基于miR-21探讨针刺对PI3K/AKT信号通路脑缺血再灌注大鼠脑区星形胶质细胞影响

Acupuncture Effects on Astrocytes in Cerebral Ischemia Reperfusion Rats under PI3K/AKT Signaling Pathway Based on miR-21

目的 观察电针对激活脑缺血再灌注(cerebralischemia reperfusion injury,CIRI)大鼠脑组织磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)信号通路和星形胶质细胞(astrocyte,AS)表达影响,进而调控神经发生过程,以及microRNA-21(miR-21)在此过程中的作用。方法 选取SD级别雌性大鼠18只,应用随机数字表法将其分成3组,即假手术组、模型组、电针组,每组6只。除假手术组外,其他两组采用Longa改良线栓法建立大鼠CIRI模型,评定造模成功后,电针组给予电针治疗,其他两组不做处理。采用动物神经功能评分(Longa评分)及神经行为学测试(Morris水迷宫实验)对各组大鼠进行评定。应用HE染色观察脑组织病理改变,免疫荧光法检测各组星形胶质细胞胶质纤维酸性蛋白(GFAP)、过氧化物酶体增殖物激活受体γ(PPARγ)表达情况,实时荧光定量反转录-聚合酶链反应(Real-time PCR,RT-PCR)法检测各组星形胶质细胞miR-21表达情况,蛋白免疫印迹(Western blot,WB)法检测各组星形胶质细胞胞内磷脂酰肌醇激酶(PI3K)、苏氨酸蛋白激酶(AKT)蛋白表达情况。结果 (1)Longa评分结果:治疗后电针组大鼠Longa评分降低,与假手术组比较,差异具有统计学意义(P<0.05)。(2)Morris水迷宫实验结果:与假手术组比较,模型组大鼠穿越逃生平台次数减少(P<0.05);治疗后电针组较模型组穿越逃生平台次数显著增加(P<0.05)。(3)脑组织病理切片结果:电针组大鼠脑组织损伤情况缓解最为显著。(4)免疫荧光检测结果:电针组较模型组星形胶质细胞数量增多(P<0.05),模型组较假手术组PPARγ阳性表达减弱(P<0.05),电针组较模型组PPARγ阳性表达增多(P<0.05)。(5)RT-PCR检测结果:与假手术组比较,模型组miR-21表达增多(P<0.05),电针组较模型组表达增多更为显著(P<0.05)。(6)WB检测结果:模型组PI3K、AKT蛋白表达较假手术组增多(P<0.05)。电针治疗干预后,与模型组相比,电针组PI3K、AKT蛋白表达增多更为显著(P<0.05)。结论电针治疗可上调PPARγ表达,活化miR-21激活PI3K/AKT信号通路表达,保护因脑缺血再灌注导致损伤的神经元,使其修复与再生,以降低脑缺血再灌注带来的不良影响。

Objective To observe the effects of electroacupuncture on activating the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway and astrocyte(AS)expression in the brain tissue of rats with cerebral ischemia-reperfusion injury(CIRI),thereby regulating the process of neurogenesis and the role of microRNA-21(miR-21)in this process.Methods Eighteen SD grade female rats were selected and randomly divided into 3 groups with 6 rats in each group,namely,sham operation group,model group,and electroacupuncture group.In addition to the sham operation group,rat models of CIRI were established using Longa's modified thread embolization method.After assessing the success of the model.The electroacupuncture group received electroacupuncture treatment,while the other two groups did not receive any treatment.The rats in each group were evaluated for neurological function(Longa score)and neurobehavioral tests(Morris water maze test,MWM),HE staining was used to observe pathological changes in brain tissue,and immunofluorescence was used to detect GFAP and PPARγexpression of astrocytes in each group:real-time fluorescence quantitative reverse transcription-polymerase chain reaction(Real-timePCR,RT-PCR)was used to detect the expression of miR-21 in astrocytes of each group,and Western blot(WB)was used to detect the expression of PI3K and AKT proteins in astrocytes of each group.Results(1)The results of Longa score:Compared with sham operated group,the Longa score of electroacupuncture group was significantly decreased,the difference was statistical significance(P<0.05).(2)The results of MWM:The results of the Morris water maze experiment showed that compared with the sham operation group,the number of times the model group rats crossed the escape platform decreased(P<0.05);After treatment,the number of times the electroacupuncture group crossed the escape platform significantly increased compared to the model group(P<0.05).(3)The results of pathological biopsy of brain tissue:The damage was significantly improved to the brain tissue in the electroacupuncture group.(4)The results of immunofluorescence analysis:Compared with model group,the astrocytes of electroacupuncture group were increased(P<0.05).Compared with sham operated group,PPARγpositive expression of model group was receded(P<0.05).Compared with model group,PPARγpositive expression of electroacupuncture group was increased(P<0.05).(5)The results of RT-PCR:Compared with sham operated group,miR-21 expression of model group was clearly increased(P<0.05).Compared with model group,miR-21 expression of electroacupuncture group was clearly increased(P<0.05).(6)The results of WB:Compared with sham operated group,P13K,AKT protein expression of model group were significantly increased(P<0.05).Compared with model group,the expression of PI3K and AKT in electroacupuncture group showed a more significant up-regulation trend than that in model group after the electroacupuncture treatment intervension(P<0.05).Conclusion Electrocupuncture treatment can up-regulate the expression of PPARγ,activate miR-21 and the expression of PI3K/AKT signaling pathway,protect damaged neurons and promote their regeneration and repair,thereby alleviating cerebral ischemia-reperfusion injury.