Notoginsenoside R1 Attenuates High Glucose-Induced Endothelial Damage In Rat Retinal Capillary Endothelial Cells By Modulating The Intracellular Redox State

1.Background and objectivesDiabetes mellitus(DM)is a global health problem.Recent estimate showed that people with diabetes is projected to increase to 522 million by 2030.Retinal small vessel disease,including microvascular lesions,is considered to play an important role in the development of diabetic complication.Over 20%of people with diabetes have signs of diavetic retinopathy(DR)or might have vision-threatening retinopathy.DR is a progressive disorder,it is the most common cause of blindness in people aged 20-74 years.However,despite many years of intensive research efforts,there is still no effective therapy for the prevention and treatment of DR.Our previous study showed that Panax notoginseng Saponins(PNS)is endowed with a significant protective function against high glucose-induced oxidative injury in RCECs.PNS can reduced intracellular ROS and lowered MDA contents.PNS also can increased the anti-oxygenic enzyme activity and enhancd anti-oxygenic ability.Furthermore,PNS reduced the glucose-induced ROS via decreasing the high glucose-induced Nox4 expression.Due to the complex physiological active and function of PNS,additional studies are needed to clarify the mechanism and its exact function.Therefore,we hypothesized that the main components of PNS,Notoginsenoside R1(NR1)might have the protect effect against high glucose-induced injury in RCECs,and then to study the possible mechanism.2.MethodsSection Ⅰ:A cell model for diabetic retinopathy research under high-glucose stimulation.Retinas were digested by 0.1%Ⅱ collagenase for 30 min at 37℃.The culture method choose highly selective endothelial cell culture medium associated gelatin adhesion which may successfully obtain the highly purified rat RCECs.Using endothelial cell culture medium to make the RCECs into suspension cell,count cells and seeded on 96 culture-bottles,then make endothelial cell culture medium of different glucose(5.5 mM,10 mM,20 mM,30 mM and 40 mM).After cultured for 24 h,48 h and 72 h in vitro,MTT method was used to detect cell viability.Section Ⅱ:Protective effects of Notoginsenoside R1 against high glucose-induced injury in RCECs.MTT method was used to detect RCECs exposed to high-level glucose solutions(30 mM)at various time periods(24,48 and 72 h)in the presence or absence of Notoginsenoside R1(NR1).Trypan blue exclusion test was to count the number of live cells.Colorimetry method was used to detect the activity of LDH in culture medium.Apoptotic cells(double-labeled with annexin V/PI)were observed by confocal laser scanning microscopy and the relative mtDNA copy number was measured by real-time quantitative PCR.Section Ⅲ:Effect of Notoginsenoside R1 against high glucose-induced injury in RCECs,analysis of its mechanisms of action.Chemiluminescence method were employed to detect NOX activity.Colorimetry method was used to detect the activitiy of CAT.In this study,we monitored ROS production with AmpliteTM ROS Red and examined the expression of 3-nitrotyrosine(3-NT,a marker of ONOO--mediated protein nitration)by immunofluorescence.Colorimetry method was used to detect the levels of NAD+and NADH,absorbance value were measured at 460 nm in a microplate reader.Chemiluminescence method were employed to detect NADPH/NADP+ratio.GSH and GSSG levels were determined using fluorescence detection assay.Section Ⅳ:Notoginsenoside R1 inhibited glucotoxicity dependent on the NAD-SIRT axis in RCECsChemiluminescence method were employed to detect PARP activity.Colorimetry method was used to detect the activitiy of total SIRT.The expression levels of SIRT-1,SIRT-3 and Nmnat-1 were determined by Western blotting assay and reverse transcription-polymerase chain reaction.3.Results(1)The culture method based on highly selective endothelial cell culture medium associated to 0.1%Ⅱ collagenas enzyme digestion and gelatin adhesion-promoting were helpful for gaining the highly purified endothelial cells.MTT assay show that after stimulated with high glucose(30 mM)for 48 h and 72 h,cell viability were decreased.While there were no significant effect of 10 mM and 20 mM glucose groups.Based on this results,high glucose(30 mM glucose)levels were used to mimic hyperglycemia condition.(2)Incubation with 30 mM glucose resulted in a significant increase in LDH release and a decrease in cell viability and the number of live cells,compared to cells incubated with 5.5 mM glucose medium.Treatment with NR1(5,10 μM)not only remarkably reduced LDH release,but also significantly increased cell viability and the number of live cells.Following staining with the annexin V/PI apoptosis kit,cells showed weak green fluorescence,whereas no red fluorescence was found in 5.5 mM glucose-treated cells.The number of cells with green and red fluorescence increased following exposure to high glucose(30 mM).Treatment with NR1(5,10 μM)resulted in lower fluorescence intensity.And compared to 5.5 mM glucose-treated cells,the mtDNA copy number decreased significantly.Treatment with 5.5 mM glucose and NR1(5,10μM)resulted in increased the mtDNA copy number.(3)Incubation with 30 mM glucose resulted in a significant increase in NOX activity,while CAT activity decreased,compared to cells incubated with 5.5 mM glucose medium.High glucose also caused a significant increased in both ROS and 3-NT levels in RCECs.Treatment with NR1(5,10 μM)not only remarkably inhibited NOX activitiy,but also increased CAT activity in RCECs.NR1 could also significantly reduced the production of ROS and decreased 3-NT expression.High glucose also decreased the ratios of NAD+/NADH,NADPH/NADP+ and GSH/GSSG.The addition of NR1 to the medium significantly increased the the ratios of NAD+/NADH,NADPH/NADP+and GSH/GSSG.(4)Incubation of RCECs with 30 mM gulcose markedly increased the activity of PARP,and decreased the activity of total SIRT,decreased the expression levels of SIRT-1 and SIRT-3.Treatment with NR1(5,10 μM)significantly decreased PARP activity,while increased the total SIRT activity and the expression levels of SIRT-1 and SIRT-3.Furthmore,incubation of RCECs with 30 mM gulcose markedly decreased the expression levels of Nmnat-1.But the expression levels of Nmnat-1 remained unchanged when treatment with NR1.4.Conclusion(1)These results demonstrate that NR1 could markly attenuate high glucose-induced cell damage in RCECs by reducing LDH release,increasing cell viability and the number of live cells and the mtDNA copy number and inhibiting of cell apoptosis.(2)NR1 reduced the high glucose-induced ROS via decreasing the high glucose-induced NOX activity.NR1 also can increased the anti-oxygenic enzyme activity and enhancd anti-oxygenic ability,increased the ratios of NAD+/NADH,NADPH/NADP+and GSH/GSSG.Therefore,NR1may exert its protective effects via mechanisms that involve changes in the cellular redox state.(3)NR1 was shown to have protect effects against high glucose-induced injury,and its operation mechanism is elucidated.This mechanism performance for the following two aspects.(Ⅰ).High glucose decrease the NAD+level in RCECs by decreased the expression of Nmnat-1 mRNA and its protein.But the results showed that NR1 could not interrupt the level of Nmnat-1 protein,which was a key enzyme in both the de novo pathway and the salvage pathway of NAD+ synthesis.(Ⅱ).NR1 inhibitied PARP activity which could mediate intracellular NAD+levels while significant DNA damage were occurs in RCECs.NR1 also increased the the expression levels of SIRT-1 and SIRT-3,which exert its protective effects via mechanisms that target molecules involve insulin resistance,mitochondrial dysfunction and oxidative stress.The present findings provide evidence for a functional role of NR1 in the prevention of DR.