Objective:To explore the effect of protein kinase C-α(PKC-α) mediated mitochondrial dysfunction on fetal neural tube defects(NTD) caused by gestational diabetes.Methods:Female C57BL/6J mice were randomly divided into control group,NTD group and NTD+sh-PKC-α group,with 6 mice in each group.In addition to control group,in NTD group and NTD+sh-PKC-α group,diabetic embryonism mouse model were established.Mice in the NTD+sh-PKC-α group were injected with 100μL carrying sh-PKC-α lentvirus (1×108 TU/mL) through the tail vein at embryonic day 1(E1),day 4(E4),and day 8(E8),respectively.At day 11.5(E11.5),the embryos were dissected from the uterus for analysis.Mitochondria in neural tube were observed by transmission electron microscope.The expression of PKC-α,anti-microtubule-associated protein 1 light chain 3(LC3) and anti-lysosomal associated membrane protein 2 (LAMP2) in embryonic neural tubes were analyzed by Western Blot.PKC-α knockdown C17.2 mouse neural stem cells were constructed in vitro and cultured under low(5 mmol/L) or high(25 mmol/L) glucose conditions.The changes of autophagosomes were detected by immunofluorescence staining.Results:In the NTD group,the mitochondria in the embryonic neural tube swelled and the ridge disappeared.Compared with the control group,the expressions of LC3Ⅱ and LAMP2 proteins in the fetal neural tube of NTD group decreased(P<0.05),and PKC-α protein up-regulated(P<0.01).By silencing the expression of PKC-α in the neural tube of mouse embryos,the expression of LC3Ⅱ and LAMP2 proteins in the embryos increased(P<0.05).In addition,the incidence of NTD in NTD+sh-PKC-α group was lower than that in NTD group(P<0.01).The expression levels of LC3Ⅱ and LAMP2 protein and the number of Cyto-ID staining points in C17.2 neural stem cells were lower than those in normal glucose conditions(P<0.05).PKC-α protein knockdown increased the expression levels of LC3Ⅱ and LAMP2 protein and the number of Cyto-ID staining spots under high glucose condition(P<0.05).Conclusion:PKC-α mediated mitochondrial dysfunction is associated with maternal diabetes induced NTD embryos,and by silencing maternal PKC-α,it attenuates the formation of NTD resulting from neural tube autophagy inhibition.
gestational diabetesprotein kinase C-αmitochondrianeural tube defectsautophagyexperimental study
维普
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