脑卒中后脑血管内皮细胞内质网应激抑制Wnt7/β-catenin通路导致血脑屏障损伤的机制研究  

Mechanism of blood-brain barrier damage caused by the inhibition of Wnt7/β-cateninpathway induced by endoplasmic reticulum stress in cerebrovascular endothelial cellsafter stroke

作　　者：董海平[1] 谢海怡 马晓晓 王震虹[1] DONG Haiping;XIE Haiyi;MA Xiaoxiao;WANG Zhenhong(Department of Anesthesiology,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200120,China)

机构地区：[1]上海交通大学医学院附属仁济医院麻醉科,上海200120

出　　处：《上海交通大学学报（医学版）》2023年第7期829-838,共10页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(82071290);上海市自然科学基金(20ZR1433400);上海市卫生健康委员会青年资助项目(20194Y0072)。

摘　　要：目的·探讨缺血性脑卒中后脑血管内皮细胞Wnt7/β-catenin信号失活是否导致血脑屏障(blood-brain barrier,BBB)完整性破坏,并研究内质网应激爆发是否介导了Wnt7/β-catenin通路的抑制。方法·采用线栓法阻断小鼠大脑中动脉建立大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型,脑缺血60 min后拔除线栓。向MCAO模型小鼠腹腔注射内质网应激抑制剂4-苯基丁酸(4-phenylbutyric acid,4-PBA)作为4-PBA+MCAO组。并设置假手术组(Sham组)。MCAO后24 h用伊文思蓝(Evans blue,EB)测定小鼠BBB的通透性,干湿法测定脑组织含水量,免疫荧光测定小鼠脑血管内皮细胞和周细胞黏附性。对人脑微细血管内皮细胞(human brain microvascular endothelial cells,HBMECs)进行氧糖剥夺(oxygen and glucose deprivation,OGD)4 h,加入4-PBA培养24 h。细胞实验分为空白对照组、OGD组和OGD+4-PBA组。采用CCK-8测定细胞活力,通过检测FITC标记的牛血清白蛋白(FITC-BSA)的通过率来评估细胞通透性;通过ELISA测定HBMECs中血小板衍生生长因子β(platelet-derived growth factorβ,PDGF-β)的分泌水平;采用Fluo-3 AM钙离子荧光探针检测细胞的荧光强度并评估细胞内钙离子浓度,通过CM-H2DCFDA荧光探针测量活性氧(reactive oxygen species,ROS)含量以明确细胞内质网应激状态;蛋白质印迹法(Western blotting)检测HBMECs中的连接蛋白[紧密连接蛋白1(zonula occludens-1,ZO-1)和密封蛋白5(claudin-5)]、内质网应激蛋白[CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)、葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、门冬氨酸特异性半胱氨酸蛋白酶12(cysteine-containing aspartate-specific proteases 12,Caspase-12)]、Wnt7和β-连环蛋白(β-catenin)的表达水平。结果·MCAO模型小鼠脑梗死区水含量较假手术组增加,EB渗出量明显增多(均P<0.05),小鼠脑血管内皮细胞和周细胞之间的黏附性下降;腹腔注射4-PBObjective·To investigate whether the inactivation of Wnt7/β-catenin signaling causes the destruction of the bloodbrain barrier(BBB)in cerebrovascular endothelial cells after ischemic stroke,and investigate whether endoplasmic reticulum stress bursts mediates the inhibition of Wnt7/β-catenin pathway.Methods·The model of middle cerebral artery occlusion(MCAO)in mice was established by a monofilament nylon suture with a round tip,which was used to temporarily occlude the middle cerebral artery for 60 min.MCAO model mice were intraperitoneally injected with the endoplasmic reticulum stress blocker 4-phenylbutyric acid(4-PBA)as 4-PBA+MCAO group.Sham surgery group(Sham group)was set.Twenty-four hours after MCAO,Evans blue(EB)was used to measure the BBB permeability.The brain water content was calculated by dry-wet weight ratio,and the adhesion of cerebrovascular endothelial cells and pericytes in mice was measured by immunofluorescence.Human brain microvascular endothelial cells(HBMECs)were used to establish an oxygen and glucose deprivation(OGD)model for 4 h,and then cultured with 4-PBA for 24 h.Cells were divided into blank control group,OGD group,and OGD+4-PBA group for CCK-8 assay to determine the cell viability.FITC-labeled bovine serum albumin(FITC-BSA)was used to detect the cell permeability.The secretion of plateletderived growth factorβ(PDGF-β)was measured by ELISA.Fluo-3 AM fluorescence probe was used to detect the fluorescence intensity of cells to assess intracellular Ca2+concentration,and reactive oxygen species(ROS)content was measured by CMH2DCFDA fluorescence probe to clarify the endoplasmic reticulum stress state.Western blotting was used to examine the expression of connexins,including zonula occludens-1(ZO-1)and claudin-5,the expression of endoplasmic reticulum stress proteins,including CCAAT/enhancer-binding protein homologous protein(CHOP),glucose-regulated protein 78(GRP78),and cysteine-containing aspartate-specific proteases-12(Caspase-12),and the expression of Wnt7/β-catenin in HBMECs.Res

关 键 词：内质网应激 Wnt7/β-catenin 血脑屏障 缺血性脑卒中 

分 类 号：R743.33[医药卫生—神经病学与精神病学]