.05),上调TNFR1浓度(P<0.05)。其中金乌健骨胶囊高剂量组各指标优于来氟米特对照组(P<0.05)。结论:金乌健骨胶囊通过抑制RA患者FLS细胞TNF-α分泌,促进TNFR1的分泌,增加Caspase8、Caspase3蛋白表达,正向调节RA-FLS细胞凋亡,抑制RA-FLS增殖和迁移。

Objective:To investigate the effects of Jinwu Jiangu Capsule on proliferation,migration and apoptosis of rheumatoid arthritis-fibroblast like synovial cells(RA-FLS)cells.Methods:The RA-FLS cells was isolated and cultured by explant adherent culture method,HE staining and immunochemisty method were used to detected the proliferation characteristic of RAFLS cells.According to the different concentration of serum containing Jinwu Jiangu Capsule,the cells were divided into blank control group,rabbit serum control group,high,medium and low dose group of Jinwu Jiangu Capsule and leflunomide control group.The healing rate of cell,the expression level of Caspase8 and Caspase3 and the concentrations of TNF-αand TNFR1 were determined by Scratch test,Western blot assay(WB)and enzyme-linked immunosorbent assay(ELISA).Results:The cells by explant adherent culture method were RA-FLS.Jinwu Jiangu Capsule could decrease the proliferation of RA-FLS(P<0.05)in 24 h and 48 h compared with the blank control group.Jinwu Jiangu Capsule could decrease the wound healing rate significantly(P<0.05),decreased the protein expression of Caspase8,Caspase3(P<0.05),decreased the content of TNF-α(P<0.05),while decrease the expression of TNFR1(P<0.05)compared with the rabbit serum control group.The indicators of high dose group of Jinwu Jiangu Capsule were better than that in leflunomide control group(P<0.05).Conclusion:Jinwu Jiangu Capsule can down regulate TNF-αproduction,upregulate the expression of TNFR1,Caspase8 and Caspase3 to inhibit the proliferation and migration of RA-FLS cells by promoting apoptosis.