RESUMO
Nitric oxide (NO) synthesized by eNOS plays a key role in regulation of endothelial barrier integrity but underlying cell signaling pathway is not fully understood at present. Here, we report opposing roles of two different redox-dependent NO metabolites; peroxynitrite (ONOO-) vs. S-nitrosoglutathione (GSNO), in cell signaling pathways for endothelial barrier disruption. In cultured human brain microvessel endothelial cells (hBMVECs), thrombin induced F-actin stress fiber formation causes barrier disruption via activating eNOS. Thrombin induced eNOS activity participated in cell signaling (e.g. RhoA and calcium influx mediated phosphorylation of myosin light chain) for F-actin stress fiber formation by increasing ONOO- levels. On the other hand, thrombin had no effect on intracellular levels of S-nitrosoglutathione (GSNO), another cellular NO metabolite. However, exogenous GSNO treatment attenuated the thrombin-induced cell signaling pathways for endothelial barrier disruption, thus suggesting the role of a shift of NO metabolism (GSNO vs. ONOO-) toward ONOO- synthesis in cell signaling for endothelial barrier disruption. Consistent with these in vitro studies, in animal models of traumatic brain injury and experimental autoimmune encephalomyelitis (EAE), ONOO- scavenger treatment as well as GSNO treatment were effective for attenuation of BBB leakage, edema formation, and CNS infiltration of mononuclear cells. Taken together, these data document that eNOS-mediated NO production and following redox-dependent NO metabolites (ONOO- vs. GSNO) are potential therapeutic target for CNS microvascular disease (traumatic and inflammatory) pathologies.
Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Células Endoteliais/metabolismo , Inflamação/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais , Células Cultivadas , Humanos , OxirreduçãoRESUMO
BACKGROUND: The nitric oxide (NO)-producing activity of endothelial nitric oxide synthase (eNOS) plays a significant role in maintaining endothelial function and protecting against the stroke injury. However, the activity of the eNOS enzyme and the metabolism of major NO metabolite S-nitrosoglutathione (GSNO) are dysregulated after stroke, causing endothelial dysfunction. We investigated whether an administration of exogenous of GSNO or enhancing the level of endogenous GSNO protects against neurovascular injury in wild-type (WT) and eNOS-null (endothelial dysfunction) mouse models of cerebral ischemia-reperfusion (IR). METHODS: Transient cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO) for 60 minutes in male adult WT and eNOS null mice. GSNO (0.1 mg/kg body weight, intravenously) or N6022 (GSNO reductase inhibitor, 5.0 mg/kg body weight, intravenously) was administered 30 minutes before MCAO in preinjury and at the reperfusion in postinjury studies. Brain infarctions, edema, and neurobehavioral functions were evaluated at 24 hours after the reperfusion. RESULTS: eNOS-null mice had a higher degree (P< .05) of injury than WT. Pre- or postinjury treatment with either GSNO or N6022 significantly reduced infarct volume, improved neurological and sensorimotor function in both WT and eNOS-null mice. CONCLUSION: Reduced brain infarctions and edema, and improved neurobehavioral functions by pre- or postinjury GSNO treatment of eNOS knock out mice indicate that GSNO can attenuate IR injury, likely by mimicking the eNOS-derived NO-dependent anti-ischemic and anti-inflammatory functions. Neurovascular protection by GSNO/N6022 in both pre- and postischemic injury groups support GSNO as a promising drug candidate for the prevention and treatment of stroke injury.
Assuntos
Álcool Desidrogenase/antagonistas & inibidores , Benzamidas/farmacologia , Encéfalo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Pirróis/farmacologia , S-Nitrosoglutationa/farmacologia , Álcool Desidrogenase/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/patologia , Edema Encefálico/enzimologia , Edema Encefálico/patologia , Edema Encefálico/prevenção & controle , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genéticaRESUMO
Traumatic brain injury (TBI) is the major cause of physical disability and emotional vulnerability. Treatment of TBI is lacking due to its multimechanistic etiology, including derailed mitochondrial and cellular energy metabolism. Previous studies from our laboratory show that an endogenous nitric oxide (NO) metabolite S-nitrosoglutathione (GSNO) provides neuroprotection and improves neurobehavioral function via anti-inflammatory and anti-neurodegenerative mechanisms. To accelerate the rate and enhance the degree of recovery, we investigated combining GSNO with caffeic acid phenethyl ester (CAPE), a potent antioxidant compound, using a male mouse model of TBI, controlled cortical impact in mice. The combination therapy accelerated improvement of cognitive and depressive-like behavior compared with GSNO or CAPE monotherapy. Separately, both GSNO and CAPE improved mitochondrial integrity/function and decreased oxidative damage; however, the combination therapy had greater effects on Drp1 and MnSOD. Additionally, while CAPE alone activated AMPK, this activation was heightened in combination with GSNO. CAPE treatment of normal animals also significantly increased the expression levels of pAMPK, pACC (activation of AMPK substrate ACC), and pLKB1 (activation of upstream to AMPK kinase LKB1), indicating that CAPE activates AMPK via LKB1. These results show that while GSNO and CAPE provide neuroprotection and improve functional recovery separately, the combination treatment invokes greater recovery by significantly improving mitochondrial functions and activating the AMPK enzyme. Both GSNO and CAPE are in human consumption without any known adverse effects; therefore, a combination therapy-based multimechanistic approach is worthy of investigation in human TBI.
Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Ácidos Cafeicos/farmacologia , Álcool Feniletílico/análogos & derivados , S-Nitrosoglutationa/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoácido Oxirredutases/metabolismo , Animais , Antioxidantes/metabolismo , Escala de Avaliação Comportamental , Lesões Encefálicas Traumáticas/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Álcool Feniletílico/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: Spinal cord injury (SCI) is one of the leading causes of disability and chronic pain. In SCI-induced pathology, homeostasis of the nitric oxide (NO) metabolome is lost. Major NO metabolites such as S-nitrosoglutathione (GSNO) and peroxynitrite are reported to play pivotal roles in regulating the activities of key cysteine proteases, calpains. While peroxynitrite (a metabolite of NO and superoxide) up regulates the activities of calpains leading to neurodegeneration, GSNO (a metabolite of NO and glutathione) down regulates the activities of calpains leading to neuroprotection. In this study, effect of GSNO on locomotor function and pain threshold and their relationship with the levels of peroxynitrite and the activity of calpain in the injured spinal cord were investigated using a 2-week rat model of contusion SCI. RESULTS: SCI animals were initially treated with GSNO at 2 h after the injury followed by a once daily dose of GSNO for 14 days. Locomotor function was evaluated by "Basso Beattie and Bresnahan (BBB) locomotor rating scale" and pain by mechanical allodynia. Peroxynitrite level, as expression of 3-nitrotyrosine (3-NT), calpain activity, as the degradation products of calpain substrate alpha II spectrin, and nNOS activity, as the expression phospho nNOS, were measured by western blot analysis. Treatment with GSNO improved locomotor function and mitigated pain. The treatment also reduced the levels of peroxynitrite (3-NT) and decreased activity of calpains. Reduced levels of peroxynitrite resulted from the GSNO-mediated inhibition of aberrant activity of neuronal nitric oxide synthase (nNOS). CONCLUSIONS: The data indicates that higher levels of 3-NT and aberrant activities of nNOS and calpains correlated with SCI pathology and functional deficits. Treatment with GSNO improved locomotor function and mitigated mechanical allodynia acutely post-injury. Because GSNO shows potential to ameliorate experimental SCI, we discuss implications for GSNO therapy in clinical SCI research.
Assuntos
Calpaína/metabolismo , Nitrosoguanidinas/farmacologia , Ácido Peroxinitroso/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo I/efeitos dos fármacos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Phospholipase A2 (PLA2)-derived proinflammatory lipid mediators such as prostaglandin E2 (PGE2), leukotrienes B4 (LTB4), lysophosphatidylcholine (LPC), and free fatty acids (FFA) are implicated in spinal cord injury (SCI) pathologies. Reducing the levels of these injurious bioactive lipid mediators is reported to ameliorate SCI. However, the specific role of the group IVA isoform of PLA2 cytosolic PLA2 (cPLA2) in lumbar spinal canal stenosis (LSS) due to cauda equina compression (CEC) injury is not clear. In this study, we investigated the role of cPLA2 in a rat model of CEC using a non-toxic cPLA2-preferential inhibitor, arachidonyl trifluoromethyl ketone (ATK). METHODS: LSS was induced in adult female rats by CEC procedure using silicone blocks within the epidural spaces of L4 to L6 vertebrae. cPLA2 inhibitor ATK (7.5 mg/kg) was administered by oral gavage at 2 h following the CEC. cPLA2-derived injurious lipid mediators and the expression/activity of cPLA2, 5-lipoxygenase (5-LOX), and cyclooxygenase-2 (COX-2) were assessed. ATK-treated (CEC + ATK) were compared with vehicle-treated (CEC + VEH) animals in terms of myelin levels, pain threshold, and motor function. RESULTS: ATK treatment of CEC animals reduced the phosphorylation of cPLA2 (pcPLA2) determined by Western blot, improved locomotor function evaluated by rotarod task, and reduced pain threshold evaluated by mechanical hyperalgesia method. Levels of FFA and LPC, along with PGE2 and LTB4, were reduced in CEC + ATK compared with CEC + VEH group. However, ATK treatment reduced neither the activity/expression of 5-LOX nor the expression of COX-2 in CEC + VEH animals. Increased cPLA2 activity in the spinal cord from CEC + VEH animals correlated well with decreased spinal cord as well as cauda equina fiber myelin levels, which were restored after ATK treatment. CONCLUSION: The data indicate that cPLA2 activity plays a significant role in tissue injury and pain after LSS. Reducing the levels of proinflammatory and tissue damaging eicosanoids and the deleterious lipid mediator LPC shows therapeutic potential. ATK inhibits cPLA2 activity, thereby decreasing the levels of injurious lipid mediators, reducing pain, improving functional deficits, and conferring protection against LSS injury. Thus, it shows potential for preclinical evaluation in LSS.
Assuntos
Ácidos Araquidônicos/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Polirradiculopatia/tratamento farmacológico , Administração Oral , Análise de Variância , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Feminino , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Leucotrieno B4/metabolismo , Locomoção/efeitos dos fármacos , Lisofosfatidilcolinas/metabolismo , Nociceptividade/efeitos dos fármacos , Polirradiculopatia/complicações , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Stroke immediately sets into motion sustained excitotoxicity and calcium dysregulation, causing aberrant activity in neuronal nitric oxide synthase (nNOS) and an imbalance in the levels of nitric oxide (NO). Drugs targeting nNOS-originated toxicity may therefore reduce stroke-induced damage. Recently, we observed that a redox-modulating agent of the NO metabolome, S-nitrosoglutathione (GSNO), confers neurovascular protection by reducing the levels of peroxynitrite, a product of aberrant NOS activity. We therefore investigated whether GSNO-mediated neuroprotection and improved neurological functions depend on blocking nNOS/peroxynitrite-associated injurious mechanisms using a rat model of cerebral ischemia reperfusion (IR). RESULTS: IR increased the activity of nNOS, the levels of neuronal peroxynitrite and phosphorylation at Ser(1412) of nNOS. GSNO treatment of IR animals decreased IR-activated nNOS activity and neuronal peroxynitrite levels by reducing nNOS phosphorylation at Ser(1412). The Ser(1412) phosphorylation is associated with increased nNOS activity. Supporting the notion that nNOS activity and peroxynitrite are deleterious following IR, inhibition of nNOS by its inhibitor 7-nitroindazole or reducing peroxynitrite by its scavenger FeTPPS decreased IR injury. GSNO also decreased the activation of AMP Kinase (AMPK) and its upstream kinase LKB1, both of which were activated in IR brain. AMPK has been implicated in nNOS activation via Ser(1412) phosphorylation. To determine whether AMPK activation is deleterious in the acute phase of IR, we treated animals after IR with AICAR (an AMPK activator) and compound c (an AMPK inhibitor). While AICAR potentiated, compound c reduced the IR injury. CONCLUSIONS: Taken together, these results indicate an injurious nNOS/peroxynitrite/AMPK cycle following stroke, and GSNO treatment of IR inhibits this vicious cycle, resulting in neuroprotection and improved neurological function. GSNO is a natural component of the human body, and its exogenous administration to humans is not associated with any known side effects. Currently, the FDA-approved thrombolytic therapy suffers from a lack of neuronal protective activity. Because GSNO provides neuroprotection by ameliorating stroke's initial and causative injuries, it is a candidate of translational value for stroke therapy.
Assuntos
Adenilato Quinase/metabolismo , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Ácido Peroxinitroso/metabolismo , S-Nitrosoglutationa/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Quinases Proteína-Quinases Ativadas por AMP , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
The spleen is an important mediator of both adaptive and innate immunity. As such, attempts to modulate the immune response provided by the spleen may be conducive to improved outcomes for numerous diseases throughout the body. Here, biomimicry is used to rationally design nanomaterials capable of splenic retention and immunomodulation for the treatment of disease in a distant organ, the postinfarct heart. Engineered senescent erythrocyte-derived nanotheranostic (eSENTs) are generated, demonstrating significant uptake by the immune cells of the spleen including T and B cells, as well as monocytes and macrophages. When loaded with suberoylanilide hydroxamic acid (SAHA), the nanoagents exhibit a potent therapeutic effect, reducing infarct size by 14% at 72 h postmyocardial infarction when given as a single intravenous dose 2 h after injury. These results are supportive of the hypothesis that RBC-derived biomimicry may provide new approaches for the targeted modulation of the pathological processes involved in myocardial infarction, thus further experiments to decisively confirm the mechanisms of action are currently underway. This novel concept may have far-reaching applicability for the treatment of a number of both acute and chronic conditions where the immune responses are either stimulated or suppressed by the splenic (auto)immune milieu.
Assuntos
Biomimética , Infarto do Miocárdio , Humanos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/patologia , Coração , Imunidade Inata , ImunomodulaçãoRESUMO
The hallmark of stroke injury is endothelial dysfunction leading to blood-brain barrier (BBB) leakage and edema. Among the causative factors of BBB disruption are accelerating peroxynitrite formation and the resultant decreased bioavailability of nitric oxide (NO). S-nitrosoglutathione (GSNO), an S-nitrosylating agent, was found not only to reduce the levels of peroxynitrite but also to protect the integrity of BBB in a rat model of cerebral ischemia and reperfusion (IR). A treatment with GSNO (3 µmol/kg) after IR reduced 3-nitrotyrosine levels in and around vessels and maintained NO levels in brain. This mechanism protected endothelial function by reducing BBB leakage, increasing the expression of Zonula occludens-1 (ZO-1), decreasing edema, and reducing the expression of matrix metalloproteinase-9 and E-selectin in the neurovascular unit. An administration of the peroxynitrite-forming agent 3-morpholino sydnonimine (3 µmol/kg) at reperfusion increased BBB leakage and decreased the expression of ZO-1, supporting the involvement of peroxynitrite in BBB disruption and edema. Mechanistically, the endothelium-protecting action of GSNO was invoked by reducing the activity of nuclear factor kappa B and increasing the expression of S-nitrosylated proteins. Taken together, the results support the ability of GSNO to improve endothelial function by reducing nitroxidative stress in stroke.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Infarto da Artéria Cerebral Média/patologia , Fármacos Neuroprotetores/farmacologia , Ácido Peroxinitroso/metabolismo , S-Nitrosoglutationa/farmacologia , Animais , Barreira Hematoencefálica/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Selectina E/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Azul Evans , Lateralidade Funcional , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas , Fatores de Tempo , Tirosina/análogos & derivados , Tirosina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Quinase Induzida por NF-kappaBRESUMO
BACKGROUND: Traumatic brain injury (TBI) induces primary and secondary damage in both the endothelium and the brain parenchyma, collectively termed the neurovascular unit. While neurons die quickly by necrosis, a vicious cycle of secondary injury in endothelial cells exacerbates the initial injury in the neurovascular unit following TBI. In activated endothelial cells, excessive superoxide reacts with nitric oxide (NO) to form peroxynitrite. Peroxynitrite has been implicated in blood brain barrier (BBB) leakage, altered metabolic function, and neurobehavioral impairment. S-nitrosoglutathione (GSNO), a nitrosylation-based signaling molecule, was reported not only to reduce brain levels of peroxynitrite and oxidative metabolites but also to improve neurological function in TBI, stroke, and spinal cord injury. Therefore, we investigated whether GSNO promotes the neurorepair process by reducing the levels of peroxynitrite and the degree of oxidative injury. METHODS: TBI was induced by controlled cortical impact (CCI) in adult male rats. GSNO or 3-Morpholino-sydnonimine (SIN-1) (50 µg/kg body weight) was administered orally two hours following CCI. The same dose was repeated daily until endpoints. GSNO-treated (GSNO group) or SIN-1-treated (SIN-1 group) injured animals were compared with vehicle-treated injured animals (TBI group) and vehicle-treated sham-operated animals (Sham group) in terms of peroxynitrite, NO, glutathione (GSH), lipid peroxidation, blood brain barrier (BBB) leakage, edema, inflammation, tissue structure, axon/myelin integrity, and neurotrophic factors. RESULTS: SIN-1 treatment of TBI increased whereas GSNO treatment decreased peroxynitrite, lipid peroxides/aldehydes, BBB leakage, inflammation and edema in a short-term treatment (4-48 hours). GSNO also reduced brain infarctions and enhanced the levels of NO and GSH. In a long-term treatment (14 days), GSNO protected axonal integrity, maintained myelin levels, promoted synaptic plasticity, and enhanced the expression of neurotrophic factors. CONCLUSION: Our findings indicate the participation of peroxynitrite in the pathobiology of TBI. GSNO treatment of TBI not only reduces peroxynitrite but also protects the integrity of the neurovascular unit, indicating that GSNO blunts the deleterious effects of peroxynitrite. A long-term treatment of TBI with the same low dose of GSNO promotes synaptic plasticity and enhances the expression of neurotrophic factors. These results support that GSNO reduces the levels of oxidative metabolites, protects the neurovascular unit, and promotes neurorepair mechanisms in TBI.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , S-Nitrosoglutationa/farmacologia , S-Nitrosoglutationa/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiopatologia , Encéfalo/anatomia & histologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Glutationa/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Peroxidação de Lipídeos , Masculino , Molsidomina/administração & dosagem , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/administração & dosagem , Ácido Peroxinitroso/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptor trkB/genética , Receptor trkB/metabolismo , S-Nitrosoglutationa/administração & dosagem , Sinaptofisina/genética , Sinaptofisina/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Traumatic spinal cord injury (SCI) enhances the activity of S-nitrosoglutathione reductase (GSNOR) and inhibits the mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity, resulting in prolonged and sustained pain and functional deficits. This study's objective was to test the hypotheses that GSNOR's specific inhibitor N6022 mitigates pain and improves functional recovery in a mouse model of SCI. Furthermore, the degree of recovery is enhanced and the rate of recovery is accelerated by an ALDH2 activator Alda-1. Using both wild-type and GSNOR-/- mice, the SCI model deployed for groups was contusion at the T9-T10 vertebral level. The enzymatic activity of GSNOR and ALDH2 was measured, and the expression of GSNOR and ALDH2 was determined by western blot analysis. Functional improvements in experimental animals were assessed with locomotor, sensorimotor, and pain-like behavior tests. Wild-type SCI animals had enhanced GSNOR activity and decreased ALDH2 activity, leading to neurovascular dysfunction, edema, and worsened functional outcomes, including locomotor deficits and pain. Compared to wild-type SCI mice, GSNOR-/- mice had better functional outcomes. Monotherapy with either GSNOR inhibition by N6022 or enhanced ALDH2 activity by Alda-1 correlated well with functional recovery and lessened pain. However, combination therapy provided synergistic pain-relieving effects and more significant functional recovery compared with monotherapy. Conclusively, dysregulations in GSNOR and ALDH2 are among the causative mechanisms of SCI injury. Either inhibiting GSNOR or activating ALDH2 ameliorates SCI. Combining the specific inhibitor of GSNOR (N6022) with the selective activator of ALDH2 (Alda-1) provides greater protection to the neurovascular unit and confers greater functional recovery. The study is novel, and the combination therapy (N6022 + Alda-1) possesses translational potential.
Assuntos
Álcool Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/enzimologia , Animais , Benzamidas/farmacologia , Benzodioxóis/farmacologia , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pirróis/farmacologiaRESUMO
Ischemia reperfusion injury (I/R injury) contributes significantly to morbidity and mortality following myocardial infarction (MI). Although rapid reperfusion of the ischemic myocardium was established decades ago as a highly beneficial therapy for MI, significant cell death still occurs after the onset of reperfusion. Mitochondrial dysfunction is closely associated with I/R injury, resulting in the uncontrolled production of reactive oxygen species (ROS). Considerable efforts have gone into understanding the metabolic perturbations elicited by I/R injury. Recent work has identified the critical role of reversible protein acetylation in maintaining normal mitochondrial biologic function and energy metabolism both in the normal heart and during I/R injury. Several studies have shown that modification of class I HDAC and/or Sirtuin (Sirt) activity is cardioprotective in the setting of I/R injury. A better understanding of the role of these metabolic pathways in reperfusion injury and their regulation by reversible protein acetylation presents a promising way forward in improving the treatment of cardiac reperfusion injury. Here we briefly review some of what is known about how acetylation regulates mitochondrial metabolism and how it relates to I/R injury.
Assuntos
Metabolismo Energético , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão/metabolismo , Acetilação , Animais , Humanos , Camundongos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
Traumatic brain injury (TBI) causes sustained disability due to compromised neurorepair mechanisms. Crucial to neurorepair and functional recovery following both TBI and stroke is hypoxia-inducible factor-1 alpha (HIF-1α). Based on reports that HIF-1α could be stabilized via S-nitrosylation, we tested the hypothesis that the S-nitrosylating agent S-nitrosoglutathione (GSNO) would stabilize HIF-1α, thereby stimulating neurorepair mechanisms and aiding in functional recovery. TBI was induced by controlled cortical impact (CCI) in adult rats. GSNO (0.05mg/kg) was administered at two hours after CCI. The treatment was repeated daily until the 14th day after CCI. Functional recovery was assessed by motor and cognitive functions, and the recovery was compared with the expression of HIF-1α. The mechanisms of GSNO-mediated S-nitrosylation of HIF-1α were determined using brain endothelial cells. While non-treated TBI animals showed sustained neurobehavioral deficits, GSNO treatment of TBI improved neurobehavioral functions. GSNO also increased the expression of HIF-1α and VEGF. The beneficial effects of GSNO on neurobehavioral functions in TBI animals were blocked by treatment with the HIF-1α inhibitor 2-methoxyestradiol (2-ME). The stimulatory effect of GSNO on VEGF was reversed not only by 2-ME but also by the denitrosylating agent dithiothreitol, confirming our hypothesis that GSNO's benefits are mediated by the stabilization of HIF-1α via S-nitrosylation. GSNO's S-nitrosylation of HIF-1α was further confirmed using a biotin switch assay in endothelial cells. The data provide evidence that GSNO treatment of TBI aids functional recovery through stabilizing HIF-1α via S-nitrosylation. GSNO is a natural component of the human brain/body, and its exogenous administration has not shown adverse effects in humans. Therefore, the translational potential of GSNO therapy in TBI is high.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fármacos Neuroprotetores/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , S-Nitrosoglutationa/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Lesões Encefálicas/psicologia , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Expressão Gênica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Masculino , Camundongos , Destreza Motora/efeitos dos fármacos , Destreza Motora/fisiologia , Estabilidade Proteica/efeitos dos fármacos , Distribuição Aleatória , Ratos Sprague-Dawley , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Recuperação de Função Fisiológica/fisiologiaRESUMO
Traumatic brain injury (TBI) derails nitric oxide (NO)-based anti-inflammatory and anti-excitotoxicity mechanisms. NO is consumed by superoxide to form peroxynitrite, leading to decreased NO bioavailability for S-nitrosoglutathione (GSNO) synthesis and regulation of neuroprotective pathways. Neuronal peroxynitrite is implicated in neuronal loss and functional deficits following TBI. Using a contusion mouse model of TBI, we investigated mechanisms for the opposed roles of GSNO versus peroxynitrite for neuroprotection and functional recovery. TBI was induced by controlled cortical impact (CCI) in adult male mice. GSNO treatment at 2h after CCI decreased the expression levels of phospho neuronal nitric oxide synthase (pnNOS), alpha II spectrin degraded products, and 3-NT, while also decreasing the activities of nNOS and calpains. Treatment of TBI with FeTPPS, a peroxynitrite scavenger, had effects similar to GSNO treatment. GSNO treatment of TBI also reduced neuronal degeneration and improved neurobehavioral function in a two-week TBI study. In a cell free system, SIN-1 (a peroxynitrite donor and 3-nitrotyrosinating agent) increased whereas GSNO (an S-nitrosylating agent) decreased calpain activity, and these activities were reversed by, respectively, FeTPPS and mercuric chloride, a cysteine-NO bond cleaving agent. These data indicate that peroxynitrite-mediated activation and GSNO-mediated inhibition of the deleterious nNOS/calpain system play critical roles in the pathobiology of neuronal protection and functional recovery in TBI disease. Given GSNO׳s safety record in other diseases, its neuroprotective efficacy and promotion of functional recovery in this TBI study make low-dose GSNO a potential candidate for preclinical evaluation.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , S-Nitrosoglutationa/farmacologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Calpaína/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Óxido Nítrico Sintase Tipo I/metabolismo , Ácido Peroxinitroso/metabolismo , Fosforilação/efeitos dos fármacos , Distribuição Aleatória , Recuperação de Função Fisiológica/fisiologiaRESUMO
Stroke is associated with significant morbidity and mortality due to the limited neuroregeneration capacity of the injured brain. Other than thrombolysis in the acute phase of the disease by tissue plasminogen activator (tPA), which offers only a short window of treatment (~3 hours), an ideal stroke therapy is not available mainly because of limited understanding of the mechanisms of neuroregeneration and functional recovery in the chronic phase. Yet many drug therapies, including S-nitrosoglutathione (GSNO), have been shown to provide neuroprotection against acute disease in animal models of transient cerebral ischemia reperfusion (IR) and permanent ischemia. GSNO was also effective in stimulating neuroregeneration-related factors in the chronic phase of the disease. In this short review, we assess the evidence supporting exogenous administration of GSNO after experimental stroke as a means to stimulate neuroregeneration and aid in functional recovery via stabilization of the HIF-1α/VEGF pathway.
RESUMO
BACKGROUND: For stroke patients, stimulating neurorepair mechanisms is necessary to reduce morbidity and disability. Our previous studies on brain and spinal cord trauma show that exogenous treatment with the S-nitrosylating agent S-nitrosoglutathione (GSNO) - a nitric oxide and glutathione metabolite of the human body - stimulates neurorepair and aids functional recovery. Using a rat model of cerebral ischemia and reperfusion (IR) in this study, we tested the hypothesis that GSNO invokes the neurorepair process and improves neurobehavioral functions through the angiogenic HIF-1α/VEGF pathway. METHODS: Stroke was induced by middle cerebral artery occlusion for 60 minutes followed by reperfusion in adult male rats. The injured animals were treated with saline (IR group, n=7), GSNO (0.25 mg/kg, GSNO group, n=7), and GSNO plus the HIF-1α inhibitor 2-methoxyestra-diol (2-ME) (0.25 mg/kg GSNO + 5.0 mg/kg 2-ME, GSNO + 2-ME group, n=7). The groups were studied for either 7 or 14 days to determine neurorepair mediators and functional recovery. Brain capillary endothelial cells were used to show that GSNO promotes angiogenesis and that GSNO-mediated induction of VEGF and the stimulation of angiogenesis are dependent on HIF-1α activity. RESULTS: IR injury increased the expression of neurorepair mediators HIF-1α, VEGF, and PECAM-1 and vessel markers to a limited degree that correlate well with significantly compromised neurobehavioral functions compared with sham animals. GSNO treatment of IR not only remarkably enhanced further the expression of HIF-1α, VEGF, and PECAM-1 but also improved functioning compared with IR. The GSNO group also had a higher degree of vessel density than the IR group. Increased expression of VEGF and the degree of tube formation (angiogenesis) by GSNO were reduced after the inhibition of HIF-1α by 2-ME in an endothelial cell culture model. 2-ME treatment of the GSNO group also blocked not only GSNO's effect of reduced infarct volume, decreased neuronal loss, and enhanced expression of PECAM-1 (P<0.001), but also its improvement of motor and neurological functions (P<0.001). CONCLUSION: GSNO stimulates the process of neurorepair, promotes angiogenesis, and aids functional recovery through the HIF-1α-dependent pathway, showing therapeutic and translational promise for stroke.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , S-Nitrosoglutationa/farmacologia , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Isquemia Encefálica/patologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Masculino , Artéria Cerebral Média/patologia , Atividade Motora/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/psicologiaRESUMO
PURPOSE: Stroke disability stems from insufficient neurorepair mechanisms. Improvement of functions has been achieved through rehabilitation or therapeutic agents. Therefore, we combined exercise with a neurovascular protective agent, S-nitrosoglutathione (GSNO), to accelerate functional recovery. METHODS: Stroke was induced by middle cerebral artery occlusion for 60 min followed by reperfusion in adult male rats. Animals were treated with vehicle (IR group), GSNO (0.25 mg/kg, GSNO group), rotarod exercise (EX group) and GSNO plus exercise (GSNO+EX group). The groups were studied for 14 days to determine neurorepair mechanisms and functional recovery. RESULTS: Treated groups showed reduced infarction, decreased neuronal cell death, enhanced neurotrophic factors, and improved neurobehavioral functions. However, the GSNO+EX showed greater functional recovery (p < 0.05) than the GSNO or the EX group. A GSNO sub group, treated 24 hours after IR, still showed motor function recovery (p < 0.001). The protective effect of GSNO or exercise was blocked by the inhibition of Akt activity. CONCLUSIONS: GSNO and exercise aid functional recovery by stimulating neurorepair mechanisms. The improvements by GSNO and exercise depend mechanistically on the Akt pathway. A combination of exercise and GSNO shows greater functional recovery. Improved recovery with GSNO, even administered 24 hours post-IR, demonstrates its clinical relevance.