Inhibitor Of Mitochondrial Permeability Transition Research Articles

Toxicity of polyhydroxylated fullerene to mitochondria

Mitochondrial dysfunction is considered as a crucial mechanism of nanomaterial toxicity. Herein, we investigated the effects of polyhydroxylated fullerene (C60(OH)44, fullerenol), a model carbon-based nanomaterial with high water solubility, on isolated mitochondria. Our study demonstrated that fullerenol enhanced the permeabilization of mitochondrial inner membrane to H+ and K+ and induced mitochondrial permeability transition (MPT). The fullerenol-induced swelling was dose-dependent and could be effectively inhibited by MPT inhibitors such as cyclosporin A (CsA), adenosine diphosphate (ADP), ruthenium red (RR) and ethylenediaminetetraacetic acid (EDTA). After treating the mitochondria with fullerenol, the mitochondrial membrane potential (MMP) was found collapsed in a concentration-independent manner. The fluorescence anisotropy of hematoporphyrin (HP) changed significantly with the addition of fullerenol, while that of 1,6-diphenyl-hexatriene (DPH) changed slightly. Moreover, a decrease of respiration state 3 and increase of respiration state 4 were observed when mitochondria were energized with complex II substrate succinate. The results of transmission electron microscopy (TEM) provided direct evidence that fullerenol damaged the mitochondrial ultrastructure. The investigations can provide comprehensive information to elucidate the possible toxic mechanism of fullerenols at subcellular level.

Tissue and sex specificities in Ca2+ handling by isolated mitochondria in conditions avoiding the permeability transition.

What is the central question of this study? The assessment of Ca(2+) handling by isolated mitochondria can be biased by dysfunctions secondary to Ca(2+) -induced mitochondrial permeability transition (MPT). As a result of this uncertainty and the differing experimental conditions between studies, the tissue and sex diversities in mitochondrial Ca(2+) transport are still unsettled questions. What is the main finding and its importance? If MPT is not prevented during Ca(2+) transport assays, some measured variables are biased. Accounting for the implied importance of preventing MPT, we observed substantial tissue specificities in the mitochondrial Ca(2+) handling, particularly in the Ca(2+) efflux pathways. The characteristics of mitochondria, including their Ca(2+) transport functions, may exhibit tissue specificity and sexual dimorphism. Given that measurements of Ca(2+) handling by isolated mitochondria may be biased by dysfunction secondary to Ca(2+) -induced mitochondrial permeability transition (MPT) pore opening, this study evaluated the extent to which MPT inhibition by ciclosporin affected the measurement of Ca(2+) transport in isolated rat liver mitochondria. The results indicate that the steady-state levels of external Ca(2+) and the rates of mitochondrial Ca(2+) efflux through the selective pathways can be overestimated by up to fourfold if MPT pore opening is not prevented. We analysed Ca(2+) transport in isolated mitochondria from the liver, skeletal muscle, heart and brain of male and female rats in incubation conditions containing MPT inhibitors, NAD-linked substrates and relevant levels of free Ca(2+), Mg(2+) and Na(+). The Ca(2+) influx rates were similar among the samples, except that the liver mitochondria displayed values fourfold higher. In contrast, the Ca(2+) efflux rates exhibited more tissue diversity, especially in the presence of Na(+). Interestingly, the Na(+)-independent Ca(2+) efflux was highest in the heart mitochondria (∼ 4 nmol mg(-1) min(-1)), thus challenging the view that cardiac mitochondrial Ca(2+) efflux relies almost exclusively on a Na(+)-dependent pathway. Sex specificity was observed in only two kinetic indexes of heart mitochondrial Ca(2+) homeostasis and in the ADP-stimulated respiration of liver mitochondria (∼ 20% higher in females). The present study shows the methodological importance of preventing MPT when measuring the properties and the physiological variability of the Ca(2+) handling by isolated mitochondria.

Uncoupling, metabolic inhibition and induction of mitochondrial permeability transition in rat liver mitochondria caused by the major long-chain hydroxyl monocarboxylic fatty acids accumulating in LCHAD deficiency

Patients with long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiency commonly present liver dysfunction whose pathogenesis is unknown. We studied the effects of long-chain 3-hydroxylated fatty acids (LCHFA) that accumulate in LCHAD deficiency on liver bioenergetics using mitochondrial preparations from young rats. We provide strong evidence that 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, the monocarboxylic acids that are found at the highest tissue concentrations in this disorder, act as metabolic inhibitors and uncouplers of oxidative phosphorylation. These conclusions are based on the findings that these fatty acids decreased ADP-stimulated (state 3) and uncoupled respiration, mitochondrial membrane potential and NAD(P)H content, and, in contrast, increased resting (state 4) respiration. We also verified that 3HTA and 3HPA markedly reduced Ca2+ retention capacity and induced swelling in Ca2+-loaded mitochondria. These effects were mediated by mitochondrial permeability transition (MPT) induction since they were totally prevented by the classical MPT inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca2+ uptake blocker. Taken together, our data demonstrate that the major monocarboxylic LCHFA accumulating in LCHAD deficiency disrupt energy mitochondrial homeostasis in the liver. It is proposed that this pathomechanism may explain at least in part the hepatic alterations characteristic of the affected patients.

Glutathione protects mitochondrion and alleviates liver ischemia-reperfusion injury in rats

Objective To investigate the protective effects of glutathione(GSH)against hepatic ischemia/reperfusion(I/R)injury and the related mechanism.Methods SD rats were randomized into Sham,I/R and GSH groups with 20 rats in each group.Rat models of segmental(70%)warm hepatic ischemia were established in I/R and GSH groups.GSH was injected through the femoral vein at the dose of 5 mg/kg 5 min before ischemia.At 24 hafter reperfusion,liver injury was evaluated by serological and histological indices.Liver cell apoptoses were evaluated by TUNEL staining.The GSH/oxidized glutathione(GSSG)ratios of tissue level were compared between different groups.Liver mitochondria were collected and the mitochondrial calcium capacity(CRC)was evaluated.Results The serum aspartate transaminanse(AST)and alanine aminotransferase(ALT)levels in GSH group were significantly decreased 6hafter reperfusion compared with I/R group(P0.05).24 hafter reperfusion,the liver injury was alleviated and the number of apoptosis cells was significantly decreased in GSH group compared with I/R group(P0.05).The GSH/GSSG ratio of tissue level in GSH group was significantly increased 6hafter reperfusion compared with I/R group(P0.05).Liver mitochondrial CRC in GSH group was significantly increased 6hafter reperfusion compared with I/R group(P0.05).Conclusion GSH preconditioning can protect liver from hepatic I/R injury,which is possibly by inhibiting oxidative response and subsequent inhibition of mitochondrial permeability transition.

GNX-4728, a novel small molecule drug inhibitor of mitochondrial permeability transition, is therapeutic in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder in humans characterized by progressive degeneration of skeletal muscle and motor neurons in spinal cord, brainstem, and cerebral cortex causing skeletal muscle paralysis, respiratory insufficiency, and death. There are no cures or effective treatments for ALS. ALS can be inherited, but most cases are not associated with a family history of the disease. Mitochondria have been implicated in the pathogenesis but definitive proof of causal mechanisms is lacking. Identification of new clinically translatable disease mechanism-based molecular targets and small molecule drug candidates are needed for ALS patients. We tested the hypothesis in an animal model that drug modulation of the mitochondrial permeability transition pore (mPTP) is therapeutic in ALS. A prospective randomized placebo-controlled drug trial was done in a transgenic (tg) mouse model of ALS. We explored GNX-4728 as a therapeutic drug. GNX-4728 inhibits mPTP opening as evidenced by increased mitochondrial calcium retention capacity (CRC) both in vitro and in vivo. Chronic systemic treatment of G37R-human mutant superoxide dismutase-1 (hSOD1) tg mice with GNX-4728 resulted in major therapeutic benefits. GNX-4728 slowed disease progression and significantly improved motor function. The survival of ALS mice was increased significantly by GNX-4728 treatment as evidence by a nearly 2-fold extension of lifespan (360 days–750 days). GNX-4728 protected against motor neuron degeneration and mitochondrial degeneration, attenuated spinal cord inflammation, and preserved neuromuscular junction (NMJ) innervation in the diaphragm in ALS mice. This work demonstrates that a mPTP-acting drug has major disease-modifying efficacy in a preclinical mouse model of ALS and establishes mitochondrial calcium retention, and indirectly the mPTP, as targets for ALS drug development.

Postconditioning mitigates cell death following oxygen and glucose deprivation in PC12 cells and forebrain reperfusion injury in rats.

Postconditioning mitigates ischemia-induced cellular damage via a modified reperfusion procedure. Mitochondrial permeability transition (MPT) is an important pathophysiological change in reperfusion injury. This study explores the role of MPT modulation underlying hypoxic postconditioning (HPoC) in PC12 cells and studies the neuroprotective effects of ischemic postconditioning (IPoC) on rats. Oxygen-glucose deprivation (OGD) was performed for 10 hr on PC12 cells. HPoC was induced by three cycles of 10-min reoxygenation/10-min rehypoxia after OGD. The MPT inhibitor N-methyl-4-isoleucine cyclosporine (NIM811) and the MPT inducer carboxyatractyloside (CATR) were administered to selective groups before OGD. Cellular death was evaluated by flow cytometry and Western blot analysis. JC-1 fluorescence signal was used to estimate the mitochondrial membrane potential (△Ψm ). Transient global cerebral ischemia (tGCI) was induced via the two-vessel occlusion and hypotension method in male Sprague Dawley rats. IPoC was induced by three cycles of 10-sec reperfusion/10-sec reocclusion after index ischemia. HPoC and NIM811 administration attenuated cell death, cytochrome c release, and caspase-3 activity and maintained △Ψm of PC12 cells after OGD. The addition of CATR negated the protection conferred by HPoC. IPoC reduced neuronal degeneration and cytochrome c release and cleaved caspase-9 expression of hippocampal CA1 neurons in rats after tGCI. HPoC protected PC12 cells against OGD by modulating the MPT. IPoC attenuated degeneration of hippocampal neurons after cerebral ischemia.

A new twist on an old idea part 2: cyclosporine preserves normal mitochondrial but not cardiomyocyte function in mini-swine with compensated heart failure.

We recently developed a clinically relevant mini‐swine model of heart failure with preserved ejection fraction (HFpEF), in which diastolic dysfunction was associated with increased mitochondrial permeability transition (MPT). Early diastolic function is ATP and Ca2+‐dependent, thus, we hypothesized chronic low doses of cyclosporine (CsA) would preserve mitochondrial function via inhibition of MPT and subsequently maintain normal cardiomyocyte Ca2+ handling and contractile characteristics. Left ventricular cardiomyocytes were isolated from aortic‐banded Yucatan mini‐swine divided into three groups; control nonbanded (CON), HFpEF nontreated (HF), and HFpEF treated with CsA (HF‐CsA). CsA mitigated the deterioration of mitochondrial function observed in HF animals, including functional uncoupling of Complex I‐dependent mitochondrial respiration and increased susceptibility to MPT. Attenuation of mitochondrial dysfunction in the HF‐CsA group was not associated with commensurate improvement in cardiomyocyte Ca2+ handling or contractility. Ca2+ transient amplitude was reduced and transient time to peak and recovery (tau) prolonged in HF and HF‐CsA groups compared to CON. Alterations in Ca2+ transient parameters observed in the HF and HF‐CsA groups were associated with decreased cardiomyocyte shortening and shortening rate. Cellular function was consistent with impaired in vivo systolic and diastolic whole heart function. A significant systemic hypertensive response to CsA was observed in HF‐CsA animals, and may have played a role in the accelerated the development of heart failure at both the whole heart and cellular levels. Given the significant detriment to cardiac function observed in response to CsA, our findings suggest chronic CsA treatment is not a viable therapeutic option for HFpEF.

Hexane fraction of Costus afer Ker Gawl leaf inhibited mitochondrial permeability transition, F1F0 ATPase and scavenged nitric oxide and hydrogen peroxide (957.1)

Recent implication of mitochondria in the etiology of inflammatory diseases especially rheumatoid arthritis led to the evaluation of in vitro and in vivo inhibitory effects of an indigenous medicinal plant, Costus afer leaf hexane fraction (CAHFL) on mitochondrial permeability transition (MPT), F1F0 ATPase, nitric oxide (NO•) and hydrogen peroxide (H2O2) using spectrophotometric methods. Results showed that 10, 20, 40 and 60 µg/ml CAHFL significantly (P<0.05) inhibited Ca2+ induced MPT in a concentration dependent manner by 85.66%, 86.93%, 87.47% and 90.47% respectively when compared with untreated mitochondria in vitro. Spermine potentiated CAHFL MPT inhibition by 88.20%, 90.20%, 92.12% and 93.51% respectively. The In vivo study showed that 50, 100 and 250 mg/kg body weight CAHFL significantly (P<0.05) inhibited MPT in arthritic rats by 52.22%, 61.11% and 65.0% respectively in a dose dependent manner compared with control group (untreated) in 7 days. Concentrations of 10, 20, 40, 60 and 80 µg/ml CAHFL significantly (P<0.05) reduced F1F0 ATPase activity in a concentration dependent manner with 3.93 ± 0.01, 3.88 ± 0.04, 3.63 ± 0.07, 3.51 ± 0.05 and 3.49 ± 0.04 mg/ml phosphate (Pi) liberated respectively compared with 10 µg/ml 2,4‐dinitrophenol (6.60 ± 0.03 mg/ml Pi.). CAHFL scavenged NO• and H2O2 in a concentration dependent manner with an IC50¬¬ of 1.77 mg/ml and 0.33 mg/ml respectively. These data indicated that CAHFL possess the ability to protect mitochondria from damage which may account for its anti‐inflammatory action, further strengthening the claim that CAHLF could be a candidate for anti‐inflammatory drug development.Grant Funding Source: Babcock University research grant [BU/RIC/003]

A lectin from Bothrops leucurus snake venom raises cytosolic calcium levels and promotes B16-F10 melanoma necrotic cell death via mitochondrial permeability transition

BlL, a galactose-binding C-type lectin purified from Bothrops leucurus snake venom, exhibits anticancer activity. The current study was designed to elucidate the cellular mechanisms by which BlL induces melanoma cell death. The viabilities of B16-F10 melanoma cells and HaCaT keratinocytes treated with BlL were evaluated. Necrotic and apoptotic cell death, cytosolic Ca2+ levels, mitochondrial Ca2+ transport and superoxide levels were assessed in B16-F10 melanoma cells exposed to BlL. We found that treatment with BlL caused dose-dependent necrotic cell death in B16-F10 melanoma cells. Conversely, the viability of non-tumorigenic HaCaT cells was not affected by similar doses of BlL. BlL-induced B16-F10 necrosis was preceded by a significant (2-fold) increase in cytosolic calcium concentrations and a significant (3-fold) increase in mitochondrial superoxide generation. It is likely that BlL treatment triggers B16-F10 cell death via mitochondrial permeability transition (MPT) pore opening because the pharmacological MPT inhibitors bongkrekic acid and Debio 025 greatly attenuated BlL-induced cell death. Experiments evaluating mitochondrial Ca2+ transport in permeabilized B16-F10 cells strongly supported the hypothesis that BlL rapidly stimulates cyclosporine A-sensitive Ca2+-induced MPT pore opening. We therefore conclude that BlL causes selective B16-F10 melanoma cell death via dysregulation of cellular Ca2+ homeostasis and Ca2+-induced opening of MPT pore.

Reply to “Letter to the editor: ‘Cyclosporin A in left ventricular remodeling after myocardial infarction’”

Reply to “Letter to the editor: ‘Cyclosporin A in left ventricular remodeling after myocardial infarction’”

Ethylmalonic Acid Induces Permeability Transition in Isolated Brain Mitochondria

Predominant accumulation of ethylmalonic acid (EMA) in tissues and biological fluids is a characteristic of patients affected by short chain acyl-CoA dehydrogenase deficiency and ethylmalonic encephalopathy. Neurological abnormalities are frequently found in these disorders, but the mechanisms underlying the brain injury are still obscure. Since hyperlacticacidemia is also found in many affected patients indicating a mitochondrial dysfunction; in the present work, we evaluated the in vitro and ex vivo effects of EMA plus Ca(2+) on mitochondrial integrity and redox balance in succinate-supported brain organelles. We verified that the evaluated parameters were disturbed only when EMA was associated with exogenous micromolar Ca(2+) concentrations. Thus, we found that this short chain organic acid plus Ca(2+) dissipated the membrane potential and provoked mitochondrial swelling, as well as impaired the mitochondrial Ca(2+) retention capacity, resulting in a rapid Ca(2+) release and decreased NAD(P)H matrix content. In contrast, EMA was not able to stimulate mitochondrial hydrogen peroxide generation. We also observed that all these effects were prevented by the mitochondrial Ca(2+) uptake inhibitor ruthenium red and the mitochondrial permeability transition (MPT) inhibitors cyclosporin A (CsA) and ADP. Furthermore, mitochondria isolated from rat brains after in vivo intrastriatal administration of EMA was more susceptible to Ca(2+)-induced swelling, which was fully prevented by CsA and ADP. Finally, EMA significantly decreased striatal slice viability, which was attenuated by CsA. The data strongly indicate that EMA reduced the mitochondrial threshold for Ca(2+)-induced MPT reinforcing the role of this cation in EMA-induced disruption of mitochondrial bioenergetics. It is, therefore, presumed that EMA acting synergistically with Ca(2+) compromises mitochondrial energy homeostasis in the central nervous system that may explain at least in part the neurologic alterations presented by patients with abnormal levels of this organic acid.

Traditional chinese medicine shuang shen ning xin attenuates myocardial ischemia/reperfusion injury by preserving of mitochondrial function.

To investigate the potential cardioprotective effects of Shuang Shen Ning Xin on myocardial ischemia/reperfusion injury. Wistar rats were treated with trimetazidine (10 mg/kg/day, ig), Shuang Shen Ning Xin (22.5, 45 mg/kg/day, ig), or saline for 5 consecutive days. Myocardial ischemia/reperfusion injury was induced by ligation of the left anterior descending coronary artery for 40 min and reperfusion for 120 min on the last day of administration. It is found that Shuang Shen Ning Xin pretreatment markedly decreased infarct size and serum LDH levels, and this observed protection was associated with reduced myocardial oxidative stress and cardiomyocyte apoptosis after myocardial ischemia/reperfusion injury. In addition, further studies on mitochondrial function showed that rats treated with Shuang Shen Ning Xin displayed decreased mitochondrial swelling and cytosolic cytochrome c levels, which were accompanied by a preservation of complex I activities and inhibition of mitochondrial permeability transition. In conclusion, the mitochondrial protective effect of Shuang Shen Ning Xin could be a new mechanism, by which Shuang Shen Ning Xin attenuates myocardial ischemia/reperfusion injury.

Evaluation of Three-Dimensional Cultured HepG2 Cells in a Nano Culture Plate System: an In Vitro Human Model of Acetaminophen Hepatotoxicity

Overdoses of acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) cause severe liver injury, yet there is no common or high throughput in vitro human APAP model. This study examined the characteristics and usefulness of HepG2 cells grown in a nano culture plate (NCP) system, a three-dimensional culture method, as an in vitro human model for APAP-induced hepatotoxicity. The NCP-cultured HepG2 cells showed higher expression of mRNA and protein levels of cytochrome P450 2E1, which metabolizes APAP to a toxic metabolite, APAP-cysteine adduct formation, and higher sensitivity against APAP-induced cell injury compared with conventionally cultured cells. We demonstrated that treatment of APAP in NCP-cultured HepG2 cells shows key mechanistic features of APAP-induced hepatotoxicity, such as decreases in intracellular glutathione and mitochondrial membrane potential, activation of JNK, and cellular injury; and pharmacological agents, such as Cyclosporine A (a mitochondrial permeability transition inhibitor) and SP600125 (a JNK inhibitor), prevented cell injury induced by APAP exposure. In addition, the antidote of APAP-induced hepatotoxicity, N-acetylcysteine, could attenuate cellular injury induced by APAP in NCP-cultured HepG2 cells. We suggest that cellular injury induced by APAP treatment using an NCP-HepG2 system is a useful human model to study mechanisms and screen drug candidates of APAP-induced hepatotoxicity. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.13135FP]

Cinnamaldehyde-Induced Apoptosis in Human Hepatoma PLC/PRF/5 Cells Involves the Mitochondrial Death Pathway and is Sensitive to Inhibition by Cyclosporin A and z-VAD-fmk

Cinnamaldehyde (CIN) has been shown to exert chemopreventive activity against several types of human cancer cells. We previously reported that CIN induced apoptosis of human hepatoma PLC/PRF/5 cells and this effect was associated with activation of the pro-apoptotic Bcl-2 family of proteins and the MAPK cascade. To further clarify the underlying mechanism of CIN-induced apoptosis, we examined in this study its relationship with the mitochondrial death pathway using the mitochondrial permeability transition (MPT) inhibitor, cyclosporin A (CsA), and the general caspase inhibitor, z-VAD-fmk. Results indicated that CIN-induced apoptosis involved enhanced ROS generation, disruption of mitochondrial potential, and the mitochondrial release of cytochrome c and Smac/DIABLO into the cytosol, which in turn promoted caspase-3 to its active form and the subsequent cleavage of PARP. Treatment with CIN also downregulated protein levels of the anti-apoptotic factors XIAP and Bcl-2 with concomitant accumulation of the pro-apoptotic Bax in a timedependent manner. These mitochondria-related apoptotic effects induced by CIN were however blocked by CsA and z-VAD-fmk pretreatments, which prevented cells from undergoing programmed cell death triggered by CIN. Furthermore, the increase of Bax and decrease of Bcl-2 and XIAP protein expression due to CIN treatment were also reversely modulated by the two inhibitors. Taken together, these results suggested that CIN is an apoptotic inducer that acts on the mitochondrial death pathway in PLC/PRF/5 cells and its effect could be blocked by CsA and z-VAD-fmk.

Mitochondrial Dysfunction and Permeability Transition in Osteosarcoma Cells Showing the Warburg Effect

Metabolic reprogramming in cancer is manifested by persistent aerobic glycolysis and suppression of mitochondrial function and is known as the Warburg effect. The Warburg effect contributes to cancer progression and is considered to be a promising therapeutic target. Understanding the mechanisms used by cancer cells to suppress their mitochondria may lead to development of new approaches to reverse metabolic reprogramming. We have evaluated mitochondrial function and morphology in poorly respiring LM7 and 143B osteosarcoma (OS) cell lines showing the Warburg effect in comparison with actively respiring Saos2 and HOS OS cells and noncancerous osteoblastic hFOB cells. In LM7 and 143B cells, we detected markers of the mitochondrial permeability transition (MPT), such as mitochondrial swelling, depolarization, and membrane permeabilization. In addition, we detected mitochondrial swelling in human OS xenografts in mice and archival human OS specimens using electron microscopy. The MPT inhibitor sanglifehrin A reversed MPT markers and increased respiration in LM7 and 143B cells. Our data suggest that the MPT may play a role in suppression of mitochondrial function, contributing to the Warburg effect in cancer.

Histones-Mediated Lymphocyte Apoptosis during Sepsis Is Dependent on p38 Phosphorylation and Mitochondrial Permeability Transition

Lymphocyte apoptosis is one reason for immunoparalysis seen in sepsis, although the triggers are unknown. We hypothesized that molecules in plasma, which are up-regulated during sepsis, may be responsible for this. In this study, peripheral lymphocyte apoptosis caused by extracellular histones was confirmed both in mouse and human primary lymphocytes, in which histones induced lymphocyte apoptosis dose-dependently and time-dependently. To identify which intracellular signal pathways were activated, phosphorylation of various mitogen-activated protein kinases (MAPKs) were evaluated during this process, and p38 inhibitor (SB203580) was used to confirm the role of p38 in lymphocyte apoptosis induced by histones. To investigate the mitochondrial injury during these processes, we analyzed Bcl2 degradation and Rhodamine 123 to assess mitochondrial-membrane stability, via cyclosporin A as an inhibitor for mitochondrial permeability transition (MPT). Then, caspase 3 activation was also checked by western-blotting. We found that p38 phosphorylation, mitochondrial injury and caspase 3 activation occurred dose-dependently in histones-mediated lymphocyte apoptosis. We also observed that p38 inhibitor SB203580 decreased lymphocyte apoptotic ratio by 49% (P<0.05), and inhibition of MPT protected lymphocytes from apoptosis. Furthermore, to investigate whether histones are responsible for lymphocyte apoptosis, various concentrations of histone H4 neutralization antibodies were co-cultured with human primary lymphocytes and plasma from cecal ligation and puncture (CLP) mice or sham mice. The results showed that H4 neutralization antibody dose-dependently blocked lymphocyte apoptosis caused by septic plasma in vitro. These data demonstrate for the first time that extracellular histones, especially H4, play a vital role in lymphocyte apoptosis during sepsis which is dependent on p38 phosphorylation and mitochondrial permeability transition. Neutralizing H4 can inhibit lymphocyte apoptosis, indicating that it could be a potential target in clinical interventions for sepsis associated immunoparalysis.

Role of mitochondrial permeability transition in human hepatocellular carcinoma Hep-G2 cell death induced by rhein

Rhein, a compound found as a glucoside in the root of rhubarb, is currently a subject of interest for its antitumor properties. The apoptosis of tumor cell lines induced by rhein was observed, and the involvement of mitochondria was established; however, the role of mitochondrial permeability transition (MPT) remains unknown. Here we report that MPT plays an important role in the apoptosis of human hepatocellular carcinoma Hep-G2 cells induced by rhein. After adding rhein to the isolated hepatic mitochondria, swelling effects and the leakage of Ca2+ were observed. These alterations were suppressed by cyclosporin A (CsA), an MPT inhibitor. Furthermore, in Hep-G2 cells, the decrease of ATP production, the loss of mitochondrial transmembrane potential (MTP), the release of cytochrome c (Cyto c), and the activation of caspase 3 were also observed. These toxic effects of rhein can also be attenuated by CsA as well. Moreover, TUNEL assay confirmed that in the presence of CsA, rhein-induced apoptosis was largely inhibited. These results suggest that MPT plays a critical role in the pathogenesis of Hep-G2 cell injury induced by rhein, and imply that MPT may contribute to the anti-cancer activity of rhein.

GW24-e3512 Pharmacological postconditioning with tanshinone IIA attenuates myocardial ischaemia-reperfusion injury in rats

ObjectivesTanshinone IIA, one of the active ingredients in danshen, which derived from the dried root or rhizome of Salviae miltiorrhizae BGE, is cardioprotective when applied before myocardial sustained ischaemia. This...

Two independent pathways of regulated necrosis mediate ischemia–reperfusion injury

Regulated necrosis (RN) may result from cyclophilin (Cyp)D-mediated mitochondrial permeability transition (MPT) and receptor-interacting protein kinase (RIPK)1-mediated necroptosis, but it is currently unclear whether there is one common pathway in which CypD and RIPK1 act in or whether separate RN pathways exist. Here, we demonstrate that necroptosis in ischemia-reperfusion injury (IRI) in mice occurs as primary organ damage, independent of the immune system, and that mice deficient for RIPK3, the essential downstream partner of RIPK1 in necroptosis, are protected from IRI. Protection of RIPK3-knockout mice was significantly stronger than of CypD-deficient mice. Mechanistically, in vivo analysis of cisplatin-induced acute kidney injury and hyperacute TNF-shock models in mice suggested the distinctness of CypD-mediated MPT from RIPK1/RIPK3-mediated necroptosis. We, therefore, generated CypD-RIPK3 double-deficient mice that are viable and fertile without an overt phenotype and that survived prolonged IRI, which was lethal to each single knockout. Combined application of the RIPK1 inhibitor necrostatin-1 and the MPT inhibitor sanglifehrin A confirmed the results with mutant mice. The data demonstrate the pathophysiological coexistence and corelevance of two separate pathways of RN in IRI and suggest that combination therapy targeting distinct RN pathways can be beneficial in the treatment of ischemic injury.

Effect of trifluoperazine on toxicity, HIF-1α induction and hepatocyte regeneration in acetaminophen toxicity in mice

Oxidative stress and mitochondrial permeability transition (MPT) are important mechanisms in acetaminophen (APAP) toxicity. The MPT inhibitor trifluoperazine (TFP) reduced MPT, oxidative stress, and toxicity in freshly isolated hepatocytes treated with APAP. Since hypoxia inducible factor-one alpha (HIF-1α) is induced very early in APAP toxicity, a role for oxidative stress in the induction has been postulated. In the present study, the effect of TFP on toxicity and HIF-1α induction in B6C3F1 male mice treated with APAP was examined. Mice received TFP (10mg/kg, oral gavage) prior to APAP (200mg/kg IP) and at 7 and 36h after APAP. Measures of metabolism (hepatic glutathione and APAP protein adducts) were comparable in the two groups of mice. Toxicity was decreased in the APAP/TFP mice at 2, 4, and 8h, compared to the APAP mice. At 24 and 48h, there were no significant differences in toxicity between the two groups. TFP lowered HIF-1α induction but also reduced the expression of proliferating cell nuclear antigen, a marker of hepatocyte regeneration. TFP can also inhibit phospholipase A2, and cytosolic and secretory PLA2 activity levels were reduced in the APAP/TFP mice compared to the APAP mice. TFP also lowered prostaglandin E2 expression, a known mechanism of cytoprotection. In summary, the MPT inhibitor TFP delayed the onset of toxicity and lowered HIF-1α induction in APAP treated mice. TFP also reduced PGE2 expression and hepatocyte regeneration, likely through a mechanism involving PLA2.