The Role And Its Molecular Mechanism Of COX5A In Neonetal Rats With Cerebral Hypoxic-ischemic Injury

Objectives:Neonatal hypoxic-ischemic encephalopathy(NHIE)is a destructive neurological disease that is one of the leading causes of neonatal death.Cytochrome c oxidase subunit 5A(COX5A)is a metabolic enzyme present in the mitochondrial respiratory chain,and its down-regulation leads to an imbalance of mitochondrial energy,thereby affecting the development of the disease.This study aimed to investigate the role of COX5A in hypoxic-ischemic(HI)brain injury in neonatal rats,and further explore the related molecules of COX5A in vitro,in order to provide a new target for the treatment of HIE.Methods:In vivo:Fifty-four seven-day-old male Sprague-Dawley rats were randomly divided into hypoxic-ischemic(HI)and Sham-operated(Sham)groups.The HI group was subjected to the right common carotid artery ligation for ischemia,and exposed to the hypoxia chamber for 2 hours.The Sham group only exposed the right common carotid artery without electrocoagulation.Then the Zea-Longa score and 2,3,5-triphenyl-tetrazolium chloride(TTC)staining were employed to the evaluation of HI model.The cell apoptosis of the right cortex was determined by TdT-mediated dUTP-biotin nick end labeling(Tunel)staining,and the histological structure and cell morphology of the right cortical injury were observed by hematoxylin-eosin staining(HE)staining.Immunofluorescent(IF)staining was performed to observe the neurons change in the righ cortex.In addition,the expression changes of COX5A were detected by quantitative real-time polymerase chain reaction(QRT-PCR),immunohistochemical staining(IHC)and western blotting(WB)techniques.In vitro:The cortical neurons of new-born rat pups(within 24 hours)were used to culture the primary cortical neurons and subjected to oxygen glucose deprivation(OGD)injury.QRT-PCR and IF staining were used to detect the mRNA and protein expression changes of COX5A after OGD.On this basis,HSV-COX5A over-expressing virus was constructed,then transfected into cortical neurons.In order to investigate the effects of COX5A ovexpression on the outgrowth of the neurons,the immunofluorescent staining of ?-? Tubulin(Tuj1)was performed,and the quantification of the length of neuronal neurites and the average area of neurons,as well as the the cell number of neurons were analyzed.Moreover,the tunel staining was used to study the the effect of COX5A overexpression on neuronal apoptosis.What's more,the molecules interacting with COX5A were screened by GeneMANIA prediction,rho-guanine dissociation inhibitor a(Rho-GD1?),superoxide dismutase 2(Sod2),glutathione s-transferaspl(Gstpl),and triosephosephate isomerase(TPI)were found.Finally,QRT-PCR and WB experiments were employed to confirm the related molecules involved in the role of COX5A on cortical neurons.Results:The HI model in vivo and the OGD model in vitro were established,it was found:In vivo:1.The Zea-Longa score showed that compared with the Sham group,the rats in HI group arised gait instability and turned to one side when walking forward at 0 h,2 h,4 h,8 h,and 16 h,and the score was significantly increased,could reach to 3 points(P<0.05).2.TTC staining showed that there was obvious white infarction in the right cortex on HI group,and quantitative analysis showed that the infarct volume of HI group increased significantly,and the difference was statistically significant(P<0.05).Combined with the results of the Zea-Longa score and TTC staining,which proved that the HI model was successfully constructed.3.The results of HE staining showed that the cell structure was tightly arranged without abnormalities in Sham group.But the right cortical cells in the HI group were disordered,the normal neurons were reduced and the nucleus was vacuolated,as well as the nucleolus disappeared occasionally.4.The results of Tunel staining showed that compared with the Sham group,the number of Tunel positive cells in the right cortex of the HI group increased,and the apoptotic rate increased significantly(P<0.01).5.The result of IF staining showed the number of NEUN-positive cells decreased significantly in comparison of the Sham group(P<0.05).6.After HI injury,the IHC,QRT-PCR and WB results showed that when compard with the Sham group,the expression of COX5A was down-regulated in the right cortex of the brain(P<0.05).7.The decrease in COX5A expression in the right cortex was positively correlated with the decline cortical neurons after HI injury.In vitro:1.The primary cortical neurons subjected to OGD could simulate the HI model in vivo.We found that compared with the Normal group,the cortical neurons of the OGD group is smaller,and the neural network connections between the axons are sparse.2.The results of QRT-PCR and IF staining showed that the expression level of COX5A was significantly decreased in the OGD group in comparison of the Normal group(P<0.05).3.After transfecting cortical neurons with HSV-COX5A over-expressing virus,QRT-PCR results showed that compared with the negative control group(NC)group,the mRNA expression of COX5A in COX5A over-expression group increased significantly under normal or OGD condition(P<0.01),which proved successfully transfected.4.To study the effect of COX5A over-expression on the outgrowth of neurites,Tuj1 staining showed that the neurite length and cell number of the OGD group were significantly decreased when compared with the Normal group(P<0.01),and the cell body area increased,suggested that the neuronal cells appear to be swollen after OGD(P<0.05).Compared with the NC group,the neurite length(P<0.01)and cell number(P<0.05)of neurons in the COX5A overexpression group were significantly increased.5.To investigate the effect of COX5A overexpression on neuronal apoptosis,Tunel staining showed that compared with NC group,the number of apoptosis decreased after COX5 A overexpression,and the apoptosis rate decreased significantly(P<0.01)6.In addition,we predicted four genes interaction with COX5A by GeneMANIA:Gstp1,TPI,Rho GDIa and Sod2,and found that Gstpl and TPI were co-expressed with COX5A,moreover,TPI was predicted as physical interactions with COX5A.The results of QRT-PCR showed compared with NC group,the expression of Rho GDIa and Sod2 was down-regulated(P<0.05),while Gstp1 and TPI was up-regulated in COX5A overexpression group(P<0.05).What's more,the WB experiment further verified the protein expression of TPI and found that the expression decreased after OGD injury,but the protein expression level of TPI was significantly increased in the COX5A overexpression group in comparison with the NC group.Therefore,we speculated TPI may be the key molecule that promoted the outgrowth of neuritis and decreased the cell apoptosis after COX5A overexpression.Conclusions:After HI injury in neonatal rats,the down-regulation of COX5A leads to neuronal apoptosis and it's positively responding to neuronal reduction.COX5A overexpression significantly promotes the outgrowth of neuronal neurites and reduces cell apoptosis;it may be closely related to the up-regulation of TPI.