运动预处理通过外泌体介导miR-146a对大鼠缺血性脑卒中炎症反应的影响

Effects of Exercise Preconditioning on the Inflammatory Response of Rats after Ischemic Stroke through Exosome-mediated miR-146a

目的:通过建立8周运动预处理模型和大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)大鼠缺血性脑卒中模型,探讨运动预处理通过外泌体介导的微RNA 146a(microRNA-146a,miR-146a)对缺血性脑卒中脑组织炎症反应的影响及相关机制。方法:将60只6周龄雄性SD大鼠随机均分为非运动组和运动组。非运动组分为假手术对照组(group control,C组)和MCAO造模组(group MCAO,M组),各15只;运动组分为单纯运动组(group exercise,E组)和运动MCAO造模组(group exercise+MCAO,EM组),各15只。E组和EM组大鼠进行8周的跑台锻炼,每周运动6天,每天运动30min。8周后,M和EM组大鼠进行MCAO手术模型的制备,C组和E组进行模拟MCAO造模的假手术。在造模24 h后进行神经行为学评分;取血浆提取外泌体;取大鼠脑组织进行2,3,5-氯化三苯基四氮唑(TTC)染色测定脑梗死体积;尼氏染色观察大鼠神经元及尼氏小体变化;实时定量聚合酶链反应(qPCR)测定血浆外泌体和脑组织中miR-146a的含量;免疫蛋白印迹(Western Blot)测定大鼠脑组织中肿瘤坏死因子受体相关因子6(TRAF6)、核因子κB(NF-κB)和肿瘤坏死因子-α(TNF-α)的表达水平;双荧光素酶报告基因检测miR-146a与TRAF6的靶向关系。结果:(1)神经行为学评分显示EM组和M组较C组评分高(P<0.01,P<0.01);EM组较M组评分低(P<0.01)。(2)TTC染色提示EM组和M组较C组和E组梗死体积大(P<0.01,P<0.01),EM组较M组脑梗死体积小(P<0.01)。(3)尼氏染色结果表明M组较C组和E组神经元排列疏松,神经元数量减少,尼氏体着色较浅且数量减少;与M组相比,EM组的神经元数量有所增加,尼氏体数量增多。(4)qPCR检测结果显示,与C组相比,EM组和M组血浆外泌体和脑组织中miR-146a的表达量更低(P<0.05,P<0.01),同时EM组高于M组(P<0.05)。(5)Western Blot检测显示,与C组相比,EM组和M组TRAF6、NF-κB和TNF-α蛋白表达更高(P<0.05,P<0.01);与M组相比,EM组脑中TRAF6、NF-κB和TNF-α蛋白表达较低(P<0.05,P<0.05)。(6)双荧光素酶报告基因检测表明miR-146a与TRAF6存在特异性结合位点。结论:8周运动预处理减少了缺血性脑卒中的脑梗死面积,减少大脑的受损程度,其作用机制可能是通过外泌体介导miR-146a,增加了脑组织内miR-146a表达,miR-146a靶向结合TRAF6,负调控TRAF6/NF-κB,同时降低TNF-α的表达,减轻脑组织炎症反应,从而对缺血性脑损伤产生保护作用。

ObjectiveTo explore the effect of exercise preconditioning on inflammatory response in ischemic stroke brain tissue which mediated by miR-146a in extracellular vesicles in rats with middle cerebral artery occlusion(MCAO),and its mechanism.MethodsSixty 6-week-old male Sprague-Dawley rats were randomly divided into a non-exercise group and an exercise group.The non-exercise group was further divided into a shamoperation control group(C,n=15)and an MCAO model group(M,n=15),while the exercise group was further divided into an exercise only group(E,n=15)and an exercise plus MCAO model group(EM,n=15).Rats in the E and EM groups underwent 8 weeks of treadmill exercise,6 days per week,30 minutes per day.Then rats in the M and EM groups received MCAO to induce ischemic stroke,while the C and E groups underwent a sham surgery.Twenty-four hours after the surgery,neurobehavioral tests were performed.Plasma was collected to extract extracellular vesicles,and brain tissue was collected to measure the volume of cerebral infarction by using the triphenyl tetrazolium chloride(TTC)staining.Moreover,the Nissl staining was conducted to observe neuronal and Nissl body.Mean while,the content of miR-146a in plasma extracellular vesicles and brain tissue was measured by using the quantitative polymerase chain reaction(q PCR),and the expression of TNF receptor associated factor 6(TRAF6),nuclear factor kappa-B(NF-κB)and tumor necrosis factor-α(TNF-α)in brain tissues were determined using Western blotting.The targeting relationship between miR-146a and TRAF6 was detected by using the dual luciferase reporter gene assay.Results(1)The neurological behavioral scores of the EM and M groups were higher than those of the C group(P<0.01 and P<0.01),with that of the EM group lower than the M group(P<0.01).(2)TTC staining showed that the infarct volume of the EM and M groups was larger than that of the other two groups(P<0.01 and P<0.01),with that of the EM group smaller than the M group(P<0.01).(3)Nissl staining results showed that the neuronal arrangement was loose,the number of neurons reduced,and the Nissl bodies were lightly stained and decreased in the M group compared with the C and E groups.Moreover,compared with the M group,the number of neurons and Nissl bodies increased in the EM group.(4)The qPCR analysis showed that the expression of miR-146a in the plasma-derived exosomes and brain tissues of the EM and M groups decreased compared with the C group(P<0.05 and P<0.01),with that of the EM group higher than the M group(P<0.05).(5)According to Western blotting,compared with the C group,the expression levels of TRAF6,NF-κB,and TNF-αproteins increased significantly(P<0.05 and P<0.01),with that of group EM signfiicantly lower than group M(P<0.05 and P<0.05).(6)Dual-luciferase reporter gene assay showed that miR-146a had a specific binding site with TRAF6.ConclusionEight weeks of exercise preconditioning reduces the infarct area and the extent of brain damage,which may be mediated by miR-146a via exosomes,increasing the expression of miR-146a in brain tissue,targeting TRAF6,negatively regulating TRAF6/NF-κB,and reducing the expression of TNF-α,thus alleviating the inflammatory response in brain tissue and exerting a protective effect on ischemic brain injury.