| Purpose：The aim is to explore potential immunological mechanism of Yiqi Huatan HuoxueFang on iodine excess-induced autoimmune thyroiditis (AIT) by regulating of Th17cell differentiation and interaction of Th17,Th1,Th2,Treg cells.Material and method：A total of80femal healthy8-wk old NOD.H-2h4mice (weight of22g) wererandomly allocated into the normal group (NG,n=20), the test group (TG, n=20),Yiqi Huatan Huoxue Fang low-dose group(ZY1, n=20) and high-dose group(ZY3, n=20).Mice in the NG were fed with sterile water, and the others received0.05%NaIfor8weeks to induce murine model of AIT. Then, mice in both low-dose andhigh-dose group were administered Yiqi Huatan Huoxue Fang orally once a day,20.8g/kg·d and62.4g/kg·d,respectively, and one day rest every six days. Allmice were anesthetized and sacrificed8weeks later. The serum TT3and TT4weremeasured by RIA, the serum TSH by IRMA, the morphology of thyroid cells wasobserved under optical microscopy, the protein and mRNAs expressions of IL-17,IL-23,IL-6,TGF-β,INF-γand IL-4were detected by immunohistochemical methodand RT-PCR respectively, the expressions of RORγt,ROR α,STAT3and Foxp3wereanalyzed by western blot, the percentage of Th17cells in splenic mononuclearcells were detected by flow cytometry.Results：1. There is no obvious statistical differences in serum levels of TSH, TT3,and TT4of all groups（P＞0.05）.2. The number of intrathyroidal lymphocyte infiltration in the TG increasedunder light microscope with H&E staining. And the incidence of AIT in the NG, theTG, the low-dose and the high-dose group is0/10，10/10，1/10and3/10, respectively. The incidence of AIT in the TG is significantly higher than those in NG（P＜0.01）;Compared to the TG, that of both low-dose and high-dose groups were lower（P＜0.01),but there is no statistical differences between them.3. The positive staining in the TG was localized in the cell membrane and/orcytoplasm of infiltrated inflammatory lymphocytes and colored in deep brown,while that in the NG were light brown. The IOD of IL-17,IL-23,IL-6and TGF-βin the TG remarkably increased compared with the NG（P＜0.01), and the IODof them in both low-dose and high-dose groups were obviously decreased comparedto the TG（P＜0.01，P＜0.05).And the IOD of IL-17,IL-23,IL-6and TGF-βin thehigh-dose group were clearly higher than that in low-dose group（P＜0.05）.4. The expressions of IL-6,TGF-β,IL-17and IL-23mRNA in the TG wereprominently up-regulated（P＜0.01).Compared to the TG, the expressions of IL-6,TGF-β,IL-17,IL-23mRNA in the low-dose and the IL-17and IL-23mRNA in the high-dose were strikingly down-regulated（P＜0.01), whereas, the IL-6and TGF-βmRNAhad no statistics changes（P＞0.05）. And the expressions of IL-6,TGF-β,IL-17and IL-23mRNA in the high-dose were statistically up-regulated（P＜0.05）.5. Compared with the NG, the expressions of RORγt,RORαand STAT3wereelevated significantly（P＜0.01); and compared with the TG, the RORα and STAT3in both low-dose and high-dose groups, together with the RORγt in low-dose wereobviously declined（P＜0.01),while the RORγt in high-dose decreased（P＜0.05）;and compared with the low-dose, the expressions of RORγt, RORαand STAT3wereall elevated（P＜0.05）.6. The percentage of Th17cells in the splenocyte population was significantlyhigher in the TG compared to the NG by Flow cytometry (P＜0.01). That of in bothlow-dose and high-dose were remarkably lower while compared to the TG（P＜0.01，P＜0.05）,and the percentage of Th17cell in the high-dose was obviously higherthan that in the low-dose（P＜0.05）.7. The positive staining in the TG was localized in the cell membrane and/orcytoplasm of infiltrated inflammatory lymphocytes cells and colored in light brown, while that in the NG were deep brown. The IOD of INF-γand IL-4in theTG remarkably decreased compared with the NG（P＜0.01), and the IOD of them inboth low-dose and high-dose groups were obviously increased compared to the TG（P＜0.01，P＜0.05).And the IOD of INF-γand IL-4in the high-dose group wereclearly lower than that in low-dose group（P＜0.05）.8. The expressions of INF-γand IL-4mRNA in the TG were prominentlydown-regulated（P＜0.01).Compared to the TG, the expressions of INF-γand IL-4mRNA in both low-dose and high-dose were strikingly up-regulated（P＜0.01，P＜0.05). And compared to the low-dose,the expressions of INF-γand IL-4mRNA inthe high-dose were statistically down-regulated（P＜0.01）.9. Compared with the NG, the expression of Foxp3in the TG was declinedsignificantly（P＜0.01); And compared with the TG, the Foxp3in both low-dose andhigh-dose groups were obviously elevated（P﹤0.01),while the RORγt in high-dosedecreased（P＜0.05）; And compared with the low-dose, the expression of Foxp3wasdecreased significantly（P＜0.05）.Conclusion：1. Iodine excess may trigger autoimmune thyroiditis.2. Yiqi Huatan Huoxue Fang could alleviate inflammatory response by reducingthe intrathyroidal lymphocyte infiltration of AIT mice.3. Th17cells may promote inflammatory reaction in the development of iodine-excess induced AIT.4. The therapeutic mechanism of Yiqi Huatan Huoxue Fang on AIT may be relatedto depress the inflammatory axis of IL-23/IL-17and down-regulate the percentageof Th17cell by down-regulating the expressions of IL-6,TGF-β,RORγt,RORαandSTAT3.5. The therapeutic mechanism of Yiqi Huatan Huoxue Fang on AIT may be relatedto up-regulate the expressions of INF-γ,IL-4and Foxp3in order to enhance theimmunosuppression function of Th1，Th2and Treg cells towards Th17cell.
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