ENT1 Regulates Adenosine A1 Receptor Expression In Rat Brain After Cerebral Ischemia And Reperfusion

Objective:To explore the animal model of MCAO pretreatment with ENT1,behavioral evaluation of rats and adenosine A1 receptor expression changes in brain tissue.Methods: In brain ischemia reperfusion,which was set up by MCAO,the groups was divided into different species at random,including normal group?control group?MCAO group(IR 3h?6h?12h?24h?72h)?NBTI(ENT1 specific inhibitor)group and DMSO(solvent control group),n = 10 in each group.After modeling,we used means of balance beam test and motor function test to assessment the neurological deficit in rats;HE staining to observe the pathological changes of brain tissue;Using immunohistochemistry to detect the expression of adenosine A1 receptor in different groups of hippocampus.Results: 1.Neurological deficit assessment: 1.1 Balance beam experiment evaluation results: We put the balance beam experiment score 2-4 points into the formal experiment.MCAO ?NBTI and DMSO group had different degrees of neurological deficits comparing with the normal group(P<0.05).However NBTI group neurological deficit score was lower than IR 24 h group(P<0.05).1.2 Motor Function Test Results: Lower tilt angles during motor function tests indicate more severe cerebral ischemia-reperfusion injury.The motor function of MCAO,NBTI and DMSO group decreased compared with the normal group(P<0.05).The NBTI group and the 24 h after MCAO motor function test tilt angle compared to the former decreased significantly(Figure 2)(P < 0.05).2.HE staining results: This part of the experiment was selected normal group,IR 24 h group and NBTI group of rat brain tissue samples HE staining,we found: the normal group of hippocampal neuronal structure neat,densely populated cells,well-defined;the gap between the nerve cells in the brain tissue was enlarged in IR24 h,edema was found,cell distribution and structural disorganization,and nuclear pyknosis.In the NBTI group,less neurons were necrotic.3.Western Bloting,immunohistochemical detection of adenosine A1 receptor in different groups of rat brain tissue hippocampus expression3.1 Western Blot results: adenosine A1 receptor expression in each group can be seen in the MCAO model group,IR 3h group adenosine A1 receptor expression than the normal group and sham operation group decreased(P< 0.05).With the extension of time,the expression of adenosine A1 receptor gradually decreased;but in IR 12 h group,the expression of A1 R was increased,and the expression level of adenosine A1 receptor was close to the baseline in IR 72 h group level(P > 0.05).The IR 24 h time point was used as the model group time point for pretreatment with ENT1.3.2 immunohistochemistry results: The positive cells of adenosine A1 receptor in experimental hippocampus were mainly neural cells(Fig.5);In addition,Compared with IR 24 h group,adenosine A1 receptor in NBTI group increased significantly.Conclusion:IR rats pretreated with NBTI,a specific inhibitor of ENT1,may improve the neurological function of rats,which may be related to the increase of adenosine A1 receptor expression by inhibiting ENT1.