P-Glycoprotein Exacerbates Brain Injury Following Experimental Cerebral Ischemia by Promoting Proinflammatory Microglia Activation

Figure 9

Glucocorticoid receptor silence abolishes the influence of P-glycoprotein manipulation on inflammatory cytokines and microglial polarization following oxygen glucose deprivation/reoxygenation. Endothelial cells (bEnd.3) were transfected with P-glycoprotein (P-gp) and glucocorticoid receptor (GR) siRNA, P-gp pcDNA3.1 plasmid and GR siRNA, individual negative control siRNA, or untransfected, and then subjected to either oxygen glucose deprivation/reoxygenation (OGD/R) treatment or normal culture conditions. Following 24 hr coculture with microglia (BV2), bEnd.3 cells were harvested for immunofluorescence assay, while BV2 cells were harvested for RT-PCR assay and medium was collected for ELISA assay. (a, b) Representative immunofluorescence staining images and quantification of GR expression (n = 3). (c, d) mRNA expression levels of M1 and M2 markers measured via RT-PCR assay as fold changes relative to control treatment (n = 4). (e) mRNA expression level of CCL2 measured via RT-PCR assay as fold changes relative to control treatment (n = 4) and content of CCL2 determined by ELISA assay (n = 3). Scale bars, 100 μm. One-way ANOVA followed by the post hoc least significant difference test or Games Howell test for (b) and (e). Mann–Whitney test for (c), (d), and (e). All data are mean ± SD; , between two groups.

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