CD137-CD137L信号通过TRAF6/JNK/AP-1通路调控小鼠血管平滑肌细胞活化T细胞核因子c1表达

CD137-CD137L signal regulates the expression of nuclear factor of activated T cell c1 in mouse vascular smooth muscle cells via TRAF6/JNK/AP-1 pathway

目的探讨CD137-CD137L信号是否通过肿瘤坏死因子受体相关因子6/c-Jun氨基末端激酶/活化蛋白1(TRAF6/JNK/AP-1)途径调控小鼠血管平滑肌细胞(VSMC)活化T细胞核因子c1(NFATc1)的表达。方法采用组织块原代培养小鼠VSMC,第3~5代细胞用于实验。VSMC分为6组:对照组、CD137刺激组、CD137抑制组、siTRAF6干预组、siJNK干预组、siAP-1干预组。Western blot检测各组TRAF6、磷酸化JNK(p-JNK)、磷酸化AP-1(p-AP-1)、NFATc1蛋白表达水平。免疫荧光法检测各组TRAF6、p-JNK、p-AP-1、NFATc1荧光表达。结果 (1)与对照组相比,CD137刺激组(CD137-CD137L信号激活)TRAF6、p-JNK、p-AP-1、NFATc1蛋白表达水平明显升高(P<0.05);与CD137刺激组相比,CD137抑制组(CD137-CD137L信号抑制)TRAF6、p-JNK、p-AP-1、NFATc1蛋白表达水平明显降低(P<0.05)。(2)采用siRNA技术分别沉默TRAF6、JNK、AP-1后,与CD137刺激组相比,siTRAF6干预组TRAF6、p-JNK、p-AP-1、NFATc1表达明显减少(P<0.05);siJNK干预组TRAF6表达无改变,而p-JNK、p-AP-1、NFATc1表达明显减少(P<0.05);siAP-1干预组TRAF6、p-JNK表达无明显改变,而p-AP-1、NFATc1表达明显减少(P<0.05)。(3)免疫荧光检测显示:CD137刺激组TRAF6、p-JNK、p-AP-1、NFATc1荧光表达量升高(P<0.05);CD137抑制组TRAF6、p-JNK、p-AP-1、NFATc1荧光表达量明显降低(P<0.05);siTRAF6干预组、siJNK干预组、siAP-1干预组TRAF6、p-JNK、p-AP-1、NFATc1荧光表达量与上述蛋白表达结果一致。结论 CD137-CD137L信号可通过TRAF6/JNK/AP-1通路影响NFATc1的表达。

Aim To investigate whether CD137-CD137 L signal through tumor necrosis factor receptor-associated factor-6 / c-Jun amino-terminal kinase / activated protein-1（ TRAF6 / JNK / AP-1） pathway to regulate the expression of nuclear factor of activated T cell c1（ NFATc1） in mouse vascular smooth muscle cell（ VSMC）. Methods Mouse primary VSMCs were cultured from aortic tissue block,and third to fifth generations of cells were used for the experiment.VSMCs were divided into 6 groups： control group, CD137 stimulation group, CD137 inhibition group, siTRAF6 intervention group,siJNK intervention group and siAP-1 intervention group. The expression levels of TRAF6,phosphorylated JNK（ p-JNK）,phosphorylated AP-1（ p-AP-1） and NFATc1 protein were detected by Western blot in each group.The fluorescence expression of TRAF6,p-JNK,p-AP-1 and NFATc1 were detected by immunofluorescence assay in each group. Results（ 1） Compared with control group,the expression levels of TRAF6,p-JNK,p-AP-1 and NFATc1 protein were significantly increased in CD137 stimulation group（ CD137-CD137 L signal activation）（ P〈0. 05）. Compared with CD137 stimulation group,the expression levels of TRAF6,p-JNK,p-AP-1 and NFATc1 protein were significantly decreased in CD137 inhibition group（ CD137-CD137 L signal inhibition）（ P〈0.05）.（ 2） The siRNA technology was used to silence TRAF6,JNK and AP-1 respectively. Compared with CD137 stimulation group,the expressions of TRAF6,pJNK,p-AP-1 and NFATc1 were significantly reduced in siT RAF6 intervention group（ P〈0.05）; The expression of TRAF6 had no change,while the expression of p-JNK,p-AP-1 and NFATc1 were significantly decreased in siJ NK intervention group（ P〈0.05）; The expressions of TRAF6 and p-JNK had no significant change,while the expressions of p-AP-1 and NFATc1 were significantly decreased in siA P-1 intervention group（ P〈0.05）.（ 3） Immunofluorescence assay showed that the fluorescent expressions of TRAF6,p-JNK,p-AP-1 and NFATc1 were increased in CD137 stimulation group（ P〈0.05）,and the fluorescent expressions of TRAF6,p-JNK,p-AP-1 and NFATc1 were significantly decreased in CD137 inhibition group（ P〈0.05）. The fluorescent expressions of TRAF6,p-JNK,p-AP-1 and NFATc1 were consistent with the above protein expressions in siT RAF6 intervention group,siJ NK intervention group and siA P-1 intervention group.