二妙散对TNF-α诱导的人类风湿关节炎成纤维样滑膜细胞功能的影响

Effect of Ermiaosan on Function of Human Fibroblast Synovial Cells Induced by TNF-α

目的:二妙散(EMS)对肿瘤坏死因子(TNF)-α诱导的人类风湿关节炎(RA)成纤维样滑膜细胞(FLS)增殖、迁移、黏附、侵袭、分泌功能的作用及其可能机制。方法:TNF-α(20μg·L^-1)体外诱导RA患者FLS,加入不同质量浓度二妙散(0.2,0.4,0.8 g·L^-1)作用后,分别采用噻唑蓝(MTT)比色法,transwell迁移、黏附及侵袭实验检测FLS细胞的增殖活性、迁移、黏附及侵袭能力,酶联免疫吸附测定(ELISA)检测细胞上清中白细胞介素(IL)^-1β的含量。提取FLS中的蛋白,采用蛋白免疫印迹法(Western blot)检测磷酸化Janus激酶1(p-JAK1),p-信号转导与转录激活因子(STAT)1,p-STAT6的蛋白表达水平。结果:与空白组比较,TNF-α(20μg·L^-1)能增加FLS细胞的增殖、迁移、黏附、侵袭能力及IL^-1β分泌(P<0.01);与TNF-α组比较,二妙散(0.2,0.4,0.8 g·L^-1)作用24 h对TNF-α诱导的FLS细胞增殖活性没有明显影响,作用24 h内还能明显降低TNF-α诱导升高的FLS细胞迁移、黏附、侵袭能力及分泌IL^-1β(P<0.05);与空白组比较,TNF-α能分别诱导p-JAK1,p-STAT1,p-STAT6在FLS中的异常升高(P<0.01),0.2,0.4,0.8 mg·L^-1的二妙散能明显降低p-JAK1,p-STAT1,p-STAT6的表达水平(P<0.05,P<0.01)。结论:二妙散可降低FLS细胞增殖、迁移、黏附、侵袭及IL^-1β的分泌,而其机制可能与JAK/STAT信号通路相关。

Objective:To study the effects of Ermiaosan on migration,adhesion and invasion of human fibroblast-liked synovial cells(FLS)and explore its mechanism.Method:Using the human FLS as the research object,the nontoxic concentration of FLS.FLS was determined by methyl thiazolyl tetrazolium(MTT)colorimetric assay for the follow-up experiment.The transwell migration,adhesion and transwell invasion test were used to detect the migration and adhesion of the different concentration of Ermiaosan on FLS,respectively.The expression of interleukin(IL)^-1 beta of FLS supernatant was detected by enzyme linked immunosorbent assay(ELISA).Protein in FLS was extracted and protein expression levels of phosphorylated Janus kinase 1(p-JAK1),p-signal transducer and activator of transcription(STAT1)and p-STAT6 were detected by Western blot.Result:Compared with control group,tumor necrosis factor(TNF)-α(20μg·L^-1)increased the proliferation,migration,adhesion,invasion and the secretion of IL^-1βof FLS(P<0.01).Ermiaosan(0.2,0.4,0.8 mg·L^-1)had no significant effect on the proliferation of FLS induced by TNF-αfor 24 h.Within 24 h,the migration,adhesion,invasion,invasion,and secretion of IL^-1βof FLS cells induced by TNF-αwere also decreased significantly(P<0.05,P<0.01).Compared with the blank group,TNF-αcould induce abnormal elevation of p-JAK1,p-STAT1and p-STAT6 in FLS(P<0.01),while Ermiaosan of 0.2,0.4,0.8 g·L^-1could significantly reduce the expression levels of p-JAK1,p-STAT1 and p-STAT6(P<0.05,P<0.01).Conclusion:Ermiaosan can inhibit the migration,adhesion and invasion of FLS,and its mechanism may be related to the inhibition of the secretion of IL^-1β,the mechanism may be related to JAK/STAT pathway.