Naringin From Exocarpium Citri Grandis Ameliorates Cerebral Ischemia-reperfusion-induced Injury Via Nrf2/HO-1 And JAK2/STAT3 Pathways

Objective:This study with Cerebral ischemia-reperfusional rats as the objects of the research,and by observing the effect of cerebral ischemia-reperfusion injurious anti-oxidative effect and related neuroprotective mechanism of Naringin from Exocarpium Citri Grandis(NAE),which offers a new diagnostic train of thoughts for the integrative treatment of cerebral ischemia-reperfusion injury.Methods:60 SPF Male Sprague-Dawley rats,weighted 280 ± 20g,are randomly assigned to 6 groups,each group of 10:(1)Sham group(2)MCAO group(3)Edaravone group(4)High NAE group(5)Medium NAE group(6)Low NAE group.The middle cerebral artery occlusion(MCAO)models are established with Modified Zea-Longa occluding suture's method,reperfused 2 hours later.Edaravone group receives injections of edaravone 6mg/kg via vena caudalis before reperfusion.High NAE,medium NAE,low NAE groups are injected NAE 30mg/kg,15mg/kg,7.5mg/kg,respectively,while the Sham group needs to be administered equal amounts of saline.24 hours after the cerebral ischemic-reperfuion,rats will be scored by Julio's neurobehavioral scoring.After all rats' sacrifices,the serum will be kept to detect the expressional levels of GSH-Px(Glutathione peroxidase),SOD(Superoxide dismutase),MDA(Malondialdehyde),CK(Creatine kinase),LDH(Lactate dehydrogenase)and CAT(Catalase).Brain tissue will be taken out to detect the cerebral infarc volumes using 2,3,5-Triphenyltetrazolium chloride(TTC)staining.Brain tissue morphological changes with HE and Nissl staining will be observed using optical microscope.All rats' cellular morphology from the damage side in hippocamal and striatal regions will also be observed respectively.Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TdT-mediated dUTP Nick-End Labeling,TUNEL)will be used to detect the apoptotic cells in cerebral infarc areas after the ischemic reperfusion.Western blot and qRT-PCR will be used to examine Nrf2,HO-1,JAK2 and STAT3 protein and mRNA expression.Results:1.The neurobehavioral scores shows that the high,medium,low NAE groups and the edaravone group are significantly higher than model rats(P<0.05).2.The results of TTC staining indicates that high dose of naringin could significantly decrease the cerebral infarct size of MCAO rats when compared with the model group.3.The oxidative stress detection from serum shows that high NAE group can significantly increase SOD's level.4.Pathological results shows that the hippocampus can be divided into 1,2,3,4 and DG regions,the pyramidal cells in each hippocampal regions of sham group has neat arrangement,distinct cellular layers,round,plentiful and obvious nucleolus,no inflammatory cell infiltration,degeneration or necrosis.Its striatum has no neural necrotic degeneration,no abnormal Nissl bodies,and no cerebral edema as well.Compared with sham group,the pyramidal cells in the hippocampus from model group are disordered with degenerative necrosis.Local tissue of the striatum is loose,cells are deformed and necrotic,also has white matter edema,with loose reticular structure,and neural or perivascular space enlargement;The pyramidal cells in edaravone's hippocampal regions arranges neatly,the structures are clear,and no abnormal Nissl bodies.Its cells in the striatum have no degeneration or necrosis,no edema,and have homogeneous white matter.The pyramidal structures of high and medium NAE groups are clear,no degenerative necrosis,and their Nissl bodies are normal.Cells from striatal regions have a little widened peripheral clearance,with white matter slight edema,and loose reticular structures.5.TUNEL results discoveres that,the TUNEL-positive cells in edaravone group,and all NAE groups are significantly decreases as compared with the model group.Meanwhile,the TUNEL-positive cells in high NAE group are a little more than the edaravone group,but still much less than the medium and low NAE groups.6.Western Blot shows that the expression of Nrf2 and HO-1 proteins' levels are significantly increases in all NAE groups,as well as edaravone group,when compared with the model group,and both JAK2 and STAT3 proteins' expression decreases.7.qRT-PCR data suggests that,when compared with the model group,the expression of Nrf2 and HO-1 mRNAs in all NAE rats and edaravone group are significantly higher than the model rats.JAK2 and STAT3 mRNA levels were significantly decreases.Conclusion:Naringin can reduce the cerebral ischemia-reperfusion injury in rats and improve their neurobehavioral disorders.The mechanism may be related to the reduction of cerebral edema,its anti-oxidative enhancement,and the protection of neural cells in the brain.It is presumed that naringin can affect Nrf2/HO-1 and JAK2/STAT3 pathways,play a role in regulating cerebral microcirculation and enhancing the function of neural cells in the ischemic areas.