RESUMEN

Ferroptosis is a form of iron-dependent regulated cell death. Evidence of its existence and the effects of its inhibitors on subarachnoid hemorrhage (SAH) is still lacking. In the present study, we found that liproxstatin-1 protected HT22 cells against hemin-induced injury by protecting mitochondrial functions and ameliorating lipid peroxidation. In in vivo experiments, we demonstrated the presence of characteristic shrunken mitochondria in ipsilateral cortical neurons after SAH. Moreover, liproxstatin-1 attenuated the neurological deficits and brain edema, reduced neuronal cell death, and restored the redox equilibrium after SAH. The inhibition of ferroptosis by liproxstatin-1 was associated with the preservation of glutathione peroxidase 4 and the downregulation of acyl-CoA synthetase long-chain family member 4 as well as cyclooxygenase 2. In addition, liproxstatin-1 decreased the activation of microglia and the release of IL-6, IL-1ß, and TNF-α. These data enhance our understanding of cell death after SAH and shed light on future preclinical studies.

Asunto(s)

Ferroptosis , Hemorragia Subaracnoidea , Animales , Quinoxalinas , Ratas , Ratas Sprague-Dawley , Compuestos de Espiro , Hemorragia Subaracnoidea/complicaciones , Hemorragia Subaracnoidea/tratamiento farmacológico

RESUMEN

BACKGROUND: The NLRP3 inflammasome is a multiprotein complex that regulates the innate immune inflammatory response by activating caspase-1 and subsequent IL-1ß and IL-18. Fluoxetine has been shown to have the anti-inflammatory properties in many disease models. However, the effects and mechanisms of these effects of fluoxetine in early brain injury after subarachnoid hemorrhage (SAH) have not been defined. METHODS: The SAH model was induced by an endovascular perforation in adult male Sprague-Dawley (SD) rats weighing 300-320 g. N-Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (AC-YVAD-CMK) was injected intraperitoneally (5 mg/kg) 1 h after SAH. Fluoxetine was administered via intravenous route 6 h after SAH. 3-Methyladenine (3-MA) was intracerebroventricularly injected 20 min before SAH. SAH grade, neurological function, brain water content, propidium iodide (PI) staining, western blot, double immunostaining, and transmission electron microscopy were performed. RESULTS: Expression of caspase-1 increased and peaked at 24 h after SAH. Caspase activation was along with the increased necrotic cells, which occurred mainly in neurons. Necrotic cell death of microglia and astrocyte were also found. Administration of AC-YVAD-CMK, a caspase-1 inhibitor, reduced the expression of IL-1ß and IL-18 and the number of PI-positive cells, attenuated brain edema, and improved neurological function, which was also observed in fluoxetine-treated rats. Furthermore, fluoxetine treatment significantly decreased the expression of NLRP3 and cleaved caspase-1 and upregulated the expression of beclin-1, a marker for autophagy. Finally, the effects of fluoxetine in NLRP3 inflammasome activation were reversed by additional 3-MA administration. CONCLUSIONS: Together, our present study indicated that NLRP3 inflammasome and caspase-1 activation play a deleterious role in early brain injury and fluoxetine mitigates NLRP3 inflammasome and caspase-1 activation through autophagy activation after SAH, providing a potential therapeutic agent for SAH treatment.

Asunto(s)

Antiinflamatorios/farmacología , Autofagia/efectos de los fármacos , Fluoxetina/farmacología , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Hemorragia Subaracnoidea/patología , Animales , Lesiones Encefálicas/inmunología , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Inflamasomas/efectos de los fármacos , Inflamasomas/inmunología , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ratas , Ratas Sprague-Dawley , Hemorragia Subaracnoidea/inmunología , Hemorragia Subaracnoidea/metabolismo

RESUMEN

Aberrant modulation of mitochondrial dynamic network, which shifts the balance of fusion and fission towards fission, is involved in brain damage of various neurodegenerative diseases including Parkinson's disease, Huntington's disease and Alzheimer's disease. A recent research has shown that the inhibition of mitochondrial fission alleviates early brain injury after experimental subarachnoid hemorrhage, however, the underlying molecular mechanisms have remained to be elucidated. This study was undertaken to characterize the effects of the inhibition of dynamin-related protein-1 (Drp1, a dominator of mitochondrial fission) on blood-brain barrier (BBB) disruption and neuronal apoptosis following SAH and the potential mechanisms. The endovascular perforation model of SAH was performed in adult male Sprague Dawley rats. The results indicated Mdivi-1(a selective Drp1 inhibitor) reversed the morphologic changes of mitochondria and Drp1 translocation, reduced ROS levels, ameliorated the BBB disruption and brain edema remarkably, decreased the expression of MMP-9 and prevented degradation of tight junction proteins-occludin, claudin-5 and ZO-1. Mdivi-1 administration also inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB), leading to decreased expressions of TNF-ɑ, IL-6 and IL-1ß. Moreover, Mdivi-1 treatment attenuated neuronal cell death and improved neurological outcome. To investigate the underlying mechanisms further, we determined that Mdivi-1 reduced p-PERK, p-eIF2α, CHOP, cleaved caspase-3 and Bax expression as well as increased Bcl-2 expression. Rotenone (a selective inhibitor of mitochondrial complexes I) abolished both the anti-BBB disruption and anti-apoptosis effects of Mdivi-1. In conclusion, these data implied that excessive mitochondrial fission might inhibit mitochondrial complex I to become a cause of oxidative stress in SAH, and the inhibition of Drp1 by Mdivi-1 attenuated early brain injury after SAH probably via the suppression of inflammation-related blood-brain barrier disruption and endoplasmic reticulum stress-based apoptosis.

Asunto(s)

Barrera Hematoencefálica/efectos de los fármacos , Dinaminas/genética , Mitocondrias/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacos , Quinazolinonas/farmacología , Hemorragia Subaracnoidea/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Claudina-5/genética , Claudina-5/metabolismo , Dinaminas/antagonistas & inhibidores , Dinaminas/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Dinámicas Mitocondriales/genética , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/metabolismo , Ocludina/genética , Ocludina/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/agonistas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Hemorragia Subaracnoidea/genética , Hemorragia Subaracnoidea/mortalidad , Hemorragia Subaracnoidea/patología , Espacio Subaracnoideo/efectos de los fármacos , Espacio Subaracnoideo/metabolismo , Espacio Subaracnoideo/patología , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

Poly (ADP-ribose) polymerases (PARPs) play an important role in a range of neurological disorders, however, the role of PARP in early brain injury after subarachnoid hemorrhage (SAH) remains unclear. This study was designed to explore the role and the potential mechanisms of PARP in early brain injury after SAH. Eighty-nine male SD rats were randomly divided into the Sham group, SAH+Vehicle group and SAH+PARP inhibitor (PJ34) group. An endovascular perforation model was used to induce SAH in rats. PJ34 (10mg/kg) or vehicle (0.9% NaCl) was intraperitoneally administered at 5min and 8h after SAH induction. Mortality, SAH grades, neurological function, evans blue extravasation, brain edema, immunofluorescence staining and western blotting were performed. PJ34 reduced BBB permeability and brain edema, improved neurological function and attenuated neuronal cell death in the rat model of SAH. Moreover, PJ34 inhibited the nuclear translocation of NF-κB, decreased the expression of the proinflammatory cytokines IL-1ß, IL-6 and TNF-α, reduced the expression of MMP-9, prevented the degradation of tight junction proteins, and decreased microglia activation. These data indicated that PARP inhibition through PJ34 might be an important therapeutic drug for SAH.

Asunto(s)

Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Hemorragia Subaracnoidea/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Claudina-5/metabolismo , Modelos Animales de Enfermedad , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Ocludina/metabolismo , Fenantrenos/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

To study the polymorphism at D7S21 locus in Hebei Han population, 124 unrelated individuals were detected rapidly by Minisatellite Variant Repeat-Polymerase Chain Reaction (MVR-PCR) and polyacrylamide gradient gel electrophoresis followed by silver staining,and digital codes were obtained. About 36 digital codes were obtained from each individual. No two unrelated individuals shared the same codes. The probability of identity in 36 digital codes was 3.48 x 10(-18). The percentage of three repeat units, a-type, t-type and 0-type was 48.5%, 49.4% and 2.1% respectively. The heterozygosity (H), excluding probability of paternity (EPP)and polymorphism information content (PIC) were 0.9876, 0.9746 and 0.9872 respectively. The results suggested that D7S21 locus has highly polymorphism in Hebei Han population. The method-polyacrylamide gradient gel electrophoresis followed by silver staining was simple,rapid and practical.