清热活血方对肿瘤坏死因子α诱导的类风湿关节炎成纤维样滑膜细胞迁移和黏附的影响

The Effect of Qingre Huoxue Fang( 清热活血方) on Migration and Adhesion of Fibroblast-like Synoviocytes of Rheumatoid Arthritis Induced by Tumor Necrosis Factorα

目的观察清热活血方治疗活动期类风湿关节炎的可能作用机制。方法分离类风湿关节炎患者的滑膜组织制备原代类风湿关节炎成纤维样滑膜细胞(RA-FLS),采用肿瘤坏死因子α(TNF-α)诱导RA-FLS的迁移和黏附。设空白组(不加药物及TNF-α干预)、诱导组(10或20 ng/ml TNF-α诱导)、清热活血方低、中、高剂量组(20、100、500 ng/ml清热活血方+TNF-α诱导)。检测各组细胞迁移距离及面积、细胞黏附OD值和黏附抑制率,并检测细胞中PI3K/Akt信号通路相关蛋白(PI3K、p-Akt、Akt)表达。结果与空白组比较,诱导组细胞迁移距离及面积增加、黏附OD值增大、PI3K及p-Akt蛋白表达升高(P<0.01);与诱导组比较,清热活血方各剂量组均可抑制细胞的迁移和黏附以及PI3K、p-Akt蛋白表达,并以500 ng/ml效果最好(P<0.05或P<0.01)。结论清热活血方可能通过下调RA-FLS中PI3K、p-Akt蛋白表达,抑制RA-FLS的迁移和黏附,从而减缓滑膜炎症和关节破坏的进展。

Objective To observe the possible functional mechanism of Qingre Huoxue Fang（ 清热活血方） in treating rheumatoid arthritis in active phase. Methods Synovial tissues of patients with rheumatoid arthritis were separated to prepare primary generation of rheumatoid arthritis fibroblast-like synoviocytes（ RA-FLS）. Tumor necrosis factor α（ TNF-α） was adopted to induce migration and adhesion of RA-FLS. A blank group（ without medicine and TNF-α intervention）,an induced group（ induced by TNF-α of 10 or 20 ng / m L）,and the Qingre Huoxue Fang low,middle and high dose groups（ Qingre Huoxue Fang of 20,100,500 ng / m L respectively and induced by TNF-α） were set. Migration distance and square,OD value of adhesion and adherence inhibition rate of cells in each group were examined. And the expression of related proteins in PI3 K / Akt signal pathway,including PI3 K,p-Akt and Akt,was tested. Results Migration distance and square,adhesion OD value,as well as the expression of PI3 K and p-Akt protein increased in the induced group compared with those in the blank group（P〈0. 01）; Compared with the induced group,each Qingre Huoxue Fang dose group could all restrain cell migration and adhesion,as well as the expression of PI3 K and p-Akt protein. It had the best effects when the dose was of 500 ng / m L（P〈0. 05 orP〈0. 01）. Conclusion By lowering the expression of protein PI3 K and p-Akt in RA-FLS,Qingre Huoxue Fang could relieve synovial inflammation and destruction of joint; thereby restrain migration and adhesion of RA-FLS.