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

doi:10.3969/j.issn.1674-8115.2023.07.005

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (7): 829-838.doi: 10.3969/j.issn.1674-8115.2023.07.005

• 论著 · 基础研究 • 上一篇

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

董海平(), 谢海怡, 马晓晓, 王震虹()

上海交通大学医学院附属仁济医院麻醉科，上海 200120

收稿日期:2023-01-29 接受日期:2023-04-20 出版日期:2023-07-28 发布日期:2023-07-28
通讯作者: 王震虹 E-mail:397297461@qq.com;18621625707@163.com
作者简介:董海平（1988—），女，主治医师，博士生；电子信箱：397297461@qq.com。
基金资助:
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)

Mechanism of blood-brain barrier damage caused by the inhibition of Wnt7/β-catenin pathway induced by endoplasmic reticulum stress in cerebrovascular endothelial cells after stroke

DONG Haiping(), XIE Haiyi, MA Xiaoxiao, WANG Zhenhong()

Department of Anesthesiology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200120, China

Received:2023-01-29 Accepted:2023-04-20 Online:2023-07-28 Published:2023-07-28
Contact: WANG Zhenhong E-mail:397297461@qq.com;18621625707@163.com
Supported by:
National Natural Science Foundation of China(82071290);Shanghai Natural Science Foundation(20ZR1433400);Youth Funding Project of Shanghai Municipal Health Commission(20194Y0072)

摘要/Abstract

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 factor β，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-PBA后MCAO模型小鼠脑水肿程度减轻，BBB的渗透性降低（均P<0.05），脑血管内皮细胞和周细胞之间的黏附性增加。与空白对照组HBMECs比较，在OGD条件下细胞活力下降，通透性增加（均P<0.05）；同时，OGD组HBMECs中连接蛋白ZO-1、claudin-5的表达水平下降，PDGF-β的分泌水平减少，钙离子浓度升高，ROS含量明显上调，内质网应激蛋白CHOP、GRP78、Caspase-12的表达水平增加，Wnt7和β-catenin的表达水平下降（均P<0.05）。而当HBMECs的OGD模型经4-PBA处理后，OGD导致的HBMECs损伤减轻，细胞中连接蛋白的表达水平增加，HBMECs的通透性降低，PDGF-β的分泌水平增加，并且Wnt7/β-catenin信号的活性明显恢复（均P<0.05）。结论·脑卒中后脑血管内皮细胞内质网应激爆发导致Wnt7/β-catenin信号失活引起的内皮细胞损伤，是BBB破坏的关键途经。

关键词: 内质网应激, Wnt7/β-catenin, 血脑屏障, 缺血性脑卒中

Abstract:

Objective ·To investigate whether the inactivation of Wnt7/β-catenin signaling causes the destruction of the blood-brain 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 platelet-derived growth factor β (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 CM-H2DCFDA 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. Results ·Compared with the Sham group, the brain water content of the infarction area of mice increased after MCAO, and the exudation of EB increased significantly (both P<0.05). The adhesion between cerebrovascular endothelial cells and pericytes in mice was reduced after the occurrence of MCAO. After intraperitoneal injection of 4-PBA in mice with MCAO, the degree of brain edema and the exudation of EB were reduced (both P<0.05), and the adhesion between cerebrovascular endothelial cells and pericytes increased. Compared with the HBMECs of the blank control group, the viability of HBMECs after OGD decreased, and the permeability of HBMECs increased (both P<0.05). OGD condition also led to decreased expression of connexins (ZO-1 and claudin-5), decreased secretion of PDGF-β in HBMECs, increased expression of endoplasmic reticulum stress proteins (CHOP, GRP78 and Caspase-12), up-regulated intracellular Ca²⁺ concentration and ROS content, and decreased expression of Wnt7 and β-catenin in HBMECs (all P<0.05). After HBMECs were cultured with 4-PBA, the damage of HBMECs caused by OGD was reduced, and the expression of connexins increased. The permeability of HBMECs was reduced, and the secretion of PDGF-β was promoted (all P<0.05). After 4-PBA treatment, the activity of Wnt7/β-catenin signaling was significantly restored in the OGD model of HBMECs (P<0.05). Conclusion ·Wnt7/β-catenin signaling inactivation caused by endothelial reticulum stress bursts leads to cerebrovascular endothelial cell damage, which is the crucial pathway of BBB destruction after stroke.

Key words: endoplasmic reticulum stress, Wnt7/β-catenin pathway, blood-brain barrier (BBB), ischemic stroke

中图分类号:

R743.33

董海平, 谢海怡, 马晓晓, 王震虹. 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[J]. 上海交通大学学报（医学版）, 2023, 43(7): 829-838.

DONG Haiping, XIE Haiyi, MA Xiaoxiao, WANG Zhenhong. Mechanism of blood-brain barrier damage caused by the inhibition of Wnt7/β-catenin pathway induced by endoplasmic reticulum stress in cerebrovascular endothelial cells after stroke[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(7): 829-838.

图/表 4

图1 小鼠脑卒中对BBB的损伤以及抑制内质网应激对该损伤的缓解Note： A. EB exudation in the brain of mice. B. Water content of mice brain. C. Adhesion of cerebrovascular endothelial cells and pericytes in mice. Red arrows indicate overlap of CD31 and CD13. ①P=0.000, compared with the Sham group; ②P=0.011, ③P=0.000, compared with the MCAO group.

Fig 1 Injury of BBB after stroke in mice and the alleviating effect of endoplasmic reticulum stress inhibition on the injury

图2 OGD引起的HBMEC损伤以及抑制内质网应激对该损伤的缓解作用Note： A. Determination of activity of HBMECs by CCK-8 assay. B. The cell permeability of HBMECs analyzed with FITC-BSA leakage. C. Determination of the PDGF-β secretion of HBMECs by ELISA. D. Determination of the expression of ZO-1 and claudin-5 in HBMECs by Western blotting. ①P=0.000, ③P=0.002, ⑤P=0.001, compared with the Blank group; ②P=0.026, ④P=0.005, ⑥P=0.042, ⑦P=0.000, ⑧P=0.001, compared with the OGD group.

Fig 2 OGD-induced HBMEC damage and the alleviating effect of inhibiting endoplasmic reticulum stress on this injury

图3 OGD以及4-PBA处理对HBMEC内质网应激的影响Note： A. Intracellular Ca2+ level in HBMECs. B. Intracellular ROS level in HBMECs. C. Expression of endoplasmic reticulum stress proteins in HBMECs. ①P=0.003, ③P=0.000, compared with the Blank group; ②P=0.033, ④P=0.003, ⑤P=0.000, compared with the OGD group.

Fig 3 Effects of OGD and 4-PBA treatment on HBMEC endoplasmic reticulum stress

图4 抑制内质网应激对OGD后HBMEC内Wnt7/β-catenin信号的影响Note： A. Wnt7 and β-catenin protein expression bands. B. Analysis of Wnt7 protein expression in HBMECs. C. Analysis of β-catenin protein expression in HBMECs. ①P=0.000, ③P=0.002, compared with the Blank group; ②P=0.000, ④P=0.023, compared with the OGD group.

Fig 4 Effect of inhibiting endoplasmic reticulum stress on Wnt7/β-catenin signaling in HBMECs after OGD

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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-catenin pathway induced by endoplasmic reticulum stress in cerebrovascular endothelial cells after stroke

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