Effects And Mechanisms Of Ischemic Preconditioning On Ca2+-CaN-NFAT Signal Pathway And Cbl-b In PC12 Cells

Objective: Cerebrovascular diseases are common diseases that pose a serious threat to human health that it mortality and disability rates are very high.together with cardiovascular disease,malignant tumor constitute the three major causes of death in human diseases.cerebrovascular disease has leapt to the first cause of death in China,in which ischemic cerebrovascular disease accounts for the vast majority.Although ischemic cerebrovascular disease has made great progress in pathogenesis,secondary prevention,gene therapy,there are still some areas of research that are not ideal.especially in the research and development of neuroprotective agents and the mechanism of endogenous brain protection,there has been no significant breakthrough.ischemia and hypoxia can be used as a kind of injurious stressors can endogenous protection mechanism of effective mobilization of organism tissue,Improving tissue tolerance to subsequent severe ischemic injury,therefore,the concepts of ischemic preconditioning and ischemic tolerance are proposed.Ca²⁺ is play a decisive role in nerve cell damage,CaN is the only known intracellular calcium and calmodulin-dependent protein phosphatase,it is the most abundant in the nervous system,and directly regulated by Ca²⁺during cellular signal transduction,which it is participate in multiple cell function regulation.nuclear factor of activated T cell,NFAT that it is CaN exclusive and the most important physiologic substrate.The effect of signaling Ca²⁺-CaN-NFAT pathway on the prognosis of heart failure?CHF?,Ca²⁺-CaN-NFATsignaling pathway can be effectively improved in chronic heart failure?CHF?.The effect of Ca²⁺-CaN-NFAT signaling pathway on the prognosis of heart failure is not known.The effect of Ca²⁺-CaN-NFAT in PC12 cells is verified by this experiment.The model of hypoxia and anoxic preconditioning was established by using PC12 cell line in this experiment,which to observation of Ca²⁺ / CaN / NFAT signaling pathway and the mechanism of Cbl-b and study on the role of oxygen glucose deprivation preconditioning in apoptosis and its possible mechanism.further verification of the role of A23187 calcium ion carrier and cyclosporine CsA calcium blocker in ischemic preconditioning.Group: 1.control?PC12 cell culture medium 10% fetal bovine serum 5% double antibody?;2.OGD?PC12 cells,cell adhered to oxygen sugar capture treatment?;3.PRE?PC12 cells,pretreated with oxygen and glucose capture after adherent to the cell wall?;4.A23187?PC12 cells,cells adhered to the wall,added A23187,cultured for 24 hours after oxygen sugar capture pretreatment?;5.CsA?PC12 cells,after attachment,CSA added,cultured for 24 hours after oxygen sugar capture pretreatment?;6.A23187+CsA?PC12 cells,cell adherent cells,A23187 CsA were added,and after 24 hours culture,oxygen sugar capture pretreatment was carried out?7.DMSO?PC12 cells,after attachment,were added to DMSO.After cultured for 24 hours,the cells were pretreated with oxygen sugar capture?Methods: 1.Cell Culture of PC12?Normal culture medium culture in 5% CO2 medium at 37 ??2.Oxygen sugar deprivation?no sugar,no serum,37 ? and 5% CO2 and 95%N2 hypoxia culture?3.Oxygen sugar deprivation pretreatment?1.5 h OGD treatment 1 hour before OGD?.4.MTT test to cell survival rate.5.Hoechst 33258 to detection of apoptotic cells by nuclear staining.6.Western blot to test the detection of the expression of NFAT ?Cbl-b and CaN protein.7.The data processing: each group of data is represented by 3-5 independent experimental results,using the expression of mean ±standard deviation.The single factor ANOVA is carried out by using SPSS13.0 statistical software.results 1.The results of MTT assay showed that the survival rate of ischemic group was significantly lower than that of normal control group?P < 0.05?,and that of ischemic group was lower than that of ischemic group?P < 0.05?,and that of ischemic group was significantly lower than that of ischemic group?P < 0.05?.The survival rate of PC12 cells in the pretreatment group was significantly higher than that in the A23187 experimental group.The survival rate of PC12 cells in the pretreatment group was significantly higher than that in the A23187 experimental group?P < 0.05?,and the survival rate of PC12 cells in the experimental group was significantly higher than that in the A23187 experimental group,and the survival rate of PC12 cells in the experimental group was significantly higher than that in the A23187 group.The difference between each group was statistically significant?P < 0.05?.2.Detection of Western blot: different protein expression levels showed that Ca2 was involved in the regulation of ischemic preconditioning,and ischemic preconditioning significantly decreased the expression of canine NFAT and Cbl-b.The expression of CaN NFAT and Cbl-b protein induced by calcium blocker CsA was significantly lower in the ischemic preconditioning group than that in the target protein of calcium vector A23187 before pretreatment.The results showed that calcium carrier A23187 significantly weakened the protective effect of ischemic preconditioning,and the difference between the two groups was statistically significant?P < 0.05?.3.Hoechst 33258 cell nuclear staining: after ischemic preconditioning,the cell apoptosis rate was increased,the number of dense and dense staining of nucleus was higher,and the cell apoptosis rate was decreased by using CsA 1 hour before ischemic preconditioning.The number of dense and dense staining in the nucleus was greatly reduced,which was counteracted by adding A23187,which showed increased apoptosis rate and synaptic injury.Conclusion: 1.In ischemic group,the expression of CaN? NFAT? CBLB increased,the cell survival rate decreased,and the nuclear apoptosis rate increased.The PC12 cells in ischemic preconditioning group reversed this process,but increased the survival rate of the cells and played a protective role on nerve cells.2.Calcium carrier A23187 and calcium carrier blocker CsA play important roles in the regulation of cell ischemia and ischemic preconditioning.It is suggested that the change of calcium concentration in cells may be the protective effect of ischemic preconditioning on nerve cells.3.Calcium ions may block the protective effect of nerve cells...