线粒体通透件转换孔在缺血/再灌注损伤中的作用

背景 线粒体作为“能量工厂”提供细胞生长及代谢所需的三磷酸腺苷（adenosine triphosphate,ATP）,同时也是细胞存活与否的重要信号管理者.线粒体通透性转换孔（mitochondrial permeability transition pore,mPTP）是横跨线粒体内外膜之间的允许相对分子质量1.5 kD以下的分子自由通过的孔道.在脑缺血/再灌注（ischemia/reperfusion,I/R）损伤中,线粒体是研究的焦点,而mPTP作为线粒体的门户更是影响着线粒体膜电位（mitochondrial membrane potential,△ψm）、线粒体Ca2＋超载及促细胞死亡物质释放等一系列过程. 目的 对mPTP在I/R损伤中可能的作用机制及治疗方法进行综述. 内容 整理和阐述了mPTP的结构、mPTP与I/R损伤的机制和预防与治疗I/R损伤的可能方法. 趋向 随着mPTP在I/R损伤中的作用机制不断被揭示,其将成为治疗I/R损伤的重要靶点.     

甲基强的松龙对脊髓损伤后伤段脊髓线粒体功能的影响

目的：探讨脊髓损伤后伤段脊髓线粒体呼吸功能和线粒体内游离钙的变化和早期使用甲基强的松龙（MP）对其的影响。方法：54只SD大鼠，随机分组为假手术组（对照组）、脊髓损伤组（SCI组，采用Allen’s打击法造成大鼠脊髓损伤模型）和脊髓损伤后应用MP治疗组（MP组），每组又分为处理后6h、12h、24h三个时间相，每个时间相6只。在各时间相处死动物后提取伤段脊髓线粒体，测定线粒体呼吸Ⅲ态（R3）、呼吸Ⅳ态（R4）、呼吸控制率（RCR）、磷氧比（P／0）和线粒体内游离Ca^2＋浓度。结果：SCI组在伤后6h、12h和24hR3、RCR和P／0显著低于对照组，R4和线粒体内游离Ca^2＋浓度显著高于对照组。差异有显著性（P〈0．01）；伤后6h和12h MP组R3、RCR和P／0高于SCI组，R4和线粒体内游离Ca^2＋浓度低于SCI组，差异有显著性（P〈0．01）；MP组R3、R4和RCR在6h和12h时与对照组之间无显著性差异，24h时R3、RCR和P／0低于正常对照组，有显著性差异（P〈0．05）。结论：脊髓损伤后伤段脊髓线粒体呼吸功能和线粒体内游离Ca^2＋浓度明显受到影响，线粒体内膜通透性增加，线粒体氧化磷酸化的偶联程度明显受到抑制。早期使用甲基强的松龙可明显改善线粒体的呼吸功能，抑制Ca^2＋内流，保护伤段脊髓线粒体的稳定性。     

线粒体呼吸链在高糖腹膜透析液诱导人腹膜间皮细胞层高通透性中的作用

目的 研究线粒体呼吸链在高糖腹膜透析液（PDS）诱导人腹膜间皮细胞（HPMC）通透性升高中的作用,探讨高糖PDS导致腹膜高通透状态的分子机制。 方法 体外培养HPMC,用不同浓度葡萄糖PDS加或不加抗氧化剂谷胱甘肽,培养24 h。采用跨细胞电阻（TER）技术测定HPMC通透性的变化;免疫荧光及Western印迹观察claudin-1的表达;线粒体活性氧（ROS）荧光探针检测细胞线粒体ROS的生成;线粒体呼吸链酶复合物活性检测试剂盒检测复合物Ⅰ~Ⅳ的活性变化。 结果 各组细胞的TER值均随时间的延长而下降,4.25% PDS组TER值24 h内从（34.7±1.5） Ω&#8226;cm2下降至（4.3±1.4） Ω&#8226;cm2;高糖PDS使细胞间连接蛋白claudin-1的表达显著下调,葡萄糖浓度越高作用越明显;同时高糖PDS导致线粒体呼吸链酶复合物Ⅲ活性较对照组下降89.2%（P < 0.01）,进而使线粒体ROS产生增加。谷胱甘肽可逆转上述指标的变化。 结论 高浓度葡萄糖PDS通过抑制线粒体呼吸链酶复合物Ⅲ的活性,引起HPMC的氧化损伤,进而导致腹膜间皮细胞层的高通透性增高。     

异丙酚对肝缺血再灌注损伤犬肝细胞线粒体的作用

目的探讨异丙酚对肝缺血再灌注损伤犬肝细胞线粒体的作用及其机制。方法20只杂种犬随机分为4组（n=5）：假手术组（S组）、缺血再灌注组（I／R组）、小剂量异丙酚组（P1组）及大剂量异丙酚组（P2组）。以10ml／min速率静脉输注6％羟乙基淀粉150～200ml，同时从股动脉缓慢放血100～150ml，使平均动脉压（MAP）不低于85mmHg。S、I／R组静脉注射3％戊巴比妥钠30mg／kg、维库溴铵0．3mg／kg麻醉诱导，P1、P2组依次静脉注射异丙酚6mg／kg、维库溴铵0．3mg／kg麻醉诱导，气管插管后控制呼吸。S、I／R组间断静脉注射戊巴比妥钠维持麻醉，P1、P2组以血浆靶浓度6、12μg／ml靶控输注异丙酚维持麻醉30min后麻醉维持同S组。缺血期间回输自体血，维持MAP不低于80mmHg。I／R、P1组、P2组气管插管后30min制备肝缺血（缺血30min）再灌注模型。分别于缺血前即刻、缺血30min、再灌注60min抽取静脉血，测定血浆谷丙转氨酶（ALT）、谷草转氨酶（AST）及肝细胞线粒体Na^＋-K^＋-ATP酶活性，并在透射电镜下观察肝细胞线粒体的超微结构。结果肝缺血再灌注可导致血浆ALT、AST活性升高，肝细胞线粒体Na^＋-K^＋-ATP酶活性降低，肝线粒体损伤，异丙酚可减弱肝缺血再灌注导致的上述改变，以大剂量效果较好。结论异丙酚可减轻肝缺血再灌注损伤犬肝细胞线粒体损伤，呈剂量依赖性。     

微管解聚剂在缺氧早期大鼠心肌细胞线粒体损害中的作用

目的了解微管解聚剂在缺氧早期大鼠心肌细胞线粒体损害中的作用。方法常规方法分离培养Wistar乳鼠心肌细胞，分为正常组、微管解聚组（培养液中加入4μmol／L秋水仙碱）、缺氧组、缺氧解聚组（4μmol／L秋水仙碱联合缺氧处理）。分别采用激光共聚焦显微镜检测4组细胞线粒体分布情况，透射电镜观察线粒体形态变化，生物氧耗呼吸仪检测呼吸调节比（RCR），高效液相色谱仪检测腺苷三磷酸（ATP）含量。缺氧组及缺氧解聚组所设时相点为缺氧后10、20、30、60min。结果正常组线粒体呈粒状、排列规则，微管解聚组较之发生轻微改变：缺氧20、30、60min，缺氧解聚组线粒体分布的无规律性及形态结构损害均较缺氧组严重；细胞RCR分别为1．58±0．37、1．51±0．32、1．12±0．11，明显低于缺氧组3．85±0．56、2．98±0．44、1．79±0．73（P〈0．01）；ATP含量分别为（419±83）、（326±73）、（295±58）ng／mg，亦明显低于缺氧组（475±68）、（397±59）、（336±67）ng／mg（P〈0．01）。结论微管解聚剂可加重缺氧引起的心肌细胞线粒体分布紊乱和形态结构损害，以及线粒体呼吸功能和能量代谢障碍，它在线粒体缺氧损害机制中具有重要作用。     

心磷脂与缺血/再灌注损伤

心磷脂（CL）是维持线粒体能量代谢所必需的一种磷脂，参与了众多重要生理过程。此文对CL的结构、合成和转化；在维持线粒体正常功能中的作用；缺血／再灌注（I／R）损伤中的改变以及和线粒体功能的关系等作了逐一阐述，说明CL与I／R损伤导致的细胞死亡关系密切，CL量或性质的改变在细胞凋亡中处于中心地位。     

吸入麻醉药对离体鼠肝线粒体呼吸功能的影响

应用离体鼠肝线粒体在体外研究了七氟醚、安氟醚、异氟醚、氟烷对其呼吸功能的影响。这四种吸入麻醉药在高剂量（〉８０μｌ／２．５ｍｌ）时均可对以琥珀酸为底物的ＡＤＰ刺激的Ⅲ态线粒体呼吸有明显的抑制作用。氟烷的抑制作用最为明显，而且在高于临床剂量时，氟烷明显降低磷氧比（Ｐ／Ｏ）和氧化磷酸化效率（ＯＰＲ），其它三种药物无类似作用。结果提示这四种药物均可抑制线粒体的呼吸功能、均具有潜在的肝毒性，并以氟烷为甚。     

肝脏缺血/再灌注后线粒体损伤与促凋亡蛋白的释放

肝脏缺血／再灌注（I／R）损伤是临床上较为常见的组织器官损伤之一，细胞凋亡在肝脏I／R损伤中起到了重要作用。近年来的研究发现，I／R导致线粒体通透性改变，由此引起线粒体膜电位的降低和促凋亡蛋白的释放等一系列变化，在细胞凋亡中发挥关键作用。     

线粒体通透性转换通道与脑缺血再灌注损伤

线粒体通透怀转换通道是近年来发现的位于线普体膜上的电压门控性通道，线粒体通透 转换通道开放后可引起线粒通生突然增加，导致线粒体 功能障碍。脑缺血再灌注损伤与线粒体通透性转换通道的开放关系密切。     

线粒体通透性转运孔道模型的研究进展

由于很多实验结果不能通过传统的mPTP模型得到圆满的解释,最近人们逐渐接受一些新的假说：线粒体内膜存在两种胛孔道——可调控的胛孔道（regulated PT pores）和不可调控的胛孔道（unregulated PT pores）,兼性蛋白簇集、CYPD及其他未明确的分子伴侣复合体构成可调控的胛孔道,当兼性蛋白簇集大量生成,分子伴侣不足以抑制其开放时,线粒体胛孔道由可调控状态变为不可调控状态。     

Mitochondrial Permeability Transition in Cardiomyocyte Apoptosis during Acute Graft Rejection

Evidence indicates that acute cardiac graft rejection is associated with cardiomyocyte apoptosis. Mitochondrial permeability transition (MPT) induces apoptotic cell death. We sought to determine whether MPT might play a role in cardiomyocyte apoptosis in the rat model of heterotopic cardiac transplantation. Syngenic and allogenic transplantations were performed, and both native and grafted hearts were harvested 3 or 5 d after transplantation for detection of acute rejection, assessment of Ca²⁺-induced MPT, and myocardial apoptosis by TUNEL staining and caspase 3 activity. Allogenic grafts developed severe acute rejection at day 5 with concomitant cardiomyocyte apoptosis (apoptotic index: 7.1 ± 1.0% vs. 1.0 ± 0.2% in syngenic hearts, and caspase 3 activity: 38 ± 25 vs. 5 ± 9 nmol/mg, in allogenic vs. syngenic grafts, respectively). At day 5, Ca²⁺-induced MPT was dramatically altered in allogenic when compared with syngenic grafts (mean Ca²⁺ overload averaged 0 ± 20 vs. 280 ± 30 μM in allogenic and syngenic grafts, respectively). NIM811, a nonimmunosuppressive derivative of cyclosporin A (CsA), that specifically inhibits the MPT pore, did not alter acute rejection, but significantly delayed Ca²⁺-induced MPT pore opening, attenuated caspase 3 activity and cardiomyocyte apoptosis in allogenic grafts. This suggests that mitochondrial permeability transition pore opening may play an important role in cardiomyocyte apoptosis associated with acute cardiac graft rejection.     

Anti-porin antibodies prevent excitotoxic and ischemic damage to brain tissue

The mitochondrial permeability transition (MPT) is a converging event for different molecular routes leading to cellular death after excitotoxic/oxidative stress, and is considered to represent the opening of a pore in the mitochondrial membrane. There is evidence that the outer mitochondrial membrane protein porin is involved in the MPT and apoptosis. We present here a proof-of-principle study to address the hypothesis that anti-porin antibodies can prevent excitotoxic/ischemia-induced cell death. We generated anti-porin antibodies and show that the F(ab)(2) fragments penetrate living cells, reduce Ca(2+)-induced mitochondrial swelling as other MPT blockers do, and decrease neuronal death in dissociated and organotypic brain slice cultures exposed to excitotoxic and ischemic episodes. These observations present direct evidence that anti-porin antibody fragments prevent cell damage in brain tissue, that porin is a crucial protein involved in mitochondrial and cell dysfunction, and that it is conceivable that antibodies can be used as therapeutic agents.     

Effects of polyamine-loaded liposomes on liver mitochondrial permeability transition

The objective of the present study was represented by the effects of polyamineloaded liposomes on hepatic mitochondrial permeability transition (MPT). MPT was appreciated through the swelling of the isolated guinea-pig liver mithocondria induced by Ca2+, at 540 nm, using a diode-array 8452 UV-VIS spectrophotometer and a General Purpose software (Hewlett-Packard). Polyamines encapsulated in liposomes might inhibit the appearance of MPT induced by Ca2+, directly proportional to electrical charge: spermine > spermidine > putrescine > cadaverine. Thus, together with some previous data, it was observed that spermine may modulate hepatic MPT from the exterior, as well as from the interior of mitochondria. Moreover, spermidine and putrescine may also modulate MPT from the interior of mitochondria at low doses, and from the exterior only at high doses. The changes in mitochondrial membranes lipid composition (as done by control liposomes) are slightly influencing the MPT. The increase in membrane rigidity through the use of 40% cholesterol-enriched liposomes is drastically decreasing the appearance and development of MPT.     

Protective effects of NIM811 in transient focal cerebral ischemia suggest involvement of the mitochondrial permeability transition

Cerebral ischemia followed by reperfusion activates numerous pathways that lead to cell death. One such pathway involves the release of large quantities of the excitatory amino acid glutamate into the synapse and activation of N-methyl-D-aspartate receptors. This causes an increase in mitochondrial calcium levels ([Ca(2+)](m)) and a production of reactive oxygen species (ROS), both of which may induce the mitochondrial permeability transition (MPT). As a consequence, there is eventual mitochondrial failure culminating in either apoptotic or necrotic cell death. Thus, agents that inhibit MPT might prove useful as therapeutic interventions in cerebral ischemia. In this study, we have investigated the neuroprotective efficacy of the novel compound NIM811. Similar in structure of its parent compound cyclosporin A, NIM811 is a potent inhibitor of the MPT. Unlike cyclosporin A, however, it is essentially void of immunosuppressive actions, allowing the role of MPT to be clarified in ischemia/reperfusion injury. The results of these studies demonstrate that NIM811 provides almost 40% protection in a model of transient focal cerebral ischemia. This was associated with a nearly 10% reduction in mitochondrial reactive species formation and 34% and 38% reduction of cytochrome c release in core and penumbra, respectively. Treatment with NIM811 also increased calcium retention capacity by approximately 20%. Interestingly, NIM811 failed to improve ischemia-induced impairment of bioenergetics. The neuroprotective effects of NIM811 were not due to drug-induced alterations in cerebral perfusion after ischemia. Activation of MPT appears to be an important process in ischemia/reperfusion injury and may be a therapeutic target.     

The immunophilin ligand FK506 attenuates axonal injury in an impact-acceleration model of traumatic brain injury.

The immunophilin ligand, cyclosporin A (CsA), is effective in reducing the axonal damage associated with traumatic brain injury (TBI). Based upon extensive ultrastructural and immunohistochemical studies, the neuroprotection afforded by CsA appeared to be mediated via mitochondrial protection, specifically, the prevention of mitochondrial swelling and inhibition of mitochondrial permeability transition (MPT). However, the potential that CsA could also be neuroprotective via the immunophilin-mediated inhibition of the protein phosphatase, calcineurin (CN) has not been directly assessed. To address this issue, the current study assessed the ability of FK506, another immunophilin ligand that inhibits CN with no effect on MPT, to attenuate axonal damage in a rat impact-acceleration model of TBI. Traumatic axonal injury (TAI), detected via an antibody against beta-amyloid precursor protein (APP), a specific marker of axonal injury, was significantly reduced at 24 hr postinjury in Sprague-Dawley rats receiving intravenous FK506 (2 mg/kg; n = 5) 30 min prior to injury compared to vehicle controls (n = 3). While not rejecting the established efficacy of CsA in providing neuroprotection via its targeting of MPT, this study does underscore the potential importance of CN in the progressive pathobiology of TAI, suggesting that CN may constitute another important therapeutic target.     

Mitochondria-targeted peptide accelerates ATP recovery and reduces ischemic kidney injury

The burst of reactive oxygen species (ROS) during reperfusion of ischemic tissues can trigger the opening of the mitochondrial permeability transition (MPT) pore, resulting in mitochondrial depolarization, decreased ATP synthesis, and increased ROS production. Rapid recovery of ATP upon reperfusion is essential for survival of tubular cells, and inhibition of oxidative damage can limit inflammation. SS-31 is a mitochondria-targeted tetrapeptide that can scavenge mitochondrial ROS and inhibit MPT, suggesting that it may protect against ischemic renal injury. Here, in a rat model of ischemia-reperfusion (IR) injury, treatment with SS-31 protected mitochondrial structure and respiration during early reperfusion, accelerated recovery of ATP, reduced apoptosis and necrosis of tubular cells, and abrogated tubular dysfunction. In addition, SS-31 reduced medullary vascular congestion, decreased IR-mediated oxidative stress and the inflammatory response, and accelerated the proliferation of surviving tubular cells as early as 1 day after reperfusion. In summary, these results support MPT as an upstream target for pharmacologic intervention in IR injury and support early protection of mitochondrial function as a therapeutic maneuver to prevent tubular apoptosis and necrosis, reduce oxidative stress, and reduce inflammation. SS-31 holds promise for the prevention and treatment of acute kidney injury.     

NIM811, a Mitochondrial Permeability Transition Inhibitor, Prevents Mitochondrial Depolarization in Small-for-Size Rat Liver Grafts

ATP decreases markedly in small-for-size liver grafts. This study tested if the mitochondrial permeability transition (MPT) underlies dysfunction of small-for-size livers. Half-size livers were implanted into recipients of about twice the donor weight, resulting in quarter-size liver grafts. NIM811 (5 microM), a nonimmunosuppressive MPT inhibitor was added to the storage solutions. Mitochondrial polarization and cell death were assessed by confocal microscopy of rhodamine 123 (Rh123) and propidium iodide (PI), respectively. After quarter-size transplantation, alanine aminotransferase (ALT), serum bilirubin and necrosis all increased. NIM811 blocked these increases by >70%. After 38 h, BrdU labeling, a marker of cell proliferation and graft weight increase were 3% and 5%, respectively, which NIM811 increased to 30% and 42%. NIM811 also increased survival of quarter-size grafts. In sham-operated livers, hepatocytes exhibited punctate Rh123 fluorescence. By contrast, in quarter-size grafts at 18 h after implantation, mitochondria of most hepatocytes did not take up Rh123, indicating mitochondrial depolarization. Nearly all hepatocytes not taking up Rh123 continued to exclude PI at 18 h, indicating that depolarization preceded cell death. NIM811 and free radical-scavenging polyphenols strongly attenuated mitochondrial depolarization. In conclusion, mitochondria depolarized after quarter-size liver transplantation. NIM811 decreased injury and stimulated regeneration, probably by inhibiting free radical-dependent MPT onset.     

Mitochondrial permeability transition in liver ischemia and reperfusion: role of c-Jun N-terminal kinase 2

The mitochondrial permeability transition (MPT) mediates hepatic necrosis after ischemia and reperfusion (I/R). Here, we studied the role of c-Jun N-terminal kinase 2 (JNK2) in MPT-induced liver injury. Wildtype (WT) and JNK2 knockout (KO) mice underwent 70% liver ischemia for 1 hr followed by reperfusion for 8 hr, after which hepatocyte injury and animal survival was assessed. Compared with WT, JNK2 KO mice had 38% less alanine transaminase release and 39% less necrosis by histology. Survival out to 14 days was also greater in JNK2 KO mice (57% vs. 11%), and overall Kaplan-Meier survival was improved. No difference in apoptosis was observed. Intravital multiphoton microscopy of potential-indicating rhodamine 123 after reperfusion revealed depolarized mitochondria in 82% of WT hepatocytes, which decreased to 43% in JNK2 KO hepatocytes. In conclusion, JNK2 contributes to hepatocellular injury and death after I/R in association with increased mitochondrial dysfunction via the MPT.     

Prevention of I/R injury in fatty livers by ischemic preconditioning is associated with increased mitochondrial tolerance: the key role of ATPsynthase and mitochondrial permeability transition

Ischemia/reperfusion (I/R) injury is a commonly encountered clinical problem and occurs probably as a consequence of irreversible mitochondrial injury. The increased susceptibility of fatty livers to ischemic injury is associated with depletion of adenosine triphosphate (ATP) content, which is preserved by preconditioning. Mitochondria being the main ATP production source for the cell, we aimed to evaluate whether ischemic preconditioning (IPC) of fatty livers prevents the impairment in mitochondrial function induced by I/R. Lean and steatotic animals were subjected to 90 min of hepatic warm ischemia and 12 h of reperfusion. IPC effect was tested in fatty livers. After reperfusion, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. Mitochondrial membrane potential, mitochondrial respiration and susceptibility to mitochondrial permeability transition (MPT) were evaluated, as well as ATPase activity and adenine nucleotides. IPC of fatty livers decreased serum AST and ALT levels. Fatty animals subjected to I/R exhibited decreased mitochondrial membrane potential and a delay in the repolarization after a phosphorylation cycle, associated with increased state 4 respiration. Increased tolerance to MPT induction, preservation of F₁F_o-ATPsynthase activity and mitochondrial bioenergetics were observed in ischemic preconditioned fatty livers. Thus, IPC is an endogenous protecting mechanism that preserves mitochondrial function and bioenergetics in fatty livers.     

缺血后处理与线粒体通透性转运孔

背景 缺血后处理(ischemic postconditioning,IPo)能明显减轻器官缺血/再灌注损伤(ischemia/reperfusion injury,I/RI),动物实验和临床研究均已经得到证实,其机制可能与增强组织抗氧化能力和抑制细胞凋亡有关.然而近些年提出线粒体通透性转换孔(mitochondria...