补阳还五汤通过miR-26a-5p激活PTEN/PI3K/Akt信号通路减轻大鼠脑缺血再灌注损伤  

Buyang Huanwu Decoction attenuates cerebral ischemia-reperfusion injury in rats via miR-26a-5p mediated PTEN/PI3K/Akt signaling pathway

作　　者：张修红 傅开龙[1] 林侃[1] 李楠[1] 姚明龙 郑关毅[1] ZHANG Xiu-hong;FU Kai-long;LIN Kan;LI Nan;YAO Ming-long;ZHENG Guan-yi(Department of Traditional Chinese Medicine,Fujian Medical University Union Hospital,Fuzhou 350001,China)

机构地区：[1]福建医科大学附属协和医院中医科,福建福州350001

出　　处：《中国中药杂志》2024年第15期4197-4206,共10页China Journal of Chinese Materia Medica

基　　金：福建省自然科学基金项目(2020J011010);“十四五”非中医医疗机构中医药科室建设项目(闽卫中医函[2023]344号)。

摘　　要：为探讨补阳还五汤抗大鼠脑缺血再灌注损伤的机制,180只SD大鼠随机分为假手术组、模型组、补阳还五汤组、补阳还五汤+miR-26a-5p激动剂(激动剂)组、补阳还五汤+激动剂阴性对照(激动剂阴性对照)组,每组36只;每组又按再灌注时间分为第3、7、14天3个亚组。除假手术组外,其余各组采用线栓法诱导大脑中动脉阻塞(MCAO)模型,缺血90 min后再灌注。补阳还五汤组、激动剂组、激动剂阴性对照组建模24 h后开始给予补阳还五汤灌胃,每日2次;假手术组、模型组灌胃等量生理盐水,直至处死的前1 d。激动剂组和激动剂阴性对照组建模24 h后分别于侧脑室注射miR-26a-5p激动剂和miR-26a-5p激动剂阴性对照剂,其他各组注射等量生理盐水。各组建模24 h后腹腔注射5-溴脱氧尿苷(BrdU),每日1次,直至处死的前1 d。采用改良版神经损伤严重程度评分(mNSS)评价神经功能缺损,2,3,5-三苯四氮唑(TTC)染色检测脑梗死体积,TUNEL染色检测脑组织细胞凋亡,免疫荧光双标记法检测BrdU/NeuN双阳性(BrdU^(+)/NeuN^(+))细胞数评估新生神经元,双荧光素酶实验验证PTEN是miR-26a-5p的靶基因,实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测PTEN及miR-26a-5p的表达,蛋白免疫印迹法(Western blot)检测PTEN、PI3K、p-PI3K、Akt、p-Akt的表达。结果显示,与模型组比较,补阳还五汤可促进脑缺血大鼠神经功能恢复,缩小脑梗死体积,增加BrdU+/NeuN+细胞数,上调miR-26a-5p的表达,调控PTEN/PI3K/Akt信号通路,促进神经元新生;侧脑室注射miR-26a-5p激动剂后加强上述效应。综上所述,补阳还五汤可促进脑缺血大鼠神经功能恢复,减小脑梗死体积,促进脑缺血大鼠神经元新生,其机制可能是通过miR-26a-5p激活PTEN/PI3K/Akt信号通路。This study aims to investigate the mechanism of Buyang Huanwu Decoction in treatment of cerebral ischemia-reperfusion injury in rats.A total of 180 SD rats were randomly divided into 5 different groups:sham group,model group,Buyang Huanwu Decoction group,Buyang Huanwu Decoction+miR-26a-5p agomir(agomir)group,Buyang Huanwu Decoction+miR-26a-5p agomir negative control(agomir NC)group.There were 36 rats in each group.Each group was then subdivided into three subgroups for the duration of reperfusion(3,7,14 d).A ligature-induced middle cerebral artery occlusion(MCAO)model was carried out on all groups other than sham group.Reperfusion was performed following ischemia for 90 min.Buyang Huanwu Decoction group,agomir group,and agomir NC group were given Buyang Huanwu Decoction twice daily by gavage 24 h after the formation of the model.Sham group and model group were given an equal amount of physiological saline by gavage until the day before sacrifice.At 24 h after ischemia induction,miR-26a-5p agomir was injected into the lateral ventricle in agomir group,miR-26a-5p NC in agomir NC group,and equal amounts of physiological saline in the other groups.24 h after ischemia induction,BrdU was intraperitoneally injected once daily until the day before sacrifice.Modified neurological severity score(mNSS)was used to evaluate neurological deficits,2,3,5-triphenyltetrazolium chloride(TTC)staining was used to determine the cerebral infarct volume,TUNEL staining was used to assess the apoptosis of parenchymal ischemic brain tissue,and double immunofluorescence staining was used to examine BrdU/NeuN double positive neurons in the parenchymal ischemic brain tissue to evaluate the neuronal regeneration.We employed a luciferase reporter assay to identify and validate that the target gene of miR-26a-5p is PTEN.Real-time quantitative polymerase chain reaction(RTqPCR)was used to assess gene expression levels of PTEN and miR-26a-5p and Western blot to assess the protein levels of PTEN,PI3K,p-PI3K,Akt,and p-Akt.The results revealed that co

关 键 词：补阳还五汤 脑缺血再灌注 神经保护 miR-26a-5p 大脑中动脉闭塞 局灶性脑缺血 PTEN/PI3K/Akt 

分 类 号：R285.5[医药卫生—中药学]