Involvement of mitochondrial permeability transition in hepatitis B virus replication

The HBx protein of human hepatitis B virus (HBV) activates a calcium-dependent kinase pathway which is essential for the viral replication. In this study, we found that HBx expression in the absence of other HBV proteins and in the context of HBV replication decreased the mitochondrial calcein-AM/CoCl₂ signals by 10% and 14% in HepG2 cells and by 15% and 10% in Huh7 cells, respectively. This indicates that HBx can induce mitochondrial permeability transition (MPT) and cause calcium effusion into the plasma. In addition, RNA interference of Cylophilin D decreased HBx-induced MPT and suppressed HBV DNA replication by 41% in HepG2 cells. Our results suggest that HBx expression can induce MPT and facilitate HBV DNA replication.     

Changes in the mitochondrial permeability transition pore in aging and age-associated diseases

Aging is a biological process associated with impairment of mitochondrial bioenergetic function, increased oxidative stress, attenuated ability to respond to stresses and increased risk in contracting age-associated diseases. When mitochondria are subjected to oxidative stress, accompanied by calcium overload and ATP depletion, they undergo “a permeability transition”, characterized by sudden induced change of the inner mitochondrial membrane permeability for water as well as for low-molecular weight solutes (≤1.5 kDa), resulting in membrane depolarization and uncoupling of oxidative phosphorylation. Research interest in the entity responsible for this phenomenon, the “mitochondrial permeability transition pore” (MPTP) has dramatically increased after demonstration that it plays a key role in the life and death decision in cells. The molecular structure and identity of MPTP is not yet known, although the pore is thought to exist as multiprotein complex. Some evidence indicate that the sensitivity of mitochondria to Ca²⁺-induced MPTP opening increases with aging; however the basis of this difference is unknown. Changes in MPTP structure and/or function may have important implications in the aging process and aged-associated diseases. This article examines data relevant to this issue. The important role of a principal lipidic counter-partner of the MPTP, cardiolipin, will also be discussed.     

辅酶Q10对大鼠再生肝细胞线粒体通透性转换的影响

目的 了解辅酶Q₁₀（CoQ₁₀）对大鼠再生肝细胞线粒体通透性转换（MPT）的影响。方法 雌性SD大鼠120只,用不同剂量CoQ₁₀灌胃后行部分肝切除术（PH）,于术后不同时间取肝组织分离线粒体,分光光度计法测定线粒体悬液的A540nm值,之后再加入钙离子诱导并检测A540nm值。结果 PH后6~48h,高剂量（60mg/kg）CoQ₁₀处理组的再生肝线粒体通透性明显低于对照组,而低剂量（6mg/kg）处理组只在48h明显低于对照组,其他时间均无显著差异。用钙离子诱导后,各组各时间点的线粒体通透性随时间延长而不同程度的增强,但PH后6~48 h的CoQ₁₀处理组的再生肝线粒体通透性稍小于对照组。结论 一定剂量的CoQ₁₀能降低大鼠再生肝线粒体通透性,作用主要体现在肝再生的细胞增殖阶段。     

Early activation of the mitochondrial apoptotic pathway in Vesicular Stomatitis virus-infected cells

Vesicular Stomatitis Virus (VSV) has been shown to induce apoptosis in a caspase-dependent manner, but the precise apoptotic pathway remains unknown. We found that caspases 9 and 3, but not caspase 8, were activated during VSV-induced apoptosis in infected Vero cells. Since caspase 9 is related to the mitochondrial apoptotic pathway, we analyzed some mitochondrial events such as changes in the mitochondrial transmembrane potential (Deltapsim) and mitochondrial release of apoptogenic proteins such as cytochrome c and the apoptosis inducing factor (AIF). We found that VSV infection triggers the dissipation of the Deltapsim and the release of both cytochrome c and AIF from the mitochondrial intermembrane space very early in the VSV infection. These results indicate that the trigger of apoptosis in VSV-infected cells occurs through the early activation of the mitochondrial apoptotic pathway. On the other hand, intracellular levels of the anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, and the pro-apoptotic protein Bax, were assessed during viral infection. These analyses showed that as viral infection proceeded, the cellular level of Bcl-xL decreased, while the levels of Bax and Bcl-2 remained unaffected. The significance of the Bcl-xL modulation is also discussed.     

Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition

Post-ischaemic reperfusion may precipitate cardiomyocyte death upon correction of intracellular acidosis due in part to mitochondrial permeability transition. We investigated whether glycine, an amino acid with poorly understood cytoprotective properties, may interfere with this mechanism. In cardiomyocyte cultures, addition of glycine during re-energization following 1 h of simulated ischaemia (NaCN/2-deoxyglucose, pH 6.4) completely prevented necrotic cell death associated with pH normalization. Glycine also protected against cell death associated with pH normalization in reoxygenated rat hearts. Glycine prevented cyclosporin-sensitive swelling and calcein release associated with re-energization in rat heart mitochondria submitted to simulated ischaemia or to Ca²⁺ stress under normoxia. NMR spectroscopy revealed a marked glycine depletion in re-energized cardiomyocytes that was reversed by exposure to 3 m m glycine. These results suggest that intracellular glycine exerts a previously unrecognized inhibition on mitochondrial permeability transition in cardiac myocytes, and that intracellular glycine depletion during myocardial hypoxia/reoxygenation makes the cell more vulnerable to necrotic death.     

转染Akt1基因对缺血再灌注大鼠心肌线粒体通透性转换的影响

目的:研究Akt1基因对缺血再灌注(I/R)损伤心肌细胞的保护作用,并探讨Akt1基因对心肌I/R损伤时线粒体通透性转换(MPT)功能的影响。方法:使用结扎/松解左冠状动脉前降支法,结扎冠状动脉前降支30min,再灌注120min,建立大鼠在体心肌I/R损伤模型。40只雄性SD大鼠随机分为5组,每组8只:假手术对照组(control组)、心肌缺血/再灌注组(I/R组)、转Akt1基因+I/R组(Ad-gene组)、空载体+I/R组(Ad-blank组)、转Akt1基因+I/R+Akt抑制剂组(Ad-inhibitor组)。Ad-gene组术前48h开胸心肌内直接分点注射脂质体Akt1质粒复合物,control组和I/R组分别注射同等体积磷酸盐缓冲液(PBS),Ad-blank组注射等体积脂质体,Ad-inhibitor组注射等体积脂质体Akt1质粒LY294002混合物。观察转染Akt1基因对大鼠I/R心脏乳酸脱氢酶(LDH)和肌酸激酶(CK)释放、心肌细胞凋亡、Akt1表达、线粒体和胞浆内细胞色素C(Cyc-C)表达以及MPT的影响。结果:Control组可见Akt1表达,但明显低于其余各组;与control组相比较,其余各组心肌细胞凋亡指数(AI)、LDH和CK均增高;与control组相比较,其余各组Cyc-C较胞浆表达增加而线粒体内表达减少。Ad-gene组Akt1蛋白表达较control组、I/R组、Ad-blank组及Ad-inhibitor组均增加;Ad-gene组AI、LDH和CK较I/R组、Ad-blank组及Ad-inhibitor组均显著减少,但仍高于control组;Ad-gene组Cyc-C较I/R组、Ad-blank组以及Ad-inhibitor组胞浆内表达减少而线粒体内表达增加;Ad-gene组较IR组、Ad-blank组和Ad-inhibitor组在540nm处吸光度值增高,但明显低于control组。I/R组、Ad-blank组以及Ad-inhibitor组Akt1、Cyc-C、AI、LDH和CK以及在540nm处吸光度值比较均无显著差异。结论:转染Akt1基因对I/R损伤心肌细胞具有保护作用,该作用可能与Akt1抑制I/R诱导的心肌线粒体膜通透性转换,从而保护线粒体的功能有关。     

Pharmacological postconditioning protects isolated rat hearts against ischemia-reperfusion injury: the role of mitochondrial permeability transition pore

Postconditioning has been verified to provide cardioprotection and is associated with the state of mitochondrial permeability transition pore. However, there are a few limitations with clinical use of classic postconditioning; therefore, the purpose of this investigation was to study whether inhibition of mitochondrial permeability transition pore opening with cyclosporine A also provided cardioprotection. Langendorff-perfused Sprague-Dawley rat hearts were perfused for 20 minutes with Krebs-Henseleit buffer followed by 30 minutes of crystalloid cardioplegia and 60 minutes of reperfusion. Control hearts (Con group) were reperfused with Krebs-Henseleit buffer. Postconditioning hearts (Ipo group) were with six cycles of 10 seconds reocclusion separated by 10 seconds perfusion before reperfusion. Cyclosporine A postconditioning hearts (CsA group) were reperfused with Krebs-Henseleit buffer containing 0.8 μmol/L cyclosporine A at first 5 minutes of reperfusion. Compared with Con group, myocardial performance was better preserved in CsA group. Mitochondrial outer membrane integrity was preserved, with less cytosolic diffusion of cytochrome C (p < 0.05) and less frequency of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling-positive myocytes in Ipo and CsA group (p < 0.05). Postconditioning prevented apoptosis-related mitochondrial permeabilization and dysfunction after cardioplegic arrest. Cyclosporine A postconditioning had a better effect than classic postconditioning in myocardial performance.     

内质网应激诱导血管反应性降低与线粒体通透性转换孔开放的关系

 目的:探讨内质网应激是否可诱导血管反应性降低及其与线粒体通透性转换孔开放的关系。方法:利用SD大鼠盲肠结扎穿孔（CLP）脓毒性休克模型,检测不同时点肠系膜上动脉内质网应激标志分子GRP78和CHOP的蛋白质表达水平,同时检测肠系膜上动脉对去甲肾上腺素的反应性;离体血管环观察毒胡萝卜素孵育对肠系膜上动脉血管反应性的影响,并观察线粒体通透性转换孔在其中的作用。结果:大鼠CLP后不同时点肠系膜上动脉GRP78和CHOP表达呈逐渐升高趋势,肠系膜上动脉的反应性降低,用内质网应激抑制剂牛磺酸能够抑制GRP78和CHOP的表达,并显著恢复血管反应性。离体血管环研究表明,毒胡萝卜素能够降低血管反应性,牛磺酸能恢复血管反应性,线粒体通透性转换孔抑制剂环孢素A能显著增加血管反应性。结论: 内质网应激能诱导血管反应性降低,且可能与线粒体通透性转换孔有关。     

Inhibition of mitochondrial permeability transition pore opening is involved in the protective effects of mortalin overexpression against beta-amyloid-induced apoptosis in SH-SY5Y cells

Mortalin (mtHsp70) is a mitochondrial heat shock protein critical for maintaining the functional integrity of mitochondrial proteins. Our previous study demonstrated that mortalin overexpression protected against Aβ-induced neurotoxicity through a mitochondria-dependent mechanism, but the molecular details remained unclear. Recent biochemical studies implicate opening of the mitochondrial permeability transition pore (mPTP) in Aβ-mediated mitochondrial dysfunction. The present study investigated the effect of mortalin overexpression on Aβ-induced mPTP activation and ensuing neuronal apoptosis. Mortalin overexpression inhibited mPTP activation and protected SH-SY5Y neurons against Aβ-induced apoptosis. Compared to controls, neurons overexpressing mortalin also demonstrated superior intracellular free calcium regulation, lower mitochondrial reactive oxygen species generation, and decreased Bax/Bcl-2 ratios in response to Aβ treatment. Mortalin overexpression suppressed activation of the mitochondrial apoptotic cascade as demonstrated by inhibition of cytochrome c release and caspase-3 activation. Our results indicate that the cytoprotective efficacy of mortalin under Aβ-induced stress is mediated, at least in part, by inhibition of mPTP opening. Demonstration of the neuroprotective action of mortalin provides additional insights into the pathogenic mechanisms of Aβ toxicity and defines possible molecular targets for therapeutic intervention.     

线粒体通透性转换作用Cyt C水平对慢加急性肝衰竭大鼠线粒体ATP生成的影响

目的 从线粒体损伤的角度探讨ACLF存在的能量代谢障碍.方法 雄性SD大鼠随机分三组,ACLF组、NAC组各15只,正常组10只,制作ACLF模型,NAC灌胃作抗氧化模型.留肝组织及血液标本,梯度离心分离线粒体,ELLSA法测线粒体Cyt C、荧光酶标法测MPTP RFU;酶标法测肝组织、血浆GSH、MDA、SOD.结果 ACLF组肝组织、血浆MDA明显高于正常及NAC组（P＜0.05）;NAC组MDA高于正常组（P ＜0.05）;ACLF组血浆GSH、SOD低于正常及NAC组（P＜0.05）;三组Cyt C为正常组最高、ACLF组最低（P＜0.05）;三组MPTP为ACLF组最高、正常组最低;MPTP与Cyt C、ATP呈负相关（r=-0.858,-0.799）;Cyt C与ATP呈正相关（r=0.78）.结论 ACLF大鼠处于氧化应激损伤,Cyt C外流入胞质可能是ACLF发生能量代谢障碍的原因之一;NAC可在一定程度上提高肝细胞的抗氧化能力.     

Effect of acrylamide on BEAS-2B normal human lung cells: Cytotoxic,oxidative, apoptotic and morphometric analysis

Due to the broad toxic relevance of acrylamide, many measures have been taken since the 1900s. These measures increased day by day when acrylamide was discovered in foods in 2002, and its toxic spectrum was found to be wider than expected. Therefore, in some countries, the products with higher acrylamide content were restricted. On the other hand, the effects of acrylamide on the respiratory system cells have yet to be well understood. In this study, we aimed at investigating the effect of acrylamide on lung epithelial BEAS-2B cells. Initially, the cytotoxic effect of acrylamide on BEAS-2B was determined by MTT assay. Then, cellular oxidative stress was measured. Flow cytometry analysis was conducted for Annexin-V and caspase 3/7. Furthermore, Bax, Bcl-2 and Nrf-2 proteins were evaluated by immunocytochemistry. Finally, acrylamide-induced cellular morphological changes were observed under confocal and TEM microscopes. According to MTT results, the IC50 concentration of acrylamide was 2.00 mM. After acrylamide treatment, oxidative stress increased dose-dependently. Annexin V-labelled apoptotic cells and caspase 3/7 activity were higher than untreated cells in acrylamide-treated cells. Immunocytochemical examination revealed a marked decrease in Bcl-2, an increase in Bax and Nrf-2 protein staining upon acrylamide treatment. Furthermore, in confocal and TEM microscopy, apoptotic hallmarks were pronounced. In the present study, acrylamide was suggested to display anti-proliferative activity, decrease viability, induce apoptosis and oxidative stress and cause morphological changes in BEAS-2B cells.     

The neuroprotection conferred by activating the mitochondrial ATP-sensitive K+ channel is mediated by inhibiting the mitochondrial permeability transition pore

In order to further explore the mechanisms by which activation of mitochondrial ATP-sensitive potassium channels (mitoKATP) confers neuroprotection, we investigated the role of the mitochondrial permeability transition pore (MPTP) in in vivo and in vitro models. Adult male Sprague-Dawley rats were exposed to 90 min of middle cerebral artery occlusion (MCAO) followed by reperfusion for 22 h, when neurological scores and infarct volumes were evaluated. Activating mitoKATP by infusion of 2 mmol/L diazoxide into the ventricles 20 min before MCAO or inhibiting the MPTP by infusion of 1 micromol/L cyclosporin A 15 min before reperfusion significantly increased functional score and reduced infarction volume. Subsequent intracerebroventricular infusion of 2 mmol/L atractyloside, the MPTP opener, 10 min before reperfusion significantly attenuated the neuroprotective effects of diazoxide and cyclosporin A. The swelling of mitochondria isolated from brain was evaluated by spectrophotometry and served as a measure of MPTP opening. In isolated mitochondria, 100 micromol/L atractyloside attenuated the decrease of mitochondrial swelling induced by 30 micromol/L diazoxide or cyclosporin A (0.5 or 1 micromol/L). Furthermore, 100 micromol/L diazoxide or 1 micromol/L cyclosporin A both attenuated the fluorescence intensity in isolated mitochondria loaded with rhod-2 acetoxymethylester, and 100 micromol/L atractyloside abolished the effects of diazoxide and cyclosporin A. These results suggest that activation of mitoKATP protects the brain against injury, and this is probably mediated by attenuating mitochondrial Ca2+ overload and thus inhibiting MPTP opening during brain ischemia and reperfusion.     

ATP-dependent potassium channels and mitochondrial permeability transition pores play roles in the cardioprotection of theaflavin in young rat

Previous studies have confirmed that tea polyphenols possess a broad spectrum of biological functions such as anti-oxidative, anti-bacterial, anti-tumor, anti-inflammatory, anti-viral and cardiovascular protection activities, as well as anti-cerebral ischemia-reperfusion injury properties. But the effect of tea polyphenols on ischemia/reperfusion heart has not been well elucidated. The aim of this study was to investigate the protective effect of theaflavin (TF1) and its underlying mechanism. Young male Sprague-Dawley (SD) rats were randomly divided into five groups: (1) the control group; (2) TF1 group; (3) glibenclamide + TF1 group; (4) 5-hydroxydecanoate (5-HD) + TF1 group; and (5) atractyloside + TF1 group. The Langendorff technique was used to record cardiac function in isolated rat heart before and after 30 min of global ischemia followed by 60 min of reperfusion. The parameters of cardiac function, including left ventricular developing pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal differentials of LVDP (±LVdP/dt _max) and coronary flow (CF), were measured. The results showed: (1) compared with the control group, TF1 (10, 20, 40 μmol/l) displayed a better recovery of cardiac function after ischemia/reperfusion in a concentration-dependent manner. At 60 min of reperfusion, LVDP, ±LVdP/dt _max and CF in the TF1 group were much higher than those in the control group, whereas left ventricular end-diastolic pressure (LVEDP) in the TF1 group was lower than that in the control group (P < 0.01). (2) Pretreatment with glibenclamide (10 μmol/l), a K_ATP antagonist, completely abolished the cardioprotective effects of TF1 (20 μmol/l). Also, most of the effects of TF1 (20 μmol/l) on cardiac function after 60 min of reperfusion were reversed by 5-HD (100 μmol/l), a selective mitochondria K_ATP antagonist. (3) Atractyloside (20 μmol/l), a mitochondrial permeability transition pore (mPTP) opener, administered at the beginning of 15 min of reperfusion completely abolished the cardioprotection of TF1 (20 μmol/l). The results indicate that TF1 protects the rat heart against ischemia/reperfusion injury through the opening of K_ATP channels, particularly on the mitochondrial membrane, and inhibits mPTP opening.     

羟基红花黄色素A对缺血/再灌注大鼠肺线粒体通透性转换功能的影响

目的: 研究羟基红花黄色素A(HSYA)对缺血/再灌注(I/R)损伤肺组织细胞的保护作用,并探讨其对肺I/R损伤时线粒体通透性转换(MPT)功能的影响。方法: 开胸阻断左肺门45 min,松开阻断带形成再灌注,复制在体肺I/R模型。健康SD大鼠50只,随机分为5组,每组10只:假手术对照组(对照组) 、缺血/再灌注1 h组(I/R 1 h组)、缺血/再灌注3 h组(I/R 3 h组)、HSYA干预+I/R 1 h组(SI 1 h组)和HSYA干预+I/R 3 h组(SI 3 h组)。SI 1 h组和SI 3 h组分别在缺血前20 min和再灌注即刻静注HSYA (2.0 mg/kg),其余步骤分别同I/R 1 h组和I/R 3 h组,对照组、I/R 1 h组和I/R 3 h组分别注射同等体积生理盐水。观察各组肺湿干重比(W/D)、肺泡损伤数定量评价指标(IQA)、肺组织细胞凋亡情况、胞浆内和线粒体基质内细胞色素C (CytC)、凋亡诱导因子(AIF)的水平以及MPT功能。结果: 与对照组相比,其余各组肺组织细胞凋亡指数(AI)、W/D和IQA均增高,CytC和AIF胞浆含量增加而线粒体内含量减少;SI各组与相应的I/R组比,AI、W/D和IQA均显著降低,但仍高于对照组;SI组CytC和AIF较相应的I/R组胞浆含量减少而线粒体内含量增加;线粒体在540 nm处的吸光度值SI 1 h较IR 1 h、SI 3 h较IR 3 h增高,但仍低于对照组。结论: 羟基红花黄色素A在一定程度上抑制I/R肺组织细胞凋亡,发挥细胞保护作用,该作用可能与其维持MPT功能、减少CytC和AIF由线粒体向胞浆内释放有关。     

补肾壮督方含药血清对髓核细胞线粒体凋亡通路的影响

文题释义：线粒体凋亡通路：又称为内源性凋亡通路,可以通过多种应力刺激激活,导致线粒体外膜改变、线粒体DNA损伤等,这些改变将最终导致线粒体外膜通透性的增加,正常情况下存在于线粒体内外膜之间的凋亡相关蛋白将扩散至细胞质中,从而导致细胞凋亡的发生。中药血清药理方法：是指动物灌胃给予中药及制剂,经吸收进入机体血液循环,在一定时间内采集血液,分离所得血清中必定含有一定量的该药物成分,此时的血药浓度反映了机体的真实血药浓度,将此血清加入到体外细胞培养体系,观察其药理作用。背景：线粒体凋亡通路是细胞凋亡中的重要通路,前期动物实验发现补肾壮督方可通过抑制线粒体凋亡通路改善椎间盘退变,但其作用机制还有待研究。目的：观察补肾壮督方含药血清对人髓核细胞线粒体凋亡通路关键蛋白的影响,探讨其改善椎间盘退变的机制。方法：①37只SD雄性大鼠,随机分为正常组、低剂量中药组[0.506 g/(kg•d)]、中剂量中药组[1.012 g/(kg•d)]及高剂量中药组[2.024 g/(kg•d)],连续灌胃2周,灌胃结束后制备含药血清;②人髓核细胞随机分为正常组、模型组、低剂量含药血清组、中剂量含药血清组、高剂量含药血清组,模型组用200 μmol/L H₂O₂处理6 h,正常组不进行任何处理,低、中、高剂量含药血清组造模后用体积分数为2%含药血清干预48 h。透射电镜观察髓核细胞的超微结构,流式细胞术检测髓核细胞凋亡率和线粒体膜电位,qPCR和Western blot检测Apaf1、Bcl-2、Bax及Cytc的表达。结果与结论：①与正常组相比,模型组出现明显的细胞凋亡形态特征,髓核细胞凋亡率显著增加(P < 0.05),Apaf1、Cytc、Bax mRNA及蛋白表达量均显著增加(P < 0.05),线粒体膜电位、Bcl-2 mRNA及其蛋白表达量显著下降(P < 0.05);②补肾壮督方含药血清干预后,髓核细胞凋亡率明显减少(P < 0.05),Apaf1、Cytc、Bax mRNA及蛋白表达量均明显下降(P < 0.05),线粒体膜电位、Bcl-2 mRNA及其蛋白表达量显著增加  (P < 0.05);③补肾壮督方含药血清可有效抑制髓核细胞凋亡,并呈剂量依赖性,可能通过减少Apaf1、Cytc、Bax蛋白及mRNA表达,增加Bcl-2蛋白及mRNA表达,抑制线粒体凋亡通路,从而改善椎间盘退变。ORCID: 0000-0002-6045-9062(林一峰)中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Infarct size reduction by cyclosporine A at reperfusion involves inhibition of the mitochondrial permeability transition pore but does not improve mitochondrial respiration

Introduction

Ischemic postconditioning (PoCo) and cyclosporine A (CysA) given prior to reperfusion reduce myocardial infarct size after ischemia/reperfusion. Ischemic postconditioning''s protection is characterized by better preservation of mitochondrial respiration and calcium retention capacity. Protection by CysA is not entirely clear. Cyclosporine A inhibits not only mitochondrial permeability transition pore (mPTP) opening but also the phosphatase calcineurin. We have investigated whether CysA mediates protection not only by mPTP inhibition but also through a more upstream inhibition of calcineurin with subsequently better preserved mitochondrial respiration.

Material and methods

Anesthetized pigs were subjected to 90 min ischemia and 10 min reperfusion initiated with either PoCo (6 × 20 s reperfusion/re-occlusion; n = 9), CysA infusion (5 mg/kg i.v.; 5 min before reperfusion; n = 4), or immediate full reperfusion (IFR; n = 8). Mitochondria were isolated from myocardial tissue for measurement of respiration and calcium retention capacity.

Results

In mitochondria from ischemic/reperfused myocardium, ADP-stimulated complex I respiration was similar between CysA (116 ±11 nmol O₂/min/mg protein) and IFR (117 ±8), but better preserved with PoCo (160 ±9; p < 0.05). Calcium retention capacity was greater with both PoCo and CysA (1096 ±45 and 1287 ±128 nmol Ca²⁺/mg protein) than with IFR (756 ±103; p < 0.05).

Conclusions

Cyclosporine A''s protection is not associated with improved mitochondrial respiration. Protection is unlikely related to an upstream calcineurin inhibition, but is indeed secondary to mPTP inhibition.     

Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition

The killing of L929 mouse fibroblasts by tumor necrosis factor-alpha (TNF-alpha) in the presence of 0.5 microg/ml actinomycin D (Act D) is prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A(2) inhibitor aristolochic acid (ArA). The MPT is accompanied by the release of cytochrome c from the mitochondria, caspase-8 and caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. The caspase-3 inhibitor z-Asp-Glu-Val-aspartic acid fluoromethyl-ketone (Z-DEVD-FMK) did not prevent the loss of viability or the redistribution of cytochrome c, but it did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. Inhibition of the MPT reduced the activation of caspase-8 to the level occurring with TNF-alpha alone (no ActD). The caspase-8 inhibitor z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone (Z-IETD-FMK) did not prevent the cell killing and decreased only slightly the translocation of Bid to the mitochondria. These data indicate that induction of the MTP by TNF-alpha causes a release of cytochrome c, caspase-3 activation with PARP cleavage and DNA fragmentation. The loss of viability is dependent on the MPT but independent of the activation of caspase-3. The activation of caspase-8 is not dependent on the MPT. There is no evidence linking this enzyme to the loss of viability. Thus, the killing of L929 fibroblasts by TNF-alpha can occur in the absence of either caspase-3 or caspase-8 activity. Alternatively, cell death can be prevented despite an activation of caspase-8.     

Survivin抑制剂YM155对视网膜母细胞瘤Y79细胞线粒体凋亡途径的影响

目的:探讨人生存素(survivin)抑制剂YM155{4,9-二氢-1-(2-甲氧基乙基)-2-甲基-4,9-二氧代-3-(2-吡嗪甲基)-1H-萘并[2,3-d]咪唑鎓溴化物}对视网膜母细胞瘤Y79细胞凋亡、线粒体膜电位(△ψ_m)和细胞色素C(Cyt C)的影响,探讨其诱导Y79细胞凋亡的线粒体机制。方法:体外培养Y79细胞,分别以0、0.5、1、2、4、8nmol/L的YM155进行处理,采用CCK-8法和溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)掺入标记法检测YM155对Y79细胞增殖的抑制作用。Y79细胞随机分为4组:对照组、阳性对照组[加入10nmol/L拓扑替康(topotecan)]和低剂量(1 nmol/L)、高剂量(2 nmol/L)YM155组。每组设3个复孔,处理24h。分别采用TUNEL染色和Annexin V-FITC/PI双染法检测凋亡的情况;采用JC-1活细胞染色检测△ψ_m的变化;采用免疫荧光分析检测Cyt C的分布;采用Western blot法检测survivin和线粒体内Cyt C的蛋白表达。结果:与对照组比较,YM155对Y79细胞增殖具有明显抑制作用,并诱导Y79细胞凋亡(P0.05);YM155显著降低Y79细胞的△ψ_m,促进Cyt C由线粒体释放至胞浆,降低线粒体内Cyt C的水平(P0.05)。结论:YM155对Y79细胞增殖具有抑制作用,并诱导细胞凋亡其机制可能是通过线粒体介导的细胞凋亡途径实现的。     

胰腺癌细胞外泌体通过激活线粒体凋亡途径促进β细胞凋亡

目的:探讨胰腺癌细胞分泌的外泌体(exosome)对胰岛β细胞存活率和功能的影响及作用机制。方法:采用外泌体提取试剂盒提取小鼠胰腺癌细胞Pan02和MPC-83上清液外泌体,经磷钨酸染色后于透射电镜下鉴定形态;外泌体经荧光标记后与小鼠胰岛瘤MIN6细胞共孵育48 h,检测外泌体分泌水平和MIN6细胞的摄取水平;MTT和葡萄糖刺激的胰岛素分泌(GSIS)实验分别检测各组细胞的存活率和胰岛素分泌功能;q PCR检测微小RNA-204(miR-204)和Bcl-2 mRNA的表达;Western blot检测线粒体凋亡信号通路相关蛋白Bcl-2、Bax、caspase-3和细胞色素C(Cyt-C)的表达。结果:透射电镜结果显示2种胰腺癌细胞均能分泌外泌体,且Pan02细胞分泌更多。荧光标记的外泌体与胰岛β细胞共孵育结果显示,β细胞能够大量摄取胰腺癌细胞分泌的外泌体。MTT和GSIS实验结果显示,外泌体处理组的MIN6细胞存活率和高糖刺激的胰岛素分泌量显著低于未处理组(P0.01)。q PCR结果显示胰腺癌细胞分泌的外泌体富含miR-204,且外泌体处理后的MIN6细胞内Bcl-2的mRNA表达显著下调(P0.01)。Western blot结果显示,外泌体处理的MIN6细胞内Bcl-2蛋白表达显著下调(P0.05),Bax、cleaved caspase-3和Cyt-C蛋白表达显著上调(P0.01)。结论:胰腺癌细胞能够分泌外泌体,且该外泌体能够被胰岛β细胞摄取。胰腺癌细胞分泌的外泌体可以降低β细胞存活率和β细胞胰岛素的分泌功能,其机制可能通过外源性上调β细胞内miR-204的表达,进而抑制Bcl-2的mRNA和蛋白表达,最终激活β细胞内线粒体凋亡信号通路。     

Regulation of permeability of the mitochondrial membrane by mitochondrial interaction factor

The effect of mitochondrial interaction factor (MIF) on swelling of the mitochondria in isoosmotic media with KNO₃, Ca(NO₃)₂, and NH₄NO₃ was studied in the presence of oxidation inhibitors. The total MIF fraction was shown to inhibit swelling of the mitochondria in all three media. By elution of MIF from a DEAE-cellulose column with a pH gradient falling from 8.7 to 6.7, three peaks of activity were obtained (fractions A, B, and C). Quantitative differences were found in the action of these three fractions on swelling of the mitochondria. fraction C inhibited swelling more strongly in Ca(NO₃)₂, whereas fractions A and B did so in media with KNO₃ and NH₄NO₃.Laboratory of Hormone Biochemistry, Institute of Biochemistry of the Uzbek SSR, Tashkent. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 4, pp. 411–413, April, 1976.