文章摘要
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
投稿时间:2023-07-30  
DOI:10.3969/j.issn.1000-0399.2024.05.002
中文关键词: 甲状腺炎  CXC趋化因子受体3  Beclin1  囊泡转运蛋白34  自噬
英文关键词: Hashimoto’s thyroiditis  CXC chemokine receptor 3  Beclin1  Vacuolar protein sorting 34  Autophagy
基金项目:河北省医学科学研究课题计划(编号:20210338)
作者单位E-mail
赵连春 050091 河北石家庄 河北医科大学附属以岭医院检验科  
牛丽霞 050091 河北石家庄 河北医科大学附属以岭医院检验科  
赵振军 050091 河北石家庄 河北医科大学附属以岭医院检验科  
侯瞻 050091 河北石家庄 河北省人民医院核医学科  
陈芳 050091 河北石家庄 河北省人民医院核医学科  
贾乾 050091 河北石家庄 河北省人民医院体检中心  
刘光霞 050091 河北石家庄 河北省人民医院核医学科 liu_gxdr@163.com 
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中文摘要:
      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 ,siCXCR3+ 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。
英文摘要:
      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.
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