S1PR5激动剂减轻H2O2诱导的脑微血管内皮细胞高通透性及氧化应激损伤

S1PR5 agonist attenuates H2O2 -induced hyperpermeability and oxidative stress injury of brain microvascular endothelial cells

目的鞘氨醇⁃1⁃磷酸酯受体5(S1PR5)在急性缺血性卒中(AIS)血脑屏障氧化应激损伤中的作用及机制尚不清楚。文章旨在探讨S1PR5在小鼠脑微血管内皮细胞(bEnd.3)氧化应激损伤中的作用及分子机制。方法将bEnd.3细胞分为对照组、H_(2)O_(2)组、H_(2)O_(2)+5μmol/L A971432(S1PR5特异性选择性激动剂)组、H_(2)O_(2)+10μmol/L A971432组、H_(2)O_(2)+20μmol/L A971432组。用CCK⁃8法检测细胞活力改变;FITC⁃Dextran渗透法检测血管内皮细胞通透性;Western Blot检测S1PR5、ZO⁃1、VE⁃Cadherin、Occludin、Bax、Bcl2、Caspase⁃3、p⁃Erk1/2、Erk1/2、Nrf2、HO⁃1、SOD1和SOD2的蛋白表达水平;免疫荧光观察ZO⁃1的荧光强度;光学显微镜下观察细胞形态变化;DCFH⁃DA探针法检测细胞活性氧水平。结果Western blot结果显示,与对照组bEnd.3细胞的S1PR5蛋白表达(1.00±0.01)相比,H_(2)O_(2)组(0.59±0.03)明显降低(P<0.01)。与对照组相比,H_(2)O_(2)显著增加血管内皮细胞通透性,减少ZO⁃1、VE⁃Cadherin、Occludin、Nrf2、HO⁃1、SOD1和SOD2蛋白表达以及ZO⁃1荧光强度,降低细胞活力,增加MMP⁃9、Bax/Bcl2、Caspase⁃3、p⁃Erk1/2/Erk1/2的蛋白表达和细胞内总活性氧(P<0.05)。与H_(2)O_(2)组[(75.33±3.76)%]相比,H_(2)O_(2)+10、20μmol/L A971432组[(93.34±0.49)%,(100.60±7.29)%]可以明显逆转H_(2)O_(2)诱导的细胞活力下降(P<0.05)。与H_(2)O_(2)组相比,H_(2)O_(2)+5、10、20μmol/L A971432组的血管内皮细胞通透性显著降低,ZO⁃1、VE⁃Cadherin、Occludin、Nrf2、HO⁃1、SOD1和SOD2蛋白表达和ZO⁃1荧光强度增加,细胞活力增加,MMP⁃9、Bax/Bcl2、Caspase⁃3、p⁃Erk1/2/Erk1/2蛋白表达和细胞内总活性氧降低(P<0.05)。结论A971432选择性激动S1PR5可减轻H_(2)O_(2)诱导的bEnd.3内皮细胞高通透性及凋亡,并可能通过激活Nrf2/HO⁃1通路发挥抗氧化损伤作用。与H_(2)O_(2)组相比,H_(2)O_(2)+10、20μmol/L A971432组可以明显逆转H_(2)O_(2)诱导的细胞活力下降(P<0.05)。

Objective The role and mechanism of sphingosine 1 phosphate receptor 5(S1PR5)in the oxidative stress injury of blood⁃brain barrier in acute ischemic stroke(AIS)remain unclear.This study aims to investigate the role and molecular mechanism of S1PR5 in oxidative stress injury of mouse brain microvascular endothelial cells(bEnd.3).Methods BEnd.3 cells were divided into control group,H_(2)O_(2) group,H_(2)O_(2)+5μmol/L A971432(S1PR5 specific selective agonist)group,H_(2)O_(2)+10μmol/L A971432 group,H_(2)O_(2)+20μmol/L A971432 group.Cell viability was detected by CCK⁃8 assay.The permeability of vascular endothelial cells was detec⁃ted by FITC⁃Dextran osmotic method.The protein expression levels of S1PR5,ZO⁃1,VE⁃cadherin,Occludin,Bax,Bcl2,Caspase⁃3,p⁃ERK1/2,Erk1/2,Nrf2,HO⁃1,SOD1 and SOD2 were detected by Western Blot.The fluorescence intensity of ZO⁃1 was observed by immunofluorescence.The changes of cell morphology were observed under light microscope.Reactive oxygen species were detected by DCFH⁃DA probe.Results The results of Western blot showed that the expression of S1PR5 protein in bEnd.3 cells in H_(2)O_(2) group(0.59±0.03)was significantly decreased compared with that in control group(1.00±0.01)(P<0.01).Compared with the control group,H_(2)O_(2) significantly increased the permeability of vascular endothelial cells,decreased the protein expressions of ZO⁃1,VE⁃cad⁃herin,Occludin,Nrf2,HO⁃1,SOD1 and SOD2,and the fluorescence intensity of ZO⁃1,and decreased the cell viability.The protein expression of MMP⁃9,Bax/Bcl2,caspase⁃3,P⁃ERK1/2/ERK1/2 and total intracellular reactive oxygen species were increased(P<0.05).Compared with H_(2)O_(2) group[(75.33±3.76)%],H_(2)O_(2)+10,20μmol/L A971432 groups[(93.34±0.49)%,(100.60±7.29)%]could significantly reverse the decrease of cell viability induced by H_(2)O_(2)(P<0.05).Compared with the H_(2)O_(2) group,the per⁃meability of vascular endothelial cells in H_(2)O_(2)+5,10,20μmol/L A971432 group was significantly decreased,the protein expression of ZO⁃1,VE⁃Cadherin,Occludin,Nrf2,HO⁃1,SOD1 and SOD2 and the fluorescence intensity of ZO⁃1 were increased,and the cell via⁃bility was increased.The protein expression of MMP⁃9,Bax/Bcl2,Caspase⁃3,P⁃ERK1/2/ERK1/2 and total intracellular reactive oxygen species were decreased(P<0.05).Conclusion Selective activation of S1PR5 by A971432 attenuates H_(2)O_(2)⁃induced hyper⁃permeability and apoptosis of bEnd.3 endothelial cells,and might play an anti⁃oxidative role by activating Nrf2/HO⁃1 pathway.Com⁃pared with H_(2)O_(2) group,H_(2)O_(2)+10 and 20μmol/L A971432 groups could significantly reverse the decrease of cell viability induced by H_(2)O_(2)(P<0.05).