温化蠲痹方对胶原诱导性关节炎大鼠外周血单核细胞DNA甲基化转移酶表达的影响

Effects of Wenhua Juanbi Recipe on the Expressions of DNA Methyltransferases in Peripheral Blood Mononuclear Cells of Collagen-Inducing Arthritis Rats

目的观察温化蠲痹方对胶原诱导性关节炎(collagen-inducing arthritis,CIA)大鼠外周血单核细胞(peripheral blood mononuclear cells,PBMCs)DNA甲基化转移酶(DNA methyltrsansferases,DNMTs)表达的影响,探讨温化蠲痹方治疗CIA的作用机制。方法将90只Wistar大鼠随机分为造模组(80只)及正常对照组(10只,简称正常组)。造模组在大鼠尾根部注射牛Ⅱ型胶原乳剂制备CIA模型。将造模成功的50只大鼠随机分为模型组、甲氨喋呤组(MTX)、温化蠲痹方低、中、高剂量组(简称中药低、中、高剂量组),每组10只。模型组给予生理盐水灌胃;中药低、中、高剂量组分别给予温化蠲痹方22.9、45.8、68.7 g/(kg·d)灌胃,均每日1次;MTX组按0.78 mg/kg剂量灌胃MTX混悬液,每周1次,连续30日。采用容积法(排水体积)评价足趾肿胀度。给药干预后提取各组大鼠PBMCs,采用实时定量PCR法检测PBMCs DNMTs(DNMT1、DNMT3a及DNMT3b)mRNA表达水平。结果与正常组比较,模型组大鼠足趾明显肿胀(P<0.01);与模型组比较,中药低、中、高剂量组及MTX组大鼠足趾肿胀明显减轻(P<0.01)。与本组治疗前比较,中药各剂量组及MTX组大鼠足趾肿胀减轻(P<0.01)。与正常组比较,模型组大鼠PBMCs DNMT1、DNMT3a及DNMT3b表达明显降低(P<0.01);与模型组比较,中药各剂量组及MTX组DNMT1、DNMT3a及DNMT3b表达水平明显升高(均P<0.01);中药各剂量组DNMT1、DNMT3a及DNMT3b表达水平比较,差异无统计学意义(P>0.05)。结论 CIA大鼠PBMCs DNMTs表达降低。温化蠲痹方上调CIA大鼠PBMCs DNMTs表达无明显剂量依赖性。通过上调DNMTs表达,调节CIA大鼠甲基化状态可能是其治疗CIA作用机制之一。

Objective To observe the effect of Wenhua Juanbi Recipe（ WJR） on the expressions of DNA methyltransferases（ DNMTs） in peripheral blood mononuclear cells（ PBMCs） of collagen-inducing arthritis（ CIA）,and to study its mechanism for treating CIA. Methods Totally 90 Wistar rats were randomly divided into the model group（n = 80） and the normal control group（n = 10）. Rats of the model group were injected with typeⅡ collagen of bovine（ BCⅡ） emulsion from the tail to establish CIA model. Successfully modeled 50 CIA rats were randomly divided into five groups,i. e.,the model group,the methotrexate（ MTX） group,the low dose WJR group,the middle dose WJR group,the high dose WJR group,10 in each group. Rats in the model group were administered with normal saline by gastrogavage,once per day. Rats in low,middle,and high dose WJR groups wereadministered with WJR by gastrogavage at the daily dose of 22. 9,45. 8,68. 7 g / kg,respectively（ once per day）.Rats in the MTX group were administered with MTX suspension（ 0. 78 mg / kg） by gastrogavage,once per week for30 successive days. The paw swelling was evaluated using volume method（ draining volume）. PBMCs were extracted from each group after intervention. mRNA expression levels of DNMTs（ DNMT1,DNMT3 a,DNMT3b） were detected by real-time quantitative PCR. Results Compared with the normal group,the paws were obviously swollen in the model group（P〈0. 01）. Compared with the model group,swollen paws were obviously alleviated in low,middle,and high dose WJR groups,and the MTX group（P〈0. 01）. Compared with before treatment in the same group,swollen paws were obviously alleviated in low,middle,and high dose WJR groups,and the MTX group（P〈0. 01）. Compared with the normal group,expression levels of DNMT1,DNMT3 a,and DNMT3 b in PBMCs were obviously lowered in the model group（P〈0. 01）. Compared with the model group,expression levels of DNMT1,DNMT3 a,DNMT3b in PBMCs were obviously elevated in low,middle,and high dose WJR groups,and the MTX group（ all P〈0. 01）. There was no significant difference in expression levels of DNMT1,DNMT3 a,or DNMT3 b in PBMCs among low,middle,and high dose WJR groups（P〉0. 05）. Conclusions Expression levels of DNMTs in PBMCs of CIA rats decreased. WJR up-regulated the expression level of DNMTs in PBMCs of CIA rats in no obvious dose dependent way. One of WJR＇s mechanisms for treating CIA might be up-regulating expression levels of DNMTs,and adjusting the state of DNA methylation.