【作者】 周颖；

【导师】 王勇军；

【作者基本信息】 中南大学 ， 临床检验诊断学， 2022， 硕士

【摘要】 目的:本研究旨在探索达格列净对血管内皮细胞衰老以及糖尿病血管内皮依赖的舒张功能的影响,并探讨达格列净影响糖尿病血管内皮舒张功能的具体机制。方法:本研究分为体内实验和体外试验两部分。体内实验部分,糖尿病小鼠（db/db）和同窝阴性对照小鼠（db/m）随机分为四组分别接受达格列净（1mg/kg/天）或生理盐水治疗,每日口服灌胃,干预8周。每周一次记录小鼠血糖、体重以及食物摄取量。体外实验部分,高糖或过氧化氢（H₂O₂）诱导脐静脉内皮细胞（Human umbilical vein endothelial cells,HUVECs）衰老模型;烟酰胺（Nicotinamide,NAM）抑制SIRT1酶活性,以评估SIRT1是达格列净发挥抗衰老作用的关键蛋白。通过检测衰老相关β-半乳糖苷酶（Senescence-associated beta-galactosidase,SA-β-gal）活性、衰老标志蛋白表达水平（p21、p53）、氧化应激、血清一氧化氮（Nitric Oxide,NO）水平以及小鼠体内循环炎症因子浓度来评估内皮功能和衰老状态。使用小鼠动脉Myograh血管张力实验检测小鼠血管舒张功能以评估达格列净的慢性血管保护作用。使用免疫共沉淀的方法验证去乙酰化酶1（Sirtuins1,SIRT1）和内皮型一氧化氮合酶（Endothelial nitric oxide synthase,e NOS）之间的相互作用。结果:体内试验部分,达格列净可以降低糖尿病小鼠血糖水平以及胰岛素抵抗指数,同时增加血清NO浓度;小鼠血管张力实验结果表明,达格列净可以显著改善糖尿病小鼠血管内皮依赖性舒张功能障碍;除此之外,达格列净还可以减少糖尿病小鼠血管内皮衰老以及血清炎症因子的释放。体外实验部分,高糖和H₂O₂诱导HUVECs衰老,表现为ROS生成增多、p21和p53的表达增多以及p-e NOS、SIRT1表达减少,达格列净干预可以逆转HUVECs衰老状态;免疫共沉淀的结果显示,达格列净可以增加e NOS与SIRT1的结合,降低e NOS乙酰化水平;而NAM处理组细胞表现出与衰老模型组细胞相似的衰老表型,并且在NAM存在时,达格列净无法发挥其抗内皮细胞衰老的作用;结论:达格列净通过SIRT1/e NOS途径减少内皮细胞衰老,改善2型糖尿病血管内皮依赖的舒张功能障碍。图33幅,表8个,参考文献56篇更多还原

【Abstract】 Objective:This study aims to figure out whether dapagliflozin attenuates endothelial senescence and improves diabetic endothelium-dependent vasorelaxation function,and explore the possible pathway affecting vasodilation.Methods:This study was divided into in vivo and in vitro experiments.In vivo experiment,the diabetic mice（db/db mice）and heterozygous littermates（db/m mice）were randomized four group.Dapagliflozin treatment groups were given a daily oral dose of dapagliflozin（1 mg/kg/day）for 8 week.The placebo groups were given a daily oral dose of vehicle（saline）as was consumed by the dapagliflozin-treated group.Blood glucose,body weight and food intake were measured weekly.In vitro experiment,high glucose or H₂O₂ induced human umbilical vein endothelial cells（HUVECs）senescence.We used a specific and potent pharmacological inhibitor of SIRT1,Nicotinamide（NAM）,to inhibit the activity of SIRT1.It was evaluated that SIRT1 is the key protein of dapagliflozin for its anti-aging effect.Endothelial dysfunction and senescence was assessed by senescence?associated beta?galactosidase（SA‐β‐gal）activity,senescence marker protein（p21,p53）,oxidative stress,nitric oxide（NO）and inflammatory cytokines.Assessment of chronic vaso-protective effect of dapagliflozin was performed in organ bath studies.The interaction between SIRT1 and e NOS was detected by co-immunoprecipitation.Results:In vivo experiment,dapagliflozin treatment decreased the level of blood glucose and insulin resistance index,while increased serum NO concentration.As for chronic vaso-protective effect of dapagliflozin,dapaglifozin treatment improved endothelium-dependent vasorelaxation such that dilation was modestly and significantly increased.Dapagliflozin treatment also decreased the senescence of tunica intima and endothelial cells senescence of the aorta,as well as inflammatory cytokines.In vitro experiment,HG or H₂O₂ treatment induced cellular senescence in HUVECs,increased p21,p53 and ROS formation,and decreased the expression of SIRT1 and p-e NOS,but exposure to dapagliflozin reversed the effect.e NOS acetylation levels as well as levels of SIRT1 associated with e NOS following co-immunoprecipitation.The e NOS acetylation was higher in HG-or H₂O₂-treated cells and was restored by dapagliflozin treatment.SIRT1 levels associated with e NOS were decreased in H₂O₂-treated cells and were restored by dapagliflozin treatment.NAM reduced the anti-senescence effects of dapagliflozin in HG-or H₂O₂-induced senescent endothelial cells（Similar to the aging model group）.Conclusions:Dapagliflozin attenuates endothelial senescence and improves endothelium-dependent vasorelaxation by activating SIRT1/e NOS signaling pathway in type 2 diabetes mellitus.Figures 33,Tables 8,References 56更多还原