外周型苯二氮（艹卓）受体在大鼠心肌细胞缺氧复氧损伤中的作用与机制探讨
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摘要
目的
在溶栓、不稳定心绞痛、心内直视手术、心肺移植、冠脉搭桥手术和心肺复苏时,心肌缺血再灌注损伤(ischemic reperfusion injury,IRI)仍无法完全避免。防治器官IRI仍是临床亟待解决的重要问题之一。
Murry等于1986年首先报道在心肌细胞中存在缺血预适应(ischemicpreconditioning,IPC)现象,相继研究发现药物预适应可产生类似于IPC的保护作用。深入研究表明,线粒体损伤是导致IRI心肌不可逆损伤的重要原因,因而线粒体在IRI器官保护中的地位越来越受到重视。尤其线粒体膜通透性改变在细胞凋亡中起重要作用。在线粒体膜通透性调节中,线粒体通透性转运孔(permeability transition pore,PTP)起决定性作用。线粒体参与细胞凋亡的早期过程是线粒体PTP开放,使线粒体内外H~+梯度消失、呼吸链脱偶联、能量产生中断、基质肿胀并导致外膜破裂,释放出细胞色素C等各种活性蛋白。从而进一步激活caspase级联瀑布式反应,导致细胞凋亡。
外周型苯二氮革类受体(peripheral benzodiazepine receptor,PBR)是一种线粒体外膜蛋白,与电压依赖性阴离子通道(voltage-dependent anionchannel,VDAC)和核苷酸腺嘌呤转位体(adenine nucleotide translocator,ANT)共同组成线粒体PTP。PBR广泛存在于心脏、肺脏、肝脏、肾脏及血液细胞中,并参与调节线粒体PTP的开放。因此,确定PBR及其配体在线粒体、细胞和心肌再灌注损伤中的作用具有重要的意义。
本研究拟在建立体外培养心肌细胞缺氧复氧损伤模型的基础上,测定PBR基因表达的变化及其对细胞凋亡的影响。并观察PBR对缺氧复氧损伤细胞内钙离子浓度,蛋白激酶C表达及细胞凋亡相关因子Bcl2/Bax、Caspase-3等蛋白表达的影响,旨在阐明PBR参与调控细胞缺血缺氧性损伤的机制。并进一步选用目前临床常用的苯二氮(艹卓)类静脉麻醉药咪达唑仑,观察其对缺氧复氧损伤所致细胞凋亡和相关因子表达的影响,及其与PBR的关系,为临床麻醉药物选择提供理论依据。
实验材料
1、实验动物
2～4d龄的Wiser大鼠,由中国医科大学实验动物中心提供。
2、实验试剂
PK11195(Alexis,美国);Ro5-4864(Biomol,美国);咪达唑仑(上海罗氏大药厂);Bcl-2/Bax兔抗鼠多克隆抗体(即用型)(Santa,美国);Caspase-3、PKC-Epsilon兔抗鼠多克隆抗体(Neomarker,美国);Histostain~(TM) PlusKits SP 9000免疫组化染色试剂盒、ZLI-9108浓缩型DAB试剂盒(北京中杉生物公司);Furo-2/AM、碘化丙啶(Sigma,美国);胰蛋白酶(1:250)(Amresco,美国);DMEM培养基(Gibco,美国);极品胎牛血清(天津灏洋生物);FITC-Annexin V(宝灵曼,德国);Trizol(Invitrogen,美国);One step RT-PCR试剂盒(Promega,美国);聚偏二氟乙烯(PVDF)蛋白印迹膜(Bio-Rad,美国);PBS等其它试剂为国产分析纯。
3、主要实验仪器
(1)振荡水浴箱(GFL THERMOLAB,美国)
(2)超低温冰箱(SANYO MDF-U,日本)
(3)旋涡振荡器(VORTEX-2 GENE,美国)
(4)水平板电泳系统(BIO-RAD Sub-cell GT,美国)
(5)通用电泳仪(BIO-RAD PowerPac200,美国)
(6)台式低温高速冷冻离心机(Sigma 3K30,美国)
(7)二氧化碳细胞培养箱(KENDRO/HERAEUS,德国)
(8)细胞培养专用超净工作台(苏州安泰空气技术有限公司,中国)
(9)倒置显微镜(OLYMPUS AX70,日本)
(10)水平摇床(GFL,美国)
(11)显微图像分析系统(Olympus AX70/Coolsnapfx/MetaMorph,日本)
(12)电热恒温鼓风干燥箱(余姚TDW,中国)
(13)自动电泳凝胶成像分析仪(Alphainnotech ChemiImager 5500,美国)
(14)小型垂直电泳仪(Bio-Rad Mini-ProteinⅢ,美国)
(15)半干转印仪(Bio-Rad Seimidry transfer system,美国)
(16)自动封膜仪(江苏仪器设备公司,中国)
(17)HEIDOLPH DIAX900型匀浆机(德国)
(18)PTC-100型PCR扩增仪(美国)
(19)FACS Calibur流式细胞仪(BD,美国)
(20)GLS-700D型数码凝胶扫描分析系统(上海,中国)
(21)A-200 Ds电子天平(美国)
(22)B-Brown微量泵(德国)
(23)超纯水装置(MILLIPORE MILLI-Q,美国)
(24)实验室制冰机(ZIGERA 2BE-70-35,德国)
(25)恒温振荡培养箱(GFL,德国)
(26)小型台式离心机(SIGMA 1-13,美国)
(27)PH计(WTW InoLab,德国)
(28)电动高压消毒锅(HIRAYAMA HVE-50,美国)
(29)HSS-1数字超级恒温浴槽(成都仪器厂,中国)
(30)倒置相差显微镜(OLYMPUS,日本)
(31)培养皿、培养板、培养瓶、离心管等(Corning,美国)
实验方法
1、心肌细胞培养和鉴定
2～4d龄的Wister大鼠,雌雄不拘,采用改良的Simpson法分离心肌细胞,胶原酶和胰蛋白酶分次消化,收集细胞,利用贴壁速度的差异去除贴壁的非心肌细胞。细胞接近融合呈整体搏动,开始实验。
心肌细胞成活检查和鉴定:采用0.4%胎盘兰染色,不着色者为存活细胞。倒置显微镜观察心肌细胞形态变化,培养4～5d后可形成细胞簇,并出现同簇细胞的同步搏动。电镜下观察可见心肌细胞肌节清楚,确认心肌细胞培养成功。
2、建立心肌细胞缺氧复氧模型
培养至融合状态的细胞先用99.9%高纯氮气饱和15min的低糖DMEM培养液换液,置于缺氧复氧装置中,充入99.99%高纯氮气置换装置内空气造成缺氧,气体通过滤过装置除去细菌等微生物,调节气体流量计使气体流速为5L/min,5min后关闭进出气阀门,放入5%CO_2培养箱37℃培养30min。复氧时迅速打开装置,用含20%胎牛血清的高糖DMEM营养液换液后置于培养箱中正常培养2h,建立缺氧复氧损伤模型。
3、实验分组和取材
将培养至融合状态的心肌细胞随机分为七组,即C组(空白对照组)、HR组(缺氧30min复氧2h组)、P组(PK11195组)、R组(Ro5-4864组)、M组(咪达唑仑组)、PR组(PK11195+Ro5-4864组)和PM组(PK11195+咪达唑仑组),P、R、M组在缺氧前30min于培养液中分别加入终浓度10~(-4)mol/L PK11195、Ro5-4864和咪达唑仑;PR组和PM组在缺氧前30min加入以上浓度的两种药物共同孵育,然后再进行缺氧复氧。模型建立成功后,培养皿内爬片细胞以4%多聚甲醛固定,-4℃冰箱保存,用于免疫组化染色,另一部分活细胞直接进行细胞内钙测定。培养瓶内的融合细胞,收集于冻存管,液氮速冻后-70℃保存,用于RT-PCR和Western Blot实验。
4、检测指标
(1)RT-PCR方法检测各组细胞PBR mRNA表达
TRIZOL裂解各组细胞后进行总RNA的提取,测定样品OD 260/280确定RNA质量。一步法RT-PCR,按Promega公司逆转录反应试剂盒使用说明操作。
PBR目的基因预扩增片段为526 bp:
上游引物5'CAGAATTCATGGCCCCGCCCTGGGTGCC3'
下游引物5'ACGGA TCCTCACTCTGGCAGCCGCCGTC3'
内对照β-actin预扩增片段为700bp:
上游引物:5'GTGGGCCGCTCTAGGCACCAA3'
下游引物:5'CTCTTTGATGTCACGCACGATTTC3'
按试剂盒说明加入反应体系。逆转录合成第一链cDNA,反应条件为42℃30min,95℃5min。直接进入第二链cDNA合成及PCR扩增,反应条件为94℃变性40s,60℃退火40s,72℃延伸60s,30个循环,最后72℃5min结束,4℃保存。取8ulPCR产物于2%的琼脂糖凝胶电泳,在溴酚蓝指示剂到达凝胶底部边缘时停止电泳。以溴化乙啶液(1ug/ul)覆盖凝胶,染色5min自动电泳凝胶成像分析仪下观察拍照。测定电泳条带密度值。
(2)免疫细胞化学染色测定Caspase-3蛋白表达
经无菌处理后的盖玻片置于24孔培养板中,细胞培养方法同前,在复氧后2小时取出生长有细胞的盖玻片,冷丙酮固定30min,SP法免疫细胞化学染色,染色步骤按试剂盒说明书进行操作。DAB显色,苏木素轻度复染,脱水,透明,封片。采用显微图像分析系统(Olympus AX70/Coolsnapfx/MetaMorph)图像处理分析仪检测,每组在高倍镜下(×400)随机选择6个视野自动计数100个细胞,测出Caspase-3蛋白表达的平均光密度。
(3)免疫蛋白印迹杂交(Western blot)测定Bcl-2、Bax和PKC蛋白的表达
分别裂解各组细胞,收集上清,测定蛋白质浓度,然后按每个电泳孔道加入50μg蛋白混合样品进行聚丙烯酰胺凝胶电泳,转膜。先后加入一抗(Bcl-2、Bax、PKC兔抗鼠多克隆抗体,稀释度为1:500)和二抗(羊抗兔单克隆抗体,稀释度为1:5000)与进行杂交。然后进行化学发光反应(ECL),洗片,在自动电泳凝胶成像分析仪上分析各电泳条带的蛋白含量。
(4)流式细胞仪FITC-Annexin V/PI法检测细胞凋亡
细胞模型建立成功后,收集细胞,孵育缓冲液洗涤,标记溶液重悬细胞,室温避光孵育10～15min,离心沉淀细胞,孵育缓冲液洗涤。加入荧光溶液(将FITC-Annexin V和PI加入到孵育缓冲液中,终浓度均为1μg/ml),4℃避光孵育20min。流式细胞仪激发光波长488nm,波长515nm的通带滤器检测FITC荧光,另一波长大于560nm的滤器检测PI,CellQUEST软件分析实验结果。
(5)Meta Flour单细胞内钙测定系统测定细胞内钙离子浓度
细胞首先用1mmol/L Fluo-2 DMSO孵育液37℃标记15min,避光温和搅拌,然后用缓冲液洗掉未结合的荧光探针,荧光倒置显微镜下观察(510nm),利用Meta Flour软件测定标记细胞的荧光强度,间接反映钙离子浓度。
5、统计学处理
实验数据采用均数±标准差((?)±s)表示,采用SPSS 12.0统计软件进行单因素方差分析,方差齐性采用LSD检验,方差不齐采用Dunnett T3检验,P＜0.05差异具有显著性。
结果
1、HR组凋亡细胞明显增加,与C组比较差异显著(P＜0.01),R组细胞凋亡明显减轻,与HR组比较差异显著(P＜0.05)。PR组细胞凋亡与R组比较差异显著(P＜0.01),与HR组比较无显著差异(P＞0.05)。M组细胞凋亡明显减轻,与HR组差异显著(P＜0.01),与PM组比较无显著差异(P＞0.05)。
2、HR组细胞PBR表达明显下调,与C组比较差异显著(P＜0.05),R组PBR表达上调,与HR组比较差异显著(P＜0.01)。PR组与R组比较差异显著(P＜0.01),与HR组比较无显著差异(P＞0.05)。M组PBR表达与HR组、PM组比较无明显差异(P＞0.05)。
3、HR组Bcl-2表达明显下调,Bax表达明显上调,与C组比较差异显著(P＜0.05)。R组Bcl-2表达上调,Bax表达下调,与HR组比较差异显著(P＜0.01)。PR组Bcl-2表达明显下调,与R组比较有显著差异(P＜0.01)。M组Bcl-2表达上调(P＜0.01),Bax表达无明显影响。PM组Bcl-2表达明显下调,与M组比较有显著差异(P＜0.01)。
4、HR组Caspase-3表达明显增强,与C组比较差异显著(P＜0.01)。P组Caspase-3表达上调,与HR组比较差异显著(P＜0.05)。R组Caspase-3表达明显下调,与HR组比较差异显著(P＜0.05)。PR组Caspase-3表达上调,与R组比较差异显著(P＜0.05)。M组Caspase-3表达下调,但与HR组比较无显著差异(P＞0.05)。
5、HR组细胞内钙离子浓度明显增加,与C组比较差异显著(P＜0.01)。R组细胞内钙离子浓度明显下降,与HR组比较差异显著(P＜0.01)。PR组细胞内钙离子浓度明显增加,与R组比较差异显著(P＜0.01),与HR组比较无明显差异(P＞0.05)。M组细胞内钙离子浓度无明显变化,与HR组、PM组比较均无明显差异(P＞0.05)。
6、HR组细胞PKC表达明显降低,与C组比较差异显著(P＜0.01)。R组细胞PKC表达增加,与HR组比较差异显著(P＜0.01)。PR组PKC表达明显降低,与R组比较差异显著(P＜0.01),与HR组比较无明显差异(P＞0.05)。M组细胞PKC表达无明显变化,与HR组比较无显著差异(P＞0.05)。
讨论
线粒体膜通透性改变,在细胞凋亡调节中起重要作用。线粒体膜通透性增加导致膜电位下降、呼吸链脱偶联、能量产生中断、ROS产生及线粒体基质肿胀,终致外膜破裂并释放出细胞色素C、凋亡诱导因子(AIF)、多种降解酶、caspase前体等各种活性蛋白,激活procaspase-3为有活性的Caspase-3。进一步水解包括细胞调节、细胞信号转导、DNA修复、组织平衡、细胞存活等环节中重要的蛋白,最终导致细胞坏死和/或凋亡。
在线粒体膜通透性调节中,线粒体通透性转运孔(permeability transitionpore,PTP)起决定性作用。外周型苯二氮(艹卓)类受体(PBR)是一种线粒体外膜蛋白,是线粒体关键膜复合物PTP的重要组成部分。PBR参与调节体内许多重要的生理病理过程,并参与调节线粒体PTP的开放和细胞凋亡。
Caspase-3又称CPP32(cysteine protease protein),是半胱氨酸蛋白酶家族的重要成员,是凋亡过程中最主要的终末执行酶。抑制Caspase酶活性可能成为防治缺氧复氧损伤的关键环节。Bcl-2亚家族可抑制线粒体释放细胞色素C,具有很强的抗凋亡作用,对Caspase依赖和非Caspase依赖的细胞凋亡均有抵抗作用。Bax亚家族可在线粒体膜上形成多聚体影响线粒体的完整性,从而促进细胞凋亡。蛋白激酶C(protein kinase C,PKC)为存在于细胞浆内由钙活化的磷脂依赖性丝/苏氨酸蛋白激酶,通过众多位于膜上和胞浆内的底物蛋白磷酸化而发挥广泛的生理作用,是IRI细胞保护作用的重要环节。而钙离子是目前所知惟一能穿过线粒体内膜传达信号的胞内第二信使,对细胞的多种功能发挥调节作用,胞质和线粒体内的钙浓度变化在细胞及膜损伤中起关键作用,[Ca~(2+)]i受很多因素的影响,PKC是关键的调节介质。
本实验应用大鼠心肌细胞缺糖缺氧及复氧损伤模型被认为能较好模拟真实心肌缺血再灌注过程,同时可排除在体和离体整体心脏缺血模型中难以控制的神经、体液、激素及心脏各类细胞的相互影响。研究显示缺氧复氧可损伤心肌细胞,引起其结构功能改变,使细胞凋亡增加。并伴有Bcl-2蛋白表达降低,Bax表达升高。Caspase-3蛋白于缺氧复氧后表达明显增强。PBR激动剂Ro5-4864对缺氧复氧损伤的心肌细胞具有明确的保护作用,可明显减少细胞凋亡。此作用与抑制Caspase-3活化、上调Bcl-2和下调Bax表达,以及活化PKC、维持钙稳态、抑制细胞内钙超载有关。其作用可被PBR的特异性拮抗剂PK11195所逆转,证明是PBR依赖性的。
PBR抗凋亡作用是通过线粒体的早期变化起作用的,包括降低线粒体膜通透性、抑制Caspase-3的活化及上调Bcl-2等。这些结果与在H_2O_2处理的造血细胞中PBR抗凋亡保护作用相一致。该研究还表明转染PBR可提高细胞对H_2O_2损伤的抵抗力。同样的研究也显示,在人淋巴细胞株U937中以H_2O_2诱导氧化应激模型,PBR激动剂具有抗凋亡保护作用。PBR特异性激动剂SSR180575和Ro5-4864能防止H_2O_2引起的氧化磷酸化损伤,对线粒体具有保护作用且具有剂量依赖性。还有研究证明,PBR参与调节IR后肾小管细胞的坏死和凋亡,并在肾功能障碍的信号转导通路中起重要作用。PBR激动剂SSR180575对鼠肾脏的IRI具有明确的保护作用。
临床常用的苯二氮(艹卓)类静脉麻醉药咪达唑仑,除具有镇静、催眠、抗焦虑和抗惊厥作用外,对缺氧复氧所致心肌细胞凋亡也有一定的保护作用。实验结果显示该作用是非PBR依赖性的,并且与细胞钙稳态和PKC信号通路无关。可能是通过抑制Caspase-3活化,上调Bcl-2表达实现的。
PBR激动剂可保持线粒体完整性、减少细胞凋亡,减轻细胞损伤,保护心脏功能。这一结果阐明了缺氧复氧所致细胞凋亡通过PBR调节的新机制,为预防和治疗IRI提供了可能的干预手段。有关PBR对应激反应的调节通路尚不完全清楚,仍需做大量的工作及深入的研究来阐明。
结论
1、PBR参与调节缺氧复氧损伤所致的心肌细胞凋亡。PBR基因表达的变化与细胞凋亡程度相反,表现出抗凋亡因子的特征。
2、PBR抗心肌细胞缺氧复氧损伤所致的细胞凋亡的与抑制Caspase-3活化,上调Bcl-2和下调Bax表达,以及活化PKC,维持钙稳态,抑制细胞内钙超载有关。
3、咪达唑仑可减轻缺氧复氧引起的心肌细胞凋亡,有一定的保护作用,其作用是非PBR依赖性的,与抑制Caspase-3活化,上调Bcl-2有关。
Introduction
Cardial ischemia reperfusion injury(IRI) is still hardly avoided when the following conditions happened such as thrombolysis,unstable angina,open heart operation,heart lung transplantation and cardiopulmonary resuscitation.The prevention of organ IRI is also an important question to be solved urgently.
The phenomenon of ischemic precondition(IPC) was firstly reported in myocardial cells by Murry and et al.The subsequent investigations found drug precondition(DPC) can produce the similar effect of IPC.Now the role of mitochondria on organ protection in IRI is known weU.The injury of mitochondria was a important factor for the irreversible injury in myocardiocytes,and the alteration of mitochondria permeability,which was regulated by permeability transition pore,played an important role in apoptosis.The early phase of apoptosis in which mitochondria participated was the opening of PTP,making the elevation of H~+ disappeared,decoupling the respiratory chain.The destroy of mitochondria membrane and inducing the release of cytoC and the other activated protein, which further triggered the cascade reaction of caspase made the cell show the character of apoptosis.
Peripherial benzodiazepine receptor(PBR) is a type of protein located in the outer membrane of mitochondria,which composed mitochondria PTP with voltage-dependent anion channel(VDAC) and adenine nucleotide translocator (ANT).PBR distributed widely,and expressed abundantly in heart,lung,liver, kidney and blood cells.Some investigations indicated that PBR took part in the regulation of opening the PTP,so determination of the effect of PBR and its ligands on mitochondria,cell and IRI is becoming a hot spot.
Our study planned to measured the expression of PBR in the model of myocardial hypoxia and reoxygenation and the effect on apoptosis.We explained the mechanism of PBR involved in ceUular IRI by observing the effect of PBR on the cellular concentration of calcium,the expression of protein kinase C(PKC) and the apoptosis involved factors such as Bcl-2/Bax,Caspase-3 and so on.We further observed the effect of PBR on apoptosis by adapting the commonly used benzodiazepines anesthetic:midazolam,and referred a potential basis for the clinical choice of anesthetics.
Materials
1.Animals:the neonate Wister rats of 2 to 4 days were provided by the experimental animal center of China Medical University.
2.Chemical and reagents:
PK11195(Alexis,USA);Ro5-4864(Biomol,USA);Midazolam(Roche, Shanghai);polyclonal antibody(Santa,USA);Caspase-3、PKC polyclonal antibody (Neomarker,USA);SP immunohistochemistry kit、DAB kit(Zhongsan, Beijing);Trypsin(1:250)(Amresco,USA);DMEM(Gibco,USA);Fetal calf serum(Haoyang,Tianjing);FITC-Annexin V(Boehringer Mannheim,German); Fura-2/AM,propidium iodide(Sigma,USA);Trizol(Invitrogen,USA); RT-PCR kit(Promega,USA);PVDF(Bio-Rad,USA).
3.Experiment instruments:
inverted phase contrast microscope(OLYMPUS,Japan);desk centrifuge (SIGMA 1-13,USA);PTC-100 PCRamplificator(USA);electrophoresis image analysator(Alphairmotech ChemiImager 5500,USA);CO_2 incubator(KENDRO/HERAEUS, German);super clean bench(Suzhou,China);PowerPac200 electrophoretic apparatus(BIO-RAD,USA);MDF-U ultra deep-freeze equipment(SANYO,Japan); micro image analysis system(Olympus AX70/Coolsnapfx/MetaMorph, Japan).
Methods
Culture and assessment of myocardial cells
The myocardial cells of neonatal rats were separated by improved Simpson method,in which 0.08%trypsin and 0.05%collagenase were used,and then the cells were cultured in DMEM medium including 20%fetal calf serum and incubated in a 95%air/5%CO_2 incubator at 37℃.To select enrich myocytes, dissociated cells were preplated for an hour to allow nonmyocytes to attach to the bottom of the culture dish.BrdU(0.1mmol/L) was added during the first two days to prevent proliferation of nonmyocytes.The cells were devided into groups randomly when grown in subsequent confluence state and showed beat in common. Cultured cells were further confirmed by the identification of myocardial inocoma under electron microscope.
The establishment of hypoxia and reoxigenation model
To induce hypoxic stress,culture medium was aspirated off and cells were replaced with hypoxic myocardial growth medium before introduced into an hypoxia chamber.The cells were removed from the hypoxic chamber after 30min, the medium was replaced by warm fresh medium and then the cells were placed in a 95%air/5%CO_2 incubator at 37℃for 2h to perform an reoxygenation phase.Normoxic control cells were treated by replacement of culture medium and incubated in a 95%air/5%CO_2 incubator at 37℃.
Experiment design
Cells were devided into seven groups,C group(control group),HR group (hypoxia for 30min and reoxygenation for 2h),P group(PK11195 group),R group(Ro5-4864 group),M group(Midazolam group),PR group(PK11195 and Ro5-4864 group) and PM group(PK11195 and Midazolam group).The groups were added PK11195,Ro5-4864 and Midazolam singlely or both respectively at a concentration of 10~(-4)mol/L before hypoxia and then treated with hypoxia and reoxygenation.One part of the cells were fixed by paraform for immunohistochemical staining and the other part were used directly for measured the intracellular calcium.Some cells were also cryopreserved for the assay by RT-PCR and Western-Blot methods.
Detection index
(1) Apoptotic cells assay
Apoptotic cells were measured by cytometry using FITC-Annexin V/PI Staining.
(2) The expression of Bcl-2/Bax and PKC
The expression of Bcl-2/Bax and PKC were measured by Western blot, and were analyzed by electrophoresis image analysator.
(3) The expression of Caspase-3
The quantitation of Caspase-3 was measured by immunohistochemistry staining and calculated the mean values by optical density detected by MetaMorph 4.5 image analysis software.
(4) The intracellular calcium concentration
Marked by fura-2,the calcium was calculated through fluorescence intensity by Meta Flour software.
(5) The expression of PBR mRNA
Quantitative reverse transcriptase-polymerase chain reaction(RT-PCR) was used.
Statistical analysis
All values in the text and figures were presented as mean SE and subjected to one-factor analysis of variance using SPSS 12.0 software.Homoscedasticity was checked by LSD method and the Dunnett T3 was adopted to check the heterogeneity of variance Differences were considered significant at P＜0.05.
Results
1.The amount of apoptosis in the HR group increased significantly compared with the C group(P＜0.01),and was attenuated in the R group compared with the HR.group(P＜0.01).PK11195 the antiapoptotic effect was blocked in the P group and the result showed a statistic difference compared with the R group(P＜0.01).midazolam decreased the apoptosis(compared with the HR group P＜0.05) and the effect can not be blocked by PK11195(P＞0.05).
2.The expression of PBR in the HR group was downregulated compared with the C group(P＜0.05),and 10~(-4)mol/L Ro5-4864 the expression of PBR was upregulated in the R group compared with the HR group(P＜0.01). PK11195 significantly blocked the upregulation compared with the R group(P＜0.01).Midazolam had no effect on the expression of PBR compared with the HR group(P＞0.05).
3.The expression of Bcl-2 was significantly downregulated after hypoxia and reoxygenation treatment compared with the C group(P＜0.05),but The expression of Bax protein was significantly upregulated compared with the C group (P＜0.05).10~(-4) mol/L Ro5-4864 significantly upregulated Bcl-2 and downregulated Bax compared with the HR group(P＜0.01),but the effect was completely reversed by PK11195(P＜0.01).midazolam upregulated the expression of Bcl-2,but did not affect the expression of Bax,and PK11195 significantly attenuated its effect on Bcl-2 compared with the M group(P＜0. 05).
4.The expression of Caspase-3 was enhanced significantly after hypoxia and reoxygenation treatment compared with the C group(P＜0.01),10~(-4)moL/L Ro5-4864 significantly downregulated Caspase-3 compared with the HR group (P＜0.05),but the effect was reversed by PK11195 compared with the R group (P＜0.05).midazolam downregulated the expression of Caspase-3,but it was not significant compared with the HR group(P＞0.05).The effect was not blocked by PK11195.
5.The concentration of intracellular calcium increased significantly induced by hypoxia and reoxygenation compared with the C group(P＜0.01). 10~(-4)mol/L Ro5-4864 significantly inhibited the increasing compared with the HR group(P＜0.01),but PK11195 blocked its effect.Midazolam had no effect on the concentration of intracellular calcium compared with the HR group(P＞0.05).
6.Hypoxia and reoxygenation decreased the expression of PKC compared with the C group(P＜0.01).10~(-4)mol/L Ro5-4864 significantly inhibited the decreasing of PKC induced by hypoxia and reoxygenation compared with the HR group(P＜0.01) and PK11195 blocked its effect compared with the R group(P ＜0.01).midazolam had no effect on the expression of PKC compared with the HR group(P＞0.05).
Discussion
Apoptosis is a kind of programmed death directed by gene,and the change of mitochondria membrane permeability played an important role.The increasing of mitochondria membrane permeability induced the breakdown of membrane potential, decoupling of respiration chain,interruption of energy production and increasing ROS production,which at last resulted in the rupture of outmembrane, cyto C release and such apoptosis induced factors.These factors can activated multiple catabolic enzymes including triggering pro-Caspase-3 to Caspase-3,which participated in several links for the cellular necrosis and/or apoptosis such as regulation,signal transduction,repair of DNA and so on.PTP played a key role in regulating the permeability of mitochondria.
PBR is a type of membrane protein located in the outmembrane of mitochondria, and is the key part of PTP.The analysis of mitochondria fragment showed VDAC and ANT connected with PBR tightly.The function of PBR related to VDAC and ANT and took part in the opening of PTP.
Caspase-3 is also called CPP32(cysteine protease protein),which pertained to the family of CPP,and became the most important final executor of apoptosis. So the inhibition of caspase may be an the effective method for prevention IRI.The sub-family of Bcl-2 can decrease the release of cyto C and showed a potential antiapoptotic character,which prevented apoptosis by an caspase-dependant and caspase-independent manner.The sub-family of Bax can form multimer,and affected the integrity of mitochondria in order to promote apoptosis.PKC is a type of phospholipids dependent serine/threonine protein kinase which activated by calcium ion in cytoplasma.It elicited a wide physiologic function by phosphorylating numerous substrate albumen located on membrane and cytoplasma,and became the important link for prevention IRI. Calcium ion is a unique second message which can go into mitochondria and regulated multiple functions of cell.The increasing concentration of calcium in mi- tochondria and cytoplasma played an important role in cell and membrane injury. It was regulated by many factors and PKC was the most important one.
We adopted the cultured neonate rat myocardial cells for inducing the model of hypoxia and reoxygenation injury,which was considered as a real manner for reflecting the course of IR,and depleted the interruption of nerve,humor, and hormone,which difficult to remove in the experiments in vivo and in vitro models.Our study showed hypoxia and reoxygenation injured myocytes,destroyed the structure and increased the apoptosis accompanied by decreased expression of Bcl-2 and increased expression of Bax.Hypoxia and reoxygenation enhanced the expression of Caspase-3.Ro5-4864,a specific agonist of PBR, prevented hypoxia and reoxygenation injury by significantly attenuating the apoptosis. These effects were related to the inhibition of Caspase-3 activation,upregulation of Bcl-2 and downregulation of Bax,activating PKC,maintaining the calcium homeostasis and inhibition of calcium overload.The protective effect was reversed by PK11195,an classic antagonist of PBR,which indicated the effects was dependent on PBR.
PBR elicited anti-apoptosis through the effects on mitochondria in the early phase including the permeability decreased,the release of cyto C,activation of Caspase-3 and breakage of DNA and et al.These results were coincide with the protection of PBR observed in the prevention of hematopoietic cell apoptosis treated by H_2O_2,which also showed that transfection of PBR increased the cellular resistance to H_2O_2.The similar investigation indicated the agonist of PBR prevented the oxidative injury induced by H_2O_2 in human lymph cell line U937. The specific agonists SSR180575 and Ro5-4864 can prevent the oxidative injury induced by H_2O_2 and protected mitochondria.Investigation also proved that PBR played an important role in the signaling passway of regulating the necrosis and apoptosis of tubular cells after IR,and SSR180575 had protective effect in rat renal IRI.
Midazolam,the clinical commonly used benzodiazepines anesthetic, showed protection in myocardial apoptosis induced by hypoxia and reoxygenation besides its sedation,hypnosis,antianxiety and anticonvulsion characters.The results indicated the effect was independent of PBR and had no effect on the concentration of intraceUular calcium and the expression of PKC.It may be related to the link of signaling passway including inhibition of Caspase-3 activation, upregulating Bcl-2 expresion and downregulating Bax.
The agonist of PBR elicited protection by maintaining the integrity of mitochondria, decreasing the injury and apoptosis.This result elucidated the mechanism of apoptosis after reperfusion was related to this receptor and referred a possible intervention for prevention and therapy of IRI.Further investigations were also demanded to explain the mechanism because the regulation signaling passway of PBR in stress was not clearly.
Conclusion
1.It was documented PBR participated in regulating the myocardial apoptosis induced by hypoxia and reoxygenation by adopting specific agonist and antagonist. The expression of PBR was opposite to the result of apoptosis and showed an anti-apoptotic character.
2.The anti-apoptotic character of PBR may be related to the mechanism of inhibiting the activation of Caspase-3,upregulating the expression of Bcl2/downregulating Bax,and activating PKC for inhibiting of the intracellular calcium overload to maintain homeostasis.
3.Midazolam prevented myocardial apoptosis induced by hypoxia and reperfusion and the effect was independent of PBR.
在溶栓、不稳定心绞痛、心内直视手术、心肺移植、冠脉搭桥手术和心肺复苏时,心肌缺血再灌注损伤(ischemic reperfusion injury,IRI)仍无法完全避免。防治器官IRI仍是临床亟待解决的重要问题之一。
Murry等于1986年首先报道在心肌细胞中存在缺血预适应(ischemicpreconditioning,IPC)现象,相继研究发现药物预适应可产生类似于IPC的保护作用。深入研究表明,线粒体损伤是导致IRI心肌不可逆损伤的重要原因,因而线粒体在IRI器官保护中的地位越来越受到重视。尤其线粒体膜通透性改变在细胞凋亡中起重要作用。在线粒体膜通透性调节中,线粒体通透性转运孔(permeability transition pore,PTP)起决定性作用。线粒体参与细胞凋亡的早期过程是线粒体PTP开放,使线粒体内外H~+梯度消失、呼吸链脱偶联、能量产生中断、基质肿胀并导致外膜破裂,释放出细胞色素C等各种活性蛋白。从而进一步激活caspase级联瀑布式反应,导致细胞凋亡。
外周型苯二氮革类受体(peripheral benzodiazepine receptor,PBR)是一种线粒体外膜蛋白,与电压依赖性阴离子通道(voltage-dependent anionchannel,VDAC)和核苷酸腺嘌呤转位体(adenine nucleotide translocator,ANT)共同组成线粒体PTP。PBR广泛存在于心脏、肺脏、肝脏、肾脏及血液细胞中,并参与调节线粒体PTP的开放。因此,确定PBR及其配体在线粒体、细胞和心肌再灌注损伤中的作用具有重要的意义。
本研究拟在建立体外培养心肌细胞缺氧复氧损伤模型的基础上,测定PBR基因表达的变化及其对细胞凋亡的影响。并观察PBR对缺氧复氧损伤细胞内钙离子浓度,蛋白激酶C表达及细胞凋亡相关因子Bcl2/Bax、Caspase-3等蛋白表达的影响,旨在阐明PBR参与调控细胞缺血缺氧性损伤的机制。并进一步选用目前临床常用的苯二氮(艹卓)类静脉麻醉药咪达唑仑,观察其对缺氧复氧损伤所致细胞凋亡和相关因子表达的影响,及其与PBR的关系,为临床麻醉药物选择提供理论依据。
实验材料
1、实验动物
2～4d龄的Wiser大鼠,由中国医科大学实验动物中心提供。
2、实验试剂
PK11195(Alexis,美国);Ro5-4864(Biomol,美国);咪达唑仑(上海罗氏大药厂);Bcl-2/Bax兔抗鼠多克隆抗体(即用型)(Santa,美国);Caspase-3、PKC-Epsilon兔抗鼠多克隆抗体(Neomarker,美国);Histostain~(TM) PlusKits SP 9000免疫组化染色试剂盒、ZLI-9108浓缩型DAB试剂盒(北京中杉生物公司);Furo-2/AM、碘化丙啶(Sigma,美国);胰蛋白酶(1:250)(Amresco,美国);DMEM培养基(Gibco,美国);极品胎牛血清(天津灏洋生物);FITC-Annexin V(宝灵曼,德国);Trizol(Invitrogen,美国);One step RT-PCR试剂盒(Promega,美国);聚偏二氟乙烯(PVDF)蛋白印迹膜(Bio-Rad,美国);PBS等其它试剂为国产分析纯。
3、主要实验仪器
(1)振荡水浴箱(GFL THERMOLAB,美国)
(2)超低温冰箱(SANYO MDF-U,日本)
(3)旋涡振荡器(VORTEX-2 GENE,美国)
(4)水平板电泳系统(BIO-RAD Sub-cell GT,美国)
(5)通用电泳仪(BIO-RAD PowerPac200,美国)
(6)台式低温高速冷冻离心机(Sigma 3K30,美国)
(7)二氧化碳细胞培养箱(KENDRO/HERAEUS,德国)
(8)细胞培养专用超净工作台(苏州安泰空气技术有限公司,中国)
(9)倒置显微镜(OLYMPUS AX70,日本)
(10)水平摇床(GFL,美国)
(11)显微图像分析系统(Olympus AX70/Coolsnapfx/MetaMorph,日本)
(12)电热恒温鼓风干燥箱(余姚TDW,中国)
(13)自动电泳凝胶成像分析仪(Alphainnotech ChemiImager 5500,美国)
(14)小型垂直电泳仪(Bio-Rad Mini-ProteinⅢ,美国)
(15)半干转印仪(Bio-Rad Seimidry transfer system,美国)
(16)自动封膜仪(江苏仪器设备公司,中国)
(17)HEIDOLPH DIAX900型匀浆机(德国)
(18)PTC-100型PCR扩增仪(美国)
(19)FACS Calibur流式细胞仪(BD,美国)
(20)GLS-700D型数码凝胶扫描分析系统(上海,中国)
(21)A-200 Ds电子天平(美国)
(22)B-Brown微量泵(德国)
(23)超纯水装置(MILLIPORE MILLI-Q,美国)
(24)实验室制冰机(ZIGERA 2BE-70-35,德国)
(25)恒温振荡培养箱(GFL,德国)
(26)小型台式离心机(SIGMA 1-13,美国)
(27)PH计(WTW InoLab,德国)
(28)电动高压消毒锅(HIRAYAMA HVE-50,美国)
(29)HSS-1数字超级恒温浴槽(成都仪器厂,中国)
(30)倒置相差显微镜(OLYMPUS,日本)
(31)培养皿、培养板、培养瓶、离心管等(Corning,美国)
实验方法
1、心肌细胞培养和鉴定
2～4d龄的Wister大鼠,雌雄不拘,采用改良的Simpson法分离心肌细胞,胶原酶和胰蛋白酶分次消化,收集细胞,利用贴壁速度的差异去除贴壁的非心肌细胞。细胞接近融合呈整体搏动,开始实验。
心肌细胞成活检查和鉴定:采用0.4%胎盘兰染色,不着色者为存活细胞。倒置显微镜观察心肌细胞形态变化,培养4～5d后可形成细胞簇,并出现同簇细胞的同步搏动。电镜下观察可见心肌细胞肌节清楚,确认心肌细胞培养成功。
2、建立心肌细胞缺氧复氧模型
培养至融合状态的细胞先用99.9%高纯氮气饱和15min的低糖DMEM培养液换液,置于缺氧复氧装置中,充入99.99%高纯氮气置换装置内空气造成缺氧,气体通过滤过装置除去细菌等微生物,调节气体流量计使气体流速为5L/min,5min后关闭进出气阀门,放入5%CO_2培养箱37℃培养30min。复氧时迅速打开装置,用含20%胎牛血清的高糖DMEM营养液换液后置于培养箱中正常培养2h,建立缺氧复氧损伤模型。
3、实验分组和取材
将培养至融合状态的心肌细胞随机分为七组,即C组(空白对照组)、HR组(缺氧30min复氧2h组)、P组(PK11195组)、R组(Ro5-4864组)、M组(咪达唑仑组)、PR组(PK11195+Ro5-4864组)和PM组(PK11195+咪达唑仑组),P、R、M组在缺氧前30min于培养液中分别加入终浓度10~(-4)mol/L PK11195、Ro5-4864和咪达唑仑;PR组和PM组在缺氧前30min加入以上浓度的两种药物共同孵育,然后再进行缺氧复氧。模型建立成功后,培养皿内爬片细胞以4%多聚甲醛固定,-4℃冰箱保存,用于免疫组化染色,另一部分活细胞直接进行细胞内钙测定。培养瓶内的融合细胞,收集于冻存管,液氮速冻后-70℃保存,用于RT-PCR和Western Blot实验。
4、检测指标
(1)RT-PCR方法检测各组细胞PBR mRNA表达
TRIZOL裂解各组细胞后进行总RNA的提取,测定样品OD 260/280确定RNA质量。一步法RT-PCR,按Promega公司逆转录反应试剂盒使用说明操作。
PBR目的基因预扩增片段为526 bp:
上游引物5'CAGAATTCATGGCCCCGCCCTGGGTGCC3'
下游引物5'ACGGA TCCTCACTCTGGCAGCCGCCGTC3'
内对照β-actin预扩增片段为700bp:
上游引物:5'GTGGGCCGCTCTAGGCACCAA3'
下游引物:5'CTCTTTGATGTCACGCACGATTTC3'
按试剂盒说明加入反应体系。逆转录合成第一链cDNA,反应条件为42℃30min,95℃5min。直接进入第二链cDNA合成及PCR扩增,反应条件为94℃变性40s,60℃退火40s,72℃延伸60s,30个循环,最后72℃5min结束,4℃保存。取8ulPCR产物于2%的琼脂糖凝胶电泳,在溴酚蓝指示剂到达凝胶底部边缘时停止电泳。以溴化乙啶液(1ug/ul)覆盖凝胶,染色5min自动电泳凝胶成像分析仪下观察拍照。测定电泳条带密度值。
(2)免疫细胞化学染色测定Caspase-3蛋白表达
经无菌处理后的盖玻片置于24孔培养板中,细胞培养方法同前,在复氧后2小时取出生长有细胞的盖玻片,冷丙酮固定30min,SP法免疫细胞化学染色,染色步骤按试剂盒说明书进行操作。DAB显色,苏木素轻度复染,脱水,透明,封片。采用显微图像分析系统(Olympus AX70/Coolsnapfx/MetaMorph)图像处理分析仪检测,每组在高倍镜下(×400)随机选择6个视野自动计数100个细胞,测出Caspase-3蛋白表达的平均光密度。
(3)免疫蛋白印迹杂交(Western blot)测定Bcl-2、Bax和PKC蛋白的表达
分别裂解各组细胞,收集上清,测定蛋白质浓度,然后按每个电泳孔道加入50μg蛋白混合样品进行聚丙烯酰胺凝胶电泳,转膜。先后加入一抗(Bcl-2、Bax、PKC兔抗鼠多克隆抗体,稀释度为1:500)和二抗(羊抗兔单克隆抗体,稀释度为1:5000)与进行杂交。然后进行化学发光反应(ECL),洗片,在自动电泳凝胶成像分析仪上分析各电泳条带的蛋白含量。
(4)流式细胞仪FITC-Annexin V/PI法检测细胞凋亡
细胞模型建立成功后,收集细胞,孵育缓冲液洗涤,标记溶液重悬细胞,室温避光孵育10～15min,离心沉淀细胞,孵育缓冲液洗涤。加入荧光溶液(将FITC-Annexin V和PI加入到孵育缓冲液中,终浓度均为1μg/ml),4℃避光孵育20min。流式细胞仪激发光波长488nm,波长515nm的通带滤器检测FITC荧光,另一波长大于560nm的滤器检测PI,CellQUEST软件分析实验结果。
(5)Meta Flour单细胞内钙测定系统测定细胞内钙离子浓度
细胞首先用1mmol/L Fluo-2 DMSO孵育液37℃标记15min,避光温和搅拌,然后用缓冲液洗掉未结合的荧光探针,荧光倒置显微镜下观察(510nm),利用Meta Flour软件测定标记细胞的荧光强度,间接反映钙离子浓度。
5、统计学处理
实验数据采用均数±标准差((?)±s)表示,采用SPSS 12.0统计软件进行单因素方差分析,方差齐性采用LSD检验,方差不齐采用Dunnett T3检验,P＜0.05差异具有显著性。
结果
1、HR组凋亡细胞明显增加,与C组比较差异显著(P＜0.01),R组细胞凋亡明显减轻,与HR组比较差异显著(P＜0.05)。PR组细胞凋亡与R组比较差异显著(P＜0.01),与HR组比较无显著差异(P＞0.05)。M组细胞凋亡明显减轻,与HR组差异显著(P＜0.01),与PM组比较无显著差异(P＞0.05)。
2、HR组细胞PBR表达明显下调,与C组比较差异显著(P＜0.05),R组PBR表达上调,与HR组比较差异显著(P＜0.01)。PR组与R组比较差异显著(P＜0.01),与HR组比较无显著差异(P＞0.05)。M组PBR表达与HR组、PM组比较无明显差异(P＞0.05)。
3、HR组Bcl-2表达明显下调,Bax表达明显上调,与C组比较差异显著(P＜0.05)。R组Bcl-2表达上调,Bax表达下调,与HR组比较差异显著(P＜0.01)。PR组Bcl-2表达明显下调,与R组比较有显著差异(P＜0.01)。M组Bcl-2表达上调(P＜0.01),Bax表达无明显影响。PM组Bcl-2表达明显下调,与M组比较有显著差异(P＜0.01)。
4、HR组Caspase-3表达明显增强,与C组比较差异显著(P＜0.01)。P组Caspase-3表达上调,与HR组比较差异显著(P＜0.05)。R组Caspase-3表达明显下调,与HR组比较差异显著(P＜0.05)。PR组Caspase-3表达上调,与R组比较差异显著(P＜0.05)。M组Caspase-3表达下调,但与HR组比较无显著差异(P＞0.05)。
5、HR组细胞内钙离子浓度明显增加,与C组比较差异显著(P＜0.01)。R组细胞内钙离子浓度明显下降,与HR组比较差异显著(P＜0.01)。PR组细胞内钙离子浓度明显增加,与R组比较差异显著(P＜0.01),与HR组比较无明显差异(P＞0.05)。M组细胞内钙离子浓度无明显变化,与HR组、PM组比较均无明显差异(P＞0.05)。
6、HR组细胞PKC表达明显降低,与C组比较差异显著(P＜0.01)。R组细胞PKC表达增加,与HR组比较差异显著(P＜0.01)。PR组PKC表达明显降低,与R组比较差异显著(P＜0.01),与HR组比较无明显差异(P＞0.05)。M组细胞PKC表达无明显变化,与HR组比较无显著差异(P＞0.05)。
讨论
线粒体膜通透性改变,在细胞凋亡调节中起重要作用。线粒体膜通透性增加导致膜电位下降、呼吸链脱偶联、能量产生中断、ROS产生及线粒体基质肿胀,终致外膜破裂并释放出细胞色素C、凋亡诱导因子(AIF)、多种降解酶、caspase前体等各种活性蛋白,激活procaspase-3为有活性的Caspase-3。进一步水解包括细胞调节、细胞信号转导、DNA修复、组织平衡、细胞存活等环节中重要的蛋白,最终导致细胞坏死和/或凋亡。
在线粒体膜通透性调节中,线粒体通透性转运孔(permeability transitionpore,PTP)起决定性作用。外周型苯二氮(艹卓)类受体(PBR)是一种线粒体外膜蛋白,是线粒体关键膜复合物PTP的重要组成部分。PBR参与调节体内许多重要的生理病理过程,并参与调节线粒体PTP的开放和细胞凋亡。
Caspase-3又称CPP32(cysteine protease protein),是半胱氨酸蛋白酶家族的重要成员,是凋亡过程中最主要的终末执行酶。抑制Caspase酶活性可能成为防治缺氧复氧损伤的关键环节。Bcl-2亚家族可抑制线粒体释放细胞色素C,具有很强的抗凋亡作用,对Caspase依赖和非Caspase依赖的细胞凋亡均有抵抗作用。Bax亚家族可在线粒体膜上形成多聚体影响线粒体的完整性,从而促进细胞凋亡。蛋白激酶C(protein kinase C,PKC)为存在于细胞浆内由钙活化的磷脂依赖性丝/苏氨酸蛋白激酶,通过众多位于膜上和胞浆内的底物蛋白磷酸化而发挥广泛的生理作用,是IRI细胞保护作用的重要环节。而钙离子是目前所知惟一能穿过线粒体内膜传达信号的胞内第二信使,对细胞的多种功能发挥调节作用,胞质和线粒体内的钙浓度变化在细胞及膜损伤中起关键作用,[Ca~(2+)]i受很多因素的影响,PKC是关键的调节介质。
本实验应用大鼠心肌细胞缺糖缺氧及复氧损伤模型被认为能较好模拟真实心肌缺血再灌注过程,同时可排除在体和离体整体心脏缺血模型中难以控制的神经、体液、激素及心脏各类细胞的相互影响。研究显示缺氧复氧可损伤心肌细胞,引起其结构功能改变,使细胞凋亡增加。并伴有Bcl-2蛋白表达降低,Bax表达升高。Caspase-3蛋白于缺氧复氧后表达明显增强。PBR激动剂Ro5-4864对缺氧复氧损伤的心肌细胞具有明确的保护作用,可明显减少细胞凋亡。此作用与抑制Caspase-3活化、上调Bcl-2和下调Bax表达,以及活化PKC、维持钙稳态、抑制细胞内钙超载有关。其作用可被PBR的特异性拮抗剂PK11195所逆转,证明是PBR依赖性的。
PBR抗凋亡作用是通过线粒体的早期变化起作用的,包括降低线粒体膜通透性、抑制Caspase-3的活化及上调Bcl-2等。这些结果与在H_2O_2处理的造血细胞中PBR抗凋亡保护作用相一致。该研究还表明转染PBR可提高细胞对H_2O_2损伤的抵抗力。同样的研究也显示,在人淋巴细胞株U937中以H_2O_2诱导氧化应激模型,PBR激动剂具有抗凋亡保护作用。PBR特异性激动剂SSR180575和Ro5-4864能防止H_2O_2引起的氧化磷酸化损伤,对线粒体具有保护作用且具有剂量依赖性。还有研究证明,PBR参与调节IR后肾小管细胞的坏死和凋亡,并在肾功能障碍的信号转导通路中起重要作用。PBR激动剂SSR180575对鼠肾脏的IRI具有明确的保护作用。
临床常用的苯二氮(艹卓)类静脉麻醉药咪达唑仑,除具有镇静、催眠、抗焦虑和抗惊厥作用外,对缺氧复氧所致心肌细胞凋亡也有一定的保护作用。实验结果显示该作用是非PBR依赖性的,并且与细胞钙稳态和PKC信号通路无关。可能是通过抑制Caspase-3活化,上调Bcl-2表达实现的。
PBR激动剂可保持线粒体完整性、减少细胞凋亡,减轻细胞损伤,保护心脏功能。这一结果阐明了缺氧复氧所致细胞凋亡通过PBR调节的新机制,为预防和治疗IRI提供了可能的干预手段。有关PBR对应激反应的调节通路尚不完全清楚,仍需做大量的工作及深入的研究来阐明。
结论
1、PBR参与调节缺氧复氧损伤所致的心肌细胞凋亡。PBR基因表达的变化与细胞凋亡程度相反,表现出抗凋亡因子的特征。
2、PBR抗心肌细胞缺氧复氧损伤所致的细胞凋亡的与抑制Caspase-3活化,上调Bcl-2和下调Bax表达,以及活化PKC,维持钙稳态,抑制细胞内钙超载有关。
3、咪达唑仑可减轻缺氧复氧引起的心肌细胞凋亡,有一定的保护作用,其作用是非PBR依赖性的,与抑制Caspase-3活化,上调Bcl-2有关。
Introduction
Cardial ischemia reperfusion injury(IRI) is still hardly avoided when the following conditions happened such as thrombolysis,unstable angina,open heart operation,heart lung transplantation and cardiopulmonary resuscitation.The prevention of organ IRI is also an important question to be solved urgently.
The phenomenon of ischemic precondition(IPC) was firstly reported in myocardial cells by Murry and et al.The subsequent investigations found drug precondition(DPC) can produce the similar effect of IPC.Now the role of mitochondria on organ protection in IRI is known weU.The injury of mitochondria was a important factor for the irreversible injury in myocardiocytes,and the alteration of mitochondria permeability,which was regulated by permeability transition pore,played an important role in apoptosis.The early phase of apoptosis in which mitochondria participated was the opening of PTP,making the elevation of H~+ disappeared,decoupling the respiratory chain.The destroy of mitochondria membrane and inducing the release of cytoC and the other activated protein, which further triggered the cascade reaction of caspase made the cell show the character of apoptosis.
Peripherial benzodiazepine receptor(PBR) is a type of protein located in the outer membrane of mitochondria,which composed mitochondria PTP with voltage-dependent anion channel(VDAC) and adenine nucleotide translocator (ANT).PBR distributed widely,and expressed abundantly in heart,lung,liver, kidney and blood cells.Some investigations indicated that PBR took part in the regulation of opening the PTP,so determination of the effect of PBR and its ligands on mitochondria,cell and IRI is becoming a hot spot.
Our study planned to measured the expression of PBR in the model of myocardial hypoxia and reoxygenation and the effect on apoptosis.We explained the mechanism of PBR involved in ceUular IRI by observing the effect of PBR on the cellular concentration of calcium,the expression of protein kinase C(PKC) and the apoptosis involved factors such as Bcl-2/Bax,Caspase-3 and so on.We further observed the effect of PBR on apoptosis by adapting the commonly used benzodiazepines anesthetic:midazolam,and referred a potential basis for the clinical choice of anesthetics.
Materials
1.Animals:the neonate Wister rats of 2 to 4 days were provided by the experimental animal center of China Medical University.
2.Chemical and reagents:
PK11195(Alexis,USA);Ro5-4864(Biomol,USA);Midazolam(Roche, Shanghai);polyclonal antibody(Santa,USA);Caspase-3、PKC polyclonal antibody (Neomarker,USA);SP immunohistochemistry kit、DAB kit(Zhongsan, Beijing);Trypsin(1:250)(Amresco,USA);DMEM(Gibco,USA);Fetal calf serum(Haoyang,Tianjing);FITC-Annexin V(Boehringer Mannheim,German); Fura-2/AM,propidium iodide(Sigma,USA);Trizol(Invitrogen,USA); RT-PCR kit(Promega,USA);PVDF(Bio-Rad,USA).
3.Experiment instruments:
inverted phase contrast microscope(OLYMPUS,Japan);desk centrifuge (SIGMA 1-13,USA);PTC-100 PCRamplificator(USA);electrophoresis image analysator(Alphairmotech ChemiImager 5500,USA);CO_2 incubator(KENDRO/HERAEUS, German);super clean bench(Suzhou,China);PowerPac200 electrophoretic apparatus(BIO-RAD,USA);MDF-U ultra deep-freeze equipment(SANYO,Japan); micro image analysis system(Olympus AX70/Coolsnapfx/MetaMorph, Japan).
Methods
Culture and assessment of myocardial cells
The myocardial cells of neonatal rats were separated by improved Simpson method,in which 0.08%trypsin and 0.05%collagenase were used,and then the cells were cultured in DMEM medium including 20%fetal calf serum and incubated in a 95%air/5%CO_2 incubator at 37℃.To select enrich myocytes, dissociated cells were preplated for an hour to allow nonmyocytes to attach to the bottom of the culture dish.BrdU(0.1mmol/L) was added during the first two days to prevent proliferation of nonmyocytes.The cells were devided into groups randomly when grown in subsequent confluence state and showed beat in common. Cultured cells were further confirmed by the identification of myocardial inocoma under electron microscope.
The establishment of hypoxia and reoxigenation model
To induce hypoxic stress,culture medium was aspirated off and cells were replaced with hypoxic myocardial growth medium before introduced into an hypoxia chamber.The cells were removed from the hypoxic chamber after 30min, the medium was replaced by warm fresh medium and then the cells were placed in a 95%air/5%CO_2 incubator at 37℃for 2h to perform an reoxygenation phase.Normoxic control cells were treated by replacement of culture medium and incubated in a 95%air/5%CO_2 incubator at 37℃.
Experiment design
Cells were devided into seven groups,C group(control group),HR group (hypoxia for 30min and reoxygenation for 2h),P group(PK11195 group),R group(Ro5-4864 group),M group(Midazolam group),PR group(PK11195 and Ro5-4864 group) and PM group(PK11195 and Midazolam group).The groups were added PK11195,Ro5-4864 and Midazolam singlely or both respectively at a concentration of 10~(-4)mol/L before hypoxia and then treated with hypoxia and reoxygenation.One part of the cells were fixed by paraform for immunohistochemical staining and the other part were used directly for measured the intracellular calcium.Some cells were also cryopreserved for the assay by RT-PCR and Western-Blot methods.
Detection index
(1) Apoptotic cells assay
Apoptotic cells were measured by cytometry using FITC-Annexin V/PI Staining.
(2) The expression of Bcl-2/Bax and PKC
The expression of Bcl-2/Bax and PKC were measured by Western blot, and were analyzed by electrophoresis image analysator.
(3) The expression of Caspase-3
The quantitation of Caspase-3 was measured by immunohistochemistry staining and calculated the mean values by optical density detected by MetaMorph 4.5 image analysis software.
(4) The intracellular calcium concentration
Marked by fura-2,the calcium was calculated through fluorescence intensity by Meta Flour software.
(5) The expression of PBR mRNA
Quantitative reverse transcriptase-polymerase chain reaction(RT-PCR) was used.
Statistical analysis
All values in the text and figures were presented as mean SE and subjected to one-factor analysis of variance using SPSS 12.0 software.Homoscedasticity was checked by LSD method and the Dunnett T3 was adopted to check the heterogeneity of variance Differences were considered significant at P＜0.05.
Results
1.The amount of apoptosis in the HR group increased significantly compared with the C group(P＜0.01),and was attenuated in the R group compared with the HR.group(P＜0.01).PK11195 the antiapoptotic effect was blocked in the P group and the result showed a statistic difference compared with the R group(P＜0.01).midazolam decreased the apoptosis(compared with the HR group P＜0.05) and the effect can not be blocked by PK11195(P＞0.05).
2.The expression of PBR in the HR group was downregulated compared with the C group(P＜0.05),and 10~(-4)mol/L Ro5-4864 the expression of PBR was upregulated in the R group compared with the HR group(P＜0.01). PK11195 significantly blocked the upregulation compared with the R group(P＜0.01).Midazolam had no effect on the expression of PBR compared with the HR group(P＞0.05).
3.The expression of Bcl-2 was significantly downregulated after hypoxia and reoxygenation treatment compared with the C group(P＜0.05),but The expression of Bax protein was significantly upregulated compared with the C group (P＜0.05).10~(-4) mol/L Ro5-4864 significantly upregulated Bcl-2 and downregulated Bax compared with the HR group(P＜0.01),but the effect was completely reversed by PK11195(P＜0.01).midazolam upregulated the expression of Bcl-2,but did not affect the expression of Bax,and PK11195 significantly attenuated its effect on Bcl-2 compared with the M group(P＜0. 05).
4.The expression of Caspase-3 was enhanced significantly after hypoxia and reoxygenation treatment compared with the C group(P＜0.01),10~(-4)moL/L Ro5-4864 significantly downregulated Caspase-3 compared with the HR group (P＜0.05),but the effect was reversed by PK11195 compared with the R group (P＜0.05).midazolam downregulated the expression of Caspase-3,but it was not significant compared with the HR group(P＞0.05).The effect was not blocked by PK11195.
5.The concentration of intracellular calcium increased significantly induced by hypoxia and reoxygenation compared with the C group(P＜0.01). 10~(-4)mol/L Ro5-4864 significantly inhibited the increasing compared with the HR group(P＜0.01),but PK11195 blocked its effect.Midazolam had no effect on the concentration of intracellular calcium compared with the HR group(P＞0.05).
6.Hypoxia and reoxygenation decreased the expression of PKC compared with the C group(P＜0.01).10~(-4)mol/L Ro5-4864 significantly inhibited the decreasing of PKC induced by hypoxia and reoxygenation compared with the HR group(P＜0.01) and PK11195 blocked its effect compared with the R group(P ＜0.01).midazolam had no effect on the expression of PKC compared with the HR group(P＞0.05).
Discussion
Apoptosis is a kind of programmed death directed by gene,and the change of mitochondria membrane permeability played an important role.The increasing of mitochondria membrane permeability induced the breakdown of membrane potential, decoupling of respiration chain,interruption of energy production and increasing ROS production,which at last resulted in the rupture of outmembrane, cyto C release and such apoptosis induced factors.These factors can activated multiple catabolic enzymes including triggering pro-Caspase-3 to Caspase-3,which participated in several links for the cellular necrosis and/or apoptosis such as regulation,signal transduction,repair of DNA and so on.PTP played a key role in regulating the permeability of mitochondria.
PBR is a type of membrane protein located in the outmembrane of mitochondria, and is the key part of PTP.The analysis of mitochondria fragment showed VDAC and ANT connected with PBR tightly.The function of PBR related to VDAC and ANT and took part in the opening of PTP.
Caspase-3 is also called CPP32(cysteine protease protein),which pertained to the family of CPP,and became the most important final executor of apoptosis. So the inhibition of caspase may be an the effective method for prevention IRI.The sub-family of Bcl-2 can decrease the release of cyto C and showed a potential antiapoptotic character,which prevented apoptosis by an caspase-dependant and caspase-independent manner.The sub-family of Bax can form multimer,and affected the integrity of mitochondria in order to promote apoptosis.PKC is a type of phospholipids dependent serine/threonine protein kinase which activated by calcium ion in cytoplasma.It elicited a wide physiologic function by phosphorylating numerous substrate albumen located on membrane and cytoplasma,and became the important link for prevention IRI. Calcium ion is a unique second message which can go into mitochondria and regulated multiple functions of cell.The increasing concentration of calcium in mi- tochondria and cytoplasma played an important role in cell and membrane injury. It was regulated by many factors and PKC was the most important one.
We adopted the cultured neonate rat myocardial cells for inducing the model of hypoxia and reoxygenation injury,which was considered as a real manner for reflecting the course of IR,and depleted the interruption of nerve,humor, and hormone,which difficult to remove in the experiments in vivo and in vitro models.Our study showed hypoxia and reoxygenation injured myocytes,destroyed the structure and increased the apoptosis accompanied by decreased expression of Bcl-2 and increased expression of Bax.Hypoxia and reoxygenation enhanced the expression of Caspase-3.Ro5-4864,a specific agonist of PBR, prevented hypoxia and reoxygenation injury by significantly attenuating the apoptosis. These effects were related to the inhibition of Caspase-3 activation,upregulation of Bcl-2 and downregulation of Bax,activating PKC,maintaining the calcium homeostasis and inhibition of calcium overload.The protective effect was reversed by PK11195,an classic antagonist of PBR,which indicated the effects was dependent on PBR.
PBR elicited anti-apoptosis through the effects on mitochondria in the early phase including the permeability decreased,the release of cyto C,activation of Caspase-3 and breakage of DNA and et al.These results were coincide with the protection of PBR observed in the prevention of hematopoietic cell apoptosis treated by H_2O_2,which also showed that transfection of PBR increased the cellular resistance to H_2O_2.The similar investigation indicated the agonist of PBR prevented the oxidative injury induced by H_2O_2 in human lymph cell line U937. The specific agonists SSR180575 and Ro5-4864 can prevent the oxidative injury induced by H_2O_2 and protected mitochondria.Investigation also proved that PBR played an important role in the signaling passway of regulating the necrosis and apoptosis of tubular cells after IR,and SSR180575 had protective effect in rat renal IRI.
Midazolam,the clinical commonly used benzodiazepines anesthetic, showed protection in myocardial apoptosis induced by hypoxia and reoxygenation besides its sedation,hypnosis,antianxiety and anticonvulsion characters.The results indicated the effect was independent of PBR and had no effect on the concentration of intraceUular calcium and the expression of PKC.It may be related to the link of signaling passway including inhibition of Caspase-3 activation, upregulating Bcl-2 expresion and downregulating Bax.
The agonist of PBR elicited protection by maintaining the integrity of mitochondria, decreasing the injury and apoptosis.This result elucidated the mechanism of apoptosis after reperfusion was related to this receptor and referred a possible intervention for prevention and therapy of IRI.Further investigations were also demanded to explain the mechanism because the regulation signaling passway of PBR in stress was not clearly.
Conclusion
1.It was documented PBR participated in regulating the myocardial apoptosis induced by hypoxia and reoxygenation by adopting specific agonist and antagonist. The expression of PBR was opposite to the result of apoptosis and showed an anti-apoptotic character.
2.The anti-apoptotic character of PBR may be related to the mechanism of inhibiting the activation of Caspase-3,upregulating the expression of Bcl2/downregulating Bax,and activating PKC for inhibiting of the intracellular calcium overload to maintain homeostasis.
3.Midazolam prevented myocardial apoptosis induced by hypoxia and reperfusion and the effect was independent of PBR.
引文
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