甲氨蝶呤治疗对类风湿性关节炎患者RORγt基因甲基化水平和Th17/Treg平衡的影响

Effects of methotrexate treatment on RORγt gene methylation level and Th17/Treg balance in patients with rheumatoid arthritis

目的探讨RA患者MTX对视黄酸相关孤儿受体γt(RORγt)启动子甲基化水平和Th17/Treg平衡的影响。方法选择2016年8月至2017年7月于我院确诊并使用MTX进行治疗的RA患者89例,所有患者均接受小剂量MTX维持治疗。分别于治疗前及开始治疗后3个月抽取外周静脉血,使用流式细胞仪检测Th17及Treg含量,并分析Th17细胞RORγt甲基化水平,而后分析二者之间的相关性。结果患者治疗后Th17(2.75%±0.83%vs 3.39%±0.74%)及Th17/Treg(0.26±0.09 vs 0.41±0.13)较治疗前显著下降(P<0.001),而Treg(7.29%±1.49%vs 6.13%±1.42%)较治疗前显著上升(P<0.001)。RORγt(mRNA)(2.98±0.69 vs 4.15±0.78)表达水平较治疗前显著下降(P<0.001)。患者治疗后RORγt序列1(32.21±4.71 vs 40.15±6.58)及序列2(20.73±3.3 vs 25.65±4.61)甲基化水平显著低于治疗前(P<0.001)。Pearson相关性分析结果显示,患者治疗前后RORγt序列1及序列2甲基化水平均与Th17/Treg呈正相关(r=0.345,0.297;P<0.05)。结论MTX能有效抑制RORγt基因的甲基化水平,改善RA患者Th17/Treg失衡的作用,提示RORγt甲基化水平可能参与对Th17/Treg平衡的调节。

Helper T 17 cell(Th17)/regulatory T cell(Treg)balance is an important factor affecting the progression of rheumatoid arthritis(RA).This study was designed to investigate the effect of methotrexate(MTX)on the methylation level of retinoic acid-related orphan receptorγt(RORγt)promoter and the balance of Th17/Treg in RA patients.This experiment enrolled total of 89 patients with RA who were diagnosed in our hospital from August2016 to July 2017 and treated with MTX(low-dose MTX maintenance therapy).Peripheral venous blood was taken before treatment and 3 months after the treatment.Flow cytometry was used to detect the content of Th17 and Treg,and the methylation level of RORγt in Th17 cells.Furthermore,the correlation between Th17/Treg and RORγt methylation level was analyzed.Data showed that after the treatment,Th17(2.75%±0.83%vs 3.39%±0.74%)and Th17/Treg(0.26±0.09 vs 0.41±0.13)were significantly decreased,while Treg(7.29±1.49 vs 6.13±1.42)was significantly increased,as compared with before treatment(all P<0.05);the expression level of RORγt(mRNA)(2.98±0.69 vs 4.15±.0.78)was significantly lower than that of before treatment(P<0.05).After treatment,the methylation level of RORγt sequence 1(32.21±4.71 vs 40.15±6.58)and sequence 2(20.73±3.38 vs 25.65±4.61)were significantly decreased,as compared with before treatment(P<0.05).Pearson correlation analysis showed that the RORγt sequence 1 and sequence 2 methylation levels were positively correlated with Th17/Treg before and after treatment(r=0.345,0.297;P<0.05).Taken together,MTX can effectively inhibit the methylation level of RORγt gene and improve the imbalance of Th17/Treg in RA patients,suggesting RORγt methylation may be involved in the regulation of Th17/Treg balance.