The Mechanism Of Cerebral Neuron Apoptosis Induced By Omi/HtrA2 Cleavage Of Mitochondrial Fusion Protein OPA1 In Brain Ischemia And Reperfusion

Background:The mechanism of the neuron damage induced by cerebral ischemia is always the focus of researcher’s attention. Some reports showed that the role of some proteins in physiological processes is not consistent with the pathological processes. The unfolded protein response in mitochondria increased is called mitochondrial stress. Omi / Htr A2 is a kind of serine protease, which is essential for mitochondrial homeostasis in mammals and play a role as the molecular chaperone. It cleaved and matured in the mitochondria and stored in the intermembrane space of mitochondria. OPA1 is one of mitochondrial fusion protein, it has some relationship with apoptotic. When the neuron was stimulated, Omi/Htr A2 is released from the mitochondrial intermembrane space into the cytosol as a pro-apoptotic protein. It can bind with anti apoptosis protein XIAP then activate Caspase 9 and Caspase3 to induce apoptotic. Therefore, our study is of great significance on reveal the dual role of Omi/Htr A2 and OPA1 in cerebral ischemia reperfusion. The study findings will provide new ideas for the treatment, and put forward a new target for treatment of brain injury. Objective:To explore the role of Omi/Htr A2 and OPA1 in cerebral neuron damage and its mechanism in the rat model of transient focal cerebral ischemia and PC12 model of oxidative stress. Methods:In vivo, The male Wistar rats were randomly divided into four group:sham,1H group, 1.5H group and 3H group(the rat exposed to 1 hour, 1.5 hours, 3 hours t MCAO followed by 24 hours of reperfusion, respectively). The rat model of transient middle cerebral artery occlusion(t MCAO) was prepared by the thread embolism method. The brain damage were detected by hematoxylin and eosin(H&E) staining. The protein expression of apoptosis related protein caspase3 and Omi/Htr A2, were analysised by Western Blot.1. In vitro, PC12 cells were exposed to different concentration of H2O2, and ucf-101(a specific inhibitor of Omi/Htr A2). The PC12 cells can be divided into 4 group: con, H2O2, H+U and ucf-101. PC12 cells were treated with these drugs for 6 hours.1) MTT method were used to detect cell viability.2) Western Blot were used to detect protein expression of Omi/Htr A2 and apoptosis-related protein cleaved-caspase 3.3) Microscope were used to observe the changes of cell morp Hpology.4) Western Blot were used to detect protein expression of Omi/Htr A2, apoptosis-related protein cleaved-caspase3, mitochondrial stress protein Clp P, CHOP and mitochondrial fusion protein OPA1.5)Mito-tracker Red staining were used to observe the changes of mitochondria morphology in PC12 cells6) Co-Immunoprecipitation were used to determine physical relationship between OPA1 and Omi/Htr A2. Result:The result in vivo showed that prolongation of ischaemia significantly increased the cortical injury observed in rats and associated with a gradual increase in the protein expression of cleaved caspase3 and Omi/Htr A2. It suggested that Omi/Htr A2 was involved in brain ischemia and reperfusion injury.The result in vitro showed that when PC12 cells exposed to H2O2,the survival rate decreased, when PC12 cells are treated with combination of ucf-101,the survival rates increased. Western Blot results showed that H2O2 can obviously increase the expression of apoptosis related proteins cleaved-caspase3 and Omi/Htr A2 which degrade XIAP to induce apoptosis at the same time. When PC12 cells treated with ucf-101 to inhibit the activity of Omi/Htr A2, the expression level of apoptosis related proteins decreased. These results suggested Omi/Htr A2 was involved in apoptosis induced by oxidative stress. The mito-tracker staining showed that punctate aggregation mitochondria increased in H2O2 group. It indicated that mitochondrial fission and fusion was broken, and the mitochondrial fusion was limited JC-1 staining results showed that the green fluorescence intensity in H2O2 group significantly increased, indicating that the potential of the membrane decreased obviously. When the activity of Omi/Htr A2 is inhibited by Ucf-101, the mitochondrial function and morp Hology is improved. The protein expression of Clp P and CHOP increased in H2O2 group. When the PC12 cell treated with combination of Ucf-101, the mitochondrial stress decreased. It suggested that Omi/Htr A2 was involved in regulation of mitochondrial stress. The direct interaction between OPA1 and mature Omi/Htr A2 was detected by Co-Immunoprecipitation. The expression of L-OPA1 will become S-OPA1 when PC12 cell exposed to H2O2. When the activity of Omi/Htr A2 is inhibited by Ucf-101, the cleavage of OPA1 is inhibited and the expression of S-OPA1 is reduced. It suggested that cytochrome C and Omi/Htr A2 release released from the mitochondria to the cytoplasm when the cleavage of OPA1 induced by Omi/Htr A2. This gives us a new direction of research to explore specifically how Omi/Htr A2 regulate the mitochondrial stress through OPA1. Conclusion:1, Omi/Htr A2 is involved in neuron damage induced by cerebral ischemia reperfusion injury.2, Omi/Htr A2 is involved in neuron apoptosis through regulating mitochondrial stress,, decreasing the mitochondrial membrane potential and cleaving the mitochondrial fusion protein OPA1,which can limit the mitochondrial fuion.