ABSTRACT
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.
Subject(s)
Benzoxepins/pharmacology , Limonins/pharmacology , Macular Degeneration/drug therapy , Oxidative Stress/drug effects , Retinal Pigment Epithelium/drug effects , Ultraviolet Rays/adverse effects , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Benzoxepins/therapeutic use , Cell Line , Cell Survival/drug effects , Cell Survival/radiation effects , DNA, Single-Stranded/drug effects , DNA, Single-Stranded/radiation effects , Disease Models, Animal , Drug Evaluation, Preclinical , Gene Expression Regulation/drug effects , Gene Knockout Techniques , Humans , Intravitreal Injections , Kelch-Like ECH-Associated Protein 1/metabolism , Limonins/therapeutic use , Lipid Peroxidation/drug effects , Lipid Peroxidation/radiation effects , Macular Degeneration/etiology , Macular Degeneration/pathology , Mice , Mitochondrial Membranes/drug effects , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oxidative Stress/genetics , Oxidative Stress/radiation effects , Primary Cell Culture , Reactive Oxygen Species/metabolism , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/radiation effects , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/radiation effectsABSTRACT
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.
