miR-134调控CREB/BDNF通路参与脑卒中后抑郁的作用机制研究  

Mechanism of miR-134 regulating CREB/BDNF pathway in post-stroke depression

作　　者：李静[1] 韩永凯[2] 苏静 朱欣茹 宋景贵[2] LI Jing;HAN Yong-kai;SU Jing;ZHU Xin-ru;SONG Jing-gui(Department of Neurology,the Third Affiliated Hospital of Xinxiang Medical College,Xinxiang 453000,China;Department of Neurology,the Second Affiliated Hospital of Xinxiang Medical College;Department of Neurology,the First Affiliated Hospital of Xinxiang Medical College)

机构地区：[1]新乡医学院第三附属医院神经内科,453000 [2]新乡医学院第二附属医院神经内科 [3]新乡医学院第一附属医院神经内科

出　　处：《天津医药》2021年第1期16-21,共6页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81471349)。

摘　　要：目的探讨微小RNA(miR)-134调节环腺苷酸应答元件结合蛋白(CREB)/脑源性神经营养因子(BDNF)通路参与脑卒中后抑郁(PSD)的作用机制。方法32只SD大鼠按照随机数字表法分为假手术组、模型组、antagomir NC组、miR-134 antagomir组,每组8只;除假手术组外,其余各组行大脑中动脉阻塞(MCAO)术,然后采用慢性不可预见温和应激(CUMS)诱导建立PSD模型,假手术组除不插入线栓外其他操作相同。MCAO术后CUMS前antagomir NC组、miR-134 antagomir组分别于大脑双侧海马区注射5μL antagomir NC、miR-134 antagomir(1μmol/L),5 d注射1次,连续5次。假手术组、模型组注射等体积生理盐水。CUMS应激前,应激第7、14、21天对大鼠称体质量,动物敞箱实验评价大鼠运动行为改变、蔗糖偏爱实验评价大鼠快感缺乏行为改变。实验结束后处死大鼠,分离海马CA1区,实时荧光定量PCR(qPCR)检测miR-134、CREB、BDNF mRNA表达,免疫组化染色检测CREB、BDNF蛋白表达情况。双荧光素酶报告基因实验鉴定CREB与miR-134的靶向关系。结果与假手术组相比,模型组大鼠体质量、水平和垂直活动得分、糖水饮用比例降低(P<0.05);与模型组、antagomir NC组相比,miR-134 antagomir组大鼠体质量、水平和垂直活动得分、糖水饮用比例升高(P<0.05),随着应激时间的延长,该效应逐渐明显。与假手术组相比,模型组海马CA1区miR-134水平升高(P<0.05),CREB、BDNF mRNA表达和阳性细胞数减少(P<0.05);与模型组、antagomir NC组相比,miR-134 antagomir组海马CA1区miR-134水平降低(P<0.05),CREB、BDNF mRNA表达和阳性细胞数增多(P<0.05)。与miR-134 mimic NC+CREB-WT相比,miR-134 mimic+CREB-WT共转染细胞荧光素酶相对活性下降(P<0.05),证明CREB序列上存在miR-134特异结合位点。结论降低miR-134可促进CREB/BDNF通路蛋白的表达,进而改善抑郁状态,实现对PSD大鼠的保护。Objective To investigate the mechanism of microRNA(miR)-134 in post-stroke depression(PSD)by regulating cyclic AMP response element-binding protein(CREB)/brain-derived neurotrophic factor(BDNF)pathway.Methods A total of 32 SD rats were randomly divided into sham operation group,model group,antagomir NC group and miR-134 antagomir group according to the random number table method,with 8 rats in each group.Except for the sham operation group,the other groups were subjected to middle cerebral artery occlusion(MCAO)and then established PSD model induced by chronic unpredictable mild stress(CUMS).The other operations were the same except that the thread plug was not inserted in the sham operation group.After MCAO,5μL(1μmol/L)antagomir NC and miR-134 antagomir were injected into the brain before CUMS in antagomir NC group and miR-134 antagomir group respectively,once every 5 days for 5 consecutive times.The sham operation group and the model group were injected with equal volume of normal saline.The rats were weighed before CUMS stress,on the 7th,14th and 21st day after stress,the exercise behavior changes of rats were evaluated by open box test,and pleasure deficiency behavior changes were evaluated by sucrose preference test.At the end of the experiment,the rats were killed and the hippocampal CA1 area was separated,the expressions of miR-134,CREB and BDNF mRNA in hippocampal CA1 area were detected by real-time fluorescence quantitative PCR(qPCR),and the protein expressions of CREB and BDNF in hippocampal CA1 area were detected by immunohistochemical staining.The targeting relationship between CREB and miR-134 was identified by double luciferase reporter gene assay.Results Compared with the sham operation group,the body mass index of rats,horizontal and vertical activity scores,and the proportion of sugar water drinking were lower in the model group(P<0.05).Compared with the model group and the antagomir NC group,the body mass index of rats,horizontal and vertical activity score,and the proportion of sugar water dr

关 键 词：卒中 抑郁症 基因沉默 大鼠 Sprague-Dawley 微RNAS CAMP反应元件结合蛋白质 脑源性神经营养因子 微小RNA-134 

分 类 号：R749.42[医药卫生—神经病学与精神病学] R743.3[医药卫生—临床医学]