The Neuroprotective Role And Mechanism Of Mas In Early Brain Injury After Subarachnoid Hemorrhage Of Rats

BackgroundAneurysmal subarachnoid hemorrhage(SAH)is an acute and critical cerebrovascular disease that is seriously harmful to human health,which accounts for about 7-10%in the whole stroke.Patients with SAH have a very high mortality and disability rate,but there is currently a lack of effective treatment.With the development of surgery and interventional therapy,we have made great sucess in the control of ruptured aneurysm,but the secondary brain injury caused by subarachnoid hemorrhage still lacks the effective treatment measures.In recent years,with the aging of the population,the incidence of the cerebrovascular disease is rising year by year,causing a serious economic burden to individuals,families,and society.Compared with cerebral ischemia,the clinical and basic research of subarachnoid hemorrhage started late,because the pathological mechanism of brain injury is not clear,and its clinical treatment options are limited greatly.Therefore,it is important to elucidate the pathological mechanism of subarachnoid hemorrhage.Early brain injury(EBI)refers to a series of brain injuries occurring within 72 hours after SAH,including mechanical brain injury,ischemia-hypoxia damage,apoptosis and necrosis,blood-brain barrier disorder,oxidative stress,and brain edema.Oxidative stress is one of the important pathological mechanism in early brain injury after subarachnoid hemorrhage.First,diffuse cerebral ischemia and hypoxia in the early stage of subarachnoid hemorrhage can cause a large amount of reactive oxygen species(ROS)in the brain,and in addition,the rupture of red blood cells releases large quantities of iron ions or iron-containing compounds into cerebrospinal fluid.Excess iron ions can also produce large amounts of free radicals through Fenton reactions.At present,apoptosis is considered as one of the main pathophysiological mechanisms of early brain injury after SAH.The apoptotic pathways confirmed in the model of subarachnoid hemorrhage include death receptor pathway,caspase-dependent and non-dependent pathways,and mitochondrial-related pathways.Studies have shown that mitochondrial and caspase-dependent pathways play a major role in cell apoptosis after subarachnoid hemorrhage.Apoptosis is an important pathological and physiological process after SAH,it is an important way to improve the prognosis of SAH patients by interrupting the cascades of apoptosis.Mas is a newly discovered RAS member,which is expressed in different tissue cells,especially in the brain.Previous studies have shown that the Mas receptors in the kidneys,peripheral vascular endothelial and liver tissue inhibit apoptosis and oxidative stress.In addition,in the study of hypertensive brain injury,it was found that the activation of Mas could reduce oxidative stress and apoptosis of vascular endothelial cells and neurons.However,its function and regulation mechanism in early brain injury after subarachnoid hemorrhage is unclear.UCP-2 is an ion channel protein on the mitochondrial membrane,which plays an important role in regulating mitochondrial homeostasis and energy metabolism.Previous studies showed that UCP-2 had the function of regulating mitochondrial ROS generation and cell death in lung,stomach,hepatocytes,and macrophages.In the model of cerebral ischemia,it was found that the cells with high expression of UCP-2 were more tolerant of oxygen-glucose deprivation(OGD).Further,in the model of middle cerebral arterial occlusion,it was found that the mitochondrial ROS level of UCP-2 transgenic mice was significantly lower than that of non-transgenic mice,and the cerebral infarct area was significantly lower than that of the control group,suggesting that UCP-2 was involved in brain protection after neuronal ischemia and hypoxia.The role of UCP-2 in regulating neuronal oxidative stress and apoptosis has been clarified,but its upstream of specific regulation mechanism is not clear.Previous study showed that the UCP-2 level was mainly related to CREB,and PKA/CREB has been confirmed as the downstream pathway of the Mas receptor,so we speculate that the PKA/CREB/UCP-2 pathway may mediate the antioxidant and anti-apoptosis roles of the Mas receptor.Recent studies have shown that the Mas receptor is a new branch of the RAS in the brain,which is a potential target for the treatment of acute cerebrovascular diseases.In the current study,we explored whether the specific agonist AVE of Mas receptor could alleviate the EBI and investigate the possible protective mechanism on anti-oxidative stress and anti-apoptosis after SAH.Part1 The Role of Mas Receptor on Oxidative Stress andNeuronal Apoptosis after SAH in RatsObjectiveIn this part of the experiment,we aimed to observe the distribution of Mas receptor in neuron adn understand the changes of Mas receptor in the brain of rats after SAH.We also aimed to elucidate the effect of Mas receptor agonist AVE on neurological function,oxidative stress,and neuronal apoptosis after SAH in rats.Methods(1)The model of SAH rat was established by internal carotid artery puncture.The co-localizaition of Mas receptor with NeuN was observed by immunofluorescence staining.The expression of Mas receptor at 3h,6h,12h,24h and 72h after SAH was detected using Western blot.(2)The experimental groups include:Sham,SAH+vehicle(10%DMSO),SAH+AVE(0.3mg/kg),SAH+AVE(0.9mg/kg)and SAH+AVE(2.7mg/kg).AVE was administered intranasally 1 h after SAH induction,and the neurological function at 24 h after SAH was evaluated(including improved Garcia score and beam balance test).TUNEL staining was performed to evaluate neuronal apoptosis.DHE staining was performed to evaluate oxidative stress injury.Western blot was performed to evaluate corresponding downstream molecular levels,including PKA,p-CREB,CREB,UCP-2,Bcl-2,Bax,Romo-1.(3)The experimental groups include:Sham,SAH+vehicle(10%DMSO),SAH+AVE(0.9mg/kg).AVE was administered intranasally 1 h after SAH induction,and Rotarod test was performed to evaluate limb coordination function at 7,14 and 21 days after SAH.Water Maze test was performed at 22-27 days after SAH to evaluate of spatial memory and learning ability.Floro-jade C test was used to evaluate neuronal degeneration in hippocampus of differenct regions,including CA1,CA2 and DG.(4)The experimental groups include:naive+vehicle(10%DMSO)and naive+AVE(0.9mg/kg)group.After 6 hours of intmasal administration of AVE,liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS)analysis was performed to further confirm that AVE can enter into the brain by intranasal administration.Results(1)The expression of Mas receptor decreased gradually after SAH,reached the lowest point at 24h,and then gradually recovered at 72h.The Mas receptor and the neuron marker(NeuN)were co-located with each other,suggesting that the Mas receptor is expressed on neuron.(2)All 3 different doses of AVE(0.3mg/kg,0.9mg/kg and 2.7mg/kg)can improve the neurological functional scores of the modified Garcia,but only the middle dose can improve both modified Garcia and beam balance test scores simultaneously.AVE significantly reduced oxidative stress and neuronal apoptosis induced by SAH.(3)AVE can obviously improve the function of limb coordination evaluated by Rotarod test at 1 week after SAH.However,there is no significant difference between the treatment group and the control group at 2 and 3 weeks after SAH.AVE can significantly improve the learning ability and spatial memory of rats after SAH.(4)LC-MS/MS indicates that the AVE can be detected in the brain after administering AVE by intranasal injection,suggesting that the drug is effective by nasal administration.ConclusionsThe Mas axis of RAS in the brain is suppressed after SAH,which may be one of the pathophysiological mechanisms causing oxidative stress and neuronal apoptosis in EBI.The regulation of Mas axis is a potential target for SAH treatment.Part 2 The Mechanism of Mas Activation Inhibiting Oxidative Stressand Neuronal ApoptosisObjectiveWe aimed to explore the role of Mas receptor in oxidative stress and neuronal apoptosis after SAH,and establish whether inhibition of Mas receptor or silence of UCP-2 could abolished the neuroprotective role of AVE on oxidative stress and neuronal apoptosis after SAH.Methods(1)The experimental groups include:SAH+AVE+NS,SAH+AVE+A779,SAH+AVE+scr siRNA and SAH+AVE+UCP-2 siRNA group.A779 or equal volume of normal saline was administered by the lateral ventricle injection at 1h before SAH induction,and the UCP-2 siRNA or scr siRNA was administerd at 48 hours before SAH induction.The treatment of AVE was started at 1 h after SAH induction,and the neurological function at 24 h after SAH was evaluated(including modified Garcia score and beam balance test).Western blot was performed to evaluate the downstream molecular level,including PKA,p-CREB,CREB,UCP-2,Bcl-2,Bax,Romo-1.(2)The experimental groups include:naive+scr siRNA,naive+UCP-2 siRNA,SAH+scr siRNA,SAH+UCP-2 siRNA.The scr siRNA or UCP-2 siRNA was administered by the lateral ventricle injection at 48 h before SAH induction.The neurological function at 24 h after SAH was evaluated(including modified Garcia score and beam balance test).Western blot was performed to evaluate the expression of UCP-2.Results(1)The inhibition of Mas by A779 can block the neuroprotective effect of AVE and reverse the expression of PKA,p-CREB,UCP-2,Bcl-2,Bax and Romo-1 induced by AVE.(2)UCP-2 siRNA can significantly inhibit the expression of UCP-2.UCP-2 knockout can block the neuroprotective effect of AVE and reverse AVE induced Bcl-2,Bax,Romo-1 expression,but there is no significant effect on the expression of PKA and p-CREB.ConclusionsMas/PKA/CREB/UCP-2 is one of signaling pathway that mediates the protective effect of AVE on early brain injury after SAH in rats.The results in this part suggested the neuroprotective role of UCP-2 in early brain injury after SAH,and provided theoretical basis for subsequent treatment of SAH targeting Mas/JCP-2.