瘦素对氧糖剥夺/复糖复氧后原代神经元的影响及相关机制  

Effects of leptin on primary neurons after oxygen-glucose deprivation and reoxygenation and related mechanism

作　　者：张艳[1] 程道宾[1] 揭春晓 胡诗俊 ZHANG Yan;CHENG Daobin;JIE Chunxiao;HU Shijun(Department of Neurology,Hainan General Hospital,Haikou 570100,China)

机构地区：[1]广西医科大学第一附属医院神经内科,南宁530000 [2]海南省人民医院神经内科

出　　处：《中国神经精神疾病杂志》2022年第6期334-341,共8页Chinese Journal of Nervous and Mental Diseases

基　　金：海南省自然科学基金青年基金项目(编号:819QN348)。

摘　　要：目的探讨瘦素预处理对原代培养神经元氧糖剥夺/复糖复氧(oxygen-glucose deprivation and reoxygenation,OGD/R)后的神经保护作用及相关机制。方法体外培养、纯化及鉴定Sprague-Dawley乳鼠皮层神经元,采用OGD/R模型,在体外模拟脑缺血/再灌注损伤。将培养7 d的神经元随机分为4组:正常组、OGD/R组、瘦素组和LY+瘦素组。采用细胞计数试剂测定细胞存活率;通过透射电镜观察各组神经元的内质网超微结构;应用细胞免疫荧光、蛋白印迹技术及RT-PCR法检测磷酸化蛋白激酶B(phsphorylated protein kinase B,p-Akt)及内质网应激相关蛋白葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、C/EBP同源蛋白(C/EBP homologous protein,CHOP)和半胱氨酸蛋白酶12(cysteinyl aspartate specific proteinase 12,Caspase12)的表达。结果与OGD/R组比较,瘦素组细胞存活率明显升高(43.04%±1.17%vs.68.61%±1.42%,P<0.001),细胞的形态损伤与内质网肿胀明显减轻,p-Akt(0.65±0.10 vs.1.10±0.21,P<0.05)和GRP78(1.04±0.06 vs.1.57±0.19,P<0.01)表达显著增加,CHOP(1.00±0.21 vs.0.59±0.11,P<0.01)和Caspase12(1.20±0.10 vs.0.86±0.10,P<0.01)表达明显减少;与瘦素组比较,LY+瘦素组细胞存活率显著降低(68.61%±1.42%vs.51.97%±2.05%,P<0.001),细胞的形态损伤与内质网肿胀明显增加,p-Akt(1.10±0.21 vs.0.76±0.23,P<0.05)和GRP78(1.57±0.19 vs.1.19±0.18,P<0.05)表达减少,CHOP(0.59±0.11 vs.0.65±0.33,P<0.05)和Caspase12(0.86±0.10 vs.1.03±0.06,P<0.05)表达显著增加。结论瘦素对OGD/R诱导的神经元损伤具有神经保护作用,其可能机制是瘦素激活PI3K/Akt信号通路,减轻内质网应激,从而促进原代神经元存活。Objective To investigate the neuroprotective effect and related mechanism of leptin pretreatment on primary cultured neurons after oxygen-glucose deprivation and reoxygenation(OGD/R).Methods The cortical neurons isolated from newborn Sprague-Dawley rats,after purification and identification,were subjected oxygen-glucose deprivation/reoxygenation(OGD/R)to establish an in vitro cerebral ischemia/reperfusion model.The neurons cultured for 7 d were randomly divided into four groups:control group,OGD/R group,leptin group and LY+leptin group.Cell counting kit-8 was used to determine the survival rate of cells;transmission electron microscopy(TEM)was used to examine the ultrastructure of endoplasmic reticulum(ER)in each group;cellular immunofluorescence,western blot and RT-PC.R were used to examine the expression of phosphorylation protein kinase B(p-Akt)and endoplasmic reticulum stress-related protein,glucose-regulated protein 78(GRP78),C/EBP-homologous protein(CHOP)and cysteinyl aspartate specific proteinases 12(Caspase 12).Results Compared with the OGD/R group,the cell survival rate was significantly increased(43.04±1.17 vs,68.61±1.42,P<0.001),and alterations of cell morphology and endoplasmic reticulum were decreased,and the expression of p-Akt(0.65±0.10 vs.1.10±0.21,P<0.05)and GRP78(1.04±0.06 vs.1.57±0.19,P<0.01)was significantly increased and the expression of CHOP(1.00±0.21 vs.0.59±0.11,P<0.01)and Caspase12(1.20±0.10 vs.0.86±0.10,P<0.01)significantly reduced in leptin group.Compared with the leptin group,the cell survival rate significantly reduced(68.61±1.42 vs.51.97±2.05,P<0.001),alterations of cell morphology and endoplasmic reticulum were increased,and the expression of p-Akt(1.10±0.21 vs.0.76±0.23,P<0.05)and GRP78(1.57±0.19 vs.1.19±0.18,P<0.05)significantly reduced and the expression of CHOP(0.59±0.11 vs.0.65±0.33,P<0.05)and Caspase12(0.86±0.10 vs.1.03±0.06,P<0.05)significantly increased in the LY+leptin group.Conclusion Leptin exhibits neuroprotective effect on OGD/R-induced neuronal dam

关 键 词：瘦素 脑缺血/再灌注损伤 PI3K/AKT信号通路 内质网应激 

分 类 号：R743[医药卫生—神经病学与精神病学]