3-(2-4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3h)-quinazolinone and Reperfusion-Injury

Dynamin-related protein 1 (Drp1) mediates mitochondrial fission and triggers NLRP3 inflammasome activation. FK866 (a NAMPT inhibitor) exerts a neuroprotective effect in ischemia/reperfusion injury through the suppression of mitochondrial dysfunction. We explored the effects of FK866 on pyroptosis and inflammation mediated by Drp1 in a cardiac arrest/cardiopulmonary resuscitation (CA/CPR) rat model.. Healthy male Sprague-Dawley rats were subjected to 7 min CA by trans-esophageal electrical stimulation followed by CPR. The surviving rats were treated with FK866 (a selective inhibitor of NAMPT), Mdivi-1 (Drp1 inhibitor), FK866 + Mdivi-1, or vehicle and then underwent 24 h reperfusion. Hematoxylin and eosin staining and immunohistochemistry (to detect NSE) were used to evaluate brain injury. We performed immunofluorescent staining to analyze NLRP3 and GSDMD expression in microglia or astrocytes and western blot to determine expression of NLRP3, IL-1β, GSDMD, Drp1, and Mfn2. Transmission electron microscopy was used to observe mitochondria.. FK866 significantly decreased pathological damage to brain tissue, inhibited the activation of NLRP3 in microglia or astrocytes, downregulated the expression of NLRP3, IL-1β, GSDMD, p-Drp1 protein, upregulated Mfn2 and improve mitochondrial morphology.. Our results demonstrated that FK866 protects the brain against ischemia-reperfusion injury in rats after CA/CPR by inhibiting pyroptosis and inflammation mediated by Drp1.

Topics: Acrylamides; Animals; Anti-Inflammatory Agents; Brain; Cardiopulmonary Resuscitation; Disease Models, Animal; Dynamins; Inflammasomes; Inflammation Mediators; Male; Mitochondria; Neuroprotective Agents; NLR Family, Pyrin Domain-Containing 3 Protein; Piperidines; Pyroptosis; Quinazolinones; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction

Hepatic ischemia reperfusion (HIR) has been found to induce brain injury and cognitive dysfunction. Dynamin-related protein 1 (Drp1) mediated mitochondrial fission involves oxidative stress, apoptosis and several neurological diseases. In this study, we investigated whether Drp1 translocation to mitochondria was implicated in HIR induced hippocampus injury in young mice, and further detected the role of calcineurin in the regulation of mitochondrial dynamics. 2-week C57BL/6 mice were chosen to make HIR model. Western blot was used to detect mitochondrial dynamics regulating proteins in whole hippocampal tissues and extracted mitochondria. Transmission electron microscopy was used to observe mitochondrial morphology. TUNEL staining and ELISA (serum S100β/NSE concentrations) were used to evaluate neurons apoptosis and brain injury respectively. Drp1 inhibitor Mdivi-1 and calcineurin inhibitor FK506 were utilized to further confirm the role of Drp1 and calcineurin. Results showed that HIR affected mitochondrial dynamics in a fission-dominant manner with translocation of Drp1 to mitochondria in hippocampus of young mice. HIR induced increased expression of calcineurin and dephosphorylation of Drp1 at Ser637 in hippocampus. Treatment with Mdivi-1 and FK506 upregulated the phosphorylation of Drp1, inhibited Drp1 translocation to mitochondria, and alleviated mitochondrial fragmentation after HIR. What's more, Mdivi-1 and FK506 restrained cytochrome c release and cleaved caspase-3 expression, ameliorated hippocampal neurons apoptosis, and decreased serum S100β/NSE concentrations as well. These data suggest that calcineurin mediated Drp1 dephosphorylation and translocation to mitochondria play a crucial role in HIR induced mitochondrial fragmentation and neurons apoptosis in hippocampus.

Topics: Animals; Apoptosis; Calcineurin; Cytochromes c; Dynamins; Hippocampus; Liver; Mice; Mice, Inbred C57BL; Mitochondria, Liver; Mitochondrial Dynamics; Oxidative Stress; Phosphorylation; Quinazolinones; Reperfusion; Reperfusion Injury; Tacrolimus

Stroke significantly affects white matter in the brain by impairing axon function, which results in clinical deficits. Axonal mitochondria are highly dynamic and are transported via microtubules in the anterograde or retrograde direction, depending upon axonal energy demands. Recently, we reported that mitochondrial division inhibitor 1 (Mdivi-1) promotes axon function recovery by preventing mitochondrial fission only when applied during ischemia. Application of Mdivi-1 after injury failed to protect axon function. Interestingly, L-NIO, which is a NOS3 inhibitor, confers post-ischemic protection to axon function by attenuating mitochondrial fission and preserving mitochondrial motility via conserving levels of the microtubular adaptor protein Miro-2. We propose that preventing mitochondrial fission protects axon function during injury, but that restoration of mitochondrial motility is more important to promote axon function recovery after injury. Thus, Miro-2 may be a therapeutic molecular target for recovery following a stroke.

Topics: Adenosine Triphosphate; Aging; Animals; Axonal Transport; Axons; Calcium; Drug Evaluation, Preclinical; Humans; Hypoxia-Ischemia, Brain; Ischemic Stroke; Mice; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Nitric Oxide Synthase Type III; Ornithine; Quinazolinones; Reperfusion Injury; rho GTP-Binding Proteins; White Matter

The aim of this study is to determine the effects of mitochondrial division inhibitor 1 (Mdivi-1), the mitochondrial fission inhibitor, on the angiogenic profiles after the ischemia reperfusion injury (IR injury) in female mice. Female mice were treated with Mdivi-1 inhibitor, 2 days prior, on the day of IR injury and 2 days after IR injury, for a period of 5 days. Both control and treatment groups underwent 30 min of ischemia and 72 h of reperfusion. On the day 3, mice were sacrificed and the ischemic and nonischemic portions of heart tissue were collected. Relative levels of 53 angiogenesis-related proteins were quantified simultaneously using Angiogenic arrays. Heart function was evaluated before and after 72 h of IR injury. Mdivi-1 treatment ameliorated IR induced functional deterioration with positive angiogenic profile. The seminal changes include suppression of Matrix metalloproteinase (MMP3), tissue inhibitor of metalloproteases (TIMP1) and chemokine (C-X-C motif) ligand 10 (CXCL10) levels and prevention of connexin 43 (Cx43) loss and downregulation in the antioxidant enzyme levels. These changes are correlated with enhanced endothelial progenitor cell marker (cluster of differentiation (CD31), endothelial-specific receptor tyrosine kinase (Tek), fMS-like tyrosine kinase 4 (Flt4) and kinase insert domain protein receptor (Kdr)) presence. Our study is the first to report the role of mitochondrial dynamics in regulation of myocardial IR-induced angiogenic responses. Inhibition of excessive mitochondrial fission after IR injury ameliorated heart dysfunction and conferred positive angiogenic response. In addition, there were improvements in the preservation of Cx43 levels and oxidative stress handling along with suppression of apoptosis activation. The findings will aid in shaping the rational drug development process for the prevention of ischemic heart disease, especially in females.

Topics: Animals; Cell Line; Chemokine CXCL10; Connexin 43; Female; Heart; Mice; Mice, Inbred C57BL; Mitochondrial Dynamics; Myocardium; Neovascularization, Physiologic; Platelet Endothelial Cell Adhesion Molecule-1; Quinazolinones; Receptor, TIE-2; Reperfusion Injury; Tissue Inhibitor of Metalloproteinase-1; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factor Receptor-3

Cardiac stem cell (CSC) therapy is a promising approach to treat ischemic heart disease. However, the poor survival of transplanted stem cells in the ischemic myocardium has been a major impediment in achieving an effective cell-based therapy against myocardial infarction. Inhibiting mitochondrial fission has been shown to promote survival of several cell types. However, the role of mitochondrial morphology in survival of human CSC remains unknown. In this study, we investigated whether mitochondrial division inhibitor-1 (Mdivi-1), an inhibitor of mitochondrial fission protein dynamin-related protein-1 (Drp1), can improve survival of a novel population of human W8B2

Topics: Antigens, Surface; Apoptosis; Cell Death; Cells, Cultured; Humans; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Dynamics; Oxidative Stress; Protective Agents; Quinazolinones; Reactive Oxygen Species; Reperfusion Injury; Stem Cells

When given prior to brain ischemia, mitochondrial division inhibitor-1 (mdivi-1) attenuates the brain damage caused by ischemia. Here, we investigated the potential effects of post-ischemia mdivi-1 treatment (1mg/kg, i.p., administered immediately after 2h of ischemia and prior to reperfusion) using a MCAO rat model. Mdivi-1 treatment decreased infarct volume and improved neurological function. In addition, cytochrome C release was attenuated, and neuronal apoptosis was decreased. The mitochondrial fission protein dynamin-related protein 1 (Drp1) was decreased in the mitochondrial fraction but increased in the cytosolic fraction. Mdivi-1 treatment augmented the increases in the mRNA expression of peroxisome proliferator-activated receptor coactivator-1α, nuclear respiratory factor-1, and mitochondrial transcriptional factor A. In conclusion, when given after ischemia and prior to reperfusion, mdivi-1 can protect against brain damage by inhibiting the mitochondria-mediated apoptosis induced by mitochondrial fission. Post-ischemia mdivi-1 treatment might promote I/R-induced mitochondrial biogenesis.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cytochromes c; Disease Models, Animal; Dynamins; Male; Mitochondria; Mitochondrial Dynamics; Neurons; Neuroprotective Agents; Organelle Biogenesis; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Quinazolinones; Rats; Rats, Wistar; Reperfusion Injury

Mitochondrial division inhibitor (mdivi-1), a selective inhibitor of a mitochondrial fission protein dynamin-related protein 1 (Drp1), has been shown to exert protective effects in heart and cerebral ischemia-reperfusion models. The present study was designed to investigate the beneficial effects of mdivi-1 against spinal cord ischemia-reperfusion (SCIR) injury and its associated mechanisms. SCIR injury was induced by glutamate treatment in cultured spinal cord neurons and by descending thoracic aorta occlusion for 20 min in rats. We found that mdivi-1 (10 μM) significantly attenuated glutamate induced neuronal injury and apoptosis in spinal cord neurons. This neuroprotective effect was accompanied by decreased expression of oxidative stress markers, inhibited mitochondrial dysfunction and preserved activities of antioxidant enzymes. In addition, mdivi-1 significantly increased the expression of the large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels, and blocking BK channels by paxilline partly ablated mdivi-1 induced protection. The in vivo experiments showed that mdivi-1 treatment (1 mg/kg) overtly mitigated SCIR injury induced spinal cord edema and neurological dysfunction with no organ-related toxicity in rats. Moreover, mdivi-1 increased the expression of BK channels in spinal cord tissues, and paxilline pretreatment nullified mdivi-1 induced protection after SCIR injury in rats. Thus, mdivi-1 may be an effective therapeutic agent for SCIR injury via activation of BK channels as well as reduction of oxidative stress, mitochondrial dysfunction and neuronal apoptosis. This article is part of a Special Issue entitled SI: Spinal cord injury.

Topics: Animals; Apoptosis; Dynamins; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Mitochondria; Neurons; Oxidative Stress; Quinazolinones; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia

Apoptosis is one of the major mechanisms of neuronal injury during ischemic-reperfusion (I/R). Mitochondrial division inhibitor (mdivi-1) is a selective inhibitor of mitochondrial fission protein Drp1. The previous experiments support that mdivi-1 reduce I/R injury in the heart model of rat, but the neuroprotective effect of the mdivi-1 is not yet clearly defined at the cellular levels in brain. In our present study, we estimated a brain model of I/R injury in vitro by subjecting oxygen and glucose deprivation (OGD) followed by reoxygenation to the cultured rat primary hippocampal cells, which aimed to find the neuroprotective mechanism of mdivi-1. The cell was pretreated with mdivi-1 for 40 minutes and then ischemia for 6 hours followed by reperfusion for 20 hours. The redox state, cell apoptosis, and expression of Drp1, Bcl-2, Bax, and cytochrome C proteins were measured. The data showed that administration of mdivi-1 at the doses of 50 μM significantly reduced oxidative stress, attenuated cell apoptosis, upregulated Bcl-2 expression, and downregulated Drp1, Bax, and cytochrome C expression. The results suggested that mdivi-1 protected brain from OGD reperfusion injury, which through suppressing the ROS initiated mitochondrial pathway.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cells, Cultured; Cytochromes c; Dynamins; Hippocampus; Mitochondria; Neurons; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Quinazolinones; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury

Cerebral ischemia-reperfusion (I-R) is a complex pathological process. Although autophagy can be evoked by ischemia, its involvement in the reperfusion phase after ischemia and its contribution to the fate of neurons remains largely unknown. In the present investigation, we found that autophagy was activated in the reperfusion phase, as revealed in both mice with middle cerebral artery occlusion and oxygen-glucose deprived cortical neurons in culture. Interestingly, in contrast to that in permanent ischemia, inhibition of autophagy (by 3-methyladenine, bafilomycin A 1, Atg7 knockdown or in atg5(-/-) MEF cells) in the reperfusion phase reinforced, rather than reduced, the brain and cell injury induced by I-R. Inhibition of autophagy either with 3-methyladenine or Atg7 knockdown enhanced the I-R-induced release of cytochrome c and the downstream activation of apoptosis. Moreover, MitoTracker Red-labeled neuronal mitochondria increasingly overlapped with GFP-LC3-labeled autophagosomes during reperfusion, suggesting the presence of mitophagy. The mitochondrial clearance in I-R was reversed by 3-methyladenine and Atg7 silencing, further suggesting that mitophagy underlies the neuroprotection by autophagy. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. PARK2 translocated to mitochondria during reperfusion and Park2 knockdown aggravated ischemia-induced neuronal cell death. In conclusion, the results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis. PARK2 may be involved in the mitophagy process.

Topics: Adenine; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Brain Ischemia; Cytochromes c; Cytoprotection; Glucose; Hypoxia; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Mitochondria; Mitophagy; Neurons; Quinazolinones; Rats; Reperfusion Injury; Ubiquitin-Protein Ligases

Mitochondrial division inhibitor (mdivi-1) is a derivative of quinazolinone that acts as a selective inhibitor of a mitochondrial fission protein Drp1. A previous study demonstrated that as a selective inhibitor of Drp1, mdivi-1 has a protective effect in an experimental model of heart ischemia/reperfusion injury. In this study, we investigated the protective effects of mdivi-1 on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that mdivi-1 (1.2mg/kg) significantly reduced cerebral damage induced by ischemia/reperfusion. This neuroprotective effect was dose-dependent. Mdivi-1 treatment blocked apoptotic cell death in cerebral ischemia/reperfusion injury, and significantly decreased the expression of Drp1 and Cytochrome C. These results suggest that mdivi-1 exerts neuroprotective effects against nerve injury after cerebral ischemia/reperfusion, and the underlying mechanism may be through the prevention of Cytochrome C release and suppression of the mitochondrial apoptosis pathway.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cytochromes c; Dynamins; Infarction, Middle Cerebral Artery; Male; Neurons; Neuroprotective Agents; Quinazolinones; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger

To determine whether acute intraocular pressure (IOP) elevation alters dynamin-related protein 1 (Drp1) as well as whether a selective inhibitor of Drp1, mdivi-1, can block apoptotic cell death and subsequently increase retinal ganglion cell (RGC) survival in ischemic mouse retina.. C57BL/6 mice received injections of mdivi-1 (50 mg/kg) or vehicle, and then transient retinal ischemia was induced by acute IOP elevation. RGC survival was measured after FluoroGold labeling. Drp1 and glial fibrillary acidic protein (GFAP) protein expression and distribution were assessed at 12 hours after ischemia-reperfusion by Western blot and immunohistochemistry. Apoptotic cell death was assessed by TUNEL staining.. Drp1 and GFAP protein expression was significantly increased in the early neurodegenerative events (within 12 hours) of ischemic mouse retina. Mdivi-1 treatment blocked apoptotic cell death in ischemic retina, and significantly increased RGC survival at 2 weeks after ischemia. In the normal mouse retina, Drp1 is expressed in the ganglion cell layer (GCL) as well as the inner plexiform layer, the inner nuclear layer (INL), and the outer plexiform layer (OPL). In the GCL, Drp1 immunoreactivity was strong in RGCs. While Drp1 protein expression was increased in the GCL of vehicle-treated ischemic retina at 12 hours. Mdivi-1 treatment did not change this increase of Drp1 protein expression but significantly decreased GFAP protein expression.. These findings suggest that altered Drp1 activity after acute IOP elevation may be an important component of a biochemical cascade leading to RGC death in ischemic retina.

Topics: Acute Disease; Animals; Apoptosis; Blood Pressure; Blotting, Western; Body Weight; Cell Count; Cell Survival; Dynamins; Glial Fibrillary Acidic Protein; GTP Phosphohydrolases; Immunohistochemistry; In Situ Nick-End Labeling; Intraocular Pressure; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Nerve Tissue Proteins; Ocular Hypertension; Quinazolinones; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; Retinal Vessels