The Effect Of Omi/HtrA2 On Neuronal Apoptosis In A Cultured Hippocampal Neuron Epilepsy Model Induced By Mg²⁺-free Media In Vitro

Background and objectiveEpilepsy is a clinical syndrome which can be caused by a lot of reasons. Seizure is a transient disfunction of brain which is characterised by an abnormal neuronal discharge. The attack forms of epilepsy is very complicated, and repeated seizures can bring suffering to patients. With the development of medicine, our understanding to epilepsy has become more and more thoroughly. Up to now, there are two million people have refractory epilepsy, though the new drugs have brought hope to patients. The pathogenesis of epilepsy is ambiguous, so it is important to investigate the pathogenesis of epilepsy. In recent years, the study of neuronal apoptosis has gained much more focus, and inhibition of neuronal apoptosis may provide a new therapeutic target. So we should try our best to study how to inhibit neuronal apoptosis at the molecular level, for the sake of protecting injured neurons and restoring their function. In recent years, the relationship between epilepsy and mitochondrial is getting more and more attention. Mitochondrial is an important organelle which mediates both intrinsic and extrinsic apoptosis signaling pathways in cell death. Its function has been studied by many researchers. What'more, the relationship between mitochondrial and epilepsy has been noted by more and more people. Some protein in mitochondrial will be released to cytoplasm or karyon during seizures and then have impact on pro-apoptotic?anti-apoptotic or necrotic process. So regulating these protein may become a new therapeutic target. A new development in the understanding of mitochondrial regulation in apoptosis is the discovery of Omi/HtrA2, which is a pro-apoptotic mitochondrial serine proease involved in both forms of apoptosis, caspase-dependent as well as caspase-independent cell death. Like cytochrome c and AIF, Omi/HtrA2 is located in mitochondrial intermembrane space, and it can be released from mitochondrial and translocate to the cytosol in response to apoptotic stimuli. In the cytosol, it can both neutralize and inactivate the XIAP, which blocks apoptotic pathways by inhibiting initiator and effector caspases. What's more, Omi/HtrA2 can also induce apoptosis in a caspase-independent manner that only relies on its protease function. Recent experimental evidence has revealed that Omi/HtrA2 plays a vital role in many diseases,such as focal cerebral ischemia/reperfusion,renal ischemia/reperfusion injury and alveolar epithelial cell apoptosis by hyperoxia. However,the impact of Omi/HtrA2 in the apoptotic cell machinery that takes place in vitro under pathological conditions such as epilepsy remains unknown. In this study, we sought to examine whether Omi/HtrA2 plays a decisive role in apoptosis observed after epileptiform discharges in hippocampal neurons from newborn SD rats. In other words,whether Omi/HtrA2 is released from mitochondrial to the cytosol, if so, whether this accumulation induced XIAP degradation and caspase-3 activation and the subsequent neuronal apoptosis. We also analysised whether Ucf-101, the specific inhibitor of Omi/HtrA2, can provide neuroprotection in vitro epilepsy model. Finally, we may provide theory basis for treatment of epilepsy. Marerials and MethodsThe experimental subject is Sprague-Dawley rats which are borned in 24 hours, disinfect them with alcohol. And then take out of the brain, put it in a petri dish with phosphate buffer. Neonatal rat hippocampal tissues were sterilely obtained to prepare monoplast suspension. The neurons were seeded in 6 well plate. Above is the method of primary culture. Growth of the neurons were observed by inverted phase contrast microscope. When the neurons were cultured to the seventh to tenth day, we can use them for the following experiment. We used the neuron-specific enolase method to identify the hippocampal neurons. The neurons were randomly divided into control group and experimental group when they were cultured to the tenth day. To establish the experimental group, the cultured neurons were exposed to zero-Mg²⁺ treatment for three hours? the Sombati method?, and then returned to normal media for 3h, 8h or 24 h. For the Ucf-101 treated group and DMSO treated group, neurons were treated with Ucf-101 or DMSO before exposed to Mg²⁺-free media. TUNEL were used to measure apoptotic neurons, and the expression of Omi/Htra2?HAX-1?caspase-3 and XIAP were detected by immunoblotting. Results1. Morphologic observation of the cultured neurons: The cells' shape is almost round when they were seeded in culture plate. Cell adherence began after three hours. After 24 hours almost all of the neurons adhere to the plate, what' more, part of the neurons began growing dendrites. While there may be some neurons die because failing to adhere to the plate. With the extending of incubation time, neuronal protrusion became more and more. 5 days later, synaptic connections were observed, but the connections were still sparse. The connections became more and more tighter when the neurons growing to the seventh day. The neurons were found as round or ellipse, having many neurites. Body of neurons were largest 2 weeks later, and the connections between neurons were tighter at this time. When the cells growing to the fourth week, there would be many cell debris in the medium.2. The purity identification of hippocampal neurons: After being cultured in vitro for the seventh day, the purity was measured by immunofluorescence cytochemistry method. The result showed that the purity of hippocampal neurons was more than 90%.3. Western blots: The results showed that the expression of Omi/HtrA2 in mitochondrion was increased after 3h post epileptiform discharges?P<0.05?. However, as time went on, Omi/Htra2 expression in the mitochondrial fraction decreased dramatically after neurons subjected to Mg²⁺-free media. In contrast, cytosolic Omi/Htra2 was markedly increased post neuronal epileptiform discharges?P<0.05?. The expression of caspase-3 was distinctively higher in 24 h post epileptiform discharges than in control group?P<0.05?. What's more, the expression of HAX-1 was significantly decreased in 24 h post epileptiform discharges group compared with control group?P<0.05?. However, there were no dramatic changes in XIAP levels have been observed between the experimental group and control group?P?0.05?. The expression of caspase-3 were significantly decreased in Ucf-101 treated group compared with 24 h post epileptiform discharges group?P?0.05?, in contrast, the expression of XIAP and HAX-1 were significantly increased after Ucf-101 treatment?P?0.05?. There were no significantly different between DMSO group and 24 h post epileptiform discharges group?P>0.05?.4. Pathology observation of neurons: The number of TUNEL positive neurons was distinctively higher in 24 h post epileptiform discharges than in control group?P?0.05?. While the number of TUNEL positive neurons was significantly decreased in Ucf-101 treated group compared with 24 h post epileptiform discharges group?P?0.05?. There was no significantly different between DMSO group and 24 h post epileptiform discharges group?P>0.05?. Conclusions1. Omi/Htra2 translocated from mitochondria into the cytosol upon neurons subjected to zero-Mg²⁺ treatment.2. Omi/HtrA2 is a pro-apoptotic mitochondrial serine proease involved in both forms of apoptosis, caspase-dependent as well as caspase-independent cell death.3. Ucf-101, a Omi/HtrA2 inhibitor, could inhibit neuronal apoptosis, downregulate the expression of caspase-3, upregulate the expression of XIAP and HAX-1, and then afford its neuroprotection.