[S1PR5 activation or overexpression enhances barrier function of mouse brain microvascular endothelial cells against OGD/R injury by modulating oxidative stress]

Abstract

Objectives: To investigate the role of sphingosine-1-phosphate receptor 5 (S1PR5) in modulating barrier function of mouse brain microvascular endothelial cells with oxygen-glucose deprivation and reoxygenation (OGD/R).

Methods: Mouse brain microvascular endothelial cells (bEnd.3) were exposed to OGD/R to induce barrier dysfunction following treatment with S1PR5-specific agonist A971432 or lentivirus-mediated transfection with a S1PR5-specific siRNA, a S1PR5-overexpressing plasmid, or their respective negative control sequences. The changes in viability and endothelial barrier permeability of the treated cells were evaluated with CCK-8 assay and FITC-dextran permeability assay; the levels of intracellular reactive oxygen species (ROS) and localization and expression levels of the proteins related with barrier function and oxidative stress were detected using immunofluorescence staining, DCFH-DA probe and Western blotting.

Results: S1PR5 activation obviously enhanced viability of bEnd.3 cells exposed to OGD/R (P<0.0001). Both activation and overexpression of S1PR5 reduced FITC-dextran leakage, while S1PR5 knockdown significantly increased FITC-dextran leakage in the exposed bEnd.3 cells. Activation and overexpression of S1PR5 both increased the cellular expressions of the barrier proteins ZO-1 and occludin, while S1PR5 knockdown produced the opposite effect. In cells exposed to OGD/R, ROS production was significantly reduced by S1PR5 activation and overexpression but increased following S1PR5 knockdown. Overexpression of S1PR5 obviously increased the expressions of the antioxidant proteins Nrf2, HO-1 and SOD2 in the exposed cells.

Conclusions: S1PR5 activation and overexpression significantly improve cell viability and reduce permeability of a mouse brain microvascular endothelial cell model of OGD/R, the mechanism of which may involve the reduction in ROS production and upregulation of the antioxidant proteins.

Keywords: blood-brain barrier dysfunction; oxidative stress; oxygen-glucose deprivation and reoxygenation; sphingosine 1-phosphate receptor 5.

图1

OGD处理降低bEnd.3细胞活性并下调细胞间紧密连接蛋白的表达 Fig.1 Oxygen-glucose deprivation (OGD) decrease viability of bEnd.3 cells and down-regulate expressions of intercellular tight junction proteins. A: Representative images of bEnd.3 cells after OGD treatment. B: Viability of b.End3 cells assessed with CCK-8 assay (n=6). C: Western blotting of tight junction proteins (ZO-1 and occludin) and VE-cadherin after OGD. D-F: Quantification of ZO-1, VE-cadherin, and occludin protein expression levels (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Con.

图2

激动S1PR5有助于恢复OGD下调的细胞活性并上调细胞间紧密连接蛋白的表达 Fig.2 Activation of S1PR5 restores viability of OGD-damaged cells and up-regulates expressions of intercellular tight junction proteins. A: CCK8 assay for assessing viability of cultured bEnd.3 cells treated with different concentrations of A971432. B: CCK8 assay for assessing viability of bEnd.3 cells exposed to OGD after treatment with different concentrations of A971432 (****P<0.0001 vs 0 μmol/L A971432). C: Schematic diagram for detecting endothelial permeability of bEnd.3 cells using FITC-Dextran. D: Detection of endothelial permeability using FITC-Dextran. E: Western blotting of ZO-1 and occluding expressions in bEnd.3 cells with OGD/R after S1PR5 activation. F, G: Quantitative analysis of the expression levels of ZO-1 and occludin. H: Changes in the fluorescence intensity of tight junction proteins caused by activation of S1PR5 in bEnd.3 cells with OGD/R (scale bar= 50 μm). *P<0.05, **P<0.01, ****P<0.0001 vs Con; ^# P<0.05, ^## P<0.01, ^#### P<0.0001 vs OGD/R.

图3

敲低S1PR5加剧OGD诱导的细胞活力下降及紧密连接蛋白的减少 Fig.3 S1PR5 knockdown exacerbates the reduction of cell viability and tight junction protein expressions in OGD-induced bEnd.3 cells. A: Efficiency of S1PR5 knockdown assessed by RT-qPCR. B: Endothelial permeability of the transfected cells detected by FITC-Dextran. C: Alterations in the fluorescence intensity of the tight junction proteins in bEnd.3 cells with S1PR5 knockdown following OGD/R. D-F: Western blotting for detecting the expression levels of ZO-1 and occluding in bEnd.3 cells with OGD/R after S1PR5 knockdown. *P<0.05, **P<0.01 vs Con; ^# P<0.05, ^### P<0.001 vs OGD/R.

图4

过表达S1PR5抑制OGD诱导的细胞活力下降及紧密连接蛋白的减少 Fig.4 Overexpression of S1PR5 enhances viability and tight junction protein expressions in OGD-induced bEnd.3 cells. A: RT-qPCR for assessing efficiency of lentivirus-mediated S1PR5 overexpression (**P<0.01 vs OENC). B: Endothelial permeability of the cells detected by FITC-Dextran. C: Changes in the fluorescence intensity of tight junction proteins in OGD-induced bEnd.3 cells with S1PR5 overexpression. D-F: Western blotting for detecting expression levels of ZO-1 and occludin in OGD-induced bEnd.3 cells with S1PR5 overexpression. *P<0.05, **P<0.01, ****P<0.0001 vs Con; ^# P<0.05, ^## P<0.01, ^### P<0.001 vs OGD/R.

图5

S1PR5介导Nrf-2/HO-1通路发挥抗氧化作用 Fig.5 S1PR5 overexpression produces antioxidant effect in OGD-induced bEnd.3 cells via the Nrf-2/HO-1 pathway. A: Effects of S1PR5 knockdown and overexpression on intracellular reactive oxygen species in OGD-induced bEnd.3 cells (scale bar=50 μm). B-E: Western blotting for detecting expression levels of Nrf2, HO-1 and SOD2 in OGD-induced bEnd.3 cells with S1PR5 knockdown or overexpression. *P<0.05 vs Con, ^## P<0.01, ^#### P<0.0001 vs OGD/R.