| High incidence of cerebrovascular disease seriously harm human health. Stroke, the most common life-threatening disease of the nervous system, is currently the third largest cause of death and the first cause of disability. Cerebral ischemic/hypoxic injury-induced neuronal death is the main reason leading to loss of neurological function. The mechanism of ischemic brain damage has not been clarified and there is a lack of clinically effective therapies. Therefore, the works of studying neuronal death mechanisms and exploring new therapeutic targets have important clinical significance.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 caspase-independent 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.Our preliminary data have found that BNIP3 expression increased and inserted into the mitochondria, and then induced AIF releasing from the mitochondria, translocation to the nucleus in OGD/R (oxygen glucose deprivation/ reoxygenation) model of primary cultured hippocampal neurons. However, whether BNIP3 is involved in the death of hippocampal neurons in cerebral ischemia/reperfusion injury and whether the nuclear translocation of AIF occurred are still unclear.BNIP3 (Bcl-2/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.But the downstream moleculars in BNIP3-induced cell death pathways are not yet clear. We have known that AIF is one of the downstream elements which can induce mitochondrial dysfunction and involved in the caspase-independent cell death pathway. AIF locates 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 CA1 neurons show cell shrinkage, nuclear fragmentation and disappear, structure unclear after seven days ischemic/reperfusion injury compared with the sham-operated group. This confirms that our model really caused the death of hippocampal neurons.Firstly, we evaluated the expression level of BNIP3 in hippocampal CA1 area 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=33.117, P= 0.000). Compared with sham group, the 6 hours,12 hours,24 hours,24 hours groups significantly increased to 1.51±0.10, 1.80±0.14,2.14±0.11,2.46±0.23-fold(P<0.05).After ischemia/reperfusion injury the expression level of BNIP3 dimmers was significantly increased (F=23.113, P= 0.000). Compared with sham group,the 6 hours,12 hours groups significantly increased to 1.82±0.13,1.86±0.10-fold (P<0.05). There was no expression of BNIP3 monomers in mitochondria, and BNIP3 dimmers significantly increased (F=30.214, P=0.000). Compared with the sham group, the 12 hours,24 hours group significantly increased to 1.74±0.13,1.54±0.04-fold (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.Then, we evaluated the expression level of AIF in hippocampal CA1 area and in its mitochondria at different time points after ischemia/reperfusion injury through Western Blot. The results showed that the AIF expression level was significantly increased in the hippocampal CA1 area after ischemia/reperfusion injury(F= 2.643, P=0.49). Compared with the sham group, the 12 hours,24 hours,48 hours group significantly increased to 1.77±0.24,1.67±0.20,2.00±0.37-fold (P< 0.05). The change of AIF expression level suggested that it may participate in the neuronal death of the hippocampus after cerebral ischemia/reperfusion injury.Finaly, the results showed that the mitochondrial AIF expression level was significantly decreased in the hippocampal CA1 area after ischemia/reperfusion injury (F= 18.612, P=0.000).It revealed that AIF may released from mitochondria and trasnlocated to nucleus and suggested that AIF may be involved in BNIP3-mediated death pathway in hippocampal neurons.Meanwhile, we observed the expression level of nuclear AIF in hippocampal CA1 area at different time points after ischemia/reperfusion injury through Western Blot. The results showed that the level of nuclear AIF expression was significantly increased in the hippocampal CA1 area after ischemia/reperfusion injury(F= 4.447, P=0.016). It revealed that AIF may released from mitochondria and trasnlocated to nucleus and suggested that AIF may be involved in BNIP3-mediated death pathway in hippocampal neurons. To further clarify whether the AIF released from mitochondria and trasnlocated to nucleus, we observed the level of cytoplasmic AIF expression. The results showed that the level of cytoplasmic AIF expression was significantly increased in the hippocampal CA1 area after ischemia/reperfusion injury (F=51.001, P=0.000). It revealed that AIF may released from mitochondria and didn't remain in cytoplasm.The results further indicated that AIF may released from mitochondria and trasnlocated to nucleus and suggested that AIF may be involved in BNIP3-mediated death pathway in hippocampal neurons.In summary, the obtained results provide the basis for further study the mechanism of BNIP3-induced caspase-independent cell death.
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