cytochrome-c-t and (melle-4)cyclosporin

Ischemic postconditioning (IPoC) modulates the reperfusion maneuver to mitigate ischemia-reperfusion (I/R) injury. This study aims to investigate the effects and protective mechanism of IPoC on intestinal I/R injury.. Intestinal I/R was induced by occluding the superior mesenteric artery for 30 min followed by reperfusion for 60 min on male Wistar rats. IPoC was elicited by three cycles of 30-sec reperfusion and reocclusion of superior mesenteric artery at the initiation of reperfusion. Carboxyatractyloside (CATR), a mitochondrial permeability transition pore (mPTP) opener, and N-methyl-4-isoleucine cyclosporine (NIM811), an mPTP inhibitor, were administered separately in selected groups. The serum and intestinal sections were collected for analysis.. IPoC and the administration of NIM811 significantly diminished the expression of intestinal-type fatty acid-binding protein and lactate dehydrogenase (3427±236.8 U/L for I/R, 1190.5±36.7 U/L for IPoC, 1399.3±295.6 U/L for I/R+NIM811, and 2002±370.9 IU/L for IPoC+CATR) in portal blood, the release of cytosolic cytochrome c, and the cleaved caspase 9 expression in intestinal mucosa after intestinal I/R injury (P<0.05). Histopathologically, IPoC and NIM811 mitigated mucosal damage after I/R as well (Chiu's score, 3.8±0.4 for I/R, 0.2±0.2 for IPoC, 0.4±0.2 for I/R+NIM811, and 4.2±0.2 for IPoC+CATR; apoptotic index, 59.5%±4.6% for I/R, 15.7%±15.7% for I/R+IPoC, 3.5%±3.5% for I/R+NIM811, and 67.1%±9.3% in IPoC+CATR). CATR negated the protection conferred by IPoC.. IPoC and NIM811 attenuate intestinal I/R injury. The addition of CATR negated the effects of IPoC, indicating that the protective mechanism of IPoC was associated with the modulation of mPTP opening.

Topics: Animals; Apoptosis; Atractyloside; Caspase 3; Cyclosporine; Cytochromes c; Disease Models, Animal; Enzyme Activation; Fatty Acid-Binding Proteins; Intestinal Mucosa; Intestine, Small; Ischemic Postconditioning; L-Lactate Dehydrogenase; Ligation; Male; Malondialdehyde; Mesenteric Artery, Superior; Mesenteric Vascular Occlusion; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Time Factors

We tested the effectiveness of ischemic postconditioning (iPoC) in mitigating ischemia-reperfusion (I/R) injury of liver and the mechanism involves inhibiting the opening of the mitochondrial permeability transition pore (mPTP).. iPoC, performed by three cycles of 1 min I/R of the liver, was tested on a partial liver I/R model on rats. The serum alanine transaminase levels, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, cytochrome c release, the formation of 4-hydroxy-2-nonnenal-modified proteins, and mitochondrial membrane potential (Δψm) were measured. Atractyloside (ATR) and NIM811, which modify the opening of mPTP, were administered in selected groups.. iPoC, and NIM811, diminished the elevation of serum alanine transaminase level after I/R injury (174.0±28.3 U/L for iPoC; 94.3±25.4 U/L for control+NIM811) when compared with others (416.3±16.7 U/L for control, 557.0±86.7 U/L for iPoC+ATR, P<0.05). The expressions of cytosolic cytochrome c after I/R injury were decreased in iPoC and control+NIM811 groups when compared with others. After I/R, the apoptosis and the 4-hydroxy-2-nonnenal-modified proteins were attenuated in iPoC group when compared (apoptotic counts/50 HPF: 723.3±98.7 for iPoC, 1274±201.2 for control, 1057.6±39 for iPoC+ATR, P<0.05). The Δψm measured by flow cytometry was better preserved in iPoC and NIM811 groups.. iPoC attenuated cell deaths after I/R injury of liver. The protective effects were negated by the addition of ATR--a mPTP opener--and mimicked by injection of NIM811--a mPTP opening inhibitor. The study indicated iPoC conferred protection by modulating mPTP.

Topics: Alanine Transaminase; Animals; Cyclosporine; Cytochromes c; Heme Oxygenase (Decyclizing); Ischemic Postconditioning; Liver; Male; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury

Massive hepatectomy (MHX) leads to failure of remnant livers. Excessive metabolic burden in remnant livers may cause mitochondrial dysfunction. This study investigated whether blockade of the mitochondrial permeability transition (MPT) with N-methyl-4-isoleucine cyclosporine (NIM811) improves the outcome of MHX.. Mice were gavaged with NIM811 (10 mg/kg before surgery and 5 mg/kg daily afterward) and underwent sham-operation or approximately 90% partial hepatectomy.. Serum alanine aminotransferase, necrosis, and apoptosis increased, respectively, to approximately 1200 U/L, 6.1%, and 7% after MHX. NIM811 decreased peak alanine aminotransferase release, necrosis, and apoptosis by 70%, 100%, and 42%, respectively. 5-Bromo-2'-deoxyuridine incorporation, proliferating cell nuclear antigen expression, and the remnant liver weights were all increased significantly by NIM811 treatment, indicating improved liver regeneration. NIM811 also blunted hyperbilirubinemia by 54%, increased serum albumin by 51%, and improved survival from 6% to 40% after MHX. Hepatic mitochondrial depolarization, cell death, and MPT were detected by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. Mitochondrial depolarization occurred in many viable hepatocytes (13 cells/high-power field), and nonviable hepatocytes increased slightly to approximately 1 cell/high-power field at 3 hr after MHX. Entry of calcein into mitochondria after MHX indicated MPT onset. Importantly, NIM811 decreased mitochondria depolarization by more than 60%, blocked MPT onset, and prevented cell death. Decreases of hepatic ATP, mitochondrial cytochrome c release, and caspase-3 activation after MHX were also partially blocked by NIM811.. NIM811 minimized liver injury and improved liver regeneration after MHX, at least in part, by preventing MPT onset and subsequent compromised energy supply and proapoptotic cytochrome c release.

Topics: Adenosine Triphosphate; Alanine Transaminase; Animals; Caspase 3; Cell Death; Cyclosporine; Cytochromes c; Fluoresceins; Hepatectomy; Liver; Liver Regeneration; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondrial Diseases; Organ Size; Proliferating Cell Nuclear Antigen

Acute liver failure is a devastating illness of various causes with considerable mortality. Hepatic stimulator substance (HSS) has been suggested for use as a protective agent against acute hepatic injury induced by chemical poisons because it has a variety of biological activities. However, the mechanism whereby HSS protects against hepatotoxins is poorly understood. In this study, we established a hepatic gene transfer system via hydrodynamic tail vein injection to deliver a naked plasmid containing the human HSS gene (hHSS) and analyzed HSS-mediated protection of the liver during fulminant hepatic failure (FHF) induced by D-galactosamine (D-gal) and lipopolysaccharide (LPS). The results showed that the reporter gene, enhanced green fluorescent protein, was efficiently expressed in the liver of BALB/c mice. Hydrodynamic-based transfection of hHSS yielded a 70% survival rate compared with 36.7% for the control group at 24 h after D-gal/LPS treatment. In addition, hHSS expression preserved liver morphology and function. It is noteworthy that hHSS hydrodynamic-based transfer ameliorated indices of the mitochondrial permeability transition (MPT) resulting from the toxic effects of d-gal/LPS on the liver such as mitochondrial swelling, mitochondrial transmembrane potential disruption, and cytochrome c translocation. Furthermore, mitochondrial morphology and ATP levels were maintained in hHSS-administered mice. HSS-mediated protection was similar to that observed with the MPT inhibitor N-methyl-4-isoleucine-cyclosporin (NIM811), indicating a possible role for HSS in the regulation of MPT. In conclusion, a single dose of hHSS plasmid protected mice from FHF, and this hepatoprotective effect seemed to correlate with the inhibition of MPT.

Topics: Adenosine Triphosphate; Animals; Blotting, Western; Cyclosporine; Cytochromes c; Cytosol; Energy Metabolism; Genetic Therapy; Green Fluorescent Proteins; Humans; Injections, Intravenous; Intercellular Signaling Peptides and Proteins; Liver; Liver Failure, Acute; Male; Membrane Potentials; Mice; Mice, Inbred BALB C; Mitochondria, Liver; Mitochondrial Swelling; Peptides; Permeability; Plasmids; Regional Blood Flow; Tail; Veins

In primary culture, hepatocytes dedifferentiate, and their cytoplasm undergoes remodeling. Here, our aim was to characterize changes of mitochondria during remodeling. Hepatocytes were cultured one to five days in complete serumcontaining Waymouth's medium. In rat hepatocytes loaded with MitoTracker Green (MTG), tetramethylrhodamine methylester (TMRM), and/or LysoTracker Red (LTR), confocal microscopy revealed that mitochondria number and mass decreased by approximately 50% between Day 1 and Day 3 of culture. As mitochondria disappeared, lysosomes/autophagosomes proliferated five-fold. Decreased mitochondrial content correlated with (a) decreased cytochrome c oxidase activity and mitochondrial number observed by electron microscopy and (b) a profound decrease of PGC-1alpha mRNA expression. By contrast, mtDNA content per cell remained constant from the first to the third day of culture, although ethidium bromide (de novo mtDNA synthesis inhibitor) caused mtDNA to decrease by half from the first to the third culture day. As mitochondria disappeared, their MTG label moved into LTR-labeled lysosomes, which was indicative of autophagic degradation. A multiwell fluorescence assay revealed a 2.5-fold increase of autophagy on Day 3 of culture, which was decreased by 3-methyladenine, an inhibitor of autophagy, and also by cyclosporin A and NIM811, both selective inhibitors of the mitochondrial permeability transition (MPT). These findings indicate that mitochondrial autophagy (mitophagy) and the MPT underlie mitochondrial remodeling in cultured hepatocytes.

Topics: Adenine; Amines; Animals; Autophagy; Biological Transport; Cell Compartmentation; Cells, Cultured; Cyclosporine; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex IV; Gene Expression Regulation; Hepatocytes; Lysosomes; Male; Mitochondria, Liver; Permeability; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phagosomes; Rats; Rats, Sprague-Dawley; RNA-Binding Proteins; RNA, Messenger; Transcription Factors

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.

Topics: Animals; Brain Infarction; Brain Ischemia; Calcium Signaling; Cell Death; Cyclosporine; Cytochromes c; Disease Models, Animal; Energy Metabolism; Male; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Inbred SHR; Reactive Oxygen Species; Treatment Outcome

Cyclosporin A (CsA) is a potent immunosuppressive drug shown to inhibit mitochondrial permeability transition (mPT). Although the therapeutic efficacy of CsA in traumatic brain injury is being investigated, CsA is highly neurotoxic and any neuroprotective effect in models of spinal cord injury (SCI) is unclear. NIM811 is a non-immunosuppressive CsA derivative that inhibits mPT, and is significantly less cytotoxic than CsA. Presently, we investigated the effects of NIM811 post-treatment on indices of apoptosis, lesion size, and tissue sparing at acute time-points following SCI. Adult rats received a "mild/moderate" contusion to the spinal cord, and were administered either 20 mg/kg NIM811 or vehicle by oral gavage 15 min later. One group of rats was euthanized at 1, 4, or 24 h post-injury, and the cytosolic levels of cytochrome c and fragmented DNA in the spinal cord were quantified. The remaining rats received an additional dose of NIM811 or vehicle at 24 h post-injury, and were euthanized on day 7 for morphometric assessments of the lesion and tissue spared. Control groups included rats that received sham surgery or no surgery. The results revealed that NIM811 post-treatment reduced the cytosolic levels of cytochrome c and fragmented DNA during the first 24 h following SCI. NIM811 also reduced the volume of the lesion, and enhanced the volumes of spared gray and white matter at 7 days post-injury. Together, these findings suggest that NIM811 treatment promoted tissue survival following SCI, in part, through inhibition of apoptotic mechanisms. This is the first study to demonstrate the therapeutic potential of NIM811 post-treatment in a model of acute SCI, and supports the need for continued investigation into NIM811 as a neuroprotective treatment for human SCI.

Topics: Animals; Apoptosis; Cyclosporine; Cytochromes c; Cytosol; DNA Fragmentation; Enzyme-Linked Immunosorbent Assay; Female; Image Processing, Computer-Assisted; Neuroprotective Agents; Rats; Rats, Long-Evans; Spinal Cord Injuries; Time