补中益气汤对实验性自身免疫性甲状腺炎大鼠免疫失衡的调节及作用机制研究

Research on the regulation and mechanism of Buzhong Yiqi Decoction for immune imbalance in experimental autoimmune thyroiditis rats

目的基于TGF-β/Smad信号通路探讨补中益气汤调节实验性自身免疫性甲状腺炎(experimental autoimmune thyroiditis,EAT)大鼠模型免疫失衡的作用。方法雌性SD大鼠40只,随机选取10只为空白组,其余大鼠使用高碘饮水联合弗氏佐剂与猪甲状腺球蛋白(pTG)免疫注射建立EAT大鼠模型,成模后随机分为模型组、硒酵母组、补中益气组3组,每组10只,连续灌胃2个月。检测大鼠血清抗甲状腺抗体,观察甲状腺组织病理切片,并通过流式细胞术检测脾脏Th17、Treg比例,RT-PCR和Western blot检测TGF-β/Smad信号通路的相关基因和蛋白表达。结果与空白组比较,模型组甲状腺过氧化物酶抗体(TPOAb)、甲状腺球蛋白抗体(TGAb)水平升高(P<0.05),光镜下模型组甲状腺组织部分滤泡破坏,可见大量淋巴细胞浸润,说明造模成功。且与空白组比较,模型组Smad2、Smad3、Foxp3 mRNA低表达(P<0.05),Foxp3蛋白低表达(P<0.05),同时Th17%升高(P<0.05)。干预后,与模型组比较,硒酵母组与补中益气组TPOAb与TGAb水平下降(P<0.05),且2组甲状腺组织病理损伤均有不同程度改善。与模型组比较,补中益气组TGF-β1、Smad2、Smad3 mRNA高表达,且Smad4蛋白、Foxp3蛋白高表达,Treg%升高(P<0.05)。与硒酵母组比较,补中益气组TGF-β1、Smad2、Smad3、Foxp3 mRNA和Smad4蛋白亦呈现高表达状态,Treg%升高(P<0.05)。与模型组比较,硒酵母组和补中益气组Th17%差异无统计学意义(P>0.05)。结论补中益气汤能够显著改善EAT大鼠的自身免疫性甲状腺炎,其作用机制可能与调节TGF-β/Smad通路,增加Foxp3表达,进而调节Th17/Treg失衡有关。

Objective To discuss the effect of Buzhong Yiqi Decoction on regulating the immune imbalance of experimental autoimmune thyroiditis(EAT)rat model based on TGF-β/Smad signal pathway.Methods Among Forty female SD rats,10 were randomly selected as the blank group,the rest were immunized with high iodine drinking water combined with Freund's adjuvant and porcine thyroglobulin(pTG)to establish EAT rat model.After the model was established,they were randomly divided into three groups(10 rats in each group),including model group,selenium yeast group,Buzhong Yiqi group,and were gavaged continuously for two months.Anti-thyroid antibody in rat serum were detected,the pathological section of thyroid tissue observed,Th17 and Treg ratio of spleen were detected by flow cytometry,the expression of genes and proteins related to TGF-β/Smad signaling pathway were detected by RT-PCR and Western blot.Results Compared with the blank group,the levels of TPOAb and TGAb in the model group were increased(P<0.05).Under the light microscope,part of the thyroid follicles in the model group were destroyed,and a large number of lymphocytes infiltration were seen,indicating that the model was successful.Compared with the blank group,the mRNA expression of Smad2,Smad3 and Foxp3 in the model group was low(P<0.05),the protein expression of Foxp3 was low(P<0.05),and the Th17%increased(P<0.05).After the intervention,compared with the model group,the levels of TPOAb and TGAb in the selenium yeast group and Buzhong Yiqi group decreased(P<0.05),and the pathological damage of thyroid tissue in the two groups were improved variously.Compared with the model group,TGF-β1,Smad2 and Smad3 mRNA in the Buzhong Yiqi group were highly expressed,Smad4 protein and Foxp3 protein were highly expressed,and Treg%increased(P<0.05).Compared with selenium yeast group,the TGF-β1,Smad2,Smad3,Foxp3 mRNA and Smad4 protein in Buzhong Yiqi group also showed high expression,and Treg%increased(P<0.05).However,compared with the model group,there was no significant difference in Th17%between the selenium yeast group and the Buzhong Yiqi group(P>0.05).Conclusion Buzhong Yiqi Decoction can significantly improve autoimmune thyroiditis in EAT rats,and its mechanism may be related to theregulation of TGF-β/Smad pathway,increase of the expression of Foxp3,and then regulation of imbalance of Th17/Treg.