补阳还五汤通过抑制脑微血管内皮细胞自噬减轻大鼠脑缺血再灌注损伤  

Buyang-Huanwu decoction attenuates rat cerebral ischemia-reperfusion injury by inhibiting autophagy of cerebral microvascular endothelial cells

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作  者:李猛 左春月 靳晓飞 张天赐 周晓红 高维娟 LI Meng;ZUO Chunyue;JIN Xiaofei;ZHANG Tianci;ZHOU Xiaohong;GAO Wei-juan(Key Laboratory of Chinese Medicine Research for Prevention and Control of Cardiovascular and Cerebrovascular Diseases,Hebei University of Traditional Chinese Medicine,Shijiazhuang 050091,China)

机构地区:[1]河北中医药大学,河北省心脑血管病中医药防治研究重点实验室,河北石家庄050091

出  处:《中国病理生理杂志》2025年第3期481-491,共11页Chinese Journal of Pathophysiology

基  金:国家自然科学基金资助项目(No.81873180);河北省自然科学基金资助项目(No.H2022423382);河北省中医药管理局科研计划项目(No.2021101);校级燕赵医学项目(No.YZZZ2023009);青年教师科研能力提升项目(No.JCYJ2022009)。

摘  要:目的:探讨补阳还五汤(Buyang-Huanwu decoction,BYHWD)是否通过调节脑微血管内皮细胞(brain microvascular endothelial cells,BMECs)自噬发挥对脑缺血再灌注损伤(cerebral ischemia-reperfusion injury,CIRI)大鼠的脑保护作用。方法:(1)制备大鼠大脑中动脉栓塞/再灌注(middle cerebral artery occlusion/reperfusion,MCAO/R)模型,分为假手术(sham)组、模型(MCAO/R)组、BYHWD组和丁苯酞(3-n-butylphthalide,NBP)组。通过Zea Longa评分法检测各组大鼠神经功能缺损情况;2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)染色测定脑梗死体积;HE染色观察皮质缺血半暗带区的病理损伤情况;伊文思蓝(Evans blue,EB)染色检测血脑屏障(blood-brain barrier,BBB)通透性;透射电镜观察BMECs的超微结构;免疫荧光双染观察自噬相关蛋白微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)在BMECs中的表达情况及阳性细胞率;Western blot法检测大鼠脑缺血半暗带皮质区紧密连接蛋白ZO-1、claudin-5和occludin的表达水平。(2)制备大鼠BMECs氧糖剥夺/复氧复糖(oxygen-glucose deprivation/reoxygenation,OGD/R)模型。(1)将大鼠BMECs分为正常对照(control,CON)组、OGD/R组、二甲基亚砜组、雷帕霉素组和3-甲基腺嘌呤组,用单丹酰尸胺(monodansylcadaverine,MDC)染色观察细胞的自噬程度;(2)将大鼠BMECs分为CON组、OGD/R组、含BYHWD血清(BYHWD-containing serum,BHDS)组和NBP组,CCK-8法检测细胞活力,MDC染色和Western blot方法检测细胞自噬程度。结果:(1)与sham组相比,MCAO/R组神经功能学评分增高(P<0.01);脑梗死体积增加(P<0.01);缺血半暗带区损伤严重,组织结构紊乱,细胞间隙增宽、结构消失;EB染料渗透严重(P<0.01);BMECs结构被破坏,细胞膜结构损伤,高尔基体肿胀,内质网囊泡化扩张,线粒体损伤;LC3+CD31+/CD31+比值显著升高(P<0.01);ZO-1、claudin-5和occludin蛋白的表达水平升高(P<0.05)。与MCAO/R组相比,BYHWD组和NBP组�AIM:This study aims to investigate the protective effect of Buyang-Huanwu decoction(BYHWD)on cerebral ischemia-reperfusion injury(CIRI)in rats,focusing on its role in regulating the autophagy of cerebral microvascular endothelial cells(BMECs).METHODS:(1)We established a rat model of middle cerebral artery occlusion/reperfusion(MCAO/R)and divided the subjects into four groups:sham group,model(MCAO/R)group,BYHWD group,and 3-n-butylphthalide(NBP)group.Neurological deficits were assessed using the Zea Longa score,while the volume of cerebral infarction was measured through 2,3,5-triphenyltetrazolium chloride(TTC)staining.Pathological damage in the ischemic penumbra was evaluated using HE staining,and blood-brain barrier(BBB)permeability was assessed by Evans blue(EB)staining.The ultrastructure of BMECs was analyzed by transmission electron microscopy,and the co-expression and positive cell rate of microtubule-associated protein 1 light chain 3(LC3)in BMECs were determined through immunofluorescence double staining.Additionally,the protein expression levels of ZO-1,claudin-5 and occludin in the cortical region of the ischemic penumbra in rats were examined using Western blot analysis.(2)A rat BMEC model of oxygen-glucose deprivation/reoxygenation(OGD/R)was also established.Rat BMECs were categorized into normal control(CON),OGD/R,dimethyl sulfoxide(DMSO),rapamycin and 3-methyladenine groups to observe autophagy levels by monodansylcadaverine(MDC)staining.Furthermore,rat BMECs were divided into CON,OGD/R,BYHWD-containing serum(BHDS)and NBP groups.The cell autophagy was assessed by MDC staining and Western blot,while cell viability was measured by CCK-8 assay.RESULTS:(1)The rats in MCAO/R group exhibited significantly higher neurological scores(P<0.01)and increased cerebral infarction volumes(P<0.01)compared with sham group.Severe damage in the ischemic penumbra was observed,characterized by disordered tissue structure,widened intercellular spaces,and compromised cellular integrity.The EB dye permeability was notably el

关 键 词:补阳还五汤 脑微血管内皮细胞 自噬 血脑屏障 脑缺血再灌注损伤 

分 类 号:R285.5[医药卫生—中药学] R743.3[医药卫生—中医学] R363

 

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