脂多糖应答基因对神经元细胞类缺血再灌注损伤的影响

The effect of lipopolysaccharide respond gene on neuron ischemia-reperfusion injury

目的研究神经元细胞中脂多糖应答基因(LRG)的表达对细胞类缺血再灌注损伤的影响。方法将小鼠神经元细胞分为对照组和处理组,处理组给予体外类缺血再灌注处理,采用定量PCR(qPCR)和Western blot的方法检测细胞中LRG基因的表达。将原代培养的神经元细胞分为空白组、空载质粒组、过表达组、非特异小干扰RNA(siRNA)组、沉默组,过表达组/沉默组转染LRG基因过表达质粒或siRNA,采用噻唑蓝(MTT)法检测细胞经类缺血再灌注后的生长状况,同时利用Western blot分析细胞中活化蛋白激酶B(pAkt)的表达。将原代培养的神经元细胞再分为阴性对照组、过表达组和抑制剂组,抑制剂组利用20μmol/L LY294002抑制细胞中磷脂酰肌醇-3激酶(PI3K)的正常功能,MTT法分析过表达LRG基因的细胞体外生长状况,同时利用Western blot方法分析细胞中活化的半胱氨酸天冬氨酸蛋白酶(caspase)3的表达。结果与对照组相比,类缺血再灌注处理显著降低细胞中LRG基因的表达(P<0.05)。过表达LRG基因可显著提高缺血再灌注损伤细胞在体外的存活(P<0.05),而沉默LRG基因则降低细胞的存活(P<0.05)。同时,过表达LRG基因上调细胞中pAkt的表达而沉默LRG基因则下调pAkt的表达(P<0.05)。抑制神经元细胞中PI3K可降低缺血再灌注损伤后细胞的存活率(P<0.05),并且提高细胞中活化caspase 3的表达(P<0.05)。结论在神经元细胞中过表达LRG能通过激活PI3K/Akt信号途径降低细胞在类缺血再灌注中的损伤。

Objective To investigate the effect of lipopolysaccharide respond gene （LRG） on neuron ischemia-reperfusion injury.Methods Cultured neurons were challenged with ischemia-reperfusion condition,and LRG expression were detected by qPCR and Western blot.LRG overexpression plasmid or specific siRNA were transferred into neurons prior to ischemia-reperfusion and cell survival was detected by 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay.Meanwhile,the expression of phosphated Akt was analyzed by Western blot.Furthermore,specific phosphatidylinositol-3-kinase （PI3K） inhibitor （LY294002,20μmol/L） was added in LRG overexpressed neurons,and cell survival was determined with MTT assay.In addition,active caspase 3 was analyzed by Western blot.Results Ischemia-reperfusion conditioning significantly decreased LRG expression in neurons （P＜ 0.05）.LRG overexpression promoted survival of neurons while knockdown of LRG led to a decreased survival （P＜ 0.05）.Furthermore,LRG overexpression evidently up-regulated the expression of phosphated Akt （P＜0.05）.Besides,inhibition of PI3K contributed to a lower survival （P＜0.05） and an augmented expression of active caspase 3 in neurons （P＜ 0.05）.Conclusions Overexpression of LRG in neuron may attenuate ischemia-reperfusion injury through PI3K/ Akt pathway.