The Natural Flavone Acacetin Confers Significant Protection Against Diabetic Atherosclerotic Vascular Injury

Introduction:Diabetes mellitus(DM)is a general systemic disease.More than 80%of diabetic patients die from vascular complications.Atherosclerosis(AS)is the main vascular complication in diabetic patients.Endothelial dysfunction is an important initiation and key factor for atherosclerosis and chronic vascular complications in type 1 diabetes.Endothelial metabolic disorders and increased oxidative stress are the internal causes on the occurrence and development of DM.How to prevent and reduce vascular complications caused by DM has remained a difficult problem in cardiovascular research field for many years.Previous studies have shown that Acacetin,a natural flavonoid,has anti-inflammatory,antioxidant and anti-tumor effects.In this study,apolipoprotein gene knockout(ApoE-/-)mice were intraperitoneally injected with streptozotocin(STZ)to establish atherosclerosis model mice with type 1 diabetes mellitus(D1TM)in order to explore whether Acacetin has protective effect on the blood vessels of ApoE-/-mice with diabetic atherosclerosis and the possible molecular mechanism.Methods:Diabetic atherosclerosis mice were established by intraperitoneal injections of STZ in ApoE-/-mice,and then subcutaneously injected with 20 mg/kg of Acacetin or control agent for 12 weeks.The degree of vascular stenosis,intimal thickness,wall elasticity and blood flow velocity were evaluated by animal magnetic resonance imaging and vascular ultrasound imaging.The lipids plaque accumulations were observed by oil red O staining.HE staining and Masson staining were used to observe intimal neogenesis,perivascular fibrosis.Additionally,blood glucose,body weight,serological changes and the expression and distribution of vascular tissue protein were also assessed.In cultured human umbilical vein endothelial cells(HUVECs),endothelial celluar injury was induced by high glucose(33 mmol/L)to determine whether Acacetin protects against high glucose exposure.MTT assay was used to determine cell activity,flow cytometry to determine cell apoptosis and intracellular reactive oxygen species(ROS)level.In addition,MDA,SOD activity and mitochondrial function were also assessed.Multiple biochemical and molecular biological methods to detect the expression of oxidoreduct protein,apoptosis-related proteins and energy metabolism-related proteins in endothelial cells damaged by high glucose exposure.Gene silencing approach was employed to detect the changes on expression of related proteins for exploring the molecular mechanism of Acacetin against endothelial cell injury induced by high glucose.Results:The results showed that in STZ-induced diabetic atherosclerosis ApoE-/mice,compared with the control group,the deposition of vascular plaques and blood flow velocity were increased,the elasticity of vascular wall was decreased,Notably,lumen stenosis,neointima thickening,peritubular fibrosis were not changed significantly in diabetic ApoE-/-mice,and Acacetin significantly antagonized the above changes.Acacetin had no significant effect on the increased random blood glucose in diabetic mice,but it could significantly alleviate the abnormal distribution of blood lipids caused in diabetes setting,significantly increase the levels of antioxidant protein SOD1,SOD2 and anti-apoptotic protein Bcl2/Bax,and increase the expression of energy metabolism related proteins SIRT1,SIRT3,p-AMPK/AMPK and PGC-1 alpha.In the model of endothelial injury induced by high glucose,Acacetin(0.3 μM,1μM,3μM)can decrease ROS level in a dose-dependent manner,reduce endothelial cell apoptosis,increase antioxidant proteins in cells and supernatants,reduce lipid peroxidation damage,improve mitochondrial function,and increase SOD1,SOD2 and Bcl2/Bax levels in cells.The expression of energy metabolism proteins SIRT1,SIRT3,p-AMPK/AMPK and PGC-1 alpha related proteins can protect endothelial cells and decrease vascular sclerosis and plaques.Through gene silencing technique in HUVECs,it was found that Acacetin could inhibit atherosclerosis which was related to the activation of SIRT1-AMPK-PGC-la-SIRT3 signaling pathway.Conclusion:Hyperglycemia can damage vascular endothelial cells,accelerate the occurrence and development of vascular atherosclerosis,while Acacetin has a protective effect on diabetic atherosclerosis,which can reduce the damage of vascular endothelial and ease endothelial atherosclerotic plaque,and can alleviate HUVECs oxidative stress,apoptosis and mitochondrial dysfunction by activating SIRT1-AMPK-PGC-la-SIRT3 signaling pathway,thus protecting the occurrence and development of diabetic atherosclerosis.The results suggest that Acacetin may be a new intervention in the treatment/prevention of diabetic atherosclerosis.