摘要
目的:研究鸟嘌呤释放蛋白1(RASGRP1)在中药温化蠲痹方影响微小RNA-146a(miRNA-146a)对胶原诱导性关节炎(CIA)大鼠外周血单个核细胞(PBMC)DNA甲基化调控中的作用,探讨温化蠲痹方治疗CIA作用机制。方法:随机将健康雌性Wistar大鼠分为造模组和正常对照组(n=10,正常组),造模组在大鼠尾根部注射牛Ⅱ型胶原乳剂,建立胶原诱导性关节炎模型。30只造模成功大鼠随机分组如下:模型组(n=10)、甲氨蝶呤组(n=10,MTX组)及中药温化蠲痹方组(n=10,中药组)。中药组大鼠按22.9 g/(kg·d)灌胃中药温化蠲痹方,模型组给予生理盐水灌胃,两组大鼠都是每天1次,MTX组灌胃MTX混悬液(0.78 mg/kg),每周1次,共观察30 d。采用容积法(排水体积)评价足趾肿胀度。给药结束后,对正常组、模型组PBMC细胞培养,模型组加入ras-MARKs阻断剂,阻断剂①SB230580、阻断剂②PD98059、阻断剂③SP600125,模型组加中药,正常组转染miRNA-146a,模型组转染反义miRNA-146a。采用实时定量PCR法检测miRNA-146a、DNMTs(DNMT1、DNMT3a、DNMT3b)表达水平,Western Blot检测RASGRP1表达。结果:(1)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);MTX组与中药组比较,大鼠足趾肿胀差异无统计学意义(P>0.05);与治疗前比较,中药组和MTX组大鼠足趾肿胀减轻,差异有统计学意义(P<0.01)。(2)体内实验:与正常组比较,模型组大鼠外周血RASGRP1表达升高,差异有统计学意义(P<0.01)。与模型组比较,中药组、MTX组RASGRP1表达降低,差异有统计学意义(P<0.01)。中药组、MTX组之间比较,差异无统计学意义(P>0.05)。体外实验:与正常组比较,模型组、正常转染miRNA-146a组PBMC RASGRP1表达升高,差异有统计学意义(P<0.01)。与模型组比较,模型加中药组、模型转染反义miRNA-146a组RASGRP1表达降低,差异有统计学意义(P<0.01)。与模型组比较,模型加阻断剂①组、加阻断剂②组、加阻断剂③组PBMC RASGRP1表达降低,差异有统计学意义(P<0.01)。(3)相关性分析:模型组、模型加阻②、③、加中药组、转染miRNA-146a组、转染反义miRNA-146a组大鼠PBMC miRNA-146a表达水平与DNMT1、DNMT3a、DNMT3b呈显著负相关。结论:温化蠲痹方可能作用于RASGRP1影响ras-MARKs通路,发挥对微小RNA-146a调控CIA大鼠PBMC DNA甲基化作用,是其治疗胶原诱导性关节炎作用机制之一。
Objective:To study the effects on miRNA-146 a regulating and controlling DNA methylation in peripheral blood mononuclear cells(PBMCs)of rats with collagen-induced arthritis(CIA)by Wenhua Juanbi Recipe(WJR)through acting on RAS guang releasing protein 1(RASGRP1)and explore the mechanism of WJR treating CIA.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 BCⅡ(type Ⅱcollegan of bovine,BCⅡ)emulsion in the tail to establish CIA model.The succeeded CIA rats were randomly divided into three groups:model group(n=10),WJR group(n=10,Chinese herbs group 1 for short),MTX group(n=10).Rats of control group,model group and Chinese herbs group were given physiological saline and WJR respectively by stomach rearing according to 22.9 g/(kg·d).MTX group was given MTX liquor according to 0.78 mg/(kg·w)in the same way.The foot swelling was evaluated using volume method(displacement volume).After treatment for 30 days, PBMCs of normal group and model group were extracted and cultured in vitro, model adding blocker 1 SB230580, blocker 2 PD98059, blocker 3 SP600125, and model adding WJR groups.PBMCs of normal and model were transfected miRNA-146 a, antisense miRNA-146 a respectively.The levels of miRNA-146 a and DNMTs(DNMT1, DNMT3 a, DNMT3b)in PBMCs were examined by quantitative PCR assay, the expression of RASGRP1 was examined by Western Blot.Results:(1)Compared with the normal group,paw swelling of the model group was significant(P<0.01).Compared with the model group,paw swelling of Chinese herbs group and MTX group reduced significantly(P<0.01).Compared with MTX group,the difference of paw swelling of Chinese herbs group was not statistically significant(P>0.05).Compared with before treatment,paw swelling of Chinese herbs group and MTX group reduced significantly(P<0.01).(2)In vivo experiments: Compared with the normal group, the expression levels of RASGRP1 in PBMCs of model group were significantly elevated(P<0.01).Compared with model group, the expression levels of RASGRP1 in PBMCs of Chinese herbs group and MTX group were significantly lower(P<0.01).There was no significant differences between MTX group and Chinese herbs groups,including Chinese herbs group(P>0.05).In vitro experiments: compared with the normal group, the expression levels of RASGRP1 in PBMCs of model group and normal transfected miRNA-146 a group were significantly elevated(P<0.01).Compared with model group, the expression levels of RASGRP1 in PBMCs of model adding WJR group and model transfected antisense miRNA-146 a group were significantly lower(P<0.01).(3)Correlational analyses: The expression of miRNA-146 a and DNMTs in PBMCs of model group, model adding WJR group, model adding blocker 2 and 3 groups, normal transfected miRNA-146 a group and model transfected antisense miRNA-146 a group showed a negative correlation.Conclusion: MiRNA-146 a regulates and controls DNA methylation in PBMCs of rats with CIA by WJR through acting on RASGRP1.It is one of possible mechanisms of treating CIA by WJR.
作者
刘喜德
冯莹莹
蔡龙
周红娟
王珊珊
叶丽红
王云卿
杜静
郑琳琳
LIU Xide;FENG Yingying;CAI Long;ZHOU Hongjuan;WANG Shanshan;YE Lihong;WANG Yunqing;DU Jing;ZHENG Linlin(Department of Athropathy,Zhejiang Hospital of Integrated Traditional and Western Medicine,Hangzhou 310003,Zhejiang,China;Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China)
出处
《中华中医药学刊》
CAS
北大核心
2019年第1期10-14,共5页
Chinese Archives of Traditional Chinese Medicine
基金
浙江省自然科学基金项目(LY12H29008
LY17H270004)
浙江省中医药科技计划项目(2012ZB121
2013ZB096
2015ZA143)
杭州市医药卫生科技计划项目(2010B027
2014A37)
杭州市科技发展计划项目(20120633B12
20160533B45)
