RESUMO
BACKGROUND: During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca2+ and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined. METHODS: Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca2+, ROS, and Fe2+ to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective. RESULTS: In the absence of Ca2+, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca2+ in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca2+ synergize to induce mPTP opening. Furthermore, this synergistic form of Ca2+- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone. CONCLUSIONS: In the present study, we have investigated the relationship between Ca2+ and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca2+ mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca2+ and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.
Assuntos
Peroxidação de Lipídeos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas , Mitocôndrias Hepáticas , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Traumatismo por Reperfusão Miocárdica , Espécies Reativas de Oxigênio , Animais , Peroxidação de Lipídeos/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Camundongos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/patologia , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Cálcio/metabolismo , Dilatação Mitocondrial/efeitos dos fármacosRESUMO
It has been reported that lipophilic statins such as atorvastatin can more readily penetrate into ß-cells and reach the mitochondria, resulting in mitochondrial dysfunction, oxidative stress, decrease in insulin release. Many studies have shown that natural products can protect mitochondrial dysfunction induced by drug in different tissue. We aimed to explore mitochondrial protection potency of hesperidin, vanillic acid, and sinapic acid as natural compounds against mitochondrial dysfunction induced by atorvastatin in pancreas isolated mitochondria. Mitochondria were isolated form rat pancreas and directly treated with toxic concentration of atorvastatin (500 µM) in presence of various concentrations hesperidin, vanillic acid, and sinapic acid (1, 10, and 100 µM) separately. Mitochondrial toxicity parameters such as the reactive oxygen species (ROS) formation, succinate dehydrogenases (SDH) activity, mitochondrial swelling, depletion of glutathione (GSH), mitochondrial membrane potential (MMP) collapse, and malondialdehyde (MDA) production were measured. Our findings demonstrated that atorvastatin directly induced mitochondrial toxicity at concentration of 500 µM and higher in pancreatic mitochondria. Except MDA, atorvastatin caused significantly reduction in SDH activity, mitochondrial swelling, ROS formation, depletion of GSH, and collapse of MMP. While, our data showed that all three protective compounds at low concentrations ameliorated atorvastatin-induced mitochondrial dysfunction with the increase of SDH activity, improvement of mitochondrial swelling, MMP collapse and mitochondrial GSH, and reduction of ROS formation. We can conclude that hesperidin, vanillic acid, and sinapic acid can directly reverse the toxic of atorvastatin in rat pancreas isolated mitochondria, which may be beneficial for protection against diabetogenic-induced mitochondrial dysfunction in pancreatic ß-cells.
Assuntos
Atorvastatina , Ácidos Cumáricos , Hesperidina , Potencial da Membrana Mitocondrial , Mitocôndrias , Dilatação Mitocondrial , Pâncreas , Espécies Reativas de Oxigênio , Ácido Vanílico , Animais , Atorvastatina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pâncreas/metabolismo , Ácidos Cumáricos/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Masculino , Dilatação Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ácido Vanílico/farmacologia , Hesperidina/farmacologia , Glutationa/metabolismo , Ratos Wistar , Succinato Desidrogenase/metabolismo , Malondialdeído/metabolismoRESUMO
Permeability transition pore (PTP) opening dissipates ion and electron gradients across the internal mitochondrial membrane (IMM), including excess Ca2+ in the mitochondrial matrix. After opening, immediate PTP closure must follow to prevent outer membrane disruption, loss of cytochrome c, and eventual apoptosis. Flickering, defined as the rapid alternative opening/closing of PTP, has been reported in heart, which undergoes frequent, large variations in Ca2+. In contrast, in tissues that undergo depolarization events less often, such as the liver, PTP would not need to be as dynamic and thus these tissues would not be as resistant to stress. To evaluate this idea, it was decided to follow the reversibility of the permeability transition (PT) in isolated murine mitochondria from two different tissues: the very dynamic heart, and the liver, which suffers depolarizations less frequently. It was observed that in heart mitochondria PT remained reversible for longer periods and at higher Ca2+ loads than in liver mitochondria. In all cases, Ca2+ uptake was inhibited by ruthenium red and PT was delayed by Cyclosporine A. Characterization of this phenomenon included measuring the rate of oxygen consumption, organelle swelling and Ca2+ uptake and retention. Results strongly suggest that there are tissue-specific differences in PTP physiology, as it resists many more Ca2+ additions before opening in a highly active organ such as the heart than in an organ that seldom suffers Ca2+ loading, such as the liver.
Assuntos
Cálcio , Mitocôndrias Cardíacas , Mitocôndrias Hepáticas , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Ratos Wistar , Animais , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Masculino , Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ratos , Consumo de Oxigênio , Fígado/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Ciclosporina/farmacologiaRESUMO
Mitochondrial dysfunction plays a key role in the development of neurodegenerative disorders. In contrast, the regulation of the endocannabinoid system has been shown to promote neuroprotection in different neurotoxic paradigms. The existence of an active form of the cannabinoid receptor 1 (CB1R) in mitochondrial membranes (mitCB1R), which might exert its effects through the same signaling mechanisms as the cell membrane CB1R, has been shown to regulate mitochondrial activity. Although there is evidence suggesting that some cannabinoids may induce protective effects on isolated mitochondria, substantial evidence on the role of cannabinoids in mitochondria remains to be explored. In this work, we developed a toxic model of mitochondrial dysfunction induced by exposure of brain mitochondria to the succinate dehydrogenase inhibitor 3-nitropropionic acid (3-NP). Mitochondria were also pre-incubated with the endogenous agonist anandamide (AEA) and the synthetic CB1R agonist WIN 55212-2 to evaluate their protective effects. Mitochondrial reduction capacity, reactive oxygen species (ROS) formation, and mitochondrial swelling were assessed as toxic markers. While 3-NP decreased the mitochondrial reduction capacity and augmented mitochondrial ROS formation and swelling, both AEA and WIN 55212-2 ameliorated these toxic effects. To explore the possible involvement of mitCB1R activation on the protective effects of AEA and WIN 55212-2, mitochondria were also pre-incubated in the presence of the selective CB1R antagonist AM281, which completely reverted the protective effects of the cannabinoids to levels similar to those evoked by 3-NP. These results show partial protective effects of cannabinoids, suggesting that mitCB1R activation may be involved in the recovery of compromised mitochondrial activity, related to reduction of ROS formation and further prevention of mitochondrial swelling.
Assuntos
Ácidos Araquidônicos , Benzoxazinas , Encéfalo , Endocanabinoides , Mitocôndrias , Morfolinas , Naftalenos , Fármacos Neuroprotetores , Nitrocompostos , Alcamidas Poli-Insaturadas , Propionatos , Ratos Wistar , Espécies Reativas de Oxigênio , Animais , Nitrocompostos/toxicidade , Propionatos/farmacologia , Propionatos/toxicidade , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Benzoxazinas/farmacologia , Ácidos Araquidônicos/farmacologia , Morfolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Alcamidas Poli-Insaturadas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Masculino , Fármacos Neuroprotetores/farmacologia , Naftalenos/farmacologia , Dilatação Mitocondrial/efeitos dos fármacos , Ratos , Receptor CB1 de Canabinoide/metabolismoRESUMO
Vasogenic brain edema, a potentially life-threatening consequence following an acute ischemic stroke, is a major clinical problem. This research aims to explore the therapeutic benefits of nimodipine, a calcium channel blocker, in mitigating vasogenic cerebral edema and preserving blood-brain barrier (BBB) function in an ischemic stroke rat model. In this research, animals underwent the induction of ischemic stroke via a 60-min blockage of the middle cerebral artery and treated with a nonhypotensive dose of nimodipine (1 mg/kg/day) for a duration of five days. The wet/dry method was employed to identify cerebral edema, and the Evans blue dye extravasation technique was used to assess the permeability of the BBB. Furthermore, immunofluorescence staining was utilized to assess the protein expression levels of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). The study also examined mitochondrial function by evaluating mitochondrial swelling, succinate dehydrogenase (SDH) activity, the collapse of mitochondrial membrane potential (MMP), and the generation of reactive oxygen species (ROS). Post-stroke administration of nimodipine led to a significant decrease in cerebral edema and maintained the integrity of the BBB. The protective effects observed were associated with a reduction in cell apoptosis as well as decreased expression of MMP-9 and ICAM-1. Furthermore, nimodipine was observed to reduce mitochondrial swelling and ROS levels while simultaneously restoring MMP and SDH activity. These results suggest that nimodipine may reduce cerebral edema and BBB breakdown caused by ischemia/reperfusion. This effect is potentially mediated through the reduction of MMP-9 and ICAM-1 levels and the enhancement of mitochondrial function.
Assuntos
Barreira Hematoencefálica , Edema Encefálico , Bloqueadores dos Canais de Cálcio , AVC Isquêmico , Metaloproteinase 9 da Matriz , Nimodipina , Animais , Nimodipina/farmacologia , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Masculino , Ratos , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Modelos Animais de Doenças , Espécies Reativas de Oxigênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ratos Sprague-Dawley , Molécula 1 de Adesão Intercelular/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/complicações , Dilatação Mitocondrial/efeitos dos fármacos , Succinato Desidrogenase/metabolismoRESUMO
Abstract Purpose: To analyze the effect of methylene blue (MB) therapy during the liver ischemia-reperfusion injury (I/R) process. Methods: Thirty-five male Wistar rats were used, (70%) submitted to partial ischemia (IR) or not (NIR) (30%) were obtained from the same animal. These animals were divided into six groups: 1) Sham (SH), 2) Sham with MB (SH-MB); 3) I/R, submitted to 60 minutes of partial ischemia and 15 minutes of reperfusion; 4) NI/R, without I/R obtained from the same animal of group I/R; 5) I/R-MB submitted to I/R and MB and 6) NI/R-MB, without I/R. Mitochondrial function was evaluated. Osmotic swelling of mitochondria as well as the determination of malondialdehyde (MDA) was evaluated. Serum (ALT/AST) dosages were also performed. MB was used at the concentration of 15mg/kg, 15 minutes before hepatic reperfusion. Statistical analysis was done by the Mann Whitney test at 5%. Results: State 3 shows inhibition in all ischemic groups. State 4 was increased in all groups, except the I/R-MB and NI/R-MB groups. RCR showed a decrease in all I/R and NI/R groups. Mitochondrial osmotic swelling showed an increase in all I/R NI/R groups in the presence or absence of MB. About MDA, there was a decrease in SH values in the presence of MB and this decrease was maintained in the I/R group. AST levels were increased in all ischemic with or without MB. Conclusions: The methylene blue was not able to restore the mitochondrial parameters studied. Also, it was able to decrease lipid peroxidation, preventing the formation of reactive oxygen species.
Assuntos
Humanos , Animais , Masculino , Traumatismo por Reperfusão/prevenção & controle , Inibidores Enzimáticos/uso terapêutico , Fígado/irrigação sanguínea , Azul de Metileno/uso terapêutico , Consumo de Oxigênio , Aspartato Aminotransferases/sangue , Valores de Referência , Fatores de Tempo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismo , Reprodutibilidade dos Testes , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Ratos Wistar , Respiração Celular , Alanina Transaminase/sangue , Inibidores Enzimáticos/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Fígado/metabolismo , Malondialdeído/análise , Azul de Metileno/farmacologia , Dilatação Mitocondrial/efeitos dos fármacosRESUMO
PURPOSE: To assess the effect of two laser wavelengths, either separate or combined, on intact rat livers. METHOD: Nineteen male Wistar rats (200-300 g) were submitted to laser irradiation at 5 different sites on the liver surface.Wavelengths 660 and 780 nm were used, with a dose of irradiation of 60 J/cm2/site.The animals were divided into the groups:control (C) and animals irradiated with 660 nm laser (L1), with 780 nm laser (L2) or withboth wavelengths (L3).Mitochondrial function, mitochondrial swelling, and hepatocellular malondialdehyde (MDA) levels were determined.Data were analyzed by the Mann-Whitney test, with the level of significance set at 5%. RESULTS: There was a reduction of ADP-activated respiration (state 3) in group L1 compared to group C (p=0.0016), whereas the values of group L2 were similar to control.Group L3 also showed a reduction of state 3 (p=0.0159).There was a reduction of RCR in group L1 compared to control (p=0.0001) and to group L2 (p=0.0040).Mitochondrial swelling only differed between group L3 and control (p=0.0286).There was a increase in MDA levels in group L3 compared to control (p=0.0476) and to group L2 (p=0.0286) and in group L1 compared to group L2 (p=0.0132). CONCLUSION: Although laser irradiation reduced mitochondrial function,it did not interfere with the hepatocellular energy status.
Assuntos
Animais , Masculino , Mitocôndrias Hepáticas/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Lasers Semicondutores , Fígado/efeitos da radiação , Doses de Radiação , Espectrofotometria , Fatores de Tempo , Ratos Wistar , Terapia com Luz de Baixa Intensidade , Relação Dose-Resposta à Radiação , Malondialdeído/análise , Dilatação Mitocondrial/efeitos dos fármacosRESUMO
BACKGROUND: Despite manifold benefits of nanoparticles (NPs), less information on the risks of NPs to human health and environment has been studied. Cobalt oxide nanoparticles (Co3O4-NPs) have been reported to cause toxicity in several organisms. In this study, we have investigated the role of Co3O4-NPs in inducing phytotoxicity, cellular DNA damage and apoptosis in eggplant (Solanum melongena L. cv. Violetta lunga 2). To the best of our knowledge, this is the first report on Co3O4-NPs showing phytotoxicity in eggplant. RESULTS: The data revealed that eggplant seeds treated with Co3O4-NPs for 2 h at a concentration of 1.0 mg/ml retarded root length by 81.5 % upon 7 days incubation in a moist chamber. Ultrastructural analysis by transmission electron microscopy (TEM) demonstrated the uptake and translocation of Co3O4-NPs into the cytoplasm. Intracellular presence of Co3O4-NPs triggered subcellular changes such as degeneration of mitochondrial cristae, abundance of peroxisomes and excessive vacuolization. Flow cytometric analysis of Co3O4-NPs (1.0 mg/ml) treated root protoplasts revealed 157, 282 and 178 % increase in reactive oxygen species (ROS), membrane potential (APm) and nitric oxide (NO), respectively. Besides, the esterase activity in treated protoplasts was also found compromised. About 2.4-fold greater level of DNA damage, as compared to untreated control was observed in Comet assay, and 73.2 % of Co3O4-NPs treated cells appeared apoptotic in flow cytometry based cell cycle analysis. CONCLUSION: This study demonstrate the phytotoxic potential of Co3O4-NPs in terms of reduction in seed germination, root growth, greater level of DNA and mitochondrial damage, oxidative stress and cell death in eggplant. The data generated from this study will provide a strong background to draw attention on Co3O4-NPs environmental hazards to vegetable crops.
Assuntos
Óxidos/toxicidade , Dano ao DNA/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Cobalto/toxicidade , Solanum melongena/efeitos dos fármacos , Nanopartículas/toxicidade , Dilatação Mitocondrial/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxidos/metabolismo , Análise de Variância , Espécies Reativas de Oxigênio/metabolismo , Cobalto/metabolismo , Ensaio Cometa , Solanum melongena/metabolismo , Microscopia Eletrônica de Transmissão , Nanopartículas/metabolismo , Citometria de Fluxo , Dilatação Mitocondrial/fisiologiaRESUMO
Desequilíbrio/acúmulo de ferro tem sido implicado em injúria oxidativa associada a diversas doenças degenerativas tais como, hemocromatose hereditária, b-talassemia e ataxia de Friedreich. As mitocôndrias são particularmente sensíveis a estresse oxidativo induzido por ferro - um carregamento alto de ferro em mitocôndrias isoladas pode causar uma extensiva peroxidação lipídica e a permeabilização de membrana. Nesse estudo, nós detectamos e caracterizamos danos do DNA mitocondrial em mitocôndrias isoladas de fígado de rato, expostas ao complexo Fe2+-citrato, um dos complexos de baixo peso molecular. A intensa fragmentação do DNA foi induzida após a incubação das mitocôndrias com o complexo de ferro. A detecção de finais 3' de fosfoglicolato nas quebras de fitas de DNA mitocondrial pelo ensaio 32P-postlabeling sugere um envolvimento de radicais hidroxila na fragmentação do DNA induzido por complexo Fe2+-citrato. Os níveis elevados de 8-oxo-7,8-diidro-2'-desoxiguanosina também sugerem que o estresse oxidativo induzido por Fe2+-citrato causa danos no DNA mitocondrial. Em conclusão, nossos resultados mostram que a peroxidação lipídica mediada por ferro esteve associada com severos danos do DNA mitocondrial derivados de ataque direto das espécies reativas de oxigênio.