A Study On The Mechanism Of Orexin-A/OX1R System Inhibiting Cerebral Ischemia-reperfusion Injury By Regulating ERK1/2 Signaling Pathway

ObjectiveIschemic stroke is a cerebrovascular disease that seriously threatens the physical and mental health of human.The incidence rate has been persistently high and is still on the rise.It is well known that there is no specific effective prevention and treatment method for ischemic stroke except thrombolysis.Furthermore,thrombolysis can further cause cerebral ischemia-reperfusion injury(CIRI),the mechanism of which is very complicated.It has found that ERK1/2 signaling pathway was activated during the development of CIRI.Orexin-A,a neuropeptide mainly secreted by the hypothalamus,can inhibit the volume of cerebral infarction via its G-protein-coupled receptor,indicating its neuroprotective effect on CIRI.But its mechanism needs further study.Therefore,this study will explore how orexin-A can play a neuroprotective role in CIRI by regulating ERK1/2 signaling pathway.The results will provide effective strategies for exploring the delay or prevention of CIRI neural injury,theoretical basis and experimental basis for clinical application.The aim is to reduce ischemia-reperfusion injury,rescue penumbra neurons and improve behavior and cognitive function.MethodsThe SPF grade wistar rats were used to prepare middle cerebral artery occlusion(MCAO)models and SH-SY5Y cells were cultured in vitro to prepare oxygen and glucose deprivation(OGD)models.The rats were randomly divided into the sham operation group(Sham),the model group(I/R),the orexin-A single intervention group(OXA),and the Orexin-A and SB334867 joint intervention group(SB+OXA)and the PD98059 individual intervention group(PD)after modeling.SH-SY5Y cells in vitro were divided into the control group,the model group(OGD/R),the Orexin-A single intervention group(OXA),the Orexin-A and SB334867 co-intervention group(SB+OXA)and U0126 single intervention group(U0126).The rats need to fast for 12 h before modeling,and take cortical tissues for testing.First,we tested the expressive difference of p-ERK1/2 after CIRI to verify that the occurrence and development of CIRI is indeed accompanied by activation of ERK1/2.Then we used Orexin-A to intervene the model group to verify its neuroprotective effect and its effect on the expression of p-ERK1/2.And the SB334867 was used to intervene in the model group before Orexin-A to test whether the protective effect of Orexin-A was weakened or disappeared to verify that Orexin-A works through OX1R.Finally,we selected p-ERK1/2 inhibitors for individual intervention,and examined the effects of inhibition of p-ERK1/2 expression on CIRI.In the experiment,we used reverse transcription PCR(RT-PCR)and Western blotting to detect the differences in RNA and protein levels of p-ERK1/2 and OX1R in each treatment group.And the TTC staining was used to detect the size of cerebral infarction volume in each treatment group.And the Tunel staining was used to detect apoptosis.Finally,the Adhesive removal test and Beam walking test were used to conduct behavioral analysis of rats in each treatment group to detect differences in movement and sensation.Results1.Both MCAO reperfusion injury in rats and reoxygenation after OGD can induce high expression of p-ERK1/2(p＜0.01,p＜0.05),indicating that ERK1/2 activation is involved in cerebral ischemia reperfusion injury.2.Orexin-A intervention group can reduce cerebral infarction volume(p＜0.05)and cell apoptosis rate(p＜0.01,p＜0.05),indicating Orexin-A has neuroprotective effect.At the same time Orexin-A can reduce the high expression of p-ERK1/2(p＜0.01,p＜0.05).3.Based on the increased expression of OX1R in RNA and protein levels after MCAO reperfusion and OGD reoxygenation injury,OX1R antagonist SB334867 was selected for intervention.Compared with Orexin-A alone intervention group,SB334867 combined intervention resulted in larger cerebral infarction volume(p＜0.01,p＜0.05)and increased cell apoptosis rate(p＜0.05).The expression of p-ERK1/2 was increased(p＜0.01,p＜0.05).The results showed that SB334867 blocked or partially blocked Orexin-A’s neuroprotective effect.4.After intervention of ERK1/2 inhibitors PD98059 and U0126 in the model group,the volume of cerebral infarction in rats(p＜0.05)and the apoptosis rate were obviously decreased(p＜0.05)compared with the model group,indicating that the decreased ERK1/2 activity could have a protective effect.ConclusionCIRI can activate the ERK1/2 pathway,and Orexin-A can reduce the phosphorylation of ERK1/2 by combining with OX1R,meanwhile,it can reduce the volume of cerebral infarction and reduce cell apoptosis,thus exerting its neuroprotection effect.After blocking ERK1/2 pathway by ERK1/2 inhibitor intervention,the cerebral infarction volume of rats became smaller and the apoptosis rate was decreased.This study is helpful to explore the regulation and intervention mechanism of Orexin-A/OX1R system on stroke,lay theoretical and experimental foundation for studying the pathogenesis and treatment methods of stroke,and provide experimental basis for exploring new drug targets and new treatment strategies.