Huang Liangliang, Chen Yan, Liu Rui, Li Binbin, Fei Xuan, Li Xiang, Liu Ge, Li Yunman, Xu Baohui, Fang Weirong
1State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, China.
2Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA.
Aging Dis. 2022 Oct 1;13(5):1546-1561. doi: 10.14336/AD.2022.0225.
P-glycoprotein (P-gp) is expressed on brain microvessel endothelial cells of blood-brain barrier (BBB) and elevated after cerebral ischemia. In this study, we explored the influence and potential mechanisms of P-gp on BBB function in experimental ischemic stroke and . Middle cerebral artery occlusion/reperfusion (MCAO/R) was created in mice. Oxygen-glucose deprivation/reoxygenation (OGD/R) was performed in brain microvascular vessel-derived endothelial cells (bEnd.3) to mimic ischemia/reperfusion injury . P-gp-specific siRNA and pharmacological inhibitor cyclosporine A were used to inhibit P-gp, whereas pcDNA3.1 was utilized to overexpress P-gp. Twenty-four hours after reperfusion, acute ischemic stroke outcome, BBB integrity and permeability, autophagic proteins and relative signaling pathways were evaluated. P-gp levels were markedly elevated in mouse brain and endothelial cells following MCAO/R and OGD/R, respectively. P-gp siRNA silencing or pharmacologically inhibiting (cyclosporine A) reduced infarct volume and brain edema, attenuated brain pathology, and improved neurological behavior in association with attenuated accumulation of neutrophils and macrophages, reduced expression levels of inflammatory cytokines (TNF-α and IL-1β), matrix metalloproteinases (MMP-2 and MMP-9) and adhesion molecules (ICAM-1 and VCAM-1). P-gp silence also counteracted BBB leakage, restored the expressions of tight junction proteins (Claudin-5, Occludin and ZO-1), activated autophagic proteins (upregulated LC3-II/LC3-I and Beclin 1, and downregulated P62), and diminished Akt/mTOR signal activity in mice following MCAO/R. In the endothelial cell OGD/R assay, P-gp silence downregulated the expressions of inflammatory cytokines and adhesion molecules, inhibited leukocytes adhesion and migration, increased tight junction protein levels, and activated autophagy, all were reversible by forceful P-gp expression. Additionally, treatment with an autophagy inhibitor (3-methyladenine) abolished protections against ischemic stroke and tight junction proteins reduction followed by P-gp silence. In conclusion, increased P-gp expression after ischemic injury resulted in BBB dysfunction and hyperpermeability by suppressing Akt/mTOR-induced endothelial autophagy.
P-糖蛋白（P-gp）在血脑屏障（BBB）的脑微血管内皮细胞上表达，且在脑缺血后升高。在本研究中，我们探讨了P-gp对实验性缺血性中风中血脑屏障功能的影响及其潜在机制。在小鼠中制造大脑中动脉闭塞/再灌注（MCAO/R）模型。在脑微血管来源的内皮细胞（bEnd.3）中进行氧糖剥夺/复氧（OGD/R）以模拟缺血/再灌注损伤。使用P-gp特异性小干扰RNA（siRNA）和药物抑制剂环孢素A抑制P-gp，而利用pcDNA3.1过表达P-gp。再灌注24小时后，评估急性缺血性中风的结局、血脑屏障的完整性和通透性、自噬蛋白及相关信号通路。在MCAO/R和OGD/R后，小鼠脑和内皮细胞中的P-gp水平分别显著升高。P-gp siRNA沉默或药物抑制（环孢素A）可减少梗死体积和脑水肿，减轻脑病理学改变，并改善神经行为，同时伴有中性粒细胞和巨噬细胞的积聚减少、炎性细胞因子（肿瘤坏死因子-α和白细胞介素-1β）、基质金属蛋白酶（MMP-2和MMP-9）及黏附分子（细胞间黏附分子-1和血管细胞黏附分子-1）的表达水平降低。P-gp沉默还可对抗血脑屏障渗漏，恢复紧密连接蛋白（Claudin-5、闭合蛋白和闭锁小带蛋白-1）的表达，激活自噬蛋白（上调微管相关蛋白1轻链3-II/微管相关蛋白1轻链3-I和Beclin 1，下调P62），并降低MCAO/R后小鼠的Akt/mTOR信号活性。在内皮细胞OGD/R实验中，P-gp沉默可下调炎性细胞因子和黏附分子的表达，抑制白细胞黏附和迁移，增加紧密连接蛋白水平，并激活自噬，而强力表达P-gp可使所有这些作用逆转。此外，用自噬抑制剂（3-甲基腺嘌呤）处理可消除P-gp沉默对缺血性中风的保护作用以及紧密连接蛋白减少的现象。总之，缺血性损伤后P-gp表达增加通过抑制Akt/mTOR诱导的内皮细胞自噬导致血脑屏障功能障碍和通透性增加。
