Erythropoietin Activates Keap1-Nrf2/ARE Pathway In Brain After Ischemia

Background：Cerebral vascular disease is one of the most common and severe diseases in ourcountry.The high incidence,recurrence rate and expenditure cause enormous social and economicburdon.CVD has been the public health problem.As the improvement of society and people，sliving standard and the tendency of population ageing,there are more younger people sufferedCVD.Ischemic cerebrovascular diease accounts60-70percent. In ischemic cerebrovasculardiease,the blood flow in intracal arteries is reduced, with the brain tissue necrotic.Studiesconfirmed that after a persist4min ischemia,the necrotic brain tissue can not be revived andgeneratied,thus it may cause severe disablity.In recent years,there are a lot of studies in diagnosisand treatment of CVD in acute stage,great improvement has obtained,however, the mechanism isstill unknown. Oxidative stress become the hot point now.Objective:Establish the permanent middle cerebral artery occlusion in rats,study the influence ofrhEPO on the expression of Nrf2and HO-1in acute cerebral ischemia of rat brain, explore themechanism of rhEPO antioxidant effect.Methods:36healthy male SD rats were randomly divided into sham operated group（n=12）, modelgroup（n=12）, rhEPO group（n=12）. The models（pMCAO） were made by left middle cerebralartery occlusion. After anesthesia,cut in middle of the neck,segregate the common carotid artery,internal carotid artery and internal carotid artery.The fish line should be in about18-20mm from the croth of common carotid artery in the model group and rhEPOgroup,however,it should be only10mm in the sham operated group,then ligate the commoncarotid artery.rhEPO(5000IU/Kg) was injected intraperitoneally2h later after the successfulischemia model in rhEPO group, while sham operated group and model group were given equalvolume saline. Rats were killed24h later, pick out6rats in every group for HE stain andimmunohistochemistry,after anesthesia by chloral hydrate(10%),exposure the heart,200mlsodium chloral was infused from the left ventricle,cut the right auricle at the same time, then200ml paraform was used for fixed.After that,it should be fixed by neutral formalin for24hours.The other6rats in every group were taken left brain cortex directly for WesternBlotting.The content of Nrf2and HO-1were detected by immunohistochemistry and WesternBlotting. Results:1、HE results：In sham operated group, the structure of the brain tissue was compact,theratio of neurons and glial cells was appropriate.The cell shape was normal,there was no edemaand vacuole in the tissue.There were a lot of necrotic areas around which the tissue was loose inmodel group,pyknosis and distorted cell bodies were visibte.Compared with the model group,thenecrotic areas were smaller,cell edema was lighter in rhEPO group.2、The immunohistochemistry results：compared with the sham operated group, the positivecells of HO-1increased in model group and rhEPO group, rhEPO group had more positive cellsthan model group(P <0.05, P <0.05). There were rare plasma-positive cells of Nrf2in shamoperated group,whereas nuclei-positive cells of Nrf2increased in model group,even in rhEPOgroup(P <0.05, P <0.05). Positive cells were mainly neurons and astrocytes in ischemic areas3、Western Blotting results：the expression of Nrf2and HO-1were enhanced in the order ofsham operated group, model group and rhEPO group. The difference was statistically significant(P <0.05).Conclusions:1、After ischemia,the level of Nrf2and HO-1were uprelated.2、Erythropoietin may exerts neuroprotective effects by activating Keap1-Nrf2/AREpathway after acute cerebral ischemia.