The Role Of Sphingosine-1-phosphate-5receptor In H₂O₂-induced Injury Of Brain Microvascular Endothelial Cells

Background: During acute ischemic stroke（AIS）,brain cells are rapidly ischemic and anoxic,which makes neurons,microglia,astrocytes and other cells carry out more anaerobic metabolism to synthesize energy,leading to a high level of intracellular oxidative stress.A great fraction of harmful substances such as reactive oxygen species（ROS）and inflammatory factors damage the integrity of the blood-brain barrier（BBB）,leading to the entry of peripheral toxic cells and molecules into brain to aggravate brain damage.Serious complications such as cerebral edema and hemorrhagic transformation may also occur due to BBB injury after reperfusion therapy.Therefore,alleviating the injury of vascular endothelial cells caused by oxidative stress is crucial for protecting the BBB function and reducing neurological damage during AIS.In recent years,it has been found that sphingosine 1-phosphate receptor 5（S1PR5）can maintain the integrity of the BBB and mediate the immune quiescence of BBB in central nervous system disease models such as multiple sclerosis in vitro and animal model of Huntington’s disease,suggesting that S1PR5 has important implications to mainten the structure and function of BBB.However,it is not clear whether S1PR5 is involved in the BBB dysfunction induced by oxidative stress in stroke and its molecular mechanism remains unknown.Objective: To investigate the role S1PR5 playing during oxidative stress injury in mouse brain microvascular endothelial cells（b End.3）in vitro.Methods: The CCK-8 assay was used to detect cell viability.The b End.3 cells were divided into control group,H₂O₂ group,H₂O₂+5 μmol/L A971432 group,H₂O₂+10 μmol/L A971432 group,H₂O₂+20 μmol/L A971432 group.The expression of S1PR5 was knocked down by transfecting b End.3 cells with siRNA,and the siRNA knockdown efficiency was detected by RT-q PCR.The endothelial permeability was evaluated using FITC-Dextran.Western blot was used to detect changes of S1PR5,ZO-1,VE-Cadherin,Occludin,Bax,Bcl2,Caspase-3,Erk1/2,p-Erk1/2,Nrf2,HO-1,SOD1 and SOD2.The immunofluorescence was used to observe the possible altering ZO-1 fluorescence intensity.We also used optical microscope to record the diversification of cell morphology.The level of cell reactive oxygen species（ROS）were measured by DCFH-DA probe.Results:（1）In comparison to the control group,b End.3 cells treated with 2 mmol/L H₂O₂ for 1.5 h could significantly reduce the cell viability,increase the permeability of vascular endothelial cells and decrease the expression of ZO-1,VE-Cadherin and Occludin,as well as the antioxidant proteins of Nrf2 and HO-1（P<0.05）.It is a stable oxidative stress injury model.Compared with the control group,the concentration of A971432 below 50 μmol/L（5,10,20 μmol/L）had no significant effect on cell viability（P>0.05）,which could be used in follow-up experiments.（2）Western Blot results showed that the expression of S1PR5 in b End.3 cells was significantly decreased after H₂O₂ treatment in comparison to the normal treatment group（P<0.05）.（3）In comparison to the H₂O₂ group,A971432 could inhibit H₂O₂-induced endothelial hyperpermeability,increase the expression of ZO-1,VE-Cadherin,and Occludin,as well reduce the MMP-9 protein expression associated with endothelial injury（P<0.05）.A971432 could also increase the fluorescence intensity of ZO-1 on cell membrane under H₂O₂ condition.（4）As against the H₂O₂ group,A971432 could increase the decrease of cell viability caused by H₂O₂,and decrease the ratio of apoptosis-related protein Bax/Bcl2 and the expression of Caspase-3（P<0.05）.Pretreatment with 20 μmol/L A971432 was found that it could significantly inhibit the increase of Erk1/2 phosphorylation induced by H₂O₂（P<0.05）.Observed under the microscope,A971432 pretreatment could maintain the cell morphology under H₂O₂ injury and reduce floating.（5）In comparison to H₂O₂ group,the intracellular ROS level detected by DCFH-DA probe showed that A971432 could reduce the total intracellular ROS level and increase the protein expression of antioxidant molecules Nrf2,HO-1,SOD1 and SOD2（P<0.05）.（6）Over against the siNC group,the si S1PR5 group significantly increased the vascular endothelial permeability and decreased the protein expressions of ZO-1,VE-Cadherin and Occludin（P<0.05）.Compared with the siNC+H₂O₂ group,the endothelial permeability of the si S1PR5+ H₂O₂ group was significantly increased,and the protein expressions of ZO-1,VE-Cadherin and Occludin were significantly decreased in the si S1PR5+H₂O₂ group（P<0.01）.Conclusion: Activation of S1PR5 with A971432 selectively can alleviate H₂O₂-induced b End.3 cells hyperpermeability and apoptosis and exert anti-oxidative effect by activating the Nrf2/HO-1 pathway possibly.However,down-regulation of S1PR5 in b End.3 cells can further aggravate vascular endothelial hyperpermeability induced by H₂O₂ injury.