Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood-brain barrier injury through the Wnt/β-catenin signalling pathway.
Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood-brain barrier injury through the Wnt/β-catenin signalling pathway.
复制标题
抑制免疫蛋白酶体 LMP2 通过 Wnt/β-catenin 信号通路改善缺血/缺氧引起的血脑屏障损伤
DOI：
10.1186/s40779-021-00356-x
复制
发表时间：
2021-12-03
影响因子：
21.1
通讯作者：
Wang YZ
中科院分区：
文献类型：
--
作者：
Chen XY;Wan SF;Yao NN;Lin ZJ;Mao YG;Yu XH;Wang YZ
中风后血脑屏障（BBB）的破坏可导致脑损伤和神经功能障碍。先前的工作证实了低分子量肽2（LMP 2）的免疫蛋白酶体亚单位参与缺血性卒中的病理生理。然而，免疫蛋白酶体LMP 2和血脑屏障之间的关系仍不清楚。成年雄性Sprague-Dawley大鼠进行短暂的大脑中动脉闭塞/再灌注（MCAO/R）。在MCAO前三天，通过立体定向注射到同侧半球区域，用慢病毒介导的LMP 2 shRNA制剂治疗大鼠。用体外培养的大鼠脑微血管内皮细胞（RBMVEC）模拟缺血状态，进行氧-糖剥夺/再灌注（OGD/R）。在体内和体外分析RNA干扰介导的LMP 2或β-连环蛋白的敲低。通过分析脑组织伊文思蓝（EB）渗出量和荧光血管造影评价血脑屏障的完整性。免疫荧光和Western blotting检测目的蛋白的表达。使用划痕迁移试验评价细胞迁移。使用ImageJ软件（版本1.53m）定量免疫荧光、Western印迹和细胞迁移的结果。使用单因素方差分析比较不同组的参数数据，然后进行最小显着差异（LSD）检验。与假手术组相比，脑缺血导致MCAO/R组血脑屏障结构成分如紧密连接蛋白（occludin、claudin-1和ZO-1）的水平降低（P < 0.001）。然而，免疫蛋白酶体LMP 2的抑制恢复了这些蛋白的表达，导致LMP 2-shRNA组中闭合蛋白、claudin-1和ZO-1的水平高于对照-shRNA组（P < 0.001）。此外，免疫蛋白酶体LMP 2的抑制有助于更高的微血管密度和降低的BBB通透性[例如，EB：LMP 2-shRNA组渗出量为（58.54 ± 7.37）μg/g，对照组为（103.74 ± 4.32）μg/g，P < 0.001]，并促进MCAO/R大鼠Wnt-3a和β-catenin蛋白表达上调。体外实验中，与正常培养条件下的对照组相比，OGD/R诱导RBMVECs中LMP 2、促凋亡蛋白Bax和切割的caspase-3表达显著上调，occludin、claudin-1、ZO-1和Bcl-2表达显著下调，Wnt/β-catenin途径Wnt-3a和β-catenin蛋白表达显著抑制（P < 0.001）。然而，沉默LMP 2基因表达逆转了这些蛋白质的变化，并促进了OGD/R后RBMVECs的增殖和迁移。与对照组相比，β-catenin-siRNA转染抑制β-catenin表达，可加重OGD/R后RBMVECs紧密连接蛋白表达的下调，抑制RBMVECs的增殖和迁移（P < 0.001）。LMP 2-siRNA和β-catenin-siRNA共转染部分抵消了沉默LMP 2-siRNA对暴露于OGD/R的RBMVECs中紧密连接蛋白水平的有益作用。这项研究表明，抑制免疫蛋白酶体LMP 2改善缺血/缺氧诱导的BBB损伤，其分子机制涉及缺血条件下免疫蛋白酶体调节的Wnt/β-catenin信号通路的激活。在线版本包含补充材料，可通过10.1186/s40779-021-00356-x获得。
Disruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear. Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53m). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test. Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins (occludin, claudin-1 and ZO-1) in the MCAO/R group compared with the sham group (P < 0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P < 0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54 ± 7.37) µg/g vs. control-shRNA group (103.74 ± 4.32) µg/g, P < 0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P < 0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P < 0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R. This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions. The online version contains supplementary material available at 10.1186/s40779-021-00356-x.
登录
查看更多内容
DOI：
10.1177/1073858408327809
发表时间：
2009-06
期刊：
The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry
影响因子：
--
作者：
Meller R
通讯作者：
Meller R
缺血性中风中的血脑屏障损伤及其内皮机械传导的调节。
影响因子：
4
作者：
Nian K;Harding IC;Herman IM;Ebong EE
通讯作者：
Ebong EE
影响因子：
3.1
作者：
Angeles, Arkhjamil;Fung, Gabriel;Luo, Honglin
通讯作者：
Luo, Honglin
免疫蛋白酶体的抑制通过增强大鼠局灶性脑缺血后缺氧诱导因子-1α的丰度来促进血管生成。
影响因子：
15.1
作者：
Chen, Xingyong;Zhang, Xu;Wang, Yinzhou
通讯作者：
Wang, Yinzhou
影响因子：
5.5
作者：
Abdullahi, Wazir;Tripathi, Dinesh;Ronaldson, Patrick T.
通讯作者：
Ronaldson, Patrick T.