miR-181a调控海马神经元突触生长在癫痫发生中的作用及机制

摘要

目的 探讨miR-181a调控海马神经元突触生长在癫痫发生中的作用及机制.方法 从出生24h内的SD大鼠分离海马神经元进行原代培养,将miR-181a-mimic、miR-181 a-inhibitor及scramble miRNA转染到神经元细胞内,未转染组作为对照组,各组细胞培养24h后,免疫荧光检测突触的长度及数目;将后续实验分为对照组、无镁组及无镁+miR-181a-mimic组,脱氧核苷酸转移酶介导三磷酸脱氧乌苷-生物素刻痕末端标记法(TUNEL)检测阳性细胞率;乳酸脱氢酶(LDH)法检测细胞毒性;根据标准曲线计算培养液中的NAD+含量;Western印迹检测蛋白激酶B(Akt)和糖原合酶激酶(GSK3β)磷酸化蛋白及总蛋白的表达.结果 miR-181a-mimic组海马神经元细胞突触的长度及分支数均显著低于对照组,而miR-181a-inhibitor组海马神经元细胞突触的长度及分支数均显著高于对照组(P＜0.01).scramble miRNA不影响突触的生长;无镁+miR-181a-mimic组在1h和24 h的阳性细胞率均显著低于无镁组(P＜0.01).无镁24h组和无镁+miR-181a-mimic组细胞漏出率均显著高于对照组,NAD+含量显著低于对照组(P＜0.01),而无镁+miR-181a-mimic组细胞漏出率显著低于无镁24h组,NAD+含量显著高于无镁24 h组(P＜0.01);无镁24h组Akt和GSK3β磷酸化蛋白表达与对照组差异无统计学意义(P＞0.05),无镁+miR-181a-mimic组Akt和GSK3β磷酸化蛋白表达显著高于对照组及无镁24 h组(P＜0.01);Akt和GSK3β总蛋白表达在三组间差异无统计学意义(P＞0.05).结论 miR-181a影响海马神经元突触生长,并通过调控PI3 K/Akt/GSK3β信号通路降低神经元细胞的凋亡及细胞漏出率,提高NAD+含量对癫痫起保护作用.%Objective To investigate the role and mechanism of miR-181a in the regulation of synaptic growth of hippocampal neurons in the development of epilepsy.Methods The hippocampal neurons were isolated and cultured from SD rats born in 24 h,miR-181amimic,miR-181a-inhibitor and scramble miRNA was transfected into neuron cells,and without transfection as the control group,the cells were cultured for 24 h,synaptic length and number were detected by immunofluorescence;follow-up experiences were divided into control group,induced by magnesium free epilepsy group and magnesium free +miR-181a-mimic group,the rate of positive cells were detected by TUNEL;cytotoxicity was detected by LDH assay;NAD+ content in culture medium was calculated according to the standard curve;the expressions of Akt and GSK3 phosphorylation protein and total protein were detected by Western blot.Results The length and number of branches of hippocampus neuron synapses in miR-181a-mimic group were significantly lower than those of control group,and the length and number of branches of hippocampus neuron synapses in miR-181a-inhibitor group were significantly higher than those of control group (P＜0.01).MiRNA scramble did not affect the growth of synapses;the positive cells rates at 1 h and 24 h were significantly lower in the miR-181a-mimic group than those in the magnesium free 24 h group (P＜0.01).The leakage rate of cells in magnesium free 24 h group and magnesium free+miR-181a-mimic group were significantly higher than those of control group,the content of NAD+ content was significantly lower than that of control group (P＜0.01),and leakage rate of cells was significantly lower in magnesium free+miR-181a-mimic group than that of magnesium free 24 h group,NAD+ content was significantly higher than that of magnesium free 24 h group (P＜0.01);the expressions of Akt and GSK3β magnesium protein in magnesium free 24 h group had no significant difference with those of control group (P＞ 0.05),while the expressions in magnesium free+miR-181 a-mimic group were significantly higher than those of control group and magnesium free 24 h group (P＜0.01);Akt and GSK3β total protein expressions had no significant differences among the three groups (P＞0.05).Conclusions miR-181a effect synaptic growth of hippocampal neurons,and reduce apoptosis of hippocampal neurons cells and the leakage rate of neurons and improve NAD+ content by regulating PI3K/Akt/GSK3β signaling pathway to protect effect on epilepsy.