The Experimental Study Of Hypertonic Saline Attenuates Ischemia And Hypoxia Injury Of Blood-brain Barrier Endothelial Cells Via EGFR Signaling Pathway

Background:Brain edema is an important cause of morbidity and mortality in critically ill patients caused by variety of brain diseases and systemic diseases.Both hypertonic saline(HS)and mannitol have been recommended as first-line drugs for the treatment of spontaneous intracerebral hemorrhage(ASA/AHA2007)guidelines.However,evidence suggests that mannitol has a "rebound" side effect and worse renal function,lower volume response and brain edema.In recent years,it has been found that HS has more advantages than traditional mannitol in treatment of brain edema and intracranial hypertension,such as better effect,more lasting effect and no intracranial pressure "rebound" phenomenon.The mechanism that HS reduces brain edema is not fully understood.The main cause of brain edema is the destruction of the blood-brain barrier(Blood Brain Barrier,BBB),yet the basic structure of BBB is endothelial cells.Endothelial cells and the tight junctions formed by them are located in the inner side of the vascular cavity,where is the direct and first contact site to HS.Therefore,protecting BBB endothelial cells is an important target for prevention and treatment of brain edema.Our research group has found that HS can reduce brain edema via inhibiting microglial cells releasing inflammatory cytokine such as TNF-alpha,IL-1 beta.However,whether HS could protect BBB endothelial cells is not clear.Objective:To study the effect and mechanism of HS on endothelial cell injury induced by ischemia and hypoxia in BBB cells.Methods:Both animal model and cell culture were appled for investigating the protective effect of HS on BBB endothelial cells by Evans blue,PCR,MTS,Western,blot,TUNEL and flow cytometry apoptosis assay,immunofluorescence,immunohistochemistry,transmission electron microscopy and recombinated adenovirus.Gene sequencing methods was further appled for determining the signal transduction pathway of endothelial cell injury.Results:Firstly,the SD rats were divided into sham operation group,model group,saline group and HS group(10%HS)after constructing the model of cerebral ischemia reperfusion injury.The results showed that,compared with ischemic group and saline group,the quantitative detection of brain tissue Evans decreased significantly(P<0.05)in 10%HS group,but still higher than that in the sham operation group(P<0.05);while there was no statistically significant between ischemia group and the saline group.Observed by transmission electron microscope,the ultrastructure of BBB endothelial cells is damaged in BBB ischemia group,such as endothelial cell shrinkage,cell vacuolization structure was destroyed,however the damage was significantly reduced in HS treatment group.Secondly,the model of oxygen glucose deprivation(OGD)was constructed by bEnd.3 endothelial cells and treated with 40mmol/L HS.A series of experimental techniques such as cell viability,flow cytometry and detection of cell apoptosis by TUNEL,transmission electron microscopy were applied and found that:compared with ODG group,HS group significantly improve endothelial cell viability(P<0.05),while decreased apoptosis(P<0.05)and the IL-1 the expression(P<0.05);observed by transmission electron microscopy showed that HS treatment can significantly reduce the mitochondrial damage and dissolution,cell shrinkage and ultrastructural.HS has obvious protective effects on BBB cells induced by ischemia and hypoxia injury.The gene sequencing method was appied to further investigate the molecular mechanism of HS on protecing BBB endothelial cells.Compared with the control group,the epidermal growth factor receptor(EGFR)gene expression was significantly up-regulated(P<0.05)in the OGD group,and the expression results are verified by Q-PCR.According to the results by sequencing,the bEnd.3 endothelial cells was treated with 40mmol/L HS and analyzed by Western Blot,immunohistochemistry and immunofluorescence method.The results showed that:compared with OGD group,the EGFR,cell apoptosis rate and expression of IL-1 beta was significantly decreased(P<0.05)in HS group.in addition,EGFR overexpression by plasmid in bEnd.3 endothelial cells can significantly increase IL-1 beta expression and apoptosis rate(P<0.05).Surprisingly,HS did not significantly reduce the expression of EGFR/p-EGFR,IL-1 expression and apoptosis rate in the EGFR overexpression models.The reason may be that the self regulatory function of BBB endothelial cells was impaired by overexpression of EGFR,and the protective ability of HS to BBB endothelial cells was lost.Concludion:This study showed that HS could reduce the damage of endothelial cells induced by ischemia and hypoxia correlated to the levels of EGFR and IL-1 beta,however,the HS probably has no protective effect for the endothelial cells with EGFR overexpression.