TRAF-6调控TGF-β1-Smad2/Smad3信号通路在Graves病免疫发病机制中的作用

Role of TRAF-6 in regulating TGF-β1-Smad2/Smad3 signaling pathway in Graves' disease

目的:探讨肿瘤坏死因子受体相关因子-6(TRAF-6)是否在Graves病(GD)的发生过程中发挥免疫耐受作用及其可能的机制。方法:纳入Graves病初诊患者(GD) 30例,GD缓解期患者(e GD) 30例及健康人群(NC) 28例。提取外周血单个核细胞(PBMCs),分别采用实时定量PCR检测维甲酸相关孤儿受体(RORγt)、叉头蛋白P3(Foxp3)、白介素-2(IL-2)、TRAF-6 mRNA的表达,Western blot检测p-Smad2/Smad3、总Smad2/Smad3、TRAF-6蛋白的表达,ELISA检测血浆中转化生长因子-β1(TGF-β1)、IL-2、IL-17A、IL-10蛋白的表达。结果:①与NC组相比,GD组中IL-2的mRNA和蛋白、Foxp3 mRNA和IL-10蛋白的表达水平均减少,而RORγt mRNA和IL-17A蛋白的表达水平均增加,以上结果差异均有统计学意义(P<0. 05)。②与NC组相比,GD组中TGF-β1蛋白的表达水平降低,差异有统计学意义(P<0. 05); GD组中p-Smad2/Smad3的蛋白表达较NC组有上升趋势(P>0. 05)。③GD组中的TRAF-6 mRNA与NC组比较表达降低,差异有统计学意义(P<0. 05); TRAF-6蛋白表达较NC组有下降趋势(P>0. 05)。④e GD组中TRAF-6 mRNA的表达水平较GD组增加,差异有统计学意义(P<0. 05),较NC组差异无统计学意义(P>0. 05);其余指标在e GD组与GD组、e GD组和NC组之间的差异均没有统计学意义(P>0. 05)。结论:GD患者中TRAF-6低表达不足以抑制TGF-β1介导的Smad2/Smad3磷酸化,导致增多的Smad2/Smad3磷酸化蛋白下调IL-2表达,进而诱导Th17细胞的分化,同时抑制Treg细胞的分化,使机体不能维持免疫耐受,促进GD的发生。

Objective:To investigate whether tumor necrosis factor receptor-associated factor-6 (TRAF-6) exerts immune tolerance during the development of Graves′ disease(GD) and its possible mechanism. Methods: 30 GD newly diagnosed patients (GD group),30 GD remission patients (eGD group) and 28 healthy subjects (NC group) were recruited in this study.Peripheral blood mononuclear cells (PBMCs) were extracted and the mRNA expression of retinoid-related orphan receptor gamma t (RORγt),forkhead box protein p3 (Foxp3),interleukine-2 (IL-2) and TRAF-6 were detected by real-time quantitative PCR (RT-PCR).TRAF-6,p-Smad2,p-Smad3,and total Smad2/3 protein expression were determined by Western blot.ELISA was used to detect the expression of transforming growth factor-β1 (TGF-β1),IL-2,IL-17A and IL-10 in plasma. Results:①The mRNA and protein expression of IL-2,Foxp3 mRNA and IL-10 protein expression in the GD group were significantly lower than those in the NC group,while RORγt mRNA and IL-17 protein expression were significantly higher than those in the NC group ( P <0.05).②The TGF-β1 protein level in the GD group was significantly lower than that in the NC group ( P <0.05).The protein expression of p-Smad/Smad3 was higher in the GD group than those in the NC group( P >0.05).③The expression of TRAF-6 mRNA in GD group was significantly lower than that in NC group ( P <0.05).The expression of TRAF-6 protein was lower than NC group ( P >0.05).④The expression level of TRAF-6 mRNA in the eGD group was significantly higher than that in the GD group ( P <0.05),but no significant difference was found between the eGD group and the NC group ( P >0.05).There was no significant difference in other indexes between eGD group and GD group,and between eGD group and NC group ( P >0.05). Conclusion: TRAF-6 may play an immunotolerant role in the development of GD.Low expression of TRAF-6 may be not sufficient to inhibit the phosphorylation of Smad2/3 by TGF-β1,while increased phosphorylation of Smad2/3 down-regulate the expression of IL-2,inducing the differentiation of Th17 cells and inhibiting the differentiation of Treg cells,which makes it impossible for the host to maintain immune tolerance and promote the development of GD.