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

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)...

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

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 (nontransfected Nthy-ori3-1 cells), IFN-γ group (IFN-γ treated 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 cotransfected 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-γ group 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 coimmunoprecipitation 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.