温阳化浊通络方调控DNA甲基转移酶对硬皮病患者Treg细胞增殖的影响

Effect of Wenyang Huazhuo Tongluo Formula regulating DNA pethyltransferases on the proliferation of Treg Cells in systemic sclerosis patients

目的:研究温阳化浊通络方(WYHZTLF)对DNA甲基转移酶(DNA methyltransferases,DNMTs)及叉头翼状螺旋转录因子3(forkhead box protein 3,FOXP3)表达的影响,探讨其调控硬皮病患者调节性T细胞(regulatory T cell,Treg)细胞增殖的作用机制。方法:用Wistar雌性大鼠制备温阳化浊通络方含药血清。收集硬皮病患者外周血,分选Treg细胞,用含药血清处理后,通过CCK-8法检测含药血清对Treg细胞增殖的影响,免疫荧光法检测FOXP3蛋白表达,qRT-PCR法和Western blot法检测DNMT1、DNMT3A和DNMT3B mRNA及蛋白表达。结果:与空白血清组相比,低、中、高剂量含药血清组均可促进Treg细胞的增殖,中、高剂量组促进作用更加显著(P<0.05,P<0.01);低、中、高剂量含药血清组均可促进FOXP3蛋白表达(P<0.01);中、高剂量含药血清组明显抑制DNMT1、DNMT3A和DNMT3B mRNA表达(P<0.05);高剂量含药血清组显著抑制DNMT1蛋白表达水平(P<0.05),低、中、高剂量组显著抑制DNMT3A和DNMT3B蛋白表达(P<0.01,P<0.01)。结论:温阳化浊通络方促进硬皮病患者Treg细胞增殖可能与抑制DNMT1、DNMT3A和DNMT3B表达从而促进FOXP3表达有关。

OBJECTIVE To investigate the effects of Wenyang Huazhuo Tongluo Formula(WYHZTLF)on the expression of DNA methyltransferase and FOXP3,and explore its mechanism of regulating the proliferation of Treg cells in patients with systemic sclerosis(SSc).METHODS Wistar female rats were used to prepare WYHZTLF-containing serum.The peripheral blood of SSc patients was collected,and Treg cells were obtained by Treg sorting kit.After treatment with drug containing serum,the effects of medicated serum on Treg cells proliferation were detected by CCK-8,FOXP3 protein expression was detected by immunofluorescence,and the mRNA and protein expression of DNMT1,DNMT3 A and DNMT3 B were detected by qRT-PCR and Western blot.RESULTS Compared with the blank serum group,the low,medium,and high-dose WYHZTLF-containing serum groups could promote the proliferation of Treg cells,and the promotion effect of the medium and high-dose groups was more significant(P<0.05,P<0.01);each WYHZTLF-containing serum group could promote the expression of FOXP3 protein(P<0.01);the medium and high-dose groups significantly inhibited the expression of DNMT1,DNMT3 A and DNMT3 B mRNA(P<0.05);high-dose group significantly inhibited DNMT1 protein(P<0.05),and low,medium and high-doses groups significantly reduced the protein expression of DNMT3 A and DNMT3 B(P<0.01,P<0.01).CONCLUSION Wenyang Huazhuo Tongluo Formula can promote the proliferation of Treg cells from SSc patients,and the mechanism may be related to the inhibition of the expression of DNMT1,DNMT3 A and DNMT3 B to promote FOXP3 expression.