亚低温上调Sirt1减少氧糖剥夺后原代神经元细胞凋亡

摘要

为探讨亚低温对氧糖剥夺/复氧后原代神经元细胞自噬和凋亡的影响,运用原代神经元培养及OGD/R模型构建,CCK8测定细胞活力,TUNEL检测细胞凋亡,Western-blot检测Sirt1、Foxo1、Rab7、Beclin1、LC3及p53等蛋白的表达,以及转染mRFP-GFP-LC3自噬双标腺病毒检测神经元自噬流等方法,成功培养原代神经元及构建OGD/R模型.结果发现OGD/R后Sirt1、P-Foxo1、Rab7、Beclin1、LC3b/LC3a表达随着时间延长逐渐减少,12 h更明显,与R6 h组相比,P＜0.05,而p53增加,P＜0.05;OGD3 h/R12 h,亚低温组Sirt1、P-Foxo1、Rab7、Beclin1及LC3b/LC3a表达均明显高于常温组,P＜0.05,而p53明显低于常温组,P＜0.05.R6h亚低温组,神经元凋亡率为20％±6.7％,低于常温组的56.8％±7.6％,P＜0.05.mRFP-GFP-LC3自噬双标观察法显示亚低温组自噬溶酶体明显增多,P ＜0.05.实验显示原代神经元OGD/R后亚低温干预可以上调Sirt1的活性,增加Foxo1、Rab7和自噬相关蛋白Beclin1、LC3b/LC3a的表达,同时抑制p53,促进神经元自噬,减少凋亡.%To investigate the primary neurons autophagy and apoptosis after Oxygen Glucose Deprivation/Re-oxygenation (OGD/R),and to explore the effect of mild hypothermia on autophagy and apoptosis,following methods were used:Primary neurons culture and OGD/R model was established;the neurons were divided into the normal temperature group (37 ℃) and the mild hypothermia group (MH,34 ℃);The cells viability were measured by CCK8;cell apoptosis were measured by TUNEL;The protein expressions of Sirt1,Foxo1,p53,Rab7 and autophagy related genes such as Beclin1,LC3 were detected by western blot at each time point;The neurons autophagy flows were detected through transfection of adenovirus mRFP-GFP-LC3.The primary neuron cultures were successfully developed,and the OGD/R models were established;Western-blot showed that the expressions of Sirt1,P-Foxo1,Rab7,Beclin1 and LC3b/LC3a were gradually reduced,especially at 12 h after OGD/R,P ＜ 0.05;However,the expression of pS3 was increased,P ＜ 0.05;In MH group,the expressions of Sirt1,P-Foxo1,Rab7,Beclin1,LC3b/LC3a were obviously higher than those in NT group,P ＜ 0.05;And the expression of pS3 was obviously lower than that in NT group,P ＜ O.05;in R6 h + MH group,the rate of neuron cells apoptosis were 20％ ±6.7％,lower than 56.8％ ±7.6％ in R6 h + NT group,P ＜0.05;mRFP-GFP-LC3 adenovirus was transfected into primary neurons,the autophagy flow was detected by fluorescence microscopy.Compared with control group,the autolysosomes were reduced,but autophagosomes were increased after OGD/R,P ＜0.05;however,compared with NT group,the autophagosomes were reduced and the autolysosomes were increased in MH group,P ＜ 0.05.In conclusion,mild hypothermia therapy could increase the expression of Sirt1,Foxo1,beclin1 and LC3b/LC3a,but decrease the expression of p53,so as to promote autophagy and reduce apoptosis.