Study On The Protective Effects Of EPO On Isofluane-induced Neuron Apoptosis In Developing Hippocampal Neurons

Objective:First,investigate the isoflurane effect on apoptosis in developing hippocampal neurons and possible mechanism.Second,protective effect of EPO on isoflurane-induced neurotoxicity in developing hippocampal neurons.Methods:Hippocampal neurons were separated from SD rat fetus at embryonic days 18-20.The neurons were determined by Neuron Specific Enolase(NSE)use immunofluorescence method in order to determine the purity of hippocampal neurons.Neurons were randomly divided into three groups:control group,isoflurane group,EPO dose group.Neurons were cultured with normal culture medium in control group.0.42 mmol/L isoflurane solution was given to neurons in isoflurane group and EPO dose group,and 1 U/mL EPO was given to EPO dose group at meanwhile.Collecting cell culture medium and neurons after 24 h to perform relevant experiments.Methyl Thiazolyl Tetrazolium(MTT)was used to detect neuron cell viability.Western Blot(WB)was used to detect the expression of apoptosis related protein(Bax,Caspase-3,Caspase-9 and Bcl-2).Flow cytometry was used to detect apoptosis rate of hippocampal neurons.CaMK ?activity in hippocampal neurons was determined by ELISA method.Hippocampal neurons was incubated with Fluo-3 fluorescent prob,and observe the Ca2+ concentrate variation under the fluorescence microscope of different groups.Immunofluorescence method was used to detect the expression microfilament microtubules of hippocampal neuronal and measure the length of axon.Results:Hippocampal neuron isolated in this study have to stick wall after 1 d,and at 8 d neuronal cell body plump,protuberant intertwined to form network structure and the halo contour is obvious at 8 d.NSE expression present strong positive in hippocampal neurons,and with high purity.MTT detecting found that the hippocampal neuron cell vitality of isoflurane group was obviously lower compared with control group(p<0.01),and hippocampus neuron cell vitality of EPO dose group was obviously higher than that of isoflurane group(p<0.05),and close to the control group.The results of WB detecting apoptosis related proteins show that the expression trend of Bax,Caspase3 and Caspase9 of pro-apoptotic proteins is consistent,both the expression of isoflurane group was obviously higher than that of control group(p<0.01),the expression of EPO dose group was significantly decreased compared with isoflurane group(p<0.01).On the contrary,the expression of anti-apoptosis protein Bcl-2 was obviously lower in isoflurane group than control group(p<0.01),and was obviously higher in EPO dose group than isoflurane group(p<0.01).The result of flow cytometry detecting cell apoptosis show that apoptosis rate is the lowest in control group,hippocampus neuron cell apoptosis rate increasing significantly in isoflurane group(p<0.01),and the apoptosis rate of EPO dose group decreasing compared with isoflurane group,but still higher than the control group.The result of flow cytometry and WB corresponding to each other.Ca2+ fluorescence intensity of hippocampus neuron in isoflurane group was significantly higher than control group(p<0.01)after incubated with fluo-3 probe.The fluorescence intensity of EPO dose group was significantly decreased compared with isoflurane group(p<0.01),but still higher than control group.CaMK II activity of hippocampus neuron cell culture medium in isoflurane group was significantly decreased compared with control group(p<0.01),and that in EPO dose group was obviously higher than that of isoflurane group(p<0.05),even restore to the basic level in control group.Neuron cell microfilament microtubules immunofluorescence test found that morphology of hippocampal neurons is better in control group,and the length of axon is longer.Irregular morphology was appeared in isoflurane group,and axon length significantly decreased(p<0.01).Cell morphology and the axon length of hippocampal neurons in EPO dose group similar to the control group,but still lower than the control group(p<0.01).Conclusions:1.Cell activity was reduced In developing hippocampal neurons,cell activity was reduced,Ca2+ concentration was increased significantly,and cell apoptosis was increased obviously in hippocampal neurons under the influence of isoflurane.It suggest that isoflurane result in the calcium excessive accumulation and activate cell apoptosis process,2.Isoflurane induce the activity of CaMK II which closely related to the axon growth cone was decreased obviously,and then influence the growth of axon,which can affect neural signal transmission.3.Multifunctional nerve protective agent EPO play an effective mitigation and protection in neuron apoptosis caused by isoflurane.