Neuroprotective Effects And Mechanism Of Orexin-A On The Focal Cerebral Ischemia Reperfusion Injury Of Rats

Stroke has the characteristics of high morbidity,high mortality,high disability and high recurrence rate.Stroke is divided into hemorrhagic stroke and ischemic stroke in which ischemic stroke accounts for 60%-80%.At present,thrombolysis is one of the most effective ways to treat ischemic stroke.It makes blood vessels recanalization.However,reperfusion will further aggravate neuronal apoptosis and cause ischemia-reperfusion injury(I/RI).The pathological mechanism of I/RI is very complex including intracellular calcium overload,excessive formation of free radicals,the occurrence and development of inflammation,neuronal apoptosis and excitatory amino acid toxicity.However,up to now,the specific molecular mechanism of I/RI is not fully understood.Therefore,it is important to pay more attention to the molecular mechanism of I/RI,which will provide important theoretical significance and clinical application value for the effective treatment of stroke.Orexin-A is an excitatory neuropeptide.And it plays an important role with bingding OX1R(orexin 1 receptor)and OX2R(orexin 2 receptor)in physiology and pharmacology via activating downstream signaling.But Orexin-A has been reported to prefer to bind OX1 R.It was found that Orexin-A plays an important role not only in appetite,sleep and wakefulness,reward and seeking,but also in cardiovascular diseases.Studies have shown that OX1 R expressed with a high level in the ischemic cortex after occlusion of the middle cerebral artery in rats.And it may have neuroprotective effect by inhibitting neuronal apoptosis.However,the specific molecular mechanism of Orexin-A in ischemia-reperfusion injury is not clear.Based on the above research,we proposed hypotheses that Orexin-A/OX1 R pathway has a protective effect on ischemia-reperfusion injury by activating the downstream signaling pathway to inhibit neuronal apoptosis.In this study,the model of middle cerebral artery occlusion(MCAO)in rats and the model of oxygen-glucose deprivation(OGD)in primary hippocampal neurons were established to explore the effect and molecular mechanism of Orexin-A in pathogenesis of stroke.This work will provide new insights underlying the pathogenesis of ischemic stroke.The experiment procedures are as follows,rats were divided into the following groups randomly,including Sham group,I 2 h group,model group(I/R),NS group,Orexin-A group,LY294002 group,and Orexin-A with LY294002 group.Firstly,the expression of OX1 R in the hippocampus was examined by immunofluorescence.Then MCAO model was made by thread method.Hippocampal tissue was isolated and extracted at different time of reperfusion(R6,12,24 h)following ischemia 2h,and we detected the expression of OX1 R both at m RNA and protein levels by reverse transcription-PCR(RT-PCR)and Western blotting respectively.After 2h of ischemia,the lateral ventricle was injected with 0.9% saline and Orexin-A.The infarct volume of rats was detected by 2,3,5-triphenyltetrazolium chloride(TTC)staining which was used to verify the neuroprotective effect of Orexin-A.In order to verify whether Orexin-A changes ERK phosphorylation in ischemia-reperfusion injury,Western blotting was performed to detect ERK phosphorylation in different groups.And we further use the PI3 K inhibitor LY294002 to detect whether Orexin-A play a protective role through the PI3K-ERK signaling pathway.In addition,we cultured the primary hippocampal neurons and made OGD model through oxygen and sugar deprivation followed by reoxygenating for 24 h(R24 h).The cells were divided into three groups: Control group,OGD/R24 h group,OGD/R24 h with Orexin-A intervention group.We used CCK-8 kit and immunofluorescent staining to observe whether Orexin-A can reduce the apoptosis of neurons and change the neuronal morphological caused by oxygen-glucose deprivation.The results were as follows.Immunofluorescent staining showed that OX1 R expressed in the hippocampus.Compared with the ischemic two hours group,OX1 R expression was higher at I/R12 h on mRNA level(p <0.05)and the highest expression was observed at I/R12 h on protein level(p <0.001).Therefore,we selected the reperfusion time of 12 h(R12h)in subsequent experiment.The above experiments showed that OX1 R was expressed in the hippocampus and participated in the process of ischemia-reperfusion injury.TTC results showed that I/R group had larger infarct volume,but the volume of cerebral infarction decreased significantly after Orexin-A intervention(p<0.001),so we can see that Orexin-A intervention can reduce infarct volume significantly caused by ischemia-reperfusion.Western blotting results showed that the expression of p-ERK protein was increased significantly in I/R group compared with sham group(p<0.05).And compared with I/R group,Orexin-A reduced the high expression of p-ERK protein induced by ischemia-reperfusion significantly(p<0.05).These data suggested that Orexin-A participated in the process of ischemia-reperfusion injury by altering the phosphorylation level of ERK protein.Compared with I/R rats,the expression of p-ERK protein was decreased in LY294002 intervention group significantly(p<0.001).Orexin-A treatment resulted in the significant increase of p-ERK level in LY294002 intervention group(p<0.001).It was suggested that the change of p-ERK caused by Orexin-A was mediated by PI3 K.In addition,CCK-8 results showed that the OD value was decreased significantly after oxygen-glucose deprivation 8h compared with control group(p<0.01).After Orexin-A stimulation,OD value was increased significantly compared with the OGD/R24 h group(p<0.05).So Orexin-A can inhibit neurons apoptosis significantly.Laser confocal results showed that the number of neurons in OGD/R24 h group was reduced significantly,while Orexin-A treatment improved the morphology and the number of neurons significantly induced by oxygen-glucose deprivation.These results indicate that Orexin-A can reduced the infarction volume significantly induced by ischemia-reperfusion and inhibit the apoptosis of neurons by activating the PI3K-ERK signaling pathway.It is suggested that Orexin-A plays an neuroprotection role in ischemia-reperfusion injury.We can draw that Orexin-A can reduce the volume of cerebral infarction and inhibit apoptosis of neurons.It can activate the PI3K-ERK signaling pathway and regulate the phosphorylation of ERK protein in the process of cerebral ischemia-reperfusion injury.This study provides detailed information on the signal transduction pathways and mechanism of neuroprotection of Orexin-A during cerebral ischemia-reperfusion injury.It has important theoretical and practical value for drug development and clinical treatment of stroke.