Effect Of Hypothermia On Insulin-degrade Enzyme Expression Of Mouse’s Neuroblastoma Cells With Oxygen-glucose Deprivation-Restoration Injury

Objective:To observe the effect of hypothermia（32℃,24h）on injury and apoptosis of cells,as wells as survival rate,apoptosis rate,caspase-3 activity,β-amyloid concentration,expression level of insulin-degrading enzyme（IDE）protein and m RNA in mouse’s neuroblastoma（N2a）cells at 24 h and 48 h after oxygen-glucose deprivation/restoration of oxygen-glucose（OGD/R）injury.In order to find out whether the mechanism of hypothermic protect cerebral protection has relationship with the expression of IDE.Methods:Mouse N2a cells in good growth state and with logarithmic growth phase is selected as the study subjects of this experiment,then divided them into 3groups equally（n=70）by a random number table method:named the control group,the OGD/R group（model group）and the hypothermia plus OGD/R group （hypothermia group）.Cells were cultured with high glucose cell culture medium（DMEM）with penicillin（100 U/m L）,streptomycin（100μg/ml）,and fetal bovine serum（10%）at normal condition（37°C with 5%CO₂,21%O₂,and 74%N₂）in a cell incubator,and cells in control group was cultured only under normal condition,apart from replacement of the culture system on-time without any other culture condition change treatment.In model group,the high-glucose DMEM was changed to sugar-free DMEM and cultured in an incubator for 3 h with 5%CO₂,95%N₂and 37°C to establish the oxygen-glucose deprivation model,then the sugar-free DMEM was changed to high-glucose DMEM culture medium and cultured under the same conditions as the normal group for 24 h and 48 h to restore oxygen and glucose.After the oxygen-glucose deprivation model was established in cells in hypothermic group,the incubator temperature was first changed to 24 h of restoration of oxygen-glucose at 32°C hypothermia condition,followed by 24 h of culture in the normal environment.Various parameters were measured at 24 h and 48 h after cell reoxygenation,including the damage and apoptosis of cells in each group observed by Hoeschst 33258 staining,the survival rate of cells detected by cell counting kit（CCK-8）,cell’s apoptosis rate counted with flow cytometry,activity of caspase-3 detected through spectrophotometry,β-amyloid concentration of cells in each group detected by enzyme-linked immune sorbent assay（Elisa）,IDE expression of in cells in each group detected by Western blot（for protein）and reverse transcription-polymerase chain reaction（q RT-PCR,upstream target m RNA of IDE protein）.Results:1.Changes of cells’apoptosis injury,rate of survival and apoptosis:Compared to cells in control group,the number of apoptosis cells was increased,the rate of survival was decreased（P<0.05）,the rate of apoptosis was increased at 24 h and 48 h after restoration of oxygen-glucose in model group.Compared to cells in model group,the number of apoptosis cells was decreased（P<0.05）,the rate of survival was increased（P<0.05）,the rate of apoptosis was decreased（P<0.05）at each time point after restoration of oxygen-glucose in the hypothermia group.2.Changes of caspase-3 activity:Compared to cells in control group,the caspase-3 activity of cells in model group and hypothermia group was increased at 24h and 48 h after restoration of oxygen-glucose（P<0.05）.Compared to cells in model group,the activity of caspase-3 in cells in hypothermia group was decreased at each time point after restoration of oxygen-glucose（P<0.05）.3.Changes ofβ-amyloid concentration in cells:Compared with cells in control group,theβ-amyloid concentration of the cells in model group and hypothermia group was increased at 24 h and 48 h after restoration of oxygen-glucose（P<0.05）.Compared with the cells in model group,theβ-amyloid concentration of the cells in the hypothermia group was decreased at each time point after restoration of oxygen-glucose（P<0.05）.4.Changes of IDE protein and m RNA in cells:Compared to cells in control group,the IDE protein and m RNA was decreased at 24 h and 48 h after restoration of oxygen-glucose（P<0.05）in cells of model group and hypothermia group;compared to cells in model group,the IDE protein and m RNA in cells of hypothermia group was increased at each time point after restoration of oxygen-glucose（P<0.05）.Conclusion:1.Hypothermia can significantly reduce the oxygen glucose deprivation/double glucose reoxygenation injury of mouse N2a cells and inhibit the cell injury and apoptosis after oxygen glucose deprivation/double glucose reoxygenation injury of mouse N2a cells,and reduce the increase and accumulation ofβ-amyloid after oxygen glucose deprivation/double glucose reoxygenation injury of mouse N2a cells.2.The mechanism by which hypothermia alleviates oxygen-glucose deprivation/reoxygenation injury and apoptosis in mouse N2a cells is associated with its promotion of IDE expression thereby inhibiting the increase and accumulation ofβ-amyloid.