预缺血通过NMDA受体抑制海马CA1区线粒体内细胞色素c向胞质释放的研究
王梅 胡晓彤 戚大石 王蕾 张芳▲
江苏省麻醉学重点实验室 徐州医科大学形态学实验教学中心 江苏省脑病重点实验室,徐州 221004
[摘要]目的 探讨脑缺血/再灌注、预缺血及预缺血前给予NMDA受体特异性抑制剂MK801对大鼠海马CA1区神经元线粒体内细胞色素c(Cyt c)向胞质释放的影响。方法 建立SD大鼠四动脉结扎全脑缺血及预缺血模型,给药组大鼠在预缺血前60min给予腹腔注射MK801 3mg/kg。将SD大鼠随机分为假手术组、脑缺血/再灌注组、脑缺血/再灌注加生理盐水组、预缺血前给予MK801组、预缺血前给予生理盐水组和预缺血组。检测线粒体的标志蛋白COXⅣ在分离后的线粒体和对应胞质组分中的分布来证明实验中线粒体和胞质是否得到成功分离,利用免疫印迹法分析不同处理组大鼠海马CA1区神经元细胞线粒体组分以及对应胞质组分中的Cyt c和COXⅣ水平。结果 线粒体和胞质成功分离,COXⅣ只存在于各组的线粒体组分中,各组的胞质组分中均未检测到COXⅣ的免疫活性。脑缺血/再灌注组线粒体内Cyt c向胞质的释放与假手术组比较显著增加,差异有统计学意义(P<0.05)。预缺血组与脑缺血/再灌注组相比,线粒体内Cyt c向胞质的释放显著降低,差异有统计学意义(P<0.05)。预缺血前给予MK801组与预缺血组相比,线粒体中Cyt c向胞质释放显著增加,差异有统计学意义(P<0.05)。结论 预缺血通过NMDA受体的介导抑制脑缺血/再灌注诱导增加线粒体内Cyt c向胞质的释放,为临床治疗脑缺血/再灌注性损伤提供了理论依据。
[关键词]脑缺血/再灌注;预缺血;细胞色素c;NMDA受体;线粒体
脑缺血/再灌注会引起严重的脑损伤,已成为21世纪三大致死性疾病之一,其内在机制的研究已被国内外学者广泛关注[1],但是具体机制尚不明确。越来越多的证据表明,参与短暂性脑缺血引起的神经元死亡的线粒体通路与线粒体中细胞色素c(cytochrome c,Cyt c)的释放有关[2],并且是受体介导的,但是短暂性脑缺血引发Cyt c释放的上游信号通路还不是很清楚。研究显示,脑缺血再灌注能增加Cyt c从线粒体向胞质的释放[3]。脑预缺血,即轻微、短时而不至于引起神经细胞死亡的缺血处理后,可使脑组织获得对再次足够强度缺血的耐受,并减少脑神经元损伤的现象,被称为脑缺血耐受[4-6],又称预缺血,其在心脏和脑缺血性损伤中普遍存在。国内外的研究发现,以足够强度的亚致死性缺血刺激大脑后,大脑会在短时间内对后续的致死性缺血刺激获得短暂的耐受,并产生对脑缺血损伤的内源性保护作用。
N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR)是中枢神经系统内一类重要的兴奋性氨基酸(excitatory aminoacid,EAA)受体[7]。NMDA亚型在兴奋性突触中是一种典型的配体门控离子通道,并且与缺血引起的神经元损伤有关[8]。功能型的NMDA受体包含NR1、NR2(2A-2D)、NR3(3A/3B)三个亚基[9-10]。NR1是NMDA受体发挥其通道活性的一个主要亚基,NR2亚基则决定受体的选择性。NMDA受体的过度激活被认为是缺血应激中的主要信号转变,但是温和地刺激NMDA受体则被认为可以在体外培养的小脑粒细胞神经元中诱导神经元对兴奋性中毒的保护[11],温和地刺激NMDA受体的活性被认为与诱导缺血耐受有关[12],而其如何导致神经保护作用的细胞信号转导途径还未研究清楚。
本实验通过对SD大鼠全脑缺血和预缺血模型的研究来揭示线粒体Cyt c向胞质的释放在缺血耐受中的变化以及与NMDA受体的关系。给予SD大鼠3min缺血作为预缺血,复灌3 d后给予6 min缺血作为致死性缺血,全脑缺血模型为给予SD大鼠6min的缺血后复灌,并应用NMDA受体抑制剂MK801来探讨NMDA受体在缺血耐受形成中的分子机制。
1 材料与方法
1.1 实验动物和试剂
由本校实验动物中心和中科院上海实验动物中心提供的SPF级雄性Sprague-Dawley(SD)大鼠,250~300 g,自由进食,昼夜交替照明。将大鼠随机分为假手术组、脑缺血/再灌注组、预缺血组、脑缺血/再灌注加生理盐水组、预缺血前给予MK801组、预缺血前给予生理盐水组。兔多克隆抗Cyt c(#4272)和兔多克隆抗COXⅣ (#4844)购于Cell Signaling Technology公司,硝酸纤维素 (NC)膜购于Amersham公司,NBT/ BCIP购于Promega公司,牛血清白蛋白(组分Ⅴ)购于Amresco公司,工具药(MK801)和一些化学试剂均从Sigma-Aldrich公司购得,其他常用试剂均为国产分析纯。
1.2 实验方法
1.2.1 大鼠脑缺血及预缺血模型的制备 采用大鼠四动脉结扎模型[13],首先以20%水合氯醛(300~350 mg/kg)腹腔注射麻醉,然后分离双侧颈总动脉,并用细绳缠绕双侧颈总动脉,以利于第2天拎起。椎动脉电凝以阻塞椎动脉血供,大鼠自由进食,术后第2天,于大鼠清醒状态下拎起颈总动脉,采用动脉夹夹闭,全脑缺血6 min后取下动脉夹开始恢复血供,再灌注6 h。预缺血组先给予3 min的预缺血后复灌注3 d再给予缺血6min,并再灌注6 h,缺血模型的可靠性以体征表现判断。假手术组前期操作同缺血动物,但不夹闭双侧颈总动脉。MK801溶于生理盐水,在预缺血前60min腹腔注射给药3 mg/kg。溶剂对照动物腹腔注射等体积生理盐水。
1.2.2 线粒体的提取 在冷库中进行下述操作:大鼠缺血复灌6 h,快速断头取脑,分离出双侧海马组织,沿海马裂分离出CA1部分,为避免冰冻引起Cytc从线粒体向胞浆释放,新鲜海马CA1区中立即加入1∶10(w/v)冰冷的匀浆缓冲液,以Glas-col匀浆器速匀浆,10 s×6次,然后以1000×g 4℃离心15 min,弃沉淀,转移上清再以17 000×g 4℃离心20min,所得上清即作为胞质部分,测蛋白后分装,-80℃冰箱保存待用;对应所得的沉淀即为与胞质对应的粗制线粒体部分,测蛋白后分装,-80℃冰箱保存待用。
1.2.3 蛋白含量测定 按照改良Lowry′s法[14],标准蛋白采用牛血清蛋白(BSA,组分ν)。
1.2.4 免疫印迹(immunob l ot,IB)取分装好的含相同蛋白量的样品,分别加入1/3体积的4×蛋白上样缓冲液,沸水浴5min变性。上样等量的100μg变性蛋白质样品与凝胶孔中,经SDS-PAGE凝胶电泳分离后,电转至NC膜上,NC膜置以封闭液室温孵育3 h后,取出放入一抗工作液中孵育,4℃过夜,以TBST洗膜(5 min×3次)后,放入AP标记的二抗工作液中孵育,室温2 h,再以TBST洗膜(3 min×5次),然后水洗,并放入以NBT/BCIP试剂盒新鲜配制AP显色液中显色,以流水洗涤终止显色。扫描NC膜上显色条带,并采用Quantity One软件分析。
1.3 统计学处理
采用 Image J 1.37、Sigma STAT32、Sigma PLOT9分析软件对数据进行处理,组间比较采用one-way ANOVA,多个实验组与一个对照组比较采用LSD,以P<0.05为差异有统计学意义。
2 结果
如图1(A,B)所示,COXⅣ只存在于各组的线粒体组分中,在各组的胞质组分中均未检测到COXⅣ的免疫活性,提示本实验中的线粒体和胞质成功分离。脑缺血/再灌注组线粒体内Cyt c向胞质的释放与假手术组比较显著增加,差异有统计学意义(P<0.05)。预缺血组与脑缺血/再灌注组相比,线粒体内Cyt c向胞质的释放显著降低,差异有统计学意义(P<0.05)。预缺血前给予MK801组与预缺血组相比,线粒体中Cyt c向胞质释放显著增加,差异有统计学意义 (P<0.05)。预缺血前给予生理盐水组相对于预缺血组无明显变化,从而提示NMDA受体介导了预缺血对线粒体中Cyt c向胞质释放的抑制作用。
图1 脑缺血/再灌注、预缺血以及预缺血联合MK801对SD大鼠海马CA1区神经元线粒体中Cyt c向胞质释放的影响
3 讨论
海马CA1区的锥体细胞对于缺血引起的损伤最为脆弱[1],但是在相对温和的预缺血刺激后,细胞能获得对接下来更为剧烈缺血损伤的耐受,即缺血预适应[4-6],由于其提供了一种研究内在保护作用机制的途径,故引起广泛关注。NMDAR在神经系统发育过程中发挥着重要的生理作用,如参与调节神经元的存活,神经元树突、轴突结构的发育以及突触可塑性的形成等[9-10],尽管NMDA受体的过度激活被认为是缺血引起的细胞死亡的主要机制,但是温和地刺激NMDA受体的活性被认为与诱导缺血耐受有关,而其如何导致神经保护作用的细胞信号转导途径还未研究清楚。研究显示,NMDA在海人藻酸诱导的小鼠海马神经元死亡中具有神经保护作用[11]。大量的研究已经证明了NMDA受体在诱导缺血耐受中的关键作用[15],本研究结果显示,NMDA受体介导缺血耐受对Cyt c从线粒体向胞质释放具有抑制作用。
脑缺血/再灌注导致大脑神经元凋亡,目前其发生机制被认为有以下几个方面:①与凋亡相关基因表达以及许多内外因素的调节有关,如凋亡发生基因的激活;②脑缺血/再灌注后大量氧自由基对神经元造成的严重损伤并诱导了凋亡的发生[16];③线粒体损伤导致的细胞能量代谢障碍导致凋亡的发生[17]。线粒体中Cyt c的释放是线粒体通路参与脑缺血导致神经元凋亡的重要途径,Cyt c是线粒体内的重要细胞凋亡因子,Cyt c释放后进入胞质,与胞质中Apaf-1和caspase-9形成复合物后激活caspase酶。Apaf/Cyt c复合物又可与ATP/dATP结合并激发其多聚化从而形成凋亡小体,导致细胞凋亡[18]。
本实验通过对大鼠全脑缺血模型的研究来揭示线粒体Cyt c从线粒体向胞质的释放在缺血耐受中的变化,给予SD大鼠3 min缺血作为预缺血,复灌3 d后给予6min缺血作为致死性缺血,应用NMDA受体抑制剂MK801来探讨NMDA受体在缺血耐受形成中的分子信号机制。本研究结果显示,Cyt c向胞质的释放在缺血复灌过程中有明显升高,而给予预缺血处理使Cyt c向胞质的释放水平明显下降。在预缺血前给予NMDA受体拮抗剂MK801拮抗了预缺血对Cyt c向胞质的释放的抑制作用,这提供了一个直接的证据,证明NMDA受体在诱导缺血耐受中发挥着关键作用,NMDA受体介导了预缺血抑制脑缺血/再灌注诱导增加的Cyt c从线粒体向胞质的释放。
[参考文献]
[1]Brose SA,Golovko SA,Golovko MY,et al.Brain 2-arachidonoylglycerol levels are dramatically and rapidly increased under acute ischemia-injury which is prevented by microwave irradiation[J].Lipids,2016,51(4):487-495.
[2]Chang JW,Hwang HS,Kim YS,et al.Protective effect of Artemisia asiatica(Pamp.)Nakaiex kitam ethanolextractagainst cisplatin-induced apoptosis of human HaCaT keratinocytes:involvement of NF-kappa B-and Bcl-2-controlled mitochondrial signaling[J].Phytomedicine,2015,22 (6):679-688.
[3]Dave KR,Bhattacharya SK,Saul I,et al.Activation of protein kinase c delta following cerebral ischemia leads to release of cytochrome c from the mitochondria via bad pathway[J].PLoSOne,2011,6(7):e22057.
[4]Yoon YE,Choi KH,Kim SY,et al.Renoprotective mechanism of remote ischemic preconditioning based on transcriptomic analysis in a porcine renal ischemia reperfusion injurymodel[J].PLoSOne,2015,10(10):e0141099.
[5]Shih YM,Shih JM,Pai MH,et al.Glutamine administration after sublethal lower limb ischemia reduces inflammatory reaction and offers organ protection in ischemia/reperfusion injury[J].JPEN JParenter Enteral Nutr,2015.[Epub ahead of print]
[6]He X,Mo Y,GengW,etal.RoleofWnt/beta-catenin in the tolerance to focal cerebral ischemia induced by electroacupuncturepretreatment[J].Neurochem Int,2016.[Epub ahead ofprint]
[7]Archer T,Garcia D.Attention-deficit/hyperactivity disorder:focus upon aberrant N-methyl-D-aspartate receptors systems[J].Curr Top Behav Neurosci,2015.[Epub ahead of print]
[8]Liu Y,Cui X,Sun YE,et al.Intrathecal injection of the peptidemyr-NR2B9c attenuates bone cancer pain via perturbing N-methyl-D-aspartate receptor-PSD-95 protein interactions inmice[J].Anesth Analg,2014,118(6):1345-1354.
[9]Sachser RM,Santana F,Crestani AP,etal.Forgetting of longterm memory requires activation of NMDA receptors,L-type voltage-dependent Ca2+channels,and calcineurin[J].SciRep,2016,6:22771.
[10]Lee CH,Lu W,Michel JC,et al.Nmda receptor structures reveal subunit arrangement and pore architecture[J].Nature,2014,511(7508):191-197.
[11]Kirino T,Tsujita Y,Tamura A,et al.Induced tolerance to ischemia ingerbilhippocampalneurons[J].JCereb Blood Flow Metab,1991,11(2):299-307.
[12]Ogita K,Okuda H,Yamamoto Y,et al.In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons[J].J Neurochem,2003,85(5):1336-1346.
[13]PulsinelliWA,Brierley JB.A new model of bilateral hemispheric ischemia in the unanesthetized rat[J].Stroke,1979,10(3):267-272.
[14]Lowry OH,Rosebrough NJ,Farr AL,et al.Proteinmeasurementwith the folin phenol reagent[J].JBiol Chem,1951,193 (1):265-275.
[15]Miao B,Yin XH,Pei DS,et al.Neuroprotective effects of preconditioning ischemia on ischemic brain injury through down-regulating activation of JNK1/2 via N-methyl-D-aspartate receptor-mediated Akt1 activation[J].JBiol Chem,2005,280(23):21693-21699.
[16]Lee S,Suk K,Kim IK,et al.Signaling pathways of bisphenol A-induced apoptosis in hippocampal neuronal cells:role of calcium-induced reactive oxygen species,mitogenactivated protein kinases,and nuclear factor-kappa B[J].J Neurosci Res,2008,86(13):2932-2942.
[17]Hoque A,Hossain MI,Ameen SS,et al.A beacon of hope in stroke therapy-blockade of pathologically activated cellular events in excitotoxic neuronal death as potential neuroprotectivestrategies[J].PharmacolTher,2016,160:159-179.
[18]Lin N,Xiong LL,Zhang RP,et al.Injection of Aβ1-40 into hippocampus induced cognitive lesion associated with neuronal apoptosis and multiple gene expressions in the tree shrew[J].Apoptosis,2016,21(5):621-640.
Ischemia preconditioning decrease Cyt c release from mitochondria to cytosol through NMDA receptor in hippocam pal CA1 region
WANGMei HU Xiao-tong QIDa-shi WANG Lei ZHANG Fang▲
Jiangsu Key Laboratory of Anesthesiology;Laboratory of Morphology,Xuzhou Medical University;Jiangsu Key Laboratory of Brain Disease Bioinformation,Xuzhou 221004,China [Abstract]Objective To explore the influence of NMDA receptor specific inhibiter MK801 on the Cyt c release from mitochondria to cytosol in hippocampal CA1 of rats in cerebral ischemia/reperfusion,ischemia preconditioning and preischemia preconditioning.Methods The whole cerebral ischemia and ischemia preconditioning model were established by 4-vessel occlusion in adultmale Sprague-Dawley(SD)rats,the rats in the administration group before ischemic preconditioning for 60minuteswas given intraperitoneal injection of MK801 3mg/kg.SD ratswere randomly divided into the sham operation group,the cerebral ischemia/reperfusion group,the cerebral ischemia/reperfusion plus normal saline group,the MK801 treatment before ischemia preconditioning group,the normal saline treatment before ischemia preconditioning group and the ischemia preconditioning group.Whether themitochondria and cytosol were successfully separated in the experiment was demonstrated by detecting the distribution of themitochondrialmarker protein COXⅣ in the isolated mitochondria and corresponding cytosolic fractions.The mitochondrial components of the CA1 neurons in the hippocampus of different treatment groups and the level of Cyt c and COXⅣin the corresponding cytosolic fractions was analyzed by using immunoblotmethod.Results Mitochondria and cytosolwere separated successfully,COXⅣwas only foundin themitochondrial fractions of each group,and the COXⅣ immune activity were not detected in the cytoplasmic fractions of each group.Compared with the sham operation group,the release of Cyt c from mitochondria to cytosol in the cerebral ischemia/reperfusion group was significantly increased,with significant difference(P<0.05).Compared with cerebral ischemia/reperfusion group,the release of from mitochondria to cytosol in the ischemia preconditioning group was significantly decreased,with significant difference (P<0.05).Compared with the ischemia preconditioning group,the release of Cyt c from mitochondria to cytosol in the MK801 treatment before ischemia preconditioning group was significantly increased,with significant difference(P<0.05).Conclusion Ischemia preconditioning can obviously depress the release of Cyt c from mitochondria to cytosol induced by cerebral ischemia/reperfusion through NMDA recepter,which provides a theoretical basis for clinical treatment of cerebral ischemia/reperfusion injury.
[Key words]Cerebral ischemia/reperfusion;Ischemia preconditioning;Cytochromec;NMDA receptor;Mitochondria
[中图分类号]R-332[文献标识码]A
[文章编号]1674-4721(2016)08(c)-0004-04
(收稿日期:2016-05-31本文编辑:祁海文)
[基金项目]国家自然科学基金青年科学基金项目(81500977);江苏省高校自然科学研究面上项目(14KJB180022);江苏省普通高校自然科学研究项目(13KJD310003);江苏省麻醉学重点实验室开放研究课题(KJS1502);江苏省脑病生物信息重点实验室开放研究课题(1505);江苏省徐州市科技计划项目(KC14SH076)
[作者简介]王梅(1982-),女,硕士,实验师,主要从事缺血性脑神经元损伤分子机制的研究
▲通讯作者:张芳(1971-),女,硕士,高级实验师,主要从事缺血性脑神经元损伤分子机制的研究 |