Notch1信号通路通过调控Th17细胞参与实验性自身免疫性甲状腺炎的发生和发展

Notch1 signaling pathway participates in the occurrence and development of experimental autoimmune thyroiditis by regulating Th17 cells

目的探讨Notch1信号通路是否通过调控辅助性T细胞17(Th17)参与实验性自身免疫性甲状腺炎(EAT)的发生和发展。方法将24只小鼠随机平均分为NC组、EAT-A组(给予猪甲状腺免疫球蛋白多点皮下注射)、EAT-B组(猪甲状腺免疫球蛋白多点皮下注射前给予γ分泌酶抑制剂腹腔注射)。HE染色观察甲状腺内淋巴细胞浸润程度;流式细胞术检测小鼠脾脏单核细胞(SMC)悬液中Th17细胞比例;ELISA法检测血清甲状腺球蛋白(TgAb)滴度和SMC培养上清白细胞介素-17A(IL-17A)浓度;实时PCR分析脾细胞中Notch1、Hes1、RORγt和IL-17A mRNA表达水平;Western blotting检测脾脏组织中IL-17A蛋白表达水平。结果EAT-A组和EAT-B组小鼠甲状腺中有不同程度淋巴细胞浸润,与NC组相比血清TgAb滴度显著升高(P<0.01)。EAT-A组和EAT-B组小鼠Notch1、Hes1、RORγt和IL-17A mRNA表达,以及SMC中Th17细胞比例、IL-17A浓度、IL-17A蛋白表达水平均高于NC组(均P<0.01)。EAT-B组上述指标显著低于EAT-A组(均P<0.01)。EAT-A组小鼠SMC中Th17细胞比例与血清TgAb滴度、脾脏Notch1 mRNA及IL-17A蛋白表达水平均呈正相关。结论EAT小鼠中Notch1信号通路的表达上调,阻断此信号通路后小鼠甲状腺炎症程度减轻,同时伴有Th17细胞比例明显降低,提示Notch1信号通路可能通过调控Th17细胞参与EAT的发生和发展。

Objective To explore whether Notch1 signaling pathway participates in the development of experimental autoimmune thyroiditis(EAT)by regulating T helper 17(Th17)cells.Methods A total of 24 mice were randomly divided into NC group,EAT-A group(porcine thyroglobulin treated),and EAT-B group(treated withγ-secretase inhibitor before the injection of porcine thyroglobulin),with n=8 per group.HE staining was performed to identify the pathological changes in the thyroid gland.The proportion of Th17 cells in the spleen mononuclear cells were analyzed by flow cytometry.ELISA was performed to quantify the thyroglobulin antibody(TgAb)titers and interleukin-17A(IL-17A)concentrations.RT-PCR was used to measure the expressions of Notch1,Hes1,RORγt,and IL-17A mRNA.Western blotting was performed to examine IL-17A protein levels.Results In the EAT-A and EAT-B groups,there were different degrees of lymphocyte infiltration in the thyroid;additionally,the serum TgAb titers increased significantly as compared to the NC group(P<0.01).Notch1,Hes1,RORγt,and IL-17A mRNA expression levels,proportion of Th17 cells,IL-17A concentrations,and IL-17A protein levels in the EAT-A and EAT-B groups were higher than those in the NC group(all P<0.01);however,the indexes mentioned above in EAT-B group were significantly reduced when compared to the EAT-A group(all P<0.01).Correlation analyses revealed that proportion of Th17 cells correlated positively with the TgAb titers,Notch1 mRNA expression levels,and IL-17A protein levels in the EAT-A group.Conclusion The expression of Notch1 signaling pathway was upregulated in EAT mice.After blocking this signaling pathway,the degree of thyroid lymphocyte inflammation and the marker of autoimmune damage were reduced,which was accompanied by a decrease in Th17 cell populations.These results suggest that the Notch1 signaling pathway may be involved in the occurrence and development of EAT mice by regulating Th17 cells.