温化蠲痹方对类风湿关节炎患者外周血单个核细胞中微小RNA-146a调控DNA甲基化的作用研究  

Effect of Wenhua Juanbi Formula on the regulation of microRNA-146a-mediated DNA methylation in peripheral blood mononuclear cells of patients with rheumatoid arthritis

作　　者：丁旭阳 刘喜德[2] 刘敏[2] 叶丽红[2] 蔡龙[3] 王珊珊 Ding Xuyang;LIU Xide;LIU Min;YE Lihong;CAI Long;WANG Shanshan(Second Clinical Medical College,Zhejiang Chinese Medical University,Hangzhou,Zhejiang 310053,China;Department of TCM,Zhejiang Provincial Hospital of Integrated Traditional and Western Medicine,Hangzhou,Zhejiang 310003,China)

机构地区：[1]浙江中医药大学第二临床医学院,杭州310053 [2]浙江省中西医结合医院中医科,杭州310003 [3]浙江省中西医结合医院临床医学检验实验中心,杭州310003

出　　处：《浙江中西医结合杂志》2025年第4期313-317,共5页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省自然科学基金(No.LY21H270001、LQ23H270014);浙江省中医药科技计划项目(No.2020ZB180);杭州市卫生科技计划重大项目(No.Z20200026);国家中医药综合改革示范区中医药科技共建项目(No.GZY-KJS-ZJ-2025-085)。

摘　　要：目的探讨温化蠲痹方对活动期类风湿关节炎(RA)患者外周血单个核细胞(PBMCs)中微小RNA-146a(miRNA-146a)和DNA甲基化转移酶(DNMTs)表达的影响。方法选取9例活动期RA患者和9名健康对照者,分别按随机数字表法分为三组。RA患者:RA组、RA转染反义miRNA-146a组、RA加中药组,各3例。健康对照者:正常组、正常转染miRNA-146a组、正常转染miRNA-146a加中药组,各3名。SD大鼠10只,按体质量灌胃温化蠲痹方浓缩液[22.9 g/(kg·d)],制备温化蠲痹方含药血清。提取各组研究对象PBMCs并进行体外培养、转染及温化蠲痹方含药血清干预。使用Trizol法提取各组PBMCs总RNA,通过荧光定量PCR检测PBMCs中miRNA-146a及DNMTs[DNA甲基化转移酶1(DNMT1)、DNA甲基化转移酶3a(DNMT3a)、DNA甲基化转移酶3b(DNMT3b)]的表达情况。结果与正常组比较,RA组和正常转染miRNA-146a组PBMCs中miRNA-146a表达显著升高[(1.44±0.49)、(0.81±0.29)比(0.24±0.19),P<0.05],DNMT1、DNMT3a和DNMT3b表达显著降低[(1.77±0.99)、(1.98±0.76)比(2.75±0.12),(1.58±0.95)、(1.90±0.56)比(2.48±0.87),(1.65±0.84)、(1.95±0.82)比(2.64±0.79),P<0.05]。与RA组比较,RA转染反义miRNA-146a组、RA加中药组PBMCs中miRNA-146a表达明显下降[(0.31±0.16)、(0.83±0.61)比(1.44±0.49),P<0.05],DNMT1、DNMT3a和DNMT3b表达显著升高[(2.46±0.91)、(2.32±0.85)比(1.77±0.99),(2.17±0.59)、(2.10±0.71)比(1.58±0.95),(2.28±0.52)、(2.23±0.62)比(1.65±0.84),P<0.05]。与正常转染miRNA-146a组比较,正常转染miRNA-146a加中药组PBMCs中miRNA-146a表达显著降低[(0.34±0.17)比(0.81±0.29),P<0.05],DNMT1、DNMT3a和DNMT3b表达显著升高[(2.31±0.63)比(1.98±0.76)、(2.17±0.52)比(1.90±0.56)、(2.24±0.42)比(1.95±0.82),P<0.05]。结论温化蠲痹方可能通过下调miRNA-146a、上调DNMTs的表达,恢复正常的DNA甲基化状态,抑制免疫和炎症异常反应,从而改善RA的临床症状及病理变化。Objective To investigate the effect of Wenhua Juanbi Formula on the expression of microRNA-146a(miRNA-146a)and DNA methyltransferases(DNMTs)in peripheral blood mononuclear cells(PBMCs)in patients with active rheumatoid arthritis(RA).Methods Nine patients with active RA and nine healthy controls were selected and randomly divided into three groups according to a random number table.The RA patients were divided into the following groups(n=3 per group):RA group,RA group transfected with antisense miRNA-146a,RA group treated with traditional Chinese medicine(TCM).Similarly,the healthy controls were assigned to three groups(n=3 per group):normal group,normal group transfected with miRNA-146a,normal group transfected with miRNA-146a plus TCM.Ten SD rats were selected and administered the concentrated liquid of Wenhua Juanbi Formula by gavage at a dose of 22.9 g/(kg·d)to prepare the drug-containing serum.PBMCs were isolated from each group of subjects and cultured in vitro,transfected,and intervened with the drug-containing serum of Wenhua Juanbi Formula.Total RNA was extracted from PBMCs of each group using the Trizol method,and the expression levels of miRNA-146a and DNMTs(DNMT1,DNMT3a,DNMT3b)in PBMCs were detected using quantitative fluorescence PCR.Results Compared to the normal group,the expression levels of miRNA-146a were significantly increased in the PBMCs of the RA group and the normal group transfected with miRNA-146a[(1.44±0.49),(0.81±0.29)vs.(0.24±0.19),P<0.05)].The expression levels of DNMT1,DNMT3a,and DNMT3b were significantly reduced[(1.77±0.99),(1.98±0.76)vs.(2.75±0.12);(1.58±0.95),(1.90±0.56)vs.(2.48±0.87);(1.65±0.84),(1.95±0.82)vs.(2.64±0.79);P<0.05)].Compared to the RA group,the expression levels of miRNA-146a in PBMCs of the RA group transfected with antisense miRNA-146a and the RA group treated with TCM were significantly decreased[(0.31±0.16),(0.83±0.61)vs.(1.44±0.49),P<0.05)].The expression levels of DNMT1,DNMT3a,and DNMT3b were significantly increased[(2.46±0.91),(2.32±0.85)vs.

关 键 词：类风湿关节炎 温化蠲痹方 微小RNA-146a DNA甲基化 

分 类 号：R259[医药卫生—中西医结合]