补中益气汤调控miR-146-5p介导TLR4/MyD88/TRAF6通路干预自身免疫性甲状腺炎免疫调控的机制研究OA

Objective To explore the intervention mechanism of Buzhong Yiqi Decoction on immune regulation in mice with autoimmune thyroiditis(AIT)based on the miR-146-5p-mediated Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/tumor necrosis factor receptor-associated factor 6(TRAF6)signaling pathway.Methods Forty-eight 8-week-old NOD.H-2h4 mice were randomly divided into six groups based on the random number table:normal,model,low-dose,medium-dose,high-dose,and selenium yeast tablets with eight mice in each group.The normal group received distilled water ad libitum,whereas the other groups were administered water containing 0.05%sodium iodide free to drink from 8 weeks of age for eight consecutive weeks to induce AIT models.After the model was successfully established,the low-,medium-,and high-dose Buzhong Yiqi Decoction groups received Buzhong Yiqi Decoction at doses of 4.10,8.19,and 16.38 g/kg,respectively,via gavage.The selenium yeast tablets group was administered 0.026 mg/kg selenium yeast tablets;the normal and model groups were administered an equal amount of distilled water.Gavage was performed for 6 days,and the mice were rested for 1 day,repeated for 8 consecutive weeks.Hematoxylin and eosin staining was used to observe the pathological morphology of thyroid tissues in mice.An enzyme-linked immunosorbent assay was used to detect the level of thyroglobulin antibody(TgAb),thyroid peroxidase antibody(TPOAb),interleukin-6(IL-6),interleukin-10(IL-10),interleukin-17A(IL-17A),and tumor necrosis factor-α(TNF-α)in mice.RT-PCR was used to detect miR-146-5p expression in thyroid tissues.Flow cytometry was used to detect the distribution of splenic helper T cell 17(Th17)and regulatory T cell(Treg)subsets,and western blotting was used to detect TLR4,MyD88,TRAF6,p38 mitogen-activated protein kinase(p38MAPK),p-p38MAPK,c-Jun N-terminal kinase(JNK),and p-JNK protein levels in thyroid tissues of mice.Results Compared with the normal group,the thyroid follicular epithelial cell structure was disrupted in the model group,with lymphocytic infiltration and elevated TgAb and TPOAb levels(P<0.05).Additionally,IL-6,IL-17A,and TNF-α levels were increased(P<0.01),whereas IL-10 level was decreased(P<0.05).MiR-146-5p expression was elevated(P<0.01),and the proportion of Th17 cells and the Th17/Treg ratio were increased(P<0.01).TLR4,MyD88,TRAF6,p-JNK/JNK,p-p38MAPK/p38MAPK,and p-p38MAPK expression levels in thyroid tissues were elevated(P<0.05).Compared with the model group,the structure of epithelial cells was improved to varying degrees in the traditional Chinese medicine and Western medicine groups,with alleviated lymphocytic infiltration;TPOAb level was decreased in the high-dose Buzhong Yiqi Decoction and selenized yeast tablet groups(P<0.05),and TgAb level was decreased in each dose group of Buzhong Yiqi Decoction and the selenized yeast tablet group(P<0.05).IL-6,IL-17A,and TNF-α levels were reduced(P<0.01),whereas the IL-10 level was increased in the medium-and high-dose Buzhong Yiqi Decoction groups and the selenized yeast tablet group(P<0.05).In the low-dose Buzhong Yiqi Decoction group,the miR-146-5p level was increased(P<0.01).In contrast,it was relatively decreased in the medium-dose Buzhong Yiqi Decoction,high-dose Buzhong Yiqi Decoction,and selenized yeast tablet groups(P<0.05).The proportion of Treg cells was increased in the high-dose Buzhong Yiqi Decoction group and the selenized yeast tablet group(P<0.05),whereas the proportion of Th17 cells and the Th17/Treg ratio were decreased in each dose group of Buzhong Yiqi Decoction and the selenized yeast tablet group(P<0.05).TLR4,TRAF6,p-p38MAPK/p38MAPK,and p-p38MAPK expression levels in thyroid tissues were also decreased in each dose group of Buzhong Yiqi Decoction and selenium yeast tablet group(P<0.05);MyD88 and p-JNK/JNK expression were additionally reduced in the medium-and high-dose Buzhong Yiqi Decoction groups and selenized yeast tablet groups(P<0.05).Conclusion Buzhong Yiqi Decoction may regulate the immune response of mice with AIT and reduce thyroid lymphocyte infiltration by intervening in the miR-146-5p-mediated TLR4/MyD88/TRAF6 pathway.