RESUMEN
Diabetic retinopathy (DR), the most common and serious ocular complication, recently has been perceived as a neurovascular inflammatory disease. However, role of adaptive immune inflammation driven by T lymphocytes in DR is not yet well elucidated. Therefore, this study aimed to clarify the role of interleukin (IL)-17A, a proinflammatory cytokine mainly produced by T lymphocytes, in retinal pathophysiology particularly in retinal neuronal death during DR process. Ins2Akita (Akita) diabetic mice 12 weeks after the onset of diabetes were used as a DR model. IL-17A-deficient diabetic mice were obtained by hybridization of IL-17A-knockout (IL-17A-KO) mouse with Akita mouse. Primarily cultured retinal Müller cells (RMCs) and retinal ganglion cells (RGCs) were treated with IL-17A in high-glucose (HG) condition. A transwell coculture of RGCs and RMCs whose IL-17 receptor A (IL-17RA) gene had been silenced with IL-17RA-shRNA was exposed to IL-17A in HG condition and the cocultured RGCs were assessed on their survival. Diabetic mice manifested increased retinal microvascular lesions, RMC activation and dysfunction, as well as RGC apoptosis. IL-17A-KO diabetic mice showed reduced retinal microvascular impairments, RMC abnormalities, and RGC apoptosis compared with diabetic mice. RMCs expressed IL-17RA. IL-17A exacerbated HG-induced RMC activation and dysfunction in vitro and silencing IL-17RA gene in RMCs abolished the IL-17A deleterious effects. In contrast, RGCs did not express IL-17RA and IL-17A did not further alter HG-induced RGC death. Notably, IL-17A aggravated HG-induced RGC death in the presence of intact RMCs but not in the presence of RMCs in which IL-17RA gene had been knocked down. These findings establish that IL-17A is actively involved in DR pathophysiology and particularly by RMC mediation it promotes RGC death. Collectively, we propose that antagonizing IL-17RA on RMCs may prevent retinal neuronal death and thereby slow down DR progression.
Asunto(s)
Retinopatía Diabética/genética , Células Ependimogliales/metabolismo , Interleucina-17/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Retinopatía Diabética/fisiopatología , Humanos , Masculino , RatonesRESUMEN
Ultra-violet (UV) radiation (UVR) causes significant oxidative injury to retinal pigment epithelium (RPE) cells. Obacunone is a highly oxygenated triterpenoid limonoid compound with various pharmacological properties. Its potential effect in RPE cells has not been studied thus far. Here in ARPE-19 cells and primary murine RPE cells, obacunone potently inhibited UVR-induced reactive oxygen species accumulation, mitochondrial depolarization, lipid peroxidation and single strand DNA accumulation. UVR-induced RPE cell death and apoptosis were largely alleviated by obacunone. Obacunone activated Nrf2 signaling cascade in RPE cells, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation. It promoted transcription and expression of antioxidant responsive element-dependent genes. Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout almost reversed obacunone-induced RPE cytoprotection against UVR. Forced activation of Nrf2 cascade, by Keap1 knockout, similarly protected RPE cells from UVR. Importantly, obacunone failed to offer further RPE cytoprotection against UVR in Keap1-knockout cells. In vivo, intravitreal injection of obacunone largely inhibited light-induced retinal damage. Collectively, obacunone protects RPE cells from UVR-induced oxidative injury through activation of Nrf2 signaling cascade.
Asunto(s)
Benzoxepinas/farmacología , Limoninas/farmacología , Degeneración Macular/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Rayos Ultravioleta/efectos adversos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Benzoxepinas/uso terapéutico , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , ADN de Cadena Simple/efectos de los fármacos , ADN de Cadena Simple/efectos de la radiación , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Inyecciones Intravítreas , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Limoninas/uso terapéutico , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/efectos de la radiación , Degeneración Macular/etiología , Degeneración Macular/patología , Ratones , Membranas Mitocondriales/efectos de los fármacos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/genética , Estrés Oxidativo/efectos de la radiación , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/efectos de la radiación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/efectos de la radiaciónRESUMEN
AMP-activated protein kinase (AMPK) signaling activation can inhibit Ultra-violet (UV) radiation (UVR)-induced retinal pigment epithelium (RPE) cell injuries. LB-100 is a novel inhibitor of protein phosphatase 2A (PP2A), the AMPKα1 phosphatase. Here, our results demonstrated that LB-100 significantly inhibited UVR-induced viability reduction, cell death and apoptosis in established ARPE-19â¯cells and primary murine RPE cells. LB-100 activated AMPK, nicotinamide adenine dinucleotide phosphate (NADPH) and Nrf2 (NF-E2-related factor 2) signalings, inhibiting UVR-induced oxidative injuries and DNA damage in RPE cells. Conversely, AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A mutation, almost blocked LB-100-induced RPE cytoprotection against UVR. Importantly, CRISPR/Cas9-mediated PP2A knockout mimicked and nullified LB-100-induced anti-UVR activity in RPE cells. Collectively, these results show that PP2A inhibition by LB-100 protects RPE cells from UVR via activation of AMPK signaling.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Piperazinas/farmacología , Proteína Fosfatasa 2/genética , Protectores Solares/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Sistemas CRISPR-Cas , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Activación Enzimática , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Edición Génica , Regulación de la Expresión Génica , Humanos , Ratones , NADP/metabolismo , Cultivo Primario de Células , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de la radiación , Transducción de Señal , Rayos Ultravioleta/efectos adversosRESUMEN
Ultra-violet (UV) radiation (UVR) to human retinas induces oxidative injury to the resident retinal pigment epithelium (RPE) cells. PF-06409577 a novel, potent and direct AMP-activated protein kinase (AMPK) activator. In ARPE-19â¯cells and primary murine RPE cells, PF-06409577 significantly inhibited UVR-induced viability reduction, cell death and apoptosis. PF-06409577 activated AMPK signaling in RPE cells by increasing AMPKα1-acetyl-CoA carboxylase phosphorylation and AMPK activity. AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A dominant negative mutation, almost abolished PF-06409577-induced RPE cytoprotection against UVR. PF-06409577 enhanced nicotinamide adenine dinucleotide phosphate (NADPH) activity and expression levels of Nrf2-dependent genes in RPE cells. Furthermore, UVR-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage were largely inhibited by the AMPK activator. In summary, PF-06409577 inhibits UVR-induced oxidative stress and RPE cell death by activating AMPK signaling.