【作者】 李敬远；

【导师】 曾因明；

【作者基本信息】 中国医科大学 ， 麻醉学， 2007， 博士

【摘要】 抑制线粒体通透性转换对心肌细胞缺氧／复氧损伤的保护作用目的线粒体功能障碍在心肌细胞应激性损伤中占有重要地位，近年研究发现线粒体的功能障碍和线粒体通透性转换孔道（mitochondrial permeability transition pore，mPTP）状态密切相关，mPTP开放导致的线粒体通透性转换（mitochondrialpermeability transition，MPT）被认为是调控细胞死亡或生存，以及凋亡或坏死的重要环节。mPTP是由位于线粒体膜上多蛋白组成的非选择性复合孔道，其主要由外膜的电压依赖性阴离子通道（VDAC），内膜的腺苷酸转位子（ANT），基质的亲环蛋白D（CyP-D）组成。最近研究认为外周苯二氮（艹卓）受体（peripheral benzodiazepinereceptor，PBR）、已糖激酶、肌酸激酶（CK）可能也参与mPTP的构成。MPT在细胞应激状态（如氧化应激、生长因子去除或暴露于细胞质分裂酶）时起到“中央执行者”的作用。MPT导致线粒体膜电位下降、氧化磷酸化解耦联、ATP损耗、小分子溶质和离子在细胞质和线粒体基质间的平衡。溶质内流入线粒体基质导致线粒体基质膨胀，外膜破裂，线粒体内膜蛋白释放，包括细胞色素C（Cyt c）、procaspase9、AIF（凋亡诱导因子）、核酸内切酶（Endo G）。MPT的调控对维持线粒体内膜的不通透性（除了对一些选择性的代谢物）起到重要作用，以维持线粒体膜电位，保证氧化磷酸化过程中ATP的合成。MPT在心肌缺血再灌注损伤中起着重要作用，随着缺血时间延长，线粒体发生MPT的潜在可能性增加，称为MPT启动（priming）阶段，然而再灌注的条件可以调节MPT能否被触发，称为MPT触发（trigger）阶段。缺血缺氧期间细胞内Ca²⁺升高，pH值下降，长链脂肪酸（LCFA）增多，ROS产生，启动MPT。MPT触发阶段，受再灌注期间MPT诱导／抑制因素(尤其是基质Ca²⁺和ROS水平)的相互作用和再生膜电位的电子传递能力的影响，而后者对缺血期间的Cyt c失和内膜通透程度高度敏感。因为mPTP的开放是电压依赖性的，当mPTP暂时关闭时电子传递再生膜电位的速率决定了mPTP保持开放还是关闭。膜间隙Cyt c量和内膜通透性是控制电子传递再生膜电位速率的决定性因素，所以Cyt c失和内膜通透性增加通过抑制膜电位恢复速率，使暂时关闭的mPTP再开放的概率增加。而且Cyt c耗加重氧化应激，促进mPTP开放。由于MPT造成的线粒体不可逆的去极化最终通过破坏能量产生而导致细胞坏死，心脏要从缺血再灌注损伤中恢复，首先必须要恢复粒体功能。因此，本研究的目的是探讨抑制MPT对心肌细胞缺氧／复氧损伤的影响。实验材料1、实验动物及主要试剂出生1-2d的新生Sprague-Dawley（SD）纯种大白鼠，雌雄不拘，由徐州医学院实验动物中心提供。环孢菌素A（CsA）购自Fluka BioChemika公司。鼠单克隆抗Cyt c抗体购自Santa Cruz公司，辣根过氧化物酶标记标记的的抗小鼠IgG购自Cell signaling公司。Calcein-AM购自美国Sigma公司，线粒体／胞浆蛋白分离试剂盒C1260购自Applygen公司，MHCα／β抗体和Tn I抗体购自福州迈新公司，FITC标记的山羊抗小鼠IgG和罗丹明标记的山羊抗小鼠IgG抗体购自中山生物科技公司，Annexin V+FITC凋亡检测试剂盒购自凯基生物制品有限公司。2、主要仪器BD FACSCalibur流式细胞仪（美国），Leica Tcs sp2激光共聚焦显微镜（德国），UV22101紫外分光光度计（Shimadazu公司，日本）；垂直电泳仪（美国Bio-Band公司）；转移槽（美国Bio-Band公司）；低温离心机（日本HITACHI）；Image MasterVDS（美国Pharmacie Biotech）。实验方法1、心肌细胞培养取新生大鼠心室肌，用胰酶和胶原酶消化，将细胞重悬于含10％新生牛血清的DMEM培养液。为纯化心肌细胞，采用差速贴壁分离法去除贴壁的非心肌细胞。将细胞稀释成适当浓度的细胞悬液，按5.0×105个／ml密度接种于用1％明胶铺底的90 mm培养皿。在开始的24～36 h加入100 mM溴脱氧尿苷抑制非心肌细胞增殖。2、实验分组将原代培养心肌细胞按孔随机分为五组。Con组：正常对照组，不予缺氧／复氧，而与其他组同步换液，所换培养基为无血清高糖DMEM；A／R组：缺氧复氧组，心肌细胞缺氧3 h，复氧2 h；CsA1组：心肌细胞缺氧前10 min给予1μMCsA（MPT抑制剂）；CsA2组：心肌细胞复氧前10 min给予1μM CsA)；CsA3组：心肌复氧后10 min给予1μM CsA。3、观察指标复氧2h后，采用流式细胞术检测心肌细胞凋亡情况、Western blotting测定心肌细胞线粒体色素C释放。复氧50 min后，采用cold-loading／warm incubation方法染色线粒体，激光共聚焦显微镜成像，观察mPTP的开放情况。4、统计学处理所有数据采用均数±标准差（（?）±s）表示，应用SPSS 12.0统计软件处理，多组间比较用单因素方差分析（ANOVA），多样本均数间两两比较用最小显著差法（LSD），以P<0.05为有统计学差异。实验结果CsA1、CsA2组的细胞凋亡指数明显低于A／R组（P<0.05）。CsA3组细胞凋亡指数与A／R组无明显差异。CsA1、CsA2组胞浆Cyt c的含量明显低于A／R组（P<0.05），CsA3组与A／R组无明显差异。CsA1、CsA2组线粒体Cyt c的含量明显高于A／R组（P<0.05），CsA3组与A／R组无明显差异。CsA1、CsA2组Calcein保留在线粒体内，胞浆荧光强度较弱，提示MPT未开放。A／R、CsA3组Calcein从线粒体释放到胞浆，胞浆荧光强度较强，提示MPT开放。结论1、缺氧前和复氧前给予CsA可减少心肌细胞凋亡，保持Cyt c含量；2、缺氧前和复氧前抑制MPT可以减轻心肌细胞缺氧／复氧损伤；3、复氧后给予CsA不能抑制MPT，MPT呈不可逆状态。外周苯二氮（艹卓）受体配体参与调控大鼠心肌线粒体通透性转换的研究目的外周苯二氮（艹卓）受体（peripheral benzodiazepine receptor，PBR）最初是发现于中枢神经系统之外，之后发现大鼠的心血管系统中富集PBR，其结构和功能完全不同于中枢苯二氮（艹卓）受体（CBR），CBR和GABA受体耦联发挥镇静、抗焦虑和抗惊厥的作用。PBR是分子量为18 kDa的疏水蛋白，有5个跨膜区，含169个氨基酸的蛋白，主要位于线粒体外膜。众所周知线粒体是电子传递和细胞内生成ATP生成的位点。PBR在线粒体内高表达，最初关于PBR的功能的研究主要围绕其对线粒体呼吸的作用。PBR配体PK11195、R05-4864和其他相关的改变线粒体呼吸的复合物与其和PBR的亲合力有潜在的关系。PBR可调控线粒体膨胀，这和琥珀酸细胞膜色素C氧化还原酶活性有关。PBR参与了体内大量生理过程，如类固醇产生、线粒体呼吸、离子通道活性、免疫调节、卟啉转运、血红素合成、凋亡及细胞的增殖、应激反应等。最近研究认为PBR能通过调控VDAC／ANT的动力学进而调控mPTP，也可能是通过调控VDAC或ANT和其他调节因素如Bcl-2蛋白家族的相互作用起到调控mPTP作用。依据受体动力学特征，PK11195被定义为PBR拮抗剂，Ro5-4864为激动剂。本研究的目的是利用PBR特异性激动剂和拮抗剂探讨PBR在调控mPTP中的作用和机制。第一部分外周苯二氮（艹卓）受体拮抗剂PK11195对大鼠心肌线粒体通透性转换的影响实验材料1、实验动物及主要试剂Sprague-Dawley大鼠，200～250 g，雌雄不拘，由徐州医学院动物中心提供。1-（2-Chlorophenyl-N-methyl-1-methylpropyl）-3-isoquinolinecarboxamide（PK11195）和枯草蛋白酶购自Sigma公司，环孢菌素A（CsA）购自Fluka BioChemika公司。鼠单克隆抗Cyt c抗体购自Santa Cruz公司，辣根过氧化物酶标记标记的的抗小鼠IgG购自Cell signaling公司。JC-1荧光探针购自Molecular Probes公司。2、主要仪器UV22101紫外分光光度计（Shimadazu公司，日本）；垂直电泳仪（美国Bio-Band公司）；转移槽（美国Bio-Band公司）；低温离心机（日本HITACHI）；ImageMaster VDS（美国Pharmacie Biotech）；H-600电子显微镜（日本）。实验方法1、大鼠心肌线粒体分离制备大鼠断头处死，立即取心脏，剪取心室肌洗净血迹后置预冷的匀浆介质中，用电动匀浆器匀浆，制成20 ml／g组织的匀浆液。在冰浴中孵育5 min后。采用差速离心法分离线粒体，制成线粒体悬液（蛋白浓度约5 mg／ml）。以上所有操作均于4℃条件下进行。电镜观察证实线粒体结构完整，Bradford法进行线粒体蛋白定量，以BSA作标准。2、实验分组将线粒体分别和50，100，200μmol／L的PBR拮抗剂PK11195孵育（50，100，200μM组），部分线粒体和100μmol／L PK11195孵育之前5 min加入0.2μmol／LMPT抑制剂CsA（PK+CsA组），不给任何处理的线粒体为阴性对照（Con组），单纯给150μmol／L Ca²⁺作阳性对照(Ca²⁺组)。3、分光光度法观察MPTMPT的变化以线粒体膨胀产生的吸光度的下降来反应。取线粒体用线粒体膨胀测定液（300 mmol／L sucrose，120 mmol／L KCl，5 mmol／L KH₂PO₄ and 10 mmol／LMOPS-Tris，pH 7.4）稀释至最终蛋白质浓度为0.5 mg／ml。通过UV22101紫外分光光度计（Shimadazu公司，日本）检测线粒体的光密度在520 nm处的变化测定MPT变化，持续观察10 min。4、电镜观察线粒体形态各组线粒体处理完毕后，离心收集线粒体沉淀，放入3％戊二醛磷酸缓冲液中固定，1％锇酸后固定，用H-600电子显微镜对标本观察并在8 000倍下进行随机拍照。5、Western blot测线粒体Cyt c的释放将各处理组线粒体悬液孵育20 min后，立即加入1 mmol／L EDTA，将线粒体悬浮液置于预冷的离心管中，于4℃，12 000 g离心10 min，分别收集上清液和线粒体沉淀行Western blot，用辣根过氧化物酶标记显色试剂盒显色条带，图像分析仪进行半定量分析。6、线粒体膜电位测量利用JC-1线粒体膜电位试剂盒检测线粒体膜电位变化。当线粒体膜电位水平低时，JC-1主要以单体形式存在，呈绿色荧光；当线粒体膜电位水平较高时，形成聚合物，发出红色的荧光。激光共聚集显微镜下观察，计算平均荧光密度，以红色荧光的光密度值／绿色荧光的光密度值表示线粒体膜电位的高低，比值下降表示线粒体膜电位的下降。7、统计学处理所有数据采用均数±标准差（（?）±s）表示，应用SPSS 12.0统计软件处理，多组间比较用单因素方差分析，多样本均数间两两比较用SNK（Student-Newman-Keuls）检验。以P<0.05为有统计学差异。实验结果1、PK11195对MPT的影响50，100，200μmol／L的PK11195剂量依赖性诱发线粒体在520 nm处的吸光度下降（P<0.05，P<0.01 vs Con）。部分线粒体在给予100μmol／L PK11195之前加入MPT抑制剂CsA则阻断了PK11195诱发的线粒体在520 nm处的吸光度下降。2、PK11195对线粒体超微结构的影响Con组心肌线粒体为均匀的电子浓缩基质，脊排列整齐。Ca²⁺和PK11195处理过的线粒体损伤严重，表现出明显的空泡变性，线粒体肿胀，线粒体脊膜破裂。给PK11195之前给予CsA则可明显减轻线粒体损伤程度，大部分线粒体结构基本完整。3、PK11195对线粒体Cyt c释放的影响PK11195诱发线粒体Cyt c释放，与Con组相比，PK11195处理的线粒体胞浆（上清）Cyt c含量明显增多（P<0.01），同时伴随线粒体里的Cyt c含量显著减少（P<0.01）；但CsA阻断了PK11195的此种效应，与100μM组相比，PK+CsA组胞浆Cyt c含量明显减少（P<0.05），线粒体里Cyt c含量显著增多（P<0.05）。4、PK11195对线粒体膜电位的影响PK11195显著诱发线粒体膜电位下降，100μM组线粒体膜电位（0.34±0.09）明显低于Con组（0.94±0.23）（P<0.01）。在给予PK11195之前加入CsA则阻断了PK11195的上述作用，PK+CsA组线粒体膜电位（0.62±0.13）显著高于100μM组（P<0.05）。结论1、PBR拮抗剂PK11195在没有外源性钙离子存在的情况下也可诱发MPT；2、PBR拮抗剂PK11195诱导MPT呈剂量依赖性；3、PK11195通过诱导MPT导致线粒体超微结构损伤和线粒体Cvt c释放、膜电位下降；4、通过调控PBR可调控病理生理状态下的MPT。第二部分外周苯二氮（艹卓）受体激动剂Ro5-4864对大鼠心肌线粒体通透性转换的影响实验材料1、实验动物及主要试剂7-chloro-5-（4-chlorophenyl）-1，3-dihydro-1-metllyl-2H-1，4-benzodiazepin-2-one（Ro5-4864），苍术苷（Atractyloside，ATR）购自Sigma公司。实验动物和其他试剂同第一部分。2、主要仪器：同第一部分。实验方法1、大鼠心肌线粒体分离制备同第一部分。2、实验分组将不同浓度（50，100，200μmol／L）的外周苯二氮（艹卓）受体激动剂Ro5-4864和线粒体在室温孵育5 min（50，100，200μM组），加入150μmol／L Ca²⁺诱发MPT。不给任何处理的线粒体为阴性对照(Con组，单纯给150μmol／L Ca²⁺作阳性对照(Ca²⁺组)。另取部分线粒体和100μmol／L Ro5-4864孵育，之前5 min加入20μmol／LATR（ATR+Ro组），同样以Ca²⁺诱发MPT。3、分光光度法观察MPT同第一部分。4、Western Blot测线粒体Cyt c的释放同第一部分。5、线粒体膜电位测量同第一部分。6、统计学处理同第一部分。实验结果1、Ro5-4864对MPT的影响50，100，200μmol／L的Ro5-4864均显著抑制Ca²⁺诱发的线粒体在520 nm处的吸光度下降（P<0.05，P<0.01）。100，200μM组与50μM组比较有显著差异（P<0.05），但100μM与200μM组间比较无显著差异。100μM组与Ro+ATR组间有显著性差异（P<0.01）。2、Ro5-4864对线粒体起微结构的影响Con组心肌线粒体为均匀的电子浓缩基质，脊排列整齐。Ca²⁺组线粒体损伤严重，表现出明显的空泡变性，线粒体肿胀，线粒体脊膜破裂。Ro5-4864可明显减轻线粒体损伤程度，大部分线粒体结构基本完整。但ATR阻断了Ro5-4864的此种效应。3、Ro5-4864对Ca²⁺诱发的线粒体Cyt c释放的影响Ro5-4864显著抑制Ca²⁺诱发的线粒体Cyt c释放。与Ca²⁺组相比，100μM组胞浆（上清）Cyt c含量明显减少（P<0.01），同时伴随线粒体里的Cyt c含量显著增加（P<0.01）；与100μM组相比，ATR+Ro组胞浆Cyt c含量明显增多（P<0.05），线粒体里cyt c含量显著减少（P<0.05）；与Con组相比，Ca²⁺组胞浆Cyt c明显增多（P<0.01），线粒体里Cyt c含量显著减少（P<0.01）。4、R05-4864对线粒体膜电位的影响R05-4864可显著抑制Ca²⁺引起的线粒体膜电位下降，100μM组线粒体膜电位（0.65±0.14）显著高于Ca²⁺组（0.25±0.05）（P<0.01）。在给予100μmol／LRo5-4864之前加入20μmol／L ATR则阻断了Ro5-4864的上述作用，ATR+Ro组线粒体膜电位（0.35±0.11）显著低于100μM组（P<0.05）。结论1、PBR激动剂Ro5-4864可抑制MPT，但未见剂量依赖性；2、PBR激动剂Ro5-4864可减轻Ca²⁺诱导的线粒体损伤；3、Ro5-4864通过抑制MPT减轻线粒体超微结构损伤、Cyt c释放和膜电位下降；4、PBR参与调控MPT，为心肌线粒体功能保护提供新的治疗靶点。更多还原

【Abstract】 Inhibition of Mitochondrial Permeability Transition Protects Cardiomyocytes Against Anoxia-Reoxygenation InjuryObjectiveMitochondria disfunction play a key role in determining cardiomyocytes fate during exposure to stress. The states of mitochondrial permeability transition pores （mPTP） is greatly associated with the mitochondria disfunction. Mitochondrial permeability transition （MPT） caused by the opening of mPTP is implicated as an important event in the control of cell death and survival and may be involved in both apoptosis and necrosis. The mPTP is considered to include the voltage-dependent anion channel （VDAC, located in mitochondrial outer-membrane）, the adenine nucleotide translocator （ANT, across the mitochondrial outer and inner membrane at mitochondrial contact site）, and the cydophilin D （CyP-D） in the matrix. Recent studies showed that peripheral benzodiazepine receptor, hexokinasc, and creatine kinase, may also be involved in the mPTP. MPT may act as a "central executioner" of cells subjected to a range of insults （such as oxidative stress, growth factor removal, or exposures to cytokines）. MPT causes dissipation of the mitochondrial membrane potential, uncoupling of oxidative phosphorylation, ATP depletion, and equilibration of small solutes and ions between the cytosol and the mitochondrial matrix. Solute influx into the matrix can cause swelling followed by rupture of the outer membrane and consequent efflux of proteins from the intermembrane space, including Cyt c, procaspase 9, apoptosis-inducing factor, and endonuclease G. The regulation of the MPT plays a key role in maintaining the impermeability of the mitochondrial inner membrane to all but a few selected metabolites, thus helping to maintain the membrane potential, which drives ATP synthesis during oxidative phosphorylation.Mounting evidence indicates that MPT may play an important a role in cardiac reperfusion injury. As ischemic time increases, latent susceptibility of mitochondria to MPT increases （the MPT priming component）, yet the conditions of reperfusion can modulate whether or not MPT is induced （the MPT trigger component）. During the ischemic period, the elevating of Ca²⁺, decreasing of pH, accumulation of long-chain fatty acids and ROS cause MPT. The MPT trigger component, is influenced by the interplay between the MPT inducers/inhibitors present during reperfusion (particularly matrix-free Ca²⁺and ROS levels) and electron transport capacity for regenerating membrane potential. The latter is highly sensitive to the extent of Cyt c loss and IM leakiness occurring during the preceding ischemia. Because PTP open probability is voltage dependent, the rapidity with which electron transport regenerates membrane potentialwhen the PTP transiently closes will play a critical role in determining whether it remains closed or reopens. Intermembrane Cyt c content and IM leakiness are both major determinants of controlling the rate at which electron transport can regenerate and maintain membrane potential, so that Cyt c loss and IM leak, by depressing the membrane potential recovery rate, will increase the probability that a transiently closed mPTP will reopen. In addition, Cyt c depletion increases oxidant stress, promoting mPTP opening, Irreversible depolarization of mitochondria by MPT ultimately induces necrotic cell death by impairing energy production. For the heart to recover from ischemia/reperfusion, its mitochondria must return to full functionality. Our study is to investigate the effect of of inhibition of mitochondrial permeability transition on cardiomyocytes anoxia- reoxygenation injury.Materials and Methods1. Reagents and animal1-to 2-day-old Sprague-Dawley（SD） rats were supplied by the Animal Center of Xuzhou Medical College, China.Cyclosporin A was obtained from Fluka Biochemical company. Mouse monoclonal anti-Cyt c antibody was purchased from Santa Cruz Biotechnology. Anti-mouse IgG and Horseradish peroxidase（HRP）-linked antibody were purchased from Cell signaling Technology. Mitochondria Isolation Kit was purchased from Applygen company, mMHCα/βand TnI were purchased from Fuzhou steps newborn thing technical development company. FITC-Labeled Goat Anti-Mouse IgG and Rhodamine- Labeled Goat Anti-Mouse IgG were purchased from Santa Cruz Biotech. Annexin V-FITC Apoptosis Detection Kit were purchased from Nanjing KeyGen Biotech.2. Primary myocyte-rich cultures of the neonatal rat myocytesVentricles from hearts of 1- to 2-day-old rats were dissociated with trypsin and collagenase. The cells were resuspended in Dulbecco’s modified Eagle’s medium（DMEM） supplemented with 10% fetal calf serum. To selectively enrich the myocytes, dissociated cells were preplated to allow nonmyocytes to attach to the bottom of the culture dish. The resultant suspension of myocytes was transferred onto collagen-coated 90-mm culture dishes. Bromodeoxyuridine （100 mM） was added during the first 24～36 h to prevent proliferation of nonmyocytes3. Experimental protocolsMyocytes were divided into the following five groups: Control （con） group. The myocytes were incubated in Krebs-Ringer-HEPES （KRH） solution with glucose during the entire experimental period. Anoxia- reoxygenation（A/R）group, Myocytes subjected 3 hours anoxia and 2 hours reoxygenation. Cyclosporin A1 （CsA1） group: The myocytes were preincubated with cyclosporin A （1μmo1/1）, an inhibitor of mitochondrial permeability transition, at 10 min before anoxia. Cyclosporin A2 （CsA2） group: The myocytes were incubated with cyclosporin A at 10 min before reoxygenation. Cyclosporin A3 （CsA3） group: The myocytes were incubated with cyclosporin A at 10 min after anoxia.4. Apoptosis was detected by flow cytometry Cyt c release from mitochondria into the cytosol was measured by Western blotting analysis. Calcein fluorescence（reflecting the opening of mPTP） was determined by using confocal microscopy.5. Statistical analysisStatistical analysis was performed by one-way analysis of variance, Least significant difference（LSD） was applied to test for the differences between individual groups. P＜0.05 was considered statistically significant.ResultsApoptosis index in CsA1、CsA2 group was lower than that in A/R group （P＜0.05）. There were no significant diffierence between CsA3 and A/R group. The content of Cyt c in cytosol in CsA1 and CsA2 groupwas lower than that in A/R group （P＜0.05）. The mitochondrial content of Cyt c in CsA1 and CsA2 group was higher than that in group A/R group （P＜0.05）. There were no significant diffierence between CsA3 and A/R group. Green calcein fluorescence was maintained inside mitochondria in CsA1 and CsA2 group, while calcein release to the cytosol in A/R and CsA3 group.Conclusions1. Treatment of CsA before anoxia and reoxgenation can reduce cardiomyocytes apoptosis, maintained the content ofmitochondria Cyt c.2. Inhibition of MPT before anoxia and reoxgenation can mitigate cardiomyocytes anoxia-reoxygenation injury.3. Treatment of CsA after reoxygenation can not inhibit MPT, which is in irreversible state. The Involvement of Peripheral Benzodiazepine Ligallds in the Regulation of Rat Cardiac Mitochondria Permcability TransitionObjectiveThe peripheral benzodiazepine receptors （PBR） was discovered as benzodiazepine binding sites outside the CNS. Thereafter, PBR were found to be abundant in the cardiovascular system of rats. The structure and function of PBR is different from the central benzodiazepine receptor （CBR）, which is coupled to GABA receptors and responsible to the classical sedative, anxiolytic and anticonvulsant effect. PBR is an evolutionarily conserved 18-kDa protein, which is highly hydrophobic, possesses five transmembrane domains a 169-amino acid protein associated with the the outer mitochondrial membrane within the cell. The mitochondria are well known as sites of electron transport and generators of cellular ATP. Because of their high level of expression in the mitochondria, initial studies of PBR function revolved around its effect on mitochondrial respiration. PBR ligands PK11195, Ro5-4864 and related compounds alter mitochondrial respiration with potencies correlated with their affinities for the PBR. Subsequent studies indicated that the PBR can regulate mitochondrial swelling, and are involved in succinate-Cyt c oxidoreductase （siteⅡ） activity. PBR has been suggested to be involved in numerous physiological processes, such as steroid production, mitochondrial respiration, and ion channel activities,immunomodulation, porphyrin transport, heme biosynthesis, apoptosis and cell proliferation and in the regulation of responses to stress. Recently, PBR is reported to regulate the opening of the MPTP by directly modulating VDAC/ANT dynamics. Alternatively, PBR may modulate VDAC or ANT interaction with other regulators such as the Bcl-2 family proteins. Based on the entropy-driven and enthalpy-driven nature of ligand receptor interactions, Isoquinoline carboxamide PK11195 has been classified as an antagonist, the benzodiazepine Ro5-4864 （7-chloro-5- （4-chlorophenyl）-1, 3-dihydro-1-methyl-2H-1, 4-benzodiazepin-2-one） an agonist with specificity for the PBR. In the current study, Based on these considerations, we decided to investigate the role of PK11195 and Ro5-4864 in the regulation of cardiac MPT. Part OneThe Effect of Peripheral Benzodiazepine Receptor Antagonist PK11195 on the Regulation of Rat Cardiac Mitochondria Permeability TransitionMaterials and Methods1. Reagents and animal1- （2-Chlorophenyl-N-methyl-1-methylpropyl）-3-isoquinolinecarboxamide （PK11195） and Subtilisin Carlsberg were purchased from the Sigma company. Cyclosporin A was obtained from Fluka Biochemical company. Mouse monoclonal anti-Cyt c antibody was purchased from Santa Cruz Biotechnology. Anti-mouse IgG and Horseradish peroxidase （HRP） - linked antibody were purchased from Cell signaling Technology. 5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’- tetraethylbenzimidazolcarbocyanine iodide （JC-1） was purchased from Molecular Probes. Male Sprague-Dawley（SD） rats weighing 200～250g were supplied by the Animal Center of Xuzhou Medical College.2. Preparation of rat heart mitochondriaRat’s hearts were removed, homogenized in ice-cold buffer and incubated on ice for 5 min. mitochondria were separated and purified by differential centrifugation. Mitochondrial pellets were resuspended in buffer and kept in ice for the rest of experiement. All mitochondrial isolation procedures were carried out at 4℃. Protein concentration was determined by modified Bradford’s method, using BSA as the standard.3. Experimental protocolsDifferent concentration of PK11195 （50, 100, 200μM） was added into incubation buffer, respectively. In additional experiments, 0.2μM cyclosporin A, an inhibitor of MPT was added 5 minutes before the addition of 100M PK11195. Calcium （150μM） induced MPT opening as positive control group.4. Determination of mitochondrial permeability transitionThe MPT was detected by the change of absorbance after chemical addition with spectrophotometer. MPT causes mitochondrial swelling, and a decrease in absorbance at 520 nm （Abs520 nm）. Fresh heart mitochondria were added to a buffer containing 300 mM sucrose, 5 mM succinate and 10 mM MOPS, pH 7.4 with Tris, and the final volume is 1.0 ml and protein concentration is 1 mg/ml. The reference cuvette has the same buffer without mitochondria. The change of absorbance was measured for 10 min with spectrophotometer at 520 nm.5. Electron microscopy microscope analysisThe mitochondria were fixed with 2.5% glutaraldehyde and were postfixed in 1% cacodylate-buffered osmium tetroxide, and observed with H-600 transmission electron microscope at a magnification of x8 000.6. Mitochondrial Cyt c releaseThe mitochondrial pellet and supernatant was separated by centrifugation. Cyt c content in the pellet and supernatant was determined using Western Blotting. The bands were detected by NBT/BCIP, Laser scanning densitometry was used for the semi-quantitative determination of the proteins.7. Mitochondrial membrane potentialThe changes in mitochondrial membrane potential were monitored with the dye. The intensity of fluorescence was determined using a laser scanning confocal microscope. JC-1 monomer emits green fluorescence, JC-1 aggregate emits red fluorescence. The ratio of red to green fluorescence for each region was calculated. The decreased ratio was interpreted as a decrease in mitochondrial membrane potential.8. Statistical analysis Statistical analysis was performed by one-way analysis of variance, Newman-Keuls was applied to test for the differences between individual groups. P＜0.05 was considered statistically significant.Results1. Effects of PK11195 on mitochondrial permeability transitionTreatment mitochondria with 50, 100, or 200μM PK11195 led to a significant decrease in absorbance at 520 nm compared to non-chemical treated mitochondria （control group）. Cyclosporin A （0.2μM） prevented MPT induced by PK11195 （100μM）.2. Effects of PK11195 on ultrastructural observation of mitochondriaThe mitochondrial samples were observed with a transmission electron microscope. Control group showed well-preserved mitochondria with an electron-dense matrix and well-arranged cristae. In the specimens treated with Ca²⁺ or 100μM PK11195, most of mitochondria showed morphological change. In particular, they showed swelling, hypertrophy, cristolysis, and matrix dilution. In the presence of 100μM PK11195 plus cyclosporin A, the most of mitochondria displayed the characteristic ultrastructure of the intact organelle.3. Effects of PK11195 on Cyt c release from mitochondrialPK11195 resulted in the translocation of Cyt c from the mitochondria to the cytosol. Compared to untreated mitochondria, PK11195 （100μM） treatment markedly decreased Cyt c content in mitochondrial pellets （P＜0.01）, whereas the content of Cyt c in cytosol was significantly increased （P＜0.01）. Cyclosporin A treatment prevented Cyt c loss from mitochondria induced by PK11195 （P＜0.05）.4. Effects of PK11195 on mitochondrial membrane potentialThe exposure of mitochondria to PK11195 caused dissipation of mitoc ondrial membrane potential significantly. Mitochondrial membrane potential in 100μM PK11195 treated mitochondria is 0.34±0.09, much lower than that in untreated group （0.94±0.23） （P＜0.01）. The mitochondrial membrane potential was maintained in PK 11195 with cyclosporin A （0.62±0.13） and higher than that in 100μM group.Conclusions1. PBR antagonist PK11195 induced MPT without any calcium addition.2. PBR antagonist PK11195 induce mitochondrial permeability transition in a concentration- dependent manner.3. PBR antagonist PK11195caused mitochondria ultrastructural abnormalities and Cyt c release via induction of MPT.4. Modulation of peripheral benzodiazepine receptor may represent an novel way to regulate MPT during pathophysiological condition. Part TwoThe Effect of Peripheral Benzodiazepine Receptor Agonist Ro5-4864 on the Regulation of Rat Cardiac Mitochondria Permeability TransitionMaterials and Methods1. Reagents and animal7-chloro-5-（4-chlorophenyl）-1, 3-dihydro-1-methy1-2H-1,4-benzodiazepin-2-one （Ro5-4864） and Atractyloside（ATR） were obtained from the Sigma company. Animal and other reagents are the same as that in part one.2. Preparation of rat heart mitochondriaThe same as that in part one.3. Experimental protocolsMitochondria were incubated with 50, 100, 200μmol/L Ro5-4864 （50, 100, 200μM group）. In additional experiments, ATR（20μmol/L）, an opener of MPT was added 5 minutes before the addition of 100μmol/L Ro5-4864 （ATR group）. Swelling was initiated by addition of 150μmol/L Ca²⁺ as positive control group (Ca²⁺ group), negative control group （Con group） was given none treatment.4. Determination of mitochondrial permeability transitionThe same as that in part one.5. Electron microscopy microscope analysisThe same as that in part one.6. Mitochondrial Cyt c releaseThe same as that in part one. 7. Mitochondrial Membrane PotentialThe same as that in part one.8. Statistical AnalysisThe same as that in part one.Results1. Cardiac mitochondrial swellingThe exposure of mitochondria to 50, 100, or 200μmol/L Ro5-4864 significantly inhibited the decrease of absorbance at 520 nm compared to that in Ca²⁺ group （P＜0.01, P＜0.05）. Compared with 50μM group, The ratio of decreasing of absorbance at 520 nm in 100μM and 200μM group were higher（P＜0.05）, but there were no significantly difference between 100μM and 200μM group（P＞0.05）. ATR, an opener of mPTP, abolished the effect of 100μmol/L Ro5-4864 against mitochondria swelling, there was significantly difference between 100μM group and ATR+Ro group（P＜0.01）.2. Ultrastructural observation of PBR antagonists Ro5-4864 exposed mitochondriaControl specimens showed well-preserved mitochondria with an electron-dense matrix and well-arranged cristae. In the specimens treated with Ca²⁺ the majority of mitochondria were grossly damaged. In particular, they showed swelling, hypertrophy, cristolysis, and matrix dilution. In the presence of 100μM Ro5-4864, the most of mitochondria displayed the characteristic ultrastructure of the intact organelle. The mitochondrial ultrastructure abnormality in ATR+Ro group is similar to Ca²⁺ group.3. Mitochondrial Cyt c releaseCa²⁺ resulted in the translocation of Cyt c from the mitochondrial pellet to the cytosol. The release of mitochondrial Cyt c was significantly inhibited by Ro5-4864, while the effect of Ro5-4864 was abolished by ATR, an opener of mPTP. Compared with Ca²⁺ group, the Cyt c in cytosol in 100μM group was lower（P＜0.05）, while the Cyt c in mitochondria was higher（P＜0.05）. In ATR+Ro group, the Cyt c in cytosol was higher（P＜0.05）, the Cyt c in mitochondria was lower（P＜0.05）, compared with 100μM group.4. Effects of PBR agonists Ro5-4864 on mitochondrial membrane potentialThe exposure of mitochondria to Ca²⁺ caused dissipation of mitochondrial membrane potential, while this effect was prevented by Ro5-4864. ATR（an opener of mPTP） abolished the effect of Ro5-4864 against dissipation of mitochondrial membrane potential. Mitochondrial membrane potential in 100μM Ro5-4864 group （0.62±0.13） is higher than that in Ca²⁺ group（0.25±0.07） and ATR +Ro group （0.31±0.09） （P＜0.05）.Conclusions1. PBR agonists Ro5-4864 inhibit MPT.2. Ro5-4864 resultes in a significant protection against mitochondria injury induced by Ca²⁺.3. Ro5-4864 prevent the release of mitochondria Cyt c and dissipation of miotochondria potential in part via induction of MPT.4. PBR involves in the regulation of MPT, represent a novel therapeutic target against cardiac mitochondrial damage.更多还原