TRPM7siRNA Regulates Neuronal Autophagy And Apoptosis Induced By Epilepsy Through The PI3K/AKT Pathway

ObjectiveTo observe the effect of gene silencing transient receptor potential melastatin 7(TRPM7)on neuronal apoptosis,autophagy related protein expression and PI3K/AKT pathway in epileptic cell model,and to explore the protective effect of gene silencing TRPM7 on neurons.Methods1、 Mouse brain neuroblastoma N2a(Neuro-2a)cells were treated by magnesium free extracellular fluid for three hours to build an in vitro epileptic cell model.2、The TRPM7 expression of Neuro-2a cells was silenced by TRPM7 si RNA transfection.There were four experimental groups: control group(Control),epileptic group(EP),epileptic transfection group(EP+si-TRPM7)and epileptic transfection negative control group(EP+si-NC).The expression of TRPM7 protein in each group was checked by Western blot.The percentage of apoptotic cells was detected by Td T-mediated d UTP Nick End Labeling(TUNEL).The expression of autophagy-related genes,ATG5,ATG7,ATG12,P62 and LC3 B was observed by immunofluorescence and Western blot.3、In order to observe the effect of transfection of TRPM7 si RNA on the PI3K/AKT pathway of neurons,the experiment was divided into six groups:control group(Control),epileptic group(EP),epileptic transfection group(EP+si-TRPM7),epileptic transfection negative control group(EP+si-NC),epilepsy and inhibitor group(EP+LY294002),epileptic transfection and inhibitor group(EP+si-TRPM7+LY294002).Western Blot technology was used to observe PI3K/AKT expression.Results1、Compared with the control group,the expression of TRPM7 protein and the percentage of TUNEL positive cells increased in the EP group.The expression of autophagy related proteins,ATG5,ATG7,ATG12 and LC3 B increased,while the expression of P62 protein decreased in the EP group.Compared with the EP+si-NC group,the expression of TRPM7 protein and the percentage of TUNEL positive cells decreased in the EP+si-TRPM7 group.The expression of autophagy related proteins,ATG5,ATG7,ATG12 and LC3 B decreased,while the expression of P62 protein increased in the EP+si-TRPM7 group.2、Compared with the Control group,the PI3K/AKT pathway in the EP group was inhibited,and the p-PI3K/PI3 K and p-AKT/AKT were significantly reduced.Compared with the EP+si-NC group,the activity of the PI3K/AKT pathway increased in the EP+si-TRPM7 group,and the p-PI3K/PI3 K and p-AKT/AKT were significantly increased.The administration of PI3 K inhibitor,LY294002,reversed the activation of TRPM7 si RNA on neuronal PI3K/AKT pathway,and p-PI3K/PI3 K and p-AKT/AKT were significantly reduced.ConclusionsGene silencing TRPM7 can up-regulate PI3K/AKT activity and reduce neuronal autophagy and apoptosis.TRPM7 si RNA may regulate neuronal autophagy and apoptosis due to epilepsy via the PI3K/AKT pathway.