S1PR5激动与过表达通过调控氧化应激增强脑微血管内皮细胞屏障功能抵抗氧糖剥夺/复氧复糖损伤

doi:10.12122/j.issn.1673-4254.2025.07.11

摘要/Abstract

摘要：

目的探讨鞘氨醇1-磷酸受体5（S1PR5）对氧糖剥夺及复氧复糖（OGD/R）诱导的小鼠脑微血管内皮细胞屏障功能的影响及相关机制。方法体外培养小鼠脑微血管内皮细胞bEnd.3，使用OGD/R诱导屏障功能障碍，分别使用S1PR5特异性激动剂A971432、敲低S1PR5的siRNA及过表达S1PR5的慢病毒进行干预。设置对照组：bEnd.3正常培养；OGD/R组：bEnd.3进行OGD/R；A971432组：OGD/R组+A971432；siNC组：转染siNC+OGD/R；siS1PR5组：转染siS1PR5+OGD/R；OE NC组：感染慢病毒LV5-NC+OGD/R；OE S1PR5组：感染慢病毒LV5-S1PR5+OGD/R。RT-qPCR法分别检测敲低或过表达S1PR5的效率；采用CCK-8检测在不同培养条件下bEnd.3细胞的活力；采用FITC-dextran渗透法检测内皮屏障渗透性；采用细胞免疫荧光法检测蛋白的定位及表达；DCFH-DA探针法检测细胞内活性氧水平；Western blotting法检测蛋白的表达水平。结果 CCK-8结果显示激动S1PR5可以增加OGD/R下调的细胞活力（P<0.0001），FITC-dextran渗透法结果显示激动和过表达S1PR5可减少FITC-dextran的渗漏（P<0.001），而敲低S1PR5增加FITC-dextran的渗漏（P<0.001）。Western blotting及免疫荧光结果显示，与OGD/R组相比，激动和过表达S1PR5可以增加屏障蛋白ZO-1（P<0.05）、Occludin（P<0.05）的表达，而敲低S1PR5会下调ZO-1和Occludin（P<0.05）的表达。ROS检测结果显示，激动和过表达S1PR5能减少ROS的产生，而敲低S1PR5会增加ROS产生。Western blotting检测发现过表达S1PR5可以增加抗氧化蛋白Nrf2（P<0.0001）、HO-1（P<0.0001）、SOD2（P<0.01）的表达。结论 S1PR5受体的激动剂干预及基因过表达可显著改善OGD/R模型诱导的活力减少及通透性增加，而基因敲低S1PR5则加剧OGD/R诱导的功能损伤，其机制可能与减少ROS，上调抗氧化蛋白表达相关。

关键词: 鞘氨醇1-磷酸受体5, 血脑屏障功能障碍, 氧化应激, 氧糖剥夺/复氧复糖损伤

Abstract:

Objective 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. Conclusion 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.

Key words: sphingosine 1-phosphate receptor 5, blood-brain barrier dysfunction, oxidative stress, oxygen-glucose deprivation and reoxygenation

王静娴, 任自敬, 周佩洋. S1PR5激动与过表达通过调控氧化应激增强脑微血管内皮细胞屏障功能抵抗氧糖剥夺/复氧复糖损伤[J]. 南方医科大学学报, 2025, 45(7): 1451-1459.

Jingxian WANG, Zijing REN, Peiyang ZHOU. S1PR5 activation or overexpression enhances barrier function of mouse brain microvascular endothelial cells against OGD/R injury by modulating oxidative stress[J]. Journal of Southern Medical University, 2025, 45(7): 1451-1459.

图/表 6

表1 S1PR5 siRNA靶向序列

Tab.1 S1PR5-specifc siRNA sequences used in this study

Gene	Targeting sequence (5'-3')
siRNA1	Sense: GCUAUUGCUCGGGGCUGCUATT Antisense: UAGCAGCCCCGAGCAAUAGCTT
siRNA2	Sense: GCUCUACGCCAAGGCCUAUTT Antisense: AUAGGCCUUGGCGUAGAGCTT
siRNA3	Sense: GCUCUACGCCAAGGCCUAUTT Antisense: ACGUGACACGUUCGGAGAATT
siNC	Sense: UUCUCCGAACGUGUCACGUTT Antisense: ACGUGACACGUUCGGAGAATT

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.

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