补中益气汤通过TGF-β/Smad信号通路对自身免疫性甲状腺炎大鼠Treg细胞的影响

Effect of Buzhong Yiqi Decoction on Treg cells in rats with autoimmune thyroiditis through TGF-β/Smad signaling pathway

探讨补中益气汤通过转化生长因子-β(TGF-β)/母对抗DPP同源物(Smad)信号通路调节实验性自身免疫性甲状腺炎(EAT)大鼠调节性T细胞(regulatory T cell,Treg)的作用。雌性SD大鼠使用高碘饮水联合弗氏佐剂与猪甲状腺球蛋白(pTG)免疫注射的方法建立EAT大鼠模型,检测血清甲状腺过氧化物酶抗体(TPOAb)、甲状腺球蛋白抗体(TGAb)水平和苏木素-伊红(HE)染色病理切片。模型成功后使用免疫磁珠法提取大鼠脾脏Treg,并用流式细胞术进行鉴定。将提取的Treg分为空白组、补中益气汤组、TGF-β组、拮抗剂(LY3200882)组、拮抗剂(LY3200882)+补中益气汤组,给药干预后,cell counting kit(CCK)-8实验检测细胞活力;蛋白免疫印迹(Western blot)法和实时荧光定量PCR(RT-qPCR)检测TGF-β/Smad信号通路相关蛋白和基因表达情况。结果显示,与空白组大鼠比较,模型大鼠TPOAb、TGAb水平显著升高,HE染色提示模型大鼠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。Treg体外给药后,CCK-8结果显示补中益气汤含药血清质量分数在20%以下促进细胞增殖。与空白血清组比较,补中益气汤含药血清组可显著增加TGF-β1、叉头框蛋白P3(FoxP3)、Smad2、Smad4蛋白表达,p-Smad2、p-Smad3、Smad3较空白血清组表达增加但差异无统计学意义;拮抗剂组加入补中益气汤含药血清后,与拮抗剂组比较,p-Smad2、Smad2、p-Smad3、Smad3、Smad4、Smad7表达无明显增加或减少。RT-qPCR结果显示,与空白血清组比较,补中益气汤组中TGF-β1、FoxP3、Smad2、Smad3、Smad4、Smad7 mRNA表达升高或降低趋势与TGF-β组趋势一致,但差异无统计学意义;拮抗剂组加入补中益气汤含药血清后,与拮抗剂组比较,TGF-β1、FoxP3、Smad2、Smad3、Smad4、Smad7 mRNA差异无统计学意义。综上,补中益气汤可通过促进TGF-β1的分泌,调节TGF-β/Smad信号通路中关键信号分子TGF-β1、Smad2及Smad4的表达促进Treg稳定及活性,进而影响辅助性T细胞17(Th17)/Treg细胞免疫平衡,抑制EAT大鼠的炎症反应。

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-βgroup,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-βgroup,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.