Effect And Mechanism Of Ca²⁺-CaN-NFAT Signaling Pathway In Oxygen-glucose Deprivation Preconditioning Of PC12 Cell

Experimental objectiveIschemic cerebrovascular disease is a common clinical disease at present,with a high rate of disability and mortality,and it is no longer simply a senile disease.Young stroke patients are more and more common in the clinic,bringing a heavy burden to the patients’families and society.Although great progress has been made in the etiology,risk factors,primary prevention,secondary prevention,drug treatment and interventional treatment in ischemic cerebrovascular disease,the effectiveness of prevention and treatment are still not satisfactory,the incidence of ICVD in China is still increasing year by year.In recent years,ischemic hypoxia preconditioning（IPC）has been found to effectively reduce focal tissue ischemia/reperfusion injury in animals,and ultimately play a protective role in the brain^[4].Brain tissue is induced to have a protective effect on subsequent fatal ischemia,and this late resistance is known as ischemic tolerance（BIT）.This provides a new direction for the prevention and treatment of ICVD.The mechanism of ischemic tolerance induced by ischemic preconditioning is still unclear,but current studies suggest that preconditioning needs to activate multiple interrelated signaling pathways to generate ischemic tolerance,so as to reduce nerve damage and promote nerve protection.One of the important factors affecting cell activity is intracellular calcium ions.Cerebral ischemia may lead to intracellular calcium overload and late death of nerve cells.The activation of calcineurin（CaN）is the key to cell injury induced by increased intracellular calcium ion concentration.Calcineurin is directly regulated by calcium ions in most cell signal transduction pathways and participates in various cell functions.One of the important physiological substrates of calcineurin is the Nuclear Factor of Activated T cell（NFAT）,which is the downstream gene of calcineurin.Ca²⁺-CaN-NFAT signaling pathway has a closely related target gene Cyclooxygenase-2（COX-2）,which is closely related to NFAT in cardiomyocytes,skin cells and lung cells.NFAT is the upstream regulatory gene of COX-2,which can regulate its protein expression.It was found that the signal transduction pathway of Ca²⁺-CaN-NFAT in skin was expressed in melanoma.Inhibition of the Ca²⁺-CaN-NFAT signaling pathway in chronic heart failure models can effectively improve the prognosis of heart failure.The Ca²⁺-CaN-NFAT signaling pathway plays an important role in different cells,but little research has been done on this neural pathway.In this study,PC12 cell lines were used to establish ischemic and hypoxic preconditioning models,in order to observe the role of Ca²⁺/CaN/NFAT signaling pathway,and to explore the role and possible mechanism of Ca²⁺/CaN/NFAT in the process of apoptosis in ischemic and hypoxic preconditioning,and to understand the role of calcium ion carrier A23187 and calcium ion blocker CyclosporinA（CsA）,in ischemic and hypoxic preconditioning.Experimental grouping1.Control group:PC12 cells,1640 medium+10%fetal bovine serum+5%100U/ml penicillin+100 U/ml streptomycin;2.OGD experimental group:Oxygen sugar deprivation treatment after PC12 cells were attached to the wall;3.Pre experimental group:PC12 cells were pretreated with oxygen deprivation after adherence;4.A23187 experimental group:PC12 cells were adhered to A23187 after adherence,and subjected to oxygen sugar deprivation pretreatment after 24 hours of culture;5.CsA experimental group:PC12 cells were adhered to CsA after adherence,and subjected to oxygen sugar deprivation pretreatment after 24 hours of culture;6.A23187+CsA experimental group:PC12 cells were adhered to A23187+CsA after adherence,and subjected to oxygen sugar deprivation pretreatment after 24 hours of culture;7.DMSO experimental group:PC12 cells were adhered to DMSO,and cultured for24 hours,followed by oxygen sugar deprivation pretreatment.Materials and methods1.Culture of PC12 cells（1640 medium+10%fetal bovine serum+5%double antibody,37℃,5%CO2 incubator）;2.Oxygen sugar deprivation treatment（OGD）（MEM medium,37℃,5%CO2 and95%N2 in an anoxic chamber）;3.Oxygen sugar deprivation pretreatment（OGDP）（1.5 h OGD treatment 24 h before OGD）;4.MTT detects cell viability;5.Real Time PCR method for semi-quantitative detection of mRNA transcription levels of CaN,NFAT and Cbl-b;6.Western blot analysis was used to detect the expression of CaN,NFAT and COX-2 proteins;7.Statistical analysis:all values were the results of 3-5 repeated experiments,and the single-factor variance test was conducted using SPSS22.0（Statistical Product and Service Solutions）statistical tool,and the values were expressed as mean standard deviation.Experimental results1.MTT assay:the survival rate of cells in ischemia-hypoxia group was lower than that in normal control group（P<0.05）.The survival rate of cells in ischemia-hypoxia group was lower than that in hypoxia-preconditioning group（P<0.05）.The survival rate of cells treated with CsA 24h before ischemia and hypoxia preconditioning was higher than that of cells treated with A23187（P<0.05）.The survival rate of CsA treated cells prior to ischemia and hypoxia preconditioning was higher than that of the preconditioning group（P<0.05）.The survival rate of cells added with A23187 before ischemia and hypoxia preconditioning was lower than that of the preconditioning group（P<0.05）.2.Real Time PCR detection:The mRNA expressions of CaN,NFAT and Cbl-b were significantly higher than that of the control group after ischemia treatment（P<0.05）.The mRNA expression of CaN,NFAT and Cbl-b decreased after pretreatment,and the difference was statistically significant（P<0.05）.The trend of the three indexes in the same group was in the same direction;3.Real Time PCR detection:After adding A23187 before ischemic preconditioning,the mRNA expressions of CaN,NFAT and Cbl-b were increased compared with the pretreatment group.The expression of CaN,NFAT and Cbl-b mRNA was simpler after CsA addition.The pretreatment group decreased,and the mRNA expression of CaN,NFAT and Cbl-b were between the former two after A23187+CsA application;4.Western blot analysis:The treatment significantly reduced the expression of NFAT and COX-2（P<0.05）.Before the ischemic preconditioning,the calcium blocker CsA was added,and the expression of CaN,NFAT and COX-2 protein was significantly decreased（P<0.05）.Calcium carrier A23187 was added before pretreatment,and the expression of CaN,NFAT and COX-2 protein was increased compared with the pure pretreatment group（P<0.05）.Experimental conclusions1.The survival rate of cells in the ischemic group was decreased,and the expressions of CaN,NFAT,Cbl-b and COX-2 were increased.Ischemic preconditioning reversed the ischemia-induced reduction in PC12 cell survival,and had a protective effect on cells,and CsA enhances the effect of protecting cells,and calcium carrier A23187 has a tendency to reverse the effect of such protection.2.A23187 and CsA play important regulatory role in ischemia and ischemic preconditioning,suggesting that changes in intracellular Ca²⁺concentration may be the inducing factor of neuroprotection during ischemic preconditioning;3.Ischemic preconditioning can protect nerve cells through the Ca²⁺-CaN-NFAT pathway.