温阳化浊通络方含药血清抑制硬皮病患者Th17细胞增殖的作用机制研究  

Study on the mechanism of Wenyang huazhuo tongluo formula contained serum inhibiting the proliferation of Th17 cells in scleroderma patients

作　　者：郭克磊 李颖利 卞博 韩立 李凯[1,2] 张红 卞华[1,2] GUO Kelei;LI Yingli;BIAN Bo;HAN Li;LI Kai;ZHANG Hong;BIAN Hua(ZHANG Zhongjing School of Chinese Medicine,Nanyang Institute of Technology,Henan Nanyang 473004,China;Henan Key Laboratory of ZHANG Zhongjing Formulae and Herbs for Immunoregulation,Nanyang Institute of Technology,Henan Nanyang 473004,China;Dept.of Rheumatism Immunity,Nanyang Central Hospital,Henan Nanyang 473000,China)

机构地区：[1]南阳理工学院张仲景国医国药学院,河南南阳473004 [2]南阳理工学院河南省张仲景方药与免疫调节重点实验室,河南南阳473004 [3]南阳市中心医院风湿免疫科,河南南阳473000

出　　处：《中国药房》2022年第15期1820-1824,1832,共6页China Pharmacy

基　　金：国家自然科学基金面上项目(No.81774300,No.82074415);河南省科技发展计划项目(No.202102310176)。

摘　　要：目的研究温阳化浊通络方含药血清对维甲酸相关孤核受体(RORγt)表达及甲基化的影响,探讨其调控辅助性T细胞17(Th17)增殖从而治疗硬皮病的作用机制。方法分别灌胃Wistar大鼠15、30、60 g/(kg·d)温阳化浊通络方药液后制备不同剂量的含药血清,同时收集硬皮病患者外周血分选Th17细胞。以空白血清作为空白对照,甲基化转移酶抑制剂地西他滨(DCA)作为阳性对照,用不同剂量含药血清处理Th17细胞后,采用CCK-8法检测Th17细胞的增殖,采用qRT-PCR法检测细胞中RORγt、白细胞介素17(IL-17)mRNA的表达,采用Western blot法检测细胞中RORγt蛋白表达,采用酶联免疫吸附测定法检测细胞上清液中IL-17蛋白表达,采用甲基化特异性PCR法检测细胞中RORγt基因启动子甲基化水平,并采用双荧光素酶法检测细胞中RORγt基因启动子的转录活性。结果与空白对照比较,各剂量温阳化浊通络方含药血清和DCA均可抑制Th17细胞的增殖(P<0.05),降低RORγt、IL-17 mRNA及其蛋白的表达水平,提高RORγt基因启动子甲基化水平,减弱RORγt基因启动子转录活性;其中除含药血清低剂量组Th17 mRNA表达水平和RORγt基因启动子甲基化水平外,其余各组上述指标差异均有统计学意义(P<0.05)。结论温阳化浊通络方可通过促进RORγt基因甲基化,抑制RORγt表达及IL-17分泌,进而抑制Th17细胞的增殖。OBJECTIVE To study the effects of Wenyang huazhuo tongluo formula contained serum on the expression and methylation of retinoic acid related nuclear orphan receptor(RORγt),and to explore the mechanism of its regulation of Th17 cell proliferation in the treatment of scleroderma.METHODS Wistar rats were given 15,30,60 g/(kg·d)Wenyang huazhuo tongluo formula intragastrically to prepare different doses of drug-contained serum,and peripheral blood of scleroderma patients were collected to sort Th17 cells.Using blank serum as blank control,methyltransferase inhibitor decitabine(DCA)as positive control,Th17 cells were treated with different concentrations of drug-contained serum,and then the proliferation of Th17 cells was detected by CCK-8;mRNA expressions of RORγt and IL-17 were detected by qRT-PCR,and the protein expression of RORγt was detected by Western blot assay;the protein expression of IL-17 in the cell supernatant was detected by ELISA,and the methylation level of RORγt gene promoter was detected by methylation-specific PCR.The transcriptional activity of RORγt gene promoter was detected by dual-luciferase assay.RESULTS Compared with blank control,Wenyang huazhuo tongluo formula contained serum and DCA could inhibit the proliferation of Th17 cells(P<0.05),reduced the mRNA and protein expressions of RORγt and IL-17,enhanced the methylation level of RORγt gene promoter and attenuated the transcription activity of RORγt gene promoter.Except for mRNA expression of Th17 and the methylation level of RORγt gene promoter in drug-contained serum low-dose group,there were statistical significance in above indexes of other groups(P<0.05).CONCLUSIONS Wenyang huazhuo tongluo formula can promote the methylation level of RORγt gene,and inhibit the expression of RORγt and the secretion of IL-17,so as to inhibit the proliferation of Th17 cells.

关 键 词：硬皮病 温阳化浊通络方 含药血清 维甲酸相关孤核受体 甲基化 Th17细胞 

分 类 号：R966[医药卫生—药理学] R285[医药卫生—药学]