Effect Of Progesterone On The Expression Of ATP-sensitive Potassium Channel Subunit Kir6.2 In Rat Primary Cortical Neurons Damaged By Aβ_25-35

Alzheimer’s disease（AD）is a degenerative disease of the central nervous system accompanied by cognitive behavior and dysfunction.It is characterized by insidious onset of progressive disease.The pathological feature is the accumulation of extracellular Aβin the brain,resulting in neurotoxicity and causing a large number of neurons to be lost.The clinical manifestations of learning and memory impairment are prominent.Studies have shown that CaMKII（calmodulin-dependent protein kinase II）acquires ATP in learning and memory.The ATP-sensitive potassium channel(K_ATP)is composed of an inward rectifier potassium channel subunit（Kir6.1/Kir6.2）and a rhythmic potassium channel sulfonylurea receptor（SUR1/SUR2）1:1Constitutive octamer,conjugated to cell electrical activity and cell metabolism,K_ATPTP channels are widely distributed in cortical neurons and hippocampal neurons.Early studies have shown that fine neuronal injury is accompanied by the loss of a large number of potassium ions,and recent pharmacology Studies have found that K_ATPTP channels may be involved in neuroprotection and may be a potential therapeutic target for AD.Because Aβcan cause mitochondrial dysfunction,ATP levels decline,and then cell metabolism occurs disorder,eventually leading to neuronal damage,and K_ATPTP channel function in the normal state to maintain the mitochondrial membrane potential of nerve cells and inhibit neuronal apoptosis.Progesterone（PROG）as an endogenous neurosteroid plays an important role in the treatment of normal neural activity and neurological diseases.The influence of progesterone on potassium channels and inhibition of neuronal apoptosis in the AD model is not yet clear.PartⅠProgesterone regulates Kir6.2 and CaMKII protein in neuronalinjury induced by Aβ_25-35Objective:To observe the protective effect of different concentrations of progesterone on primary cultured neurons and to explore the mechanism of progesterone regulation of K_ATPTP channels.Methods:Neonatal SD neonatal rats were taken within 24h.Cortical neurons were obtained from cortical cultures in a sterile clean bench and neuron purity was determined by immunocytochemical staining.The AD cell model was established using the Aβactive fragment Aβ_25-35,MTT method and Hoechst33258 staining method were used to detect the effect of different concentrations of progesterone on neuronal activity and apoptosis rate.Western blot was used to detect the effect of progesterone on Aβ_25-35-induced neuron Kir6.2 protein expression.After the action of pinacidil on neuronal cells,the expression of Kir6.2 and p-CaMKII/CaMKII protein was observed.Results:1.Identification of neuronal purityImmunofluorescence double staining showed that the neuron purity was90.5%.2.Effect of progesterone on the survival rate of neurons treated with Aβ25-35After MTT assay,Compared with the Aβ_25-355-35 group,there were no significant difference in the Aβ_25-35+PROG（0.5μM）group and Aβ_25-35+PROG（1μM）group.The cell viability of the Aβ_25-35+PROG（2μM）and Aβ_25-35+PROG（4μM）groups was significantly higher than that of the Aβ_25-35group（P<0.05）、（P<0.01）.3.Effect of progesterone on apoptosis of neurons induced by Aβ_25-35Hoechst33258 staining revealed,the apoptosis of Aβ_25-35+PROG（0.5μM）and Aβ_25-35+PROG（1μM）group was no significant difference than that of Aβ_25-355-35 group.The apoptosis of Aβ_25-35+PROG（2μM）and Aβ_25-35+PROG（4μM）groups was significantly lower than that of Aβ_25-355-35 group（P<0.01）、（P<0.01）.4.Effect of progesterone on Kir6.2 protein expression in neurons treated with Aβ_25-35Western Blot showed,compared with the Aβ_25-355-35 group,Aβ_25-35+PROG（0.5μM）group and Aβ_25-35+PROG（1μM）group were no significant difference in Kir6.2 protein expression,but Kir6.2 protein expression was significantly decreased in Aβ_25-35+PROG（2μM）and Aβ_25-35+PROG（4μM）groups（P<0.05）、（P<0.01）.5.Effect of progesterone on expression of CaMKII protein in neurons treated with Aβ_25-35Western Blot showed,compared with the Aβ_25-355-35 group.The protein expression of Kir6.2 in Aβ_25-35+PROG（2μM）group was decreased（P<0.05）,and the expression of p-CaMKII/CaMKII protein was significantly increased（P<0.05）.The expression of Kir6.2 protein was significantly increased（P<0.05）in Aβ_25-35+PROG（2μM）+Pin（10μM）group compared with Aβ_25-35+PROG（2μM）group.When elevated,the CaMKII/CaMKII protein expression was significantly reduced（P<0.05）.PartⅡEffect of progesterone gene regulation and non-gene regulationon Kir6.2 protein expressionObjective:Exploring the Mediation of Progesterone Membrane Receptor and Nuclear Receptor on Progesterone Regulation of ATP Sensitive Potassium ChannelsMethods:The effect of pretreatment with progesterone membrane antagonist AG205 and progesterone antagonist RU486 on neuronal activity and apoptotic rate was examined by MTT method and Hoechst 33258 staining method.The expression of Kir6.2 protein was detected by western blot.Variety.Results:1.Effect of progesterone receptor antagonist on cell viability of injured Aβ_25-355-35 neuronsAfter MTT assay,compared with the Aβ_25-355-35 group,the cell viability was significantly increased（P<0.01）in the Aβ_25-35+PROG（2μM）group.There was no significant difference between Aβ_25-35+PROG（2μM）group and Aβ_25-35+PROG（2μM）+RU486（10μM）group.Compared with Aβ_25-35+PROG（2μM）group the cell viability was significantly increased（P<0.05）in the Aβ_25-35+PROG（2μM）+AG205（10μM）.2.Effects of progesterone receptor antagonists on apoptosis of neurons induced by Aβ_25-35Hoechst33258 staining revealed,the apoptosis rate of Aβ_25-35+PROG（2μM）group was significantly lower（P<0.01）than that of Aβ_25-35group.There was no significant difference in apoptosis rate between Aβ_25-35+PROG（2μM）+RU486（10μM）group and Aβ_25-35+PROG（2μM）group.Compared with Aβ_25-35+PROG（2μM）group,apoptosis significantly increased（P<0.05）in the Aβ_25-35+PROG（2μM）+AG205（10μM）group.3.Effect of progesterone receptor antagonist on expression of Kir6.2protein in Aβ_25-355-35 injured neuronsWestern Blot showed,compared with the Aβ_25-355-35 group,the expression of Kir6.2 protein was significantly decreased（P<0.01）in the Aβ_25-35+PROG（2μM）group.compared with Aβ_25-35+PROG（2μM）group the Kir6.2protein expression was increased（P<0.05）in Aβ_25-35+PROG（2μM）+AG205（10μM）group,There was no significant difference in Kir6.2 protein expression between Aβ_25-35+PROG（2μM）+RU486（10μM）group and Aβ_25-35+PROG（2μM）group.Conclusions:1.Progesterone significantly reduces Aβ-induced neuronal apoptosis and exerts neuroprotective effects.2.Progesterone significantly down-regulates the expression of Kir6.2protein and significantly increases the phosphorylation level of CaMKII protein,which may be one of the molecular mechanisms by which progesterone exerts neuroprot-ective effects.3.The downregulation of Kir6.2 protein expression by progesterone can be reversed by the PGRMC1 receptor antagonist.