Study On Mechanism Of Neuron Apoptosis And MT/NAC Anti-apoptosis During Mimic Ischemia-reperfusion Injury In Vitro

Objective To further explore the pathway of neuron apoptosis induced by imitative ischemia-reperfusion in vitro, we studied the mechanisms of neuron apoptosis and melatonin and N-acetylcysteine against apoptosis to supply new theoretical gists for foundation research of brain neuron apoptosis induced by ischemia-reperfusion and clinical therapy of aged vascular dementia(VD).Methods Deprived of glucose and oxygen for 90 minutes then reperfusion for different time in vitro,SHY5Y cells were normally cultured to establish neuron cell injury model.The experiment was divided into control, reperfusion and MT/NAC groups. SHY5Y cells apoptosis condition was detected by DNA agarose gel electrophoresis , flow cytometer(FCM) and transmission electron microscope, the cells damage was evaluated by MTT assay , intracellular reactive oxygen species (ROS) was assessed by fluorescence-spectrophotometry , excitatory neurotransmitter glutamate(Glu) release was assessed by HLPC , caspase-3 and lactate dehydrogenase (LDH) activity were assessed by spectrophotometry , Cytochrome C(Cyt.C) release from mitochondria was assessed by Western blotting and ELISA , mitochondrial trans-membrane potential change was measured by fluorescence-spectrophotometry assay and was observed by laser co-focus microscope . It was observed that the process of neuron apoptosis induced by imitative ischemia-reperfusion in vitro whether to pass the approach for mitochondria apoptosis and that melatonin(MT) and N-aceylcysteine(NAC) whether to have the action for neuron protection .Results Cells damage were not observed after mimic ischemia 60 minutes then reperfusion 24 hours , cells apoptosis and DNA fragmentations were observed after mimic ischemia 90 minutes then reperfusion 24 hours . Cells necrosis occurred after mimic ischemia for 120 minutes then reperfusion for 12,24 hours , MT and/or NAC can reduced the generation of apoptotic neurons . Excitatory neurotransmitter glutamates(Glu) largely released from the neurons after mimic ischemia reperfusion and their activity was 70.2% higher than control(P<0.01). Lactate dehydrogenases(LDH) largely released from the neurons and their content increased 41.7% comparing with control(P<0.01). The intracellular ROS sharply increased within reperfusion 1 minute , reached to peak for reperfusion 30 minutes(P<0.01 compared with control) and then decreased gradually , MT and/or NAC can significantly decrease the generation of ROS(P<0.01) . The fluorescence intensity of mitochondrial trans-membrane potential obviously decreased after ischemia-reperfusion 1 hour(P<0.01 compared with control) , evidently increased for reperfusion 3 hours and then continued reducing , MT and/or NAC can prevent the decrease of mitochondrial trans-membrane potential(P<0.01) . Mitochondrial cytochrome C reduced and cytosolic cytochrome C increased after ischemia for 90 minutes then reperfusion 3 hours and reached to peak for reperfusion 6 hours(P<0.01 compared with control) , MT and/or NAC can inhibit the release of cytochrome C from mitochondria(P<0.01) . Caspase-3 activity increased along with the prolong of reperfusion time from 0 to 24 hours , MT and/or NAC can inhibit the activity of caspase-3(P<0.01) . In a word , MT and/or NAC can protect the function and structure of the mitochondria and enhance the survival vigor of SHY5Y cells .Conclusion Mitochondria mediated pathway is one of the mechanisms both neuron apoptosis induced by imitative ischemia-reperfusion and its signal transduction . MT and NAC can protect human neurons from apoptosis induced by imitative ischemia-reperfusion through its anti-oxidation and anti-apoptosis actions .