CXC趋化因子受体3对甲状腺细胞自噬与炎症的影响及机制

Effect and mechanism of CXC chemokine receptor 3 on autophagy and inflammation of thyroid cells and its mechanisms

目的探讨CXC趋化因子受体3(CXCR3)对干扰素-γ(IFN-γ)诱导的甲状腺滤泡上皮细胞(Nthy-ori3-1)自噬及炎症的影响及相关机制。方法Nthy-ori3-1细胞分为正常组(不做任何处理)、IFN-γ组(500 U/mLIFN-γ处理24 h)、si-NC组[转染小干扰RNA(siRNA)]、si-CXCR3组(转染CXCR3 siRNA)、si-CXCR3+si-NC组(转染CXCR3 siRNA+转染siRNA)、si-CXCR3+si-Beclin1组(转染CXCR3 siRNA+转染Beclin1 siRNA)和si-CXCR3+3MA组(转染CXCR3 siRNA+3-MA自噬抑制剂)。实时荧光定量PCR检测各组CXCR3、CXC趋化因子配体9(CXCL9)的mRNA表达情况,免疫印迹法检测各组CXCR3、CXCL9、微管相关蛋白1轻链3-I(LC3I)、LC3II、囊泡转运蛋白34(Vps34)及Beclin1蛋白表达情况,免疫荧光染色观察各组细胞内自噬标志物LC3斑点数;酶联免疫吸附法试剂盒检测各组白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)和IL-1β水平;免疫共沉淀检测CXCR3与Beclin1相互作用。结果与正常组相比,IFN-γ组CXCR3、CXCL9表达水平显著升高(P<0.01);与si-NC组相比,si-CXCR3组Beclin1和Vps34表达及LC3II与LC3I比值(LC3II/LC3I)显著升高(P<0.01),LC3斑点数显著增加(P<0.01)。同时,与si-NC组相比,si-CXCR3组炎症因子IL-6、TNF-α及IL-1β的水平显著降低(P<0.01)。免疫共沉淀结果表明,CXCR3与Beclin1存在相互作用。与si-CXCR3+si-NC组相比,si-CXCR3+si-Beclin1组及si-CXCR3+3-MA组细胞的Beclin1、Vps34表达及LC3II/LC3I显著降低(P<0.01),LC3斑点数明显减弱(P<0.05);IL-6、TNF-α和IL-1β显著升高(P<0.05)。结论CXCR3可抑制Beclin1/Vps34信号通路,通过降低自噬进而促进甲状腺滤泡上皮细胞炎症。

Objective To investigate the effect of CXC chemokine receptor 3(CXCR3)on interferon-γ(IFN-γ)-induced autophagy and inflammation of thyroid cells and the underlying mechanisms.Methods Altogether 500 U/mL of IFN-γ was administered to Nthy-ori3-1 cells for 24 hours toestablish the injury modelof thyroid follicular epithelial cells.The cells were divided as follows:control group(non-transfected Nthy-ori3-1 cells),IFN-γ group(IFN-γtreated Nthy-ori3-1 cells),si-NC group(negative control small interfering RNA transfected cells),si-CXCR3 group(CXCR3 siRNA transfected cells),si-CXCR3+si-NC group(CXCR3 siRNA and negative control siRNA co-transfected cells),si-CXCR3+si-Beclin1 group(CXCR3 siRNA and Beclin1 siRNA co-transfected cells)and si-CXCR3+3-MA group(CXCR3 siRNA transfected and 3-MA treated cells).The mRNA expression of CXCR3 and C-X-C motifligand 9(CXCL9)were detected by real-time quantitative PCR(RT-qPCR).The protein expression of CXCR3,CXCL9,microtubule-associated protein 1 light chain 3(LC3I),LC3II,vacuolar protein sorting 34(Vps34)and Beclin1 was detected using Western blot.The number of LC3 puncta in Nthy-ori3-1 cells was assessed using immunofluorescence staining inflammatory cytokines interleukin-6(IL-6),tumor necrosis factor α(TNF-α)and interleukin-1β(IL-1β)were detected using ELISA.The interaction between CXCR3 and beclin1 was identified by co-immunoprecipitation.Results CXCR3 and CXCL9 expression was significantly increased in IFN-γgroup when compared with the control group(P<0.01).Compared with the si-NC group,the expression of beclin1,Vps34,LC3 II/LC3 I and LC3 puncta markedly increased in the si-CXCR3 group(P<0.05).Furthermore,compared with the si-NC group,IL-6,TNF-α and IL-1β levels were significantly decreased in the si-CXCR3 group(P<0.01).The co-immunoprecipitation assay reveled that CXCR3 interacted with beclin1.Compared with the si-CXCR3+si-NC group,the expression of beclin1,Vps34,LC3 II/LC3 I and LC3 puncta was significantly decreased,while levels of IL-6,TNF-α and IL-1β were significantly increased in the si-CXCR3+si-beclin1 group and si-CXCR3+3-MA group(P<0.01).Conclusions CXCR3 inhibited the Beclin1/Vps34 signaling pathway,reducing autophagy and promoting inflammation of thyroid follicular epithelial cells.