The Inhabition Effect Of Anthocyanins On Generation Of AGEs And Damage Of HUVEC Cells

With the improvement of living standards,the number of patients with diabetes(diabetes mellitus,DM)is increasing steadily in the world.Diabetic vascular complications are the leading cause of disability and death of diabetes.In vivo sustained hyperglycemia,the formation of advanced glycation end products(AGEs)is accelerated and is difficult to eliminate in the blood vessel wall.It has been shown that the interaction between AGEs and their receptor RAGE elicits intracellular oxidative stress and then leads to abnormal secretion of various cytokines and disturbance of cellular metabolism,and activation of intracellular signaling pathways.This in turn causes structural changes and dysfunction of endothelial cells.Reducing or inhibiting the formation of AGEs may effectively delay and prevent the progression of diabetes complications.Many chemicals have been used to prevent diabetes complications and treatment-related diseases.However,long-term intake of chemical drugs may appear side effects such as drug dependence,liver poisoning.Therefore,natural antioxidants will be the most promising drugs to prevent the formation of AGEs and the treatment of diabetic complications.Preliminary laboratory researches have proved that anthocyanin from Padus racemosaand fruits(APRF)anthocyanin from Coreopsis tinctoria Nutt(ACTN)have anti-oxidant activity.This study will further investigate the ability of these compounds to antagonise AGEs in vitro.The HUVECs damaged model was established,and then the protective effects and mechanisms of these compounds on HUVECs damaged by AGEs were discussed.In this experiment,AGEs were preparated in vitro,and UV spectrophotometer,fluorescence spectrometer and SDS-PAGE were usedtoidentify AGEs.The fluorescence spectrophotometer,circular dichroism spectroscopy and SDS-PAGE were conducted to observe the effects of APRF(0.0125 mg/mL?0.25 mg/mL?0.5 mg/mL)and ACTN(0.075 mg/mL?0.15 mg/mL?0.3 mg/mL).Examining the cell morphology after incubating by APRF(0.2 mg/mL?0.4 mg/mL)and ACTN(0.5 mg/mL?1.0 mg/mL)under the microscope.DCFH-DA assay was used to indicate the intracellular reactive oxygen species(ROS)levels in AGEs(200 ?g/mL)treated cells were detected for 24 h and indicate the influence of APRF(0.2 mg/mL,0.4 mg/mL)and ACTN(0.5 mg/mL,1.0 mg/mL)on HUVECs.Real-time quantitative PCR method was used todetect the the mRN A expression of IC AM-1?VC AM-1?IL-1??IL-18?CRP?NLRP3?Caspase-1?MTH1,and PARP-1 in HUVECs.The expressin proteins level of each group related associated proteins Nrf2,p38 MAPK,P-I?B,NF-?B p65 phosphorylation were measured by Western blot.The results showed that:AGEs were successfully generated after 6 weeks..Fluorescence spectrophotometer?Circular dichroism and SDS-PAGE results showed that two kinds of anthocyanins decreased in a dose-dependent manner compared with the AGEs group after intervention with dense anthocyanins and chrysanthemum glycosides respectively.DCFH-DA result showed that anthocyanin's inhibitory capacity can be attributable to its ability to scavenge free radicals.The microscopic observation showed that there was a significant inhibitory effect on the HUVECs of AGEs after the pretreatment of anthocyanins.Compared with the control group,AGEs upregulate the intracellular related factors significantly.When added anthocyanins to AGEs group,the intracellular related factors attenuated significantly compared with AGEs treatment group.It showed that the protein expression of p-p38 MAPK,NF-?B p65 phosphorylation and nucleus Nrf2 were significant different between these groups,as t-p38 MAPK,t-Nrf2,t-NF-?B p65 and t-I?B had no change.Compared with the control group,AGEs activated Nrf2?p-p3 8 MAPK?NF-?B p65 phosphorylation(p<0.05),but it could be inhibited by anthocyanins.The results of this experiment showed that two kinds of anthocyanins can inhibit the production of AGEs in vitro and inhibit the oxidative stress induced by AGEs,reduce inflammatory cytokines,block cell signaling pathways,protect vascular cells from injury.Therefore,they can be used to treat diabetic vascular disease.