Study On The Molecular Mechanism Of ARPE-19 Cellular Metabolic Disorder Caused By Malnutrition And The Protection Of Taurine

Age-related macular degeneration(AMD)is a progressive retinal degenerative disease affecting more than 150 million people in the world.Disruption of retinal pigment epithelium(RPE)is one of the major pathological changes in AMD.Some factors are believed to be involved in the development of RPE damage,such as apoptosis,mitochondrial dysfunction,lack of nutrients and oxygen,and calcium overload.However,the exact mechanism is still unknown.Cells with nutrient deficiencies have been found to be hypersensitive to various cell death stimuli,such as autophagy,loss of calcium homeostasis,endoplasmic reticulum(ER)stress,and apoptosis.Increasing evidence indicates oxidative stress is associated with many devastating ocular diseases,including glaucoma,AMD,and diabetic retinopathy.Taurine is one of the most abundant amino acids in ocular tissues,such as the cornea,iris,lens,and retina.The lack of taurine in cat food causes the cat to go blind.Taurine can carry out multiple functions in cells:ER stress response,autophagy modulation,anti-apoptosis,and so on.Another important function of taurine is the modulation of calcium influx,which may contribute to its neuroprotective effects.However,recent studies have not demonstrated the relationship between taurine and Earle’s balanced salt solution(EBSS)-induced cell injuries.In this study,the relationship of Calpains,autophagy,endoplasmic reticulum(ER)stress,and apoptosis in ARPE-19 cells also were investigated.Nutrition deprivation of RPE cells in culture produces several responses that seem to mimic steps in the AMD cascade.Western blot,flow cytometry,immunofluorescence staining were performed to assess autophagy,ER stress,apoptosis and Calpain activity in ARPE-19 cells.We first confirmed that autophagy,ER stress,apoptosis and oxidative stress in ARPE-19 cells were induced by Earle’s balanced salt solution(EBSS)through starvation to induce RPE metabolic stress.Secondly,inhibition of ER stress by 4-phenyl butyric acid(4-PBA)alleviated autophagy and apoptosis,and suppression of autophagy by 3-methyl adenine(3-MA)reduced the cell apoptosis,but the ER stress was minimally affected.Finally,the apoptosis,ER stress and autophagy were inhibited by gene silencing of Calpain-2 and overexpression of Calpain-1,respectively.Nicotinamide adenine dinucleotide phosphate(NADPH)oxidases(NOXs)are key transmembrane proteins leading to reactive oxygen species(ROS)overproduction.However,the detailed roles of NOXs in retinal pigment epithelial(RPE)cells induced by Earle’s balanced salt solution(EBSS)through starvation to induce cell metabolic stress remain unclear.We investigated what roles NOXs play in accommodation of Calpain activity,endoplasmic stress(ER),autophagy,and apoptosis during metabolic stress in ARPE-19 cells.We first found that EBSS induced NOX2,NOX4,p22phox,and NOX5 increase rather than NOX1.Secondly,inhibition of NOXs suppressed ER stress and autophagy,decreased ROS generation,and alleviated cell apoptosis.Thirdly,NOX4,NOX5,p22phox were involved in EBSS-induced cell damage.Silencing these three NOXs resulted in reduced levels of cell damage.Finally,taurine supplement suppressed both the changes of the important upstream regulators(Calpain-1 and Calpain-2)and the activation of ER stress,autophagy and apoptosis.Taurine also critically mediated NOXs in response to EBSS stress.In conclusion,this study demonstrated for the first time that NOX oxidases are the upstream regulators of Calpain-2,ER stress,autophagy,and apoptosis.Furthermore,the protective effect of taurine is mediated by reduction of NOX-derived ROS,leading to sequential inhibition of Calpain activity,ER stress,autophagy,and apoptosis.In conclusion,we have shown that nutrition deprivation of RPE cells in culture produces several responses that seem to mimic steps in the AMD cascade.Our observations show the regulation relationships of Calpain,ER stress,autophagy,and apoptosis.Moreover,oxidative stress is the main causative for cell injury,and taurine attenuates ER stress,autophagy,and apoptosis in ARPE-19 cells via an inhibition of NADPH oxidase-derived reactive oxygen species-mediated Calpain activation pathway.These results further expand our understanding of the AMD pathological mechanism,and also to determine taurine’s therapeutic potential to treat multiple neurodegenerative diseases associated with cell death.