温阳化浊通络方抑制FOXP3甲基化促进Treg增殖治疗系统性硬化病的作用机制

Mechanism of Wenyang Huazhuo Tongluo Formula inhibiting FOXP3 methylation to promote Treg cells proliferation in treatment of systemic sclerosis

目的:研究温阳化浊通络方对FOXP3甲基化的影响,探讨其调控Treg细胞增殖治疗系统性硬化病(SSc)的作用机制。方法:收集SSc患者外周血,采用密度梯度离心法分离外周血单核细胞,用试剂盒分选Treg细胞,同时制备温阳化浊通络方含药血清。含药血清处理Treg细胞后,分别采用CCK-8检测Treg细胞增殖,qRTPCR检测FOXP3和IL-10 mRNA表达,Western blot检测FOXP3蛋白表达,ELISA法检测IL-10水平,甲基化特异性PCR检测FOXP3基因启动子区和TSDR区甲基化水平。结果:与空白组比较,温阳化浊通络方低、中、高剂量组显著促进Treg细胞的增殖(P<0.05,P<0.01),促进IL-10 mRNA和FOXP3蛋白表达(P<0.05,P<0.01);温阳化浊通络方高剂量组显著促进FOXP3 mRNA表达(P<0.01),抑制FOXP3 TSDR区甲基化水平(P<0.05);温阳化浊通络方中、高剂量组显著增加IL-10水平(P<0.05,P<0.01)。结论:温阳化浊通络方可促进Treg细胞的增殖,其机制可能与抑制FOXP3甲基化、促进FOXP3表达有关。

Objective:To investigate the effect of Wenyang Huazhuo Tongluo formula(WYHZTLF) on the methylation of FOXP3 and the proliferation of Treg cell,so that to explore the mechanism of WYHZTLF in the treatment of systemic sclerosis(SSc).Methods:Peripheral blood of SSc patients was collected,peripheral blood mononuclear cells were separated by density gradient centrifugation.Treg cells were sorted by sorting kit.At the same time,the WYHZTLF drug-containing serum was prepared.After treating Treg cells with drug-containing serum,CCK-8 was used to detect the proliferation of Treg cells,qRT-PCR was used to detect the mRNA expression of FOXP3 and IL-10,Western blot was used to detect the protein expression of FOXP3,ELISA were used to detect the level of IL-10,methylation-specific PCR was used to detect the methylation levels of FOXP3 gene promoter and TSDR region.Results:Compared with the blank group,the WYHZTLF low,middle and high dose groups significantly promoted the proliferation of Treg cells(P<0.05,P<0.01),and promoted the expression of IL-10 mRNA and FOXP3(P<0.05,P<0.01);the WYHZTLF high dose group significantly promoted the expression of FOXP3 mRNA(P<0.01),and inhibited the methylation level of FOXP3 TSDR region(P<0.05);the WYHZTLF middle and high dose groups significantly increased the level of IL-10(P<0.05,P<0.01).Conclusion:WYHZTLF can promote the proliferation of Treg cells,and its mechanism may be related to inhibiting the methylation of FOXP3 and promoting the expression of FOXP3.