温化蠲痹方对胶原诱导性关节炎大鼠外周血单个核细胞微小RNA-146a表达的影响

Effects on Expression of MicroRNA-146a in Peripheral Blood Mononuclear Cell of Rats with Collagen-Inducing Arthritis by Wenhua Juanbi Recipe

目的:研究中药温化蠲痹方对胶原诱导性关节炎(CIA)大鼠外周血单个核细胞(PBMC)微小RNA-146a(miRNA-146a)表达的影响,探讨温化蠲痹方治疗CIA的作用机制。方法:选用健康雌性Wistar大鼠,随机分为造模组、正常对照组(10只,简称正常组),造模组采用牛Ⅱ型胶原乳剂于大鼠尾根部注射,建立CIA模型。再选取造模成功大鼠50只随机分为模型组(10只)、MTX组(10只)、温化蠲痹方低剂量组(n=10,简称中药1组)、温化蠲痹方中剂量组(n=10,简称中药2组)、温化蠲痹方高剂量组(n=10,简称中药3组)。中药1、2、3组分别给予温化蠲痹方22.9 g/kg·d、45.8 g/kg·d、68.7 g/kg·d灌胃,模型组灌胃生理盐水,均每天1次,MTX组按0.78 mg/kg剂量灌胃MTX混悬液,每周1次,连续30 d。采用容积法(排水体积)评价足趾肿胀度。给药结束后,提取各组大鼠外周单个核细胞,采用实时定量PCR法检测miRNA-146a表达水平。结果:与正常组比较,模型组大鼠足趾肿胀明显,差异有统计学意义(P<0.01);与模型组比较,中药1、2、3组和MTX组大鼠足趾肿胀明显减轻,差异有统计学意义(P<0.01);与MTX组比较,中药1、2、3组大鼠足趾肿胀差异无统计学意义(P>0.05);与治疗前比较,中药1、2、3组和MTX组大鼠足趾肿胀减轻,差异有统计学意义(P<0.01)。与正常组比较,模型组大鼠PBMC miRNA-146a表达明显升高,差异有统计学意义(P<0.01);与模型组比较,中药1、2、3组和MTX组大鼠PBMC miRNA-146a表达均明显降低,差异有统计学意义(P<0.05);与MTX比较,中药1、2、3组大鼠PBMC miRNA-146a表达差异无统计学意义(P>0.05);中药1、2、3组比较,大鼠PBMC miRNA-146a表达差异无统计学意义(P>0.05)。结论:CIA大鼠PBMC miRNA-146a表达升高。中药温化蠲痹方下调CIA大鼠PBMC miRNA-146a表达无剂量依赖性,其可能通过下调miRNA-146a表达,而调节免疫细胞分化、信号转导等达到治疗CIA的作用。

Objective ： To study expression of microRNA - 146a（miRNA） in peripheral blood mononuclear cell （ PBMC ） of rats with collagen -inducing arthritis（CIA） by Wenhua Juanbi Recipe（WJR）. Methods Female Wistar rats were randomly divided into making model group and normal control group（ n = 10）. Rats of making model group were injected 0. 3 mg BC Ⅱ （ type Ⅱ collegan of bovine, BC Ⅱ ） emulsion in the tail to establish CIA model. The successful CIA rats were randomly divided into five groups, which were model group （n = 10）, W JR low -dose group （n = 10, Chinese herbs group 1 for short）, W JR middle - dose group （ n = 10, Chinese herbs group 2 for short）, W JR high, dose group （ n = 10, Chinese herbs group 3 for short） ,MTX group（n = 10）. Rats of control group,model group and Chinese herbs group 1,2,3 were given physiological saline and WJR respectively by stomach rearing according to 22.9 g/（ kg · d） ,45.8 g/（ kg · d） ,68. 7 g/（ kg·d） respectively. Rats of MTX group were given MTX liquor according to 0.78 mg,/（ kg· w） in the same way. The paw swelling was evaluated using volume method（ displacement volume）. After treatment for 30 days,the level of mi- eroRNA - 146a in PBMCs was examined by quantitative PCR assay. Results ： Compared with the normal group, paw swell- ing of the model group rats was significant （P 〈 0.01 ）. Compared with the model group, paw swelling of Chinese herbs group 1,2,3 and MTX group reduced signifieantly（P 〈0. 01 ）. Compared with MTX group,the difference of paw swelling of Chinese herbs group 1,2,3 was not statistically significant （ P 〉 0.05 ）. Compared with that before treatment, paw swelling of Chinese herbs group 1,2,3 and MTX group reduced significantly （ P 〈 0.01 ）. Compared with the normal group, expression level of miRNA - 146a in PBMCs of model group was significantly elevated （ P 〈 0.01 ）. Compared with model group,expression level of miRNA - 146a in PBMCs of Chinese herbs group 1,2,3 and MTX group was significantly lower（P 〈0.01 ）. There was no significant difference beiween MTX group and Chinese herbs groups as well as among Chinese herbs group 1,2,3 （P 〉0.05）. Conclusion：Expression level of miRNA - 146a in PBMCs of rats with CIA ele- vates. There is no dose dependent manner in WJR decreasing the level of miRNA - 146a. It＇s one of possible mechanisms that WJR decreases the level of miRNA - 146a in PBMCs of CIA rats and adjusts immune cell differentiation , signal transduction and so on.