Autophagy-mediated Preventive Effect Induced By EGCG In Retinal Müller Cells Under High-glucose Conditions

Background Diabetic retinopathy(DR)is a leading cause of vision loss worldwide.Retinal neurodegeneration is an early feature in the pathogenesis of diabetic retinopathy(DR)and contributes to the development of retinal microvasculopathy.Müller glial cells play a central role in the formation of DR.Müller cells are originally located in the retina,extending from the outer limiting membrane to the inner limiting membrane and for ming end feet at the inner retinal perimeter.The retinal Müller glial cells also maintain neurons by releasing trophic factors,recycling neurotransmitters,and controlling the ionic balance in the extracellular space.A variety of studies have exa mined the possible roles of autophagy in diabetic retinopathy.Autophagy is an evolutionarily conserved mechanism that allows cells to degrade damaged proteins and intracellular organelles,maintaining cell homeostasis against nutrient deprivation and cellular stress.Epigallocatechin gallate(EGCG)is a major polyphenol in green tea that has beneficial effects in diabetic.Epigallocatechin-3-gallate(EGCG)has shown health-associated benefits by activating autophagy against various diseases,and apoptosis of retinal neurons could be effectively inhibited with EGCG.EGCG in the regulation of autophagy was recently highlighted,however,it is not currently known whether EGCG can regulate Müller cell autophagy.Here,we are bent on EGCG Epigallocatechin-3-gallate attenuates apoptosis and NF-?B in retinal Müller cells under high-glucose conditions.Part1 Effects of autophagy on Müller cell induced by high glucosePurpose:This study aimed to investigate whether high glucose(HG)can regulate the autophagy in Müller cellMethods:Primary rat retinal Müller cells were exposed to normal or high glucose(3?6?12?24?24?36 h),and Müller cells were exposed to NG(5.5 mM)and HG(20,30,40,50 mM)for 24 h,LC3,P62,and BECN1 protein expression was exa mined by western blot analysis in various experimental group.And in fluorescence microscopy experiments,autophagy was evaluated by the autophagy marker LC3 and P62.Next,we observed the formation of autophagosomes and autolysosomes by electron microscopy.CCK-8 and TUNEL assay was used in Müller cell damage and proliferation.Result:Beclin-1 and LC3?/LC3? protein expression showed a significant up-regulation,reached the peak at HG(6 h),and then gradually declined to the baseline from 24 h to 36 h.At the same time,the expression of P62 protein markedly accumulated in the cells exposed to HG at 24 h to 36 h.There was significant statistical significance,*P<0.05.Time-dependent decrease in cell viability was observed when the Mtiller cell were exposed to HG after 24 h,cell viability showed a significant down-regulation.but it did not show significant effect to restrain cell damage in the early stage(before 12 h).And there was a clear increase TUNEL-positive cells in HG for 24 h conditions but 6 h,it was significant statistical significance,*P<0.05.However,HG-Müller cell exposure to 3MA resulted in increased cell apoptosis,there was significant statistical significance,*P<0.05.Part 2 Epigallocatechin-3-Gallate stimulates autophagy in retinal Müller cells under high-glucose conditionsPurpose:This study aimed to investigate whether EGCG can regulate autophagy is involved in Müller cell response under high glucose(HG).Methods:Primary rat retinal Müller cells were exposed to normal or high glucose in and out of presence of EGCG,pharmacologic inhibitors and activators.1)LC3,P62,and BECN1 protein expression was exa mined by western blot analysis in various experimental group.2)In fluorescence microscopy experiments,autophagy was evaluated by the autophagy marker LC3 and P62.3)Next,we observed the formation of autophagosomes and autolysosomes by electron microscopy.4)To investigate the role of lysosomes in Müller cells under diabetic conditions with or without of the presence of EGCG,we assessed lysosomal activity using acridine orange(AO).Results:We show that EGCG increases autophagy by promoting the formation of autophagosomes,increasing lysosomal acidification,and stimulating autophagic flux in Müller cell,HG induced a decrease of autophagy,However,Retinal Müller cell in HG treated with EGCG show autophagy machinery activation and reestablishment of cargo degradation.Part 3 Epigallocatechin-3-Gallate reduces apoptosis levels in retinal Müller cells under high-glucose conditions Purpose:This study aimed to investigate whether EGCG can reduces apoptosis levels by stimulate autophagy in Müller cell response under high glucose(HG).Methods:Primary rat retinal Müller cells were exposed to normal or high glucose in and out of presence of EGCG,pharmacologic inhibitors and activators.1)The rMC-1 culture under different conditions was evaluated by representative photographs 2)Cleaved caspase-3 protein expression was exa mined by western blot analysis in various experimental group.3)TUNEL assay was used in Müller cell damage 4)CCK-8,5-Ethynyl-2'-Deoxyuridine(EdU)was used in Müller cell proliferation.5)In fluorescence microscopy experiments,GFAP was analysis.6)Experimental diabetes was induced in male rats by intraperitoneal injections of streptozotocin(60 mg/kg),and values>15 mmol/1 were considered diabeticResults:1)Dose-dependent decrease in cell viability was observed when the Müller cells were exposed to different doses of EGCG for 24 h,and cell viability showed a significant upregulation,reaching the peak with EGCG(20-30?M)and then decreasing.There was significant statistical significance,*P<0.05.2)There was a clear increase in cleaved-cas3 activity and TUNEL-positive cells under HG conditions,Surprisingly,activation of autophagy with EGCG decreased TUNEL-positive cells and caspase 3 activity,compared to untreated cells.There was significant statistical significance,*P<0.05.3)A strong GFAP immunostaining was detected in sections of retinae from DR rats,whereas the signal was very weak in retinae from control rats and rats treated with EGCG at the same age.There was significant statistical significance,*P<0.05.Part 4 Epigallocatechin-3-gallate attenuates NF-?B in retinal Müller cells under high-glucose conditionsPurpose:This study aimed to investigate whether EGCG can regulate the autophagy and NF-?B involved in Müller cell response under high glucose(HG)conditions.Methods:Primary rat retinal Müller cells were exposed to normal or high glucose in and out of the presence of EGCG,pharmacologic inhibitors and activators,Müller cells were exposed to NG(5.5 mM)and HG(40mM)for 6h or 24 h1)p-NF-?B,nucl-NF-?B and cyto-NF-?B protein expression was exa mined by western blot analysis in various experimental group.2)we observed the IL-6 expression by enzyme linked immunosorbent assay(ELLSA).3)To investigate the effects of HG on the NF-?B activation nuclear translocation level,we evaluated the expression of NF-?B by fluorescence microscopy experiments4)Experimental diabetes was induced in male rats by intraperitoneal injections of streptozotocin(60 mg/kg),and values>15 mmol/l were considered diabetic.Control,DM,EGCG(40mg/kg/day).Result:1)HG treatment for 6 h no induced nuclear translocation of NF-?B,and the intracellular activated NF-?B level increased obviously for 24 h.There was significant statistical significance,*P<0.05.2)And treatment of cells with EGCG significantly increased HG-induced nuclear import of NF-?B in Müller for 24 h.There was significant statistical significance,*P<0.05.Conclusion:Our data suggested that HG increases Müller cell autophagy before 6 h and decreases after 24 h and inhibition of autophagy restrained cell proliferation and increased apoptosis in Müller cell exposed to HG High glucose downregulates autophagy and accumulates p62/SQTSM1 cargo due to lysosomal dysfunction,which was restored by the treatment with EGCG.These data showed that Müller cells under HG conditions were at increased risk of apoptosis,but EGCG could improve the condition.And EGCG reduced the reactive gliosis of Müller cells in diabetic rats.Our results showed that the ad ministration of this neuroprotectant to HG-treated retinal Müller cells inhibited mTOR and reactivated autophagy,thus increasing autophagic flux.The levels of HG-induced expression of p-NF-?B and IL6 were increased.The autophagy pathway was stimulated by EGCG can reduce NF-?B signaling and generate chronic inflammation.This might be valuable for developing a novel therapeutic strategy to treat DR.In summary,our study provided a better understanding of EGCG's actions on Müller cells,and it strongly suggested that EGCG-induced autophagy might mediate some of its therapeutic effects.