Neuroprotection And Mechanisms Of Orexin A On Secondary Brain Injury After Intracerebral Hemorrhage

BackgroundHemorrhagic stroke accounts for 20%-30%of all strokes.Though less than one third,its more acute onset,more fatal course,higher mortality and morbidity make it a thorny issue for clinical treatment.Spontaneous intracerebral hemorrhage(SICH)which is often referred to as intracerebral hemorrhage(ICH)by clinicians,is defined as non-traumatic hemorrhage in the brain,and it contributes to 60%-75%of hemorrhagic stroke.There are many causes for ICH,but the most important one is hypertension.With the development of economic society and the improvement of living conditions,and the booming of the incidence and prevalence of hypertension and metabolic syndrome worldwide,the vast of cases of intracerebral hemorrhage are followed.This is especially prominent in the developing countries with a huge population like C hina.According to the data and reports from CDC,the National Health and Family Planning Commission,and all the provinces,currently,ICH is the leading cause of death for all urban and rural citizens,and the main cause of disability and immobilization for adults.ICH poses a serious threat to the health of Chinese citizens,and in the meantime puts tremendous pressure on the public health system and national stability.Prevention and treatment of hemorrhagic stroke,especially ICH,is an arduous task for medical doctors and researchers in China and all the world.Unfortunately,there are currently no effective treatments or drugs for ICH,and all treatment attempts based on previous research results and clinical experience have failed.In recent years,there has been progress in the study of pathophysiology of ICH,and the new treatment based on this has shown some progress in experimental animal models and clinically cases.The latest research suggests that brain and nerve function damage caused by ICH mainly includes primary injury and secondary injury.The mechanical damage lead to by hematoma in the brain parenchyma is primary injury;the toxicity of hematoma metabolites,immune injury,oxidative stress,inflammatory reaction and neuronal apoptosis are secondary injuries.In the past,neurosurgery was ardently advocated,and many efforts was spared to improve the surgical technique to remove hematoma.However,it did not significantly improve the survival or prognosis of patients.It is currently believed that secondary brain injury is the fundamental cause of poor prognosis for patients.Therefore,the basic and clinical research in recent years has focused on preventing and improving secondary brain injury.Orexin(OX),also known as hypocretin(HCRT),which is secreted by specific neurons in the hypothalamus,are widely projected to and act on multiple brain regions and peripheral nervous systems.This neuropeptide hormone regulates a variety of physiological processes such as appetite,energy metabolism,neuroendocrine and sleep-wake cycles.OX has two subtypes,type A and B,and are called orexin A(OXA)and orexin B(OXB),respectively.The receptors of orexin are expressed on various cell surfaces in the central nervous system,both belong to G protein-coupled receptors(GPCRs)family,and can also be categorized into 2 subtypes,orexin receptor 1(OXR1)and orexin 2 receptor 2(OXR2),respectively.OXA has the same affinity to both types of receptors,while OXB mainly acts on the type 2 receptor.Recent studies have shown that the OX/OXR system plays a regulatory and protective role in the pathophysiology of various diseases of the nervous system such as sleep disorders,depression,substance addiction,Alzheimer’s disease,and ischemic stroke.Some scholars have reported that OXA can rapidly pass through the blood-brain barrier and activate the Akt-dependent cell survival pathway and exerts an anti-apoptotic effect.In addition,the OXA/OXR system also activates the downstream AMPK-dependent anti-inflammatory pathway by binding to its receptors,thereby inhibiting the inflammatory response.Based on the above-mentioned results,we conclude that the OXA/OXR system can play a protective role in ICH.To this end,this study used experimental mice to establish models of spontaneous intracerebral hemorrhage,using gross assessment and molecular biological techniques to explore the role of OXA/OXR system and its mechanisms.MethodsPart 1CD-1 mice were catheterized into the femoral artery to obtain autologous blood,and by stereotactic injection of the blood into the basal ganglia,models of ICH were established.The sham group(all the operations were equivalent to ICH group except for the actual blood injection)was established as the control.The research cross-sections after ICH were set at 3 hours(ICH 3h),6 hours(ICH 6h),12 hours(ICH 12h),24 hours(ICH 24h)and 72 hours(ICH 72h),respectively.The western blot(WB)technique was used to determine the expression of OXA,OXR1 and OXR2 at specific time points after ICH,in order to clarify the dynamic changes of the expression of OXA and its receptors after ICH.During the acute phase,one time point in which OXA/OXR 1/OXR2 was change significantly,immunofluorescence(IF)was performed to study the expression of OXA/OXR in neurons,microglia and glial cells after intracerebral hemorrhage.Part 2Random method was employed to establish the Sham group,intracerebral hemorrhage plus solvent control group(ICH+Vehicle),intracerebral hemorrhage plus orexin A treatment group(ICH+OXA),and to explore the therapeutic effect of orexin A after ICH.Using gross assessment such as hematoma size determination,neurobehavioral assessment,blood-brain barrier integrity assessment,and brain water content determination to study the outcome of ICH after treatment with orexin A in CD-1 mice,in order to confirm whether OX/OXR plays neuroprotective role in ICH.Part 3CD-1 mice were randomly assigned into Sham group,ICH+Vehicle group,ICH+OXA group,intracerebral hemorrhage plus orexin A plus Akt inhibitor group(ICH+OXA+MK2206),and intracerebral hemorrhage plus orexin A plus AMPK inhibitor group(ICH+OXA+Dorsomorphin)to study the molecular signaling pathway involving orexin A’s effect on improving inflammation and inhibiting apoptosis.Moreover,intracerebral hemorrhage plus orexin A plus orexin receptor 1 inhibitor(ICH+OXA+SB-334867)group and intracerebral hemorrhage plus orexin A plus orexin 2 inhibitor(ICH+OXA+JNJ-10397049)group were established.Whether the two subtypes of orexin receptors play a different role in the signaling pathway of the brain protection was also studied.ResultsPart 1WB results showed that OXA was moderately expressed in brain tissue.It began to decrease significantly 3 h after ICH and continued to decrease during the 72-hour study period.Conversely,the level of OXR1 and OXR2 in brain tissue gradually increased.Results from IF showed that OXA,OXR1 and OXR2 were widely expressed in neurons,microglia and astrocytes;OXA level was significantly decreased.24 h after ICH,while the expression of OXR1 and OXR2 was significantly increased,which was consistent with WB results.Part 2Compared with the Sham group,the short-term neurobehavioral outcome of the ICH+Vehicle group was significantly damaged,Evans blue dye extravasation and the brain water content were significantly increased.However,these indicators were improved in the ICH+OXA group,suggesting that OXA could significantly ameliorate neurological function,blood-brain barrier integrity and brain edema.The results of long-term behavioral assessment also showed that OXA can effectively improve the motor,sensory function and advanced neurological functions such as memory and learning in mice after ICH,indicating that the protective effect of OXA on secondary brain injury is not only transient.Part 3Compared with the ICH+OXA group,the apoptotic indexes were significantly increased in ICH+OXA+MK2206 group,suggesting that the anti-apoptotic effect of OXA is mainly through the Akt-dependent signaling pathway;the expression of inflammatory factors was also significantly increased in the ICH+OXA+Dorsomorphin group,suggesting that the anti-inflammatory effect of OXA is mainly through the AMPK signaling pathway.After using OXR1 selective inhibitor SB-334867,the anti-apoptotic effect of OXA was reversed,while the anti-inflammatory effect was not significantly affected.After using OXR2 selective inhibitor JNJ-10397049,the anti-inflammatory effect of OXA was offset,whereas the anti-apoptotic effect was not significantly changed.These results suggest that the anti-apoptotic effect of OXA is mainly through the OXR1 receptor-dependent signaling pathway,and the anti-inflammatory effect of OXA is mainly through the activation of the downstream signaling pathway through the OXR2 receptor.Conclusions1.The expression of OXA after ICH was significantly reduced,and its level in brain tissue gradually decreased during the whole acute phase(≤72h);the expression of orexin receptor 1 and receptor 2 was,on the contrary,gradually increased during the period.OXA and its receptor 1 and 2 are widely expressed in the central nervous system,moderately in neurons,microglia and astrocytes.2.Orexin A can significantly improve neurological function after ICH in mice,and this improvement covers not only short-term but also long-term outcome.Though OXA does not change the size of hematoma after ICH,it can significantly reduce the degree of damage of the blood-brain barrier and the degree of cerebral edema in the injured side.3.OXA can significantly increase p-Akt,BCL2/BAX ratio and reduce the expression of Caspase-3,showing anti-apoptotic effect;OXA can also significantly increase p-AMPK and reduce the expression of inflammatory factors such as NFkB,IL-1β and TNFa,suggesting it has anti-inflammatory effects.The anti-apoptotic effect of OXA can be effectively blocked by the Akt inhibitor MK2206.The use of AMPK inhibitor Dorsomorphin can effectively block the anti-inflammatory effect of OXA.Blocking of OXR1 can reverse the anti-apoptotic effect of OXA,and blockade of OXR2 can offset the anti-inflammatory effect of OXA.Therefore,the results suggest that OXA acts as an anti-apoptotic agent by binding to OXR1 and activating the Akt/BCL2/BAX pathway;binding to OXR2 to activate the AMPK/NFkB pathway to exert an anti-inflammatory effect.