决银方通过降低WIF-1基因甲基化改善寻常型银屑病血热证

Jueyin Decoction Alleviate Psoriasis Vulgaris with Blood-heat Syndrome by Hypomethylation of Wnt Inhibitory Factor-1 Gene

目的探究决银方治疗寻常型银屑病血热证的分子机制。方法将患者分为非银屑病受试者组、寻常型银屑病非血热证组、寻常型银屑病血热证组、决银方治疗愈显组和决银方治疗无效组。分别于局部浸润麻醉下运用皮肤病理活检术切取1.0 cm×0.5 cm皮损组织进行HE染色、实时荧光定量聚合酶链式反应(RT-PCR)、蛋白质印迹法(Western blotting)、亚硫酸盐扩增子测序、甲基化特异性PCR等技术检测皮损区组织病理改变、Wnt抑制因子-1(WIF-1)基因表达及甲基化水平。结果决银方治疗后,与决银方治疗无效组比,决银方治疗愈显组患者皮损组织病理切片明显改善;皮损区WIF-1信使核糖核酸(mRNA)水平明显升高(P<0.01);与寻常型银屑病血热证患者比较,决银方治疗愈显组患者皮损区WIF-1蛋白表达明显升高。亚硫酸盐扩增子测序结果显示决银方治疗愈显组患者WIF-1基因CpG岛位点甲基化水平明显降低(P<0.05)。细胞实验中,转染WIF-1短发夹核糖核酸(shRNA)后,人永生化角质形成细胞(HaCaT)中WIF-1蛋白水平降低;去甲基化药物处理细胞后,其蛋白水平升高;转染WIF-1 shRNA及用去甲基化药物处理细胞后,决银方治疗愈显组患者血清干预的细胞中WIF-1蛋白水平升高,基因甲基化程度降低。结论决银方治疗寻常型银屑病血热证患者通过降低WIF-1基因启动子区甲基化水平,提高其基因表达水平来实现。

Objective In order to explore the molecular mechanism of Jueyin decoction in treating blood-heat syndrome of psoriasis vulgaris.Methods Patients were divided into non-psoriatic group,non-blood-heat syndrome of psoriasis vulgaris group,blood-heat syndrome of psoriasis vulgaris group,Jueyin decoction effective group and Jueyin decoction invalid group.Skin pathological biopsy was used to extract 1.0 cm×0.5 cm skin lesions from five groups of patients under local anesthesia.HE staining,immunohistochemistry,real-time fluorescence quantitative polymerase chain reaction(RT-PCR),Western blotting,sulfite amplicon sequencing and methylation specific PCR were performed to detect histopathological changes,Wnt inhibitory factor-1(WIF-1)gene expression and its methylation level in the skin lesions.Results Compared with patients in non-psoriatic group,the cytosine-guanine(CpG)island site of the WIF-1 gene promoter region in the skin lesion area of sufferers in psoriasis vulgaris blood-heat syndrome group showed a hyper ethylated state,while the level of WIF-1 protein and mRNA was declined obviously(P<0.01).After treated with Jueyin decoction,compared with Jueyin decoction invalid group,the pathological sections of the skin lesions of the patients in the Jueyin decoction effective group were improved significantly;the level of WIF-1 mRNA in the skin lesion area was increased significantly(P<0.01).Compared with patients in the blood-heat syndrome group of psoriasis,the expression of WIF-1 protein in the skin lesions of patients in Jueyin treatment effective group was increased significantly.The sulfite amplicon sequencing results showed that the methylation level of the CpG island site in WIF-1 gene in the Jueyin treatment effective group was significantly reduced(P<0.05).In cell experiments,after transfection of WIF-1 short hairpin RNA(shRNA),the level of WIF-1 protein in HaCaT cells decreased.After the cells were treated with demethylation drugs,the protein level increased.WIF-1 shRNA was transfected and demethylation drugs were used after the cells were treated,the level of WIF-1 protein in the serum-intervened cells of the patients in the Jueyin decoction group was increased,and the degree of gene methylation decreased.Conclusion Jueyin decoction can alleviate blood-heat syndrome of psoriasis vulgaris by reducing the methylation level of WIF-1 gene promoter region and increasing its gene expression level.