Chen Yan, Fei Xuan, Liu Ge, Li Xiang, Huang Liangliang, Yang Lele Zixin, Li Yunman, Xu Baohui, Fang Weirong
State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing 210009, Jiangsu, China.
Penn State University, University Park, State College, PA 16802, USA.
Oxid Med Cell Longev. 2023 Dec 16;2023:6916819. doi: 10.1155/2023/6916819. eCollection 2023.
Microglia are activated following cerebral ischemic insult. P-glycoprotein (P-gp) is an efflux transporter on microvascular endothelial cells and upregulated after cerebral ischemia. This study evaluated the effects and possible mechanisms of P-gp on microglial polarization/activation in mice after ischemic stroke. P-gp-specific siRNA and adeno-associated virus (p-AAV) were used to silence and overexpress P-gp, respectively. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) were performed in mice and cerebral microvascular endothelial cells (bEnd.3) , respectively. OGD/R-injured bEnd.3 cells were cocultured with mouse microglial cells (BV2) in Transwell. Influences on acute ischemic stroke outcome, the expression of inflammatory cytokines, and chemokines and chemokines receptors, microglial polarization, glucocorticoid receptor (GR) nuclear translocation, and GR-mediated mRNA decay (GMD) activation were evaluated via reverse transcription real-time polymerase chain reaction, western blot, or immunofluorescence. Silencing P-gp markedly alleviated experimental ischemia injury as indicated by reduced cerebral infarct size, improved neurological deficits, and reduced the expression of interleukin-6 (IL-6) and IL-12 expression. Silencing P-gp also mitigated proinflammatory microglial polarization and the expression of C-C motif chemokine ligand 2 (CCL2) and its receptor CCR2 expression, whereas promoted anti-inflammatory microglia polarization. Additionally, P-gp silencing promoted GR nuclear translocation and the expression of GMD relative proteins in endothelial cells. Conversely, overexpressing P-gp via p-AAV transfection offset all these effects. Furthermore, silencing endothelial GR counteracted all effects mediated by silencing or overexpressing P-gp. Elevated P-gp expression aggravated inflammatory response and brain damage after ischemic stroke by augmenting proinflammatory microglial polarization in association with increased endothelial CCL2 release due to GMD inhibition by P-gp.
脑缺血损伤后小胶质细胞被激活。P-糖蛋白（P-gp）是微血管内皮细胞上的一种外排转运蛋白，脑缺血后上调。本研究评估了P-gp对缺血性脑卒中后小鼠小胶质细胞极化/激活的影响及可能机制。分别使用P-gp特异性小干扰RNA（siRNA）和腺相关病毒（p-AAV）使P-gp沉默和过表达。分别在小鼠和脑微血管内皮细胞（bEnd.3）中进行大脑中动脉闭塞/再灌注（MCAO/R）和氧糖剥夺/复氧（OGD/R）。将OGD/R损伤的bEnd.3细胞与小鼠小胶质细胞（BV2）在Transwell中共培养。通过逆转录实时聚合酶链反应、蛋白质免疫印迹或免疫荧光评估对急性缺血性脑卒中结局、炎性细胞因子及趋化因子和趋化因子受体表达、小胶质细胞极化、糖皮质激素受体（GR）核转位以及GR介导的mRNA降解（GMD）激活的影响。沉默P-gp可显著减轻实验性缺血损伤，表现为脑梗死体积减小、神经功能缺损改善以及白细胞介素-6（IL-6）和IL-12表达降低。沉默P-gp还减轻了促炎性小胶质细胞极化以及C-C基序趋化因子配体2（CCL2）及其受体CCR2的表达，而促进了抗炎性小胶质细胞极化。此外，P-gp沉默促进了GR核转位以及内皮细胞中GMD相关蛋白的表达。相反，通过p-AAV转染过表达P-gp抵消了所有这些作用。此外，沉默内皮细胞中的GR可抵消沉默或过表达P-gp介导的所有作用。P-gp表达升高通过增强促炎性小胶质细胞极化并伴随因P-gp抑制GMD导致内皮细胞CCL2释放增加，加重了缺血性脑卒中后的炎症反应和脑损伤。
Int J Stroke. 2022-1
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