The Changes In Expression Of Caspase-Independent Apoptosis-Associated Proteins In Hippocampus After Cerebral Ischemia

Cerebrovascular disease has a high incidence and is harmful to human health,it is a leading cause of death and the third cause of disability.The loss of neurological functions following stroke is caused by massive loss of neurons resulting from hypoxic-ischemic insults.The mechanism underling the brain ischemic injury is unclear and there is no effective clinical treatment yet.Stroke results in acute loss of neurons in the ischemic core region.The acute neuronal damage is followed by a second round of neuronal injury that occurs hours to days after brain ischemia,called delayed neuronal death,in the neighboring areas. Evidence suggests that the delayed cell death occurs primarily through an apoptosis mechanism.Apoptosis is a genetically regulated cell death pathway. The caspase family（cysteinylaspartate-specific protease） plays an important role in apoptosis.However,a growing number of studies support that caspaseindependent pathway also involved in the delayed neuronal death in stroke. Although the mechanism of caspase-independent apoptosis is not clear yet,but some caspase-independent apoptosis-related molecules have been found,such as BNIP3,AIF,EndoG,and PARP.BNIP3（Bclo2/adenovirus E1B-Nineteen KDa interacting protein 3） is a pro-apoptotic protein and it belongs to BH3-only subfamily of Bcl-2 family,a lot of studies have show that BNIP3 participate in the death pathway of several kind of cells,including epithelial cells,myocardial cells,glial cells,neurons and so on. Endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane during induction of cell death.Surprisingly,BNIP3-mediated cell death is independent of cytochrome c release and Apaf-1,caspase activation.This cell death is characterized by early plasma membrane and mitochondrial damage.These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore,proton electrochemical gradient suppression,and increased reactive oxygen species production.But it is unknown that whether and how the BNIP3 participate in the rat hippocampal neuronal cell death in the cerebral ischemia/reperfusion injury.Hypoxia-inducing factor-1（HIF-1） is an important upstream molecular,it can upregulate the BNIP3 expression.But the downstream moleculars in BNIP3-induced cell death pathways are not yet clear.We have known that AIF and EndoG are two downstream elements which can induce mitochondrial dysfunction and involved in the caspase-independent cell death pathway.AIF and EndoG locate in the mitochondrial intermembrane space and when stimulated by apoptotic signals,they release from the mitochondria and translocate to nuclear through mitochondrial permeability pores（permeability transition pores,MPTP） opened,then caused the large fragment cleavage of DNA.But it have indicate that AIF itself cannot cleave DNA,so this function is conducted by other nuclease. EndoG maybe work together with AIF.AIF and EndoG participate in a variety of cell death pathway,but whether they are the downstream elements of BNIP3 induced cell death pathway and the mechanism of they participant in the BNIP3 pathway is not clear. Our experiment first to determine the expression changes of BNIP3 in rat hippocampus after brain ischemia/reperfusion injury,then to further explore its downstream mechanism.First,transient forebrain ischemia（15 min） was induced by the modified 4-vessel occlusion method and the hippocampal neuronal survival condition was studied by Nissl staining.From Nissl staining,it is can be found the hippocampal CAl neurons show cell shrinkage,nuclear fragmentation and disappear,structure unclear after seven days of ischemic/reperfusion injury compared with the sham-operated group.This confirms that our model really caused the death of hippocampal neurons.And then,we evaluated the expression level of BNIP3 in hippocampus and its mitochondria at different time points after ischemia/reperfusion injury through Western Blot.After the model was successfully made,the rats were anesthetized and the whole-cell and mitochondrial samples were made at different time points after ischemia-reperfusion injury The results showed that BNIP3 monomer significantly increased(F_（6,14） =5.330,P =0.005 ).Compared with sham group the 1 hour group significantly increased to 2.26（P＜0.05）,the 2 hours,6 hours,12 hours groups significantly increased to 2.60,2.93,2.61（P＜0.01 ）.There was no expression of BNIP3 monomers in mitochondfia,and BNIP3 dimmers significantly increased(F_（6,28）= 2.699,P = 0.034).Compared with the sham group,the 24 hours group significantly increased（P＜0.05）.The change of BNIP3 expression level suggested that it may participate in the neuronal death of the hippocampus after cerebral ischemia/reperfusion injury.Finally,we evaluated the expression level of AIF and EndoG in hippocampus and in its mitochondria at different time points after ischemia/reperfusion injury through Western Blot.The results showed that the AIF expression had no significant changes in the hippocampus and in its mitochondria after ischemia/reperfusion injury (F_（6,21） = 1.070,P = 0.411:F_（6,14） = 1.089,P = 0.415).Andthe expression of EndoG significantly increased(F_{（6,5.333）}= 38.831,P = 0.000:F_（6,5333） = 27.050, P = 0.001 ),Compared to sham group the 24 hours group significantly increased after ischemia/reperfusion injury（P＜0.05）,and this is inconsistent with the expected results（the expression of EndoG in mitochondrial should be reduced after it translocated to nuclear）.Then we must further evaluate the expression level of Endo G in the nucleus after ischemia/reperfusion injury to determine whether EndoG transfer to nuclear.