UBIAD1通过抑制nNOS/NO途径减轻氧糖剥夺再灌注损伤

UBIAD1 protects against oxygen-glucose deprivation/reoxygenation injury via nNOS/NO pathway

目的:缺血性脑卒中的发病率和致残率均极高,缺血再灌注损伤(ischemia reperfusion injury,IRI)是治疗的关键。UbiA类异戊烯转移酶结构域1蛋白(UbiA prenyltransferase domain containing 1 protein,UBIAD1)是一类具有多种生物学功能的酶,参与线粒体呼吸链电子传递、脂代谢、氧化应激等,这些过程均与IRI有关。本研究探讨UBIAD1在脑IRI中的作用及其作用机制。方法:以小鼠成神经细胞瘤(mouse neuroblastoma Neuro2a,N2a)细胞为研究对象,构建氧糖剥夺再灌注(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤模型,以过表达UBIAD1的慢病毒载体转染N2a细胞,构建稳定过表达UBIAD1的细胞模型。第1部分实验将N2a细胞分为5组:未经OGD处理(non-OGD)组、经OGD处理4 h后分别再灌注0、4、12、24 h组;第2部分实验将N2a细胞分为6组:正常细胞(Con)+non-OGD组、转染空载体细胞(EV)+non-OGD组、UBIAD1过表达细胞(OE)+non-OGD组、Con+OGD处理(OGD/R)组、EV+OGD/R组、OE+OGD/R组;第3部分将N2a细胞分为8组:Con+non-OGD组、OE+non-OGD组、Con+non-OGD+NOS特异性抑制剂7-硝基吲唑(7-nitroindazole,7-NI)组、OE+non-OGD+7-NI组、Con+OGD/R组、OE+OGD/R组、Con+OGD/R+7-NI组、OE+OGD/R+7-NI组。使用激光共聚焦扫描显微镜观察高尔基体形态改变,流式细胞术检测细胞凋亡率,MTT法检测细胞活力,Griess试剂盒检测NO的释放,分别通过real-time PCR和蛋白质印迹法检测细胞UBIAD1、分泌途径衍生钙离子转运ATP酶1(secretory pathway Ca^(2+)-ATPase isoform 1,SPCA1)、NOS的mRNA和蛋白质表达水平。结果:与non-OGD组相比,经OGD处理4 h后再灌注0、4、12及24 h的各组N2a细胞中UBIAD1 mRNA和蛋白质表达水平均明显下调(P<0.05或P<0.01),且再灌注时间越长,UBIAD1表达水平下调越显著。与Con+OGD/R组、EV+OGD/R组相比,OE+OGD/R组UBIAD1 mRNA和蛋白质表达水平均明显上调(均P<0.01),细胞凋亡率均下降(均P<0.01),细胞活力均增高(均P<0.01),高尔基体碎裂较少,形态保存较完整,SPCA1 mRNA和蛋白质表达水平均明显上调(均P<0.05)。OE+non-OGD组与Con+non-OGD组相比,OE+OGD/R组与Con+OGD/R组相比,内皮型NOS(endothelial NOS,eNOS)及神经元型NOS(neuronal NOS,nNOS)的mRNA和蛋白质表达均下调(P<0.05或P<0.01),NO含量均减少(均P<0.01);Con+OGD/R+7-NI组与Con+OGD/R组相比,OE+OGD/R+7-NI组与OE+OGD/R组相比,NO含量均减少(均P<0.01),N2a细胞凋亡率均下降(均P<0.01)。结论:UBIAD1可减轻神经细胞OGD/R损伤,改善OGD/R后高尔基体功能,其机制可能与抑制nNOS/NO途径有关。

Objective:Cerebral infarction is a subtype of stroke with high incidence and disability rate.Ischemia reperfusion injury(IRI)is the key point of cerebral infarction treatment.UbiA prenyltransferase domain containing 1(UBIAD1)is a kind of enzyme with various biological functions including electron transport in mitochondrial respiratory chain,lipid metabolism,and oxidative stress which are related to IRI.The purpose of this study aims to determine the neuroprotective effects and the underlying mechanisms of UBIAD1 in cerebral IRI.Methods:We employed oxygen-glucose deprivation/reoxygenation(OGD/R)model in mouse neuroblastoma Neuro2a(N2a)cells to mimic cerebral IRI.Lentivirus vector over expressed UBIAD1 was transfacted into N2a cells to maintain high and stable expression of UBIAD1.In the first part of the experiment,N2a cells were divided into 5 groups:A non-OGD(N2a cells without exposure to OGD)group,groups of reoxygenation 0,4,12 and 24 h after 4 h of OGD,respectively.In the second part of the experiment,N2a cells were divided into 6 groups:A Con(normal cell)+non-OGD group,an EV(cell transfected with empty vector)+non-OGD group,an OE(over-expressed UBIAD1)+non-OGD group,a Con+OGD/R group,an EV+OGD/R group,and an OE+OGD/R group.In the third part,the N2a cells were divided into 8 groups:A Con+non-OGD group,an OE+non-OGD group,a Con+non-OGD+nNOS inhibitior 7-nitroindazole(7-NI)group,an OE+non-OGD+7-NI group,a Con+OGD/R group,an OE+OGD/R group,a Con+OGD/R+7-NI group,and an OE+OGD/R+7-NI group.The morphological changes of Golgi apparatus were observed under the confocal laser scanning microscope.The mRNA and protein levels of UBIAD1,secretory pathway Ca^(2+) -ATPase isoform 1(SPCA1),and NOS were determined by real-time PCR and Western blotting,respectively.Cell apoptosis rate was detected with flow cytometry;cell viability was detected with MTT assay,and NO release was determined with Griess assay.Results:Compared with the non-OGD group,the expression levels of UBIAD1 mRNA and protein in N2a cells in the groups of 0,4,12 and 24 h reoxygenation after OGD 4 h decreased significantly(P<0.05 or P<0.01),and the longer the reoxygenation time,the more significant the reduction of UBIAD1 expression.Compared with the Con+OGD/R group and the EV+OGD/R group,mRNA and protein levels of UBIAD1 and SPCA1 were increased(P<0.05 or P<0.01),the apoptosis rate was decreased(all P<0.01),and the cell viability was increased(all P<0.01)in the OE+OGD/R group.The Golgi fragmentation was less in the OE+OGD/R group than that in the Con+OGD/R group and the EV+OGD/R group.The mRNA and protein levels of endothelial NOS(eNOS)and neuronal NOS(nNOS)were decreased(P<0.05 or P<0.01),and the level of NO was decreased(all P<0.01)in the groups over-expressed UBIAD1(OE+non-OGD group vs Con+non-OGD group,OE+OGD/R group vs Con+OGD/R group).The level of NO and apoptosis rate of N2a cells were decreased(all P<0.01)in the the groups pretreated with 7-NI(Con+OGD/R+7-NI group vs Con+OGD/R group,OE+OGD/R+7-NI group vs OE+OGD/R group).Conclusion:UBIAD1 may exerts protective effects on OGD/R induced N2a cells by ameliorating Golgi apparatus dysfunction via the nNOS/NO pathway.