To investigate the effect of Buzhong Yiqi Decoction on regulatory T cells(Treg) in experimental rats with autoimmune thyroiditis(EAT) through the transforming growth factor-β(TGF-β)/Smad signaling pathway. Female SD rats were immunized with iodine-rich drinking water combined with Freund's adjuvant and porcine thyroglobulin(pTG) to establish the EAT model of rats, and the levels of serum thyroperoxidase antibody(TPOAb) and thyroglobulin antibody(TGAb) were detected. Pathological sections by hematoxylin-eosin(HE) staining were observed. Treg in the rats' spleen were extracted by immunomagnetic beads after the successful modeling and identified by flow cytometry. The extracted Treg were divided into blank group, Buzhong Yiqi Decoction group, TGF-β group, antagonist(LY3200882), and antagonist(LY3200882)+Buzhong Yiqi Decoction group. After the intervention, the cell counting kit-8(CCK-8) experiment was conducted to detect cell viability. Western blot and quantitative real-time PCR(RT-qPCR) were used to detect the expression of TGF-β/Smad signaling pathway-related proteins and genes. The results showed that the levels of TPOAb and TGAb increased in the rats in the model group compared to the rats in the blank group. HE staining showed that part of the follicles in the thyroid tissue of the rats in the model group were destroyed, and a large number of lymphocytes were infiltrated, indicating that the modeling was successful. After Treg were administered in vitro, CCK-8 results showed that the serum concentration of Buzhong Yiqi Decoction was below 40% to promote cell proliferation. The Buzhong Yiqi Decoction-containing serum group could increase the protein expression of TGF-β1, FoxP3, Smad2, and Smad4 compared with the blank serum group, while the expression of p-Smad2, p-Smad3, and Smad3 increased compared with the blank serum group, but the difference was not statistically significant. Compared with the antagonist group, the protein expressions of p-Smad2, Smad2, p-Smad3, Smad3, Smad4, and Smad7 did not significantly increase or decrease in the antagonist group after adding Buzhong Yiqi Decoction-containing serum. RT-qPCR showed that compared with the blank serum group, the mRNA expression of TGF-β1, FoxP3, Smad2, Smad3, Smad4, and Smad7 in the Buzhong Yiqi Decoction group increased or decreased in the same trend as that in the TGF-β group, but there was no statistical significance. After Buzhong Yiqi Decoction-containing serum was added to the antagonist group, the mRNA levels of TGF-β1, FoxP3, Smad2, Smad3, Smad4, and Smad7 were not statistically significant. In conclusion, Buzhong Yiqi Decoction could promote the stability and activity of Treg cells by promoting the secretion of TGF-β1 and regulating the expression of key signaling molecules TGF-β1, Smad2, and Smad4 in the TGF-β/Smad signaling pathway, thus affecting the immune balance of Th17/Treg and inhibiting the inflammatory response of rats with EAT.
Keywords: Buzhong Yiqi Decoction; Hashimoto′s thyroiditis; TGF-β/Smad signaling pathway; regulatory T cell(Treg).