红景天含药血清对肿瘤坏死因子α诱导的人类风湿关节炎成纤维样滑膜细胞增殖的影响

Effect of Serum Containing Herba Rhodiolae on the Proliferation of Human Fibroblast-like Synoviocytesrheumatoid Arthritis Induced by Tumor Necrosis Factor-α

目的探讨红景天含药血清对肿瘤坏死因子α(TNF-α)诱导的人类风湿关节炎成纤维样滑膜细胞(HFLS-RA)增殖的影响。方法制备空白组、模型组、雷公藤多苷组(9.45 mg·kg^(-1))及红景天低、中、高剂量组(1.05、2.1、4.2 g·kg^(-1))大鼠含药血清;体外培养HFLS-RA,以20 ng·mL^(-1)TNF-α及10%不同组含药血清对HFLS-RA进行干预。采用MTT法检测各组含药血清对HFLS-RA细胞的增殖作用;ELISA法检测细胞上清液中白细胞介素6(IL-6)、白细胞介素17(IL-17)、转化生长因子β(TGF-β)、白细胞介素10(IL-10)的含量;PCR法检测细胞中维甲酸受体相关孤儿受体γt(ROR-γt)、信号转导及转录激活因子3(Stat3)、叉头样转录因子3(Foxp3)mRNA表达;Western Blot法检测细胞中ROR-γt、Stat3、Foxp3蛋白表达。结果与空白组比较,模型组培养HFLS-RA 24、48 h后细胞增殖水平明显升高(P<0.01),ROR-γt、Stat3 mRNA及蛋白表达上调(P<0.01),IL-6、IL-17水平升高(P<0.01),Foxp3 mRNA及蛋白表达下调(P<0.01),TGF-β、IL-10水平降低(P<0.01)。与模型组比较,红景天各剂量组HFLS-RA细胞增殖水平降低(P<0.01),ROR-γt、Stat3mRNA及蛋白表达下调(P<0.05,P<0.01),IL-6、IL-17水平降低(P<0.01),Foxp3 mRNA及蛋白表达上调(P<0.01),TGF-β、IL-10水平升高(P<0.01)。结论红景天含药血清可抑制TNF-α诱导的HFLS-RA细胞的异常增殖,其机制可能与下调ROR-γt、Stat3表达及上调Foxp3表达有关。

ObjectiveTo explore the effect of serum containing Herba Rhodiolae on the proliferation of human fibroblast-like synoviocyte-rheumatoid arthritis(HFLS-RA)induced by tumor necrosis factor-α(TNF-α).Methods HFLS-RA cultured in vitro was divided into blank group,model group,tripterygium glycosides group(9.45 mg·kg^(-1))and low-,medium-and high-dose groups of serum containing Herba Rhodiolae(1.05、2.1、4.2 g·kg^(-1)),with20 ng·mL^(-1)TNF-α and different groups of medicated serum HFLS-RA intervened. MTT method was used to detect the proliferation effect of serum containing Herba Rhodiolae on HFLS-RA;ELISA method was used to detect the content of interleukin-6(IL-6),interleukin-17(IL-17),transforming growth factor-β(TGF-β)and interleukin-10(IL-10)in the cell supernatant. The PCR method was used to detect the expression of RAR-related orphan receptor gamma(ROR-γt),signal transducers and activators of transcription 3(Stat3)and Foxhead box protein 3(Foxp3)mRNA in the cells,and the Western Blot method was used to detect the expression of ROR-γt,Stat3,and Foxp3 protein in the cells.ResultsCompared with the blank group,the cell proliferation level was significantly increased in the model group after 24-hour and 48-hour of HFLS-RA culture(P<0.01),ROR-γt,Stat3 mRNA and protein expression were upregulated(P<0.01), IL-6 and IL-17 levels were increased(P<0.01), Foxp3 mRNA and protein expression were downregulated(P<0.01), TGF-β and IL-10 levels were reduced(P<0.01);Compared with the model group,the proliferation activity of HFLS-RA in each dose group of the Herba Rhodiolae decreased(P<0.01),the transcription factor ROR-γt,Stat3 mRNA and protein expression decreased(P<0.05,P<0.01),the levels of IL-6 and IL-17 decreased(P<0.01), and Foxp3 mRNA and protein expression upregulated(P<0.01),and the level of TGF-β and IL-10 increased(P<0.01).ConclusionSerum containing Herba Rhodiolae is able to inhibit the abnormal proliferation of HFLS-RA induced by TNF-α,and its mechanism may be related to the down-regulation of ROR-γt expression and Stat3,and the up-regulation of Foxp3 expression.