The Research Of ENT1 Inhibitor Regulates CREB Pathway Involved In The Neuroprotective Effects Of Cerebral Ischemia-reperfusion In Rats Model

Objective: The present study aims to investigate the role of ENT1 on neurological function in cerebral ischemia-reperfusion rats model,and the potential mechanism mediated by CREB pathway.Methods: In this study,133 SD male adult rats were selected.The cerebral ischemia-reperfusion rats model was induced by 2 hours of middle cerebral artery occlusion(MCAO)followed by reperfusion.SD male rats were randomly divided into sham operation group,MCAO group,DMSO control group and ENT1 inhibitor group,7per group.The ENT1 inhibitor NBTI and DMSO control group were given by intraperitoneal injection 30 minutes after MCAO,and repeated administration every 12 hours.The protein expression of ENT1 and p-CREB were detected by Western blot in each group,the immunofluorescence double-labeling technique were used to test the morphological localization,the Garcia score and beam balance test were used to evaluate the neurological function at 24 hs and 72 hs after MCAO.The cerebral infarction volume was observed by TTC staining.Results: The results of western blot showed that both ENT1 and p-CREB were expressed in the sham operation group,the expression of ENT1 significantly increased at 3h and 12 h and 24 h after MCAO,gradually returned to baseline level until 72 h and 1 week.Compared with the control group,the expression level of p-CREB was significantly decreased at 12 h and 24 h after MCAO,obvious recovery after 72 hours,and gradually returned to baseline level until 1 week.The ENT1 inhibitors significantly increased the expression of p-CREB at 24 h after MCAO.The results of TTC staining showed that there were no obvious infarctions in the sham operation group.Compared with the MCAO group and the DMSO control group,the ENT1 inhibitor could significantly reduce the cerebral infarction volume.At the same time,the use of ENT1 inhibitor intervention can also significantly improve the neurological score in the Garcia test and beam balance test both at 24 and 72 hours after MCAO,suggesting that ENT1 inhibitors have neuro-protective effects.The results of immuno-fluorescence double staining showed that the expression of ENT1 and p-CREB in the hippocampus was spatially consistent with the neuronal marker molecule NeuN at 24 hours after MCAO,and the immuno-fluorescence intensity of ENT1 showed strong expression in hippocampus neurons.Conclusion: ENT1 inhibitor NBTI significantly improves neurological outcome and reduces the infarct volume through up-regulation of CREB pathway.Our results indicate that ENT1 inhibitors may be potential therapeutic target to protect neurological deficits after cerebral ischemia.