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Published on: November 14, 2020
P-Glycoprotein Exacerbates Brain Injury Following Experimental Cerebral Ischemia by Promoting Proinflammatory
1State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing 210009, Jiangsu, China.
Abstract:
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) in vitro, 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.
Insights
Silencing P-glycoprotein (P-gp) reduces brain damage after ischemic stroke by decreasing inflammation and promoting beneficial microglial changes. This involves enhancing glucocorticoid receptor (GR) signaling in endothelial cells.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia activation is a key response to cerebral ischemia.
- P-glycoprotein (P-gp), an efflux transporter, is upregulated after ischemic stroke.
- P-gp's role in microglial polarization post-stroke is not fully understood.
Purpose of the Study:
- To investigate the effects of P-glycoprotein (P-gp) on microglial polarization and activation after ischemic stroke.
- To elucidate the underlying mechanisms involving glucocorticoid receptor (GR) and GR-mediated mRNA decay (GMD).
Main Methods:
- Utilized P-gp-specific siRNA and adeno-associated virus (p-AAV) for gene manipulation in mouse models of ischemic stroke (MCAO/R) and in vitro cell cultures (OGD/R).
- Assessed stroke outcomes, inflammatory markers (IL-6, IL-12, CCL2), microglial polarization (pro- vs. anti-inflammatory), GR nuclear translocation, and GMD activation.
- Employed techniques including RT-qPCR, Western blot, and immunofluorescence.
Main Results:
- P-gp silencing significantly reduced cerebral infarct size and improved neurological deficits.
- Silencing P-gp decreased pro-inflammatory cytokines (IL-6, IL-12) and chemokines (CCL2/CCR2), while promoting anti-inflammatory microglial polarization.
- P-gp silencing enhanced endothelial GR nuclear translocation and GMD activation; conversely, P-gp overexpression reversed these effects.
- Endothelial GR silencing counteracted the effects of P-gp manipulation.
Conclusions:
- Elevated P-gp expression exacerbates brain damage and inflammation post-ischemic stroke.
- P-gp aggravates the inflammatory response by promoting pro-inflammatory microglial polarization.
- This occurs via increased endothelial CCL2 release due to P-gp-mediated inhibition of GMD, highlighting P-gp as a therapeutic target.
