达格列净改善糖尿病动脉粥样硬化模型小鼠斑块的作用机制  

Mechanism of dapagliflozin alleviating plaque in diabetic atherosclerotic model mice

作　　者：方岩 张唯薇 蒋梦婷 宋玲 刘杰[2] 易丽 李泱 高磊[1,2] FANG Yan;ZHANG Weiwei;JIANG Mengting;SONG Ling;LIU Jie;YI Li;LI Yang;GAO Lei(Chinese PLA Medical School,Beijing,100853,China;Medical Department of Cardiovascular Disease,the Sixth Medical Center,Chinese PLA General Hospital,Beijing 100853,China;Department of Cardiology,the First Medical Center,Chinese PLA General Hospital,Beijing,100853,China)

机构地区：[1]解放军医学院,北京100853 [2]解放军总医院第六医学中心心血管病医学部,北京100853 [3]解放军总医院第一医学中心心内科,北京100853

出　　处：《解放军医学院学报》2022年第2期186-192,198,共8页Academic Journal of Chinese PLA Medical School

基　　金：国家自然科学基金面上项目(81671731,81970443)。

摘　　要：背景近年来多项研究显示,钠-葡萄糖共转运体2(sodium-glucose cotransporter-2,SGLT2)抑制剂可以降低2型糖尿病患者心血管事件的发生率,相关机制有待研究。目的 探究SGLT2抑制剂达格列净对糖尿病动脉粥样硬化(atherosclerosis,AS)斑块的影响及其机制。方法 构建Apo E-/-小鼠糖尿病AS模型,分别设置AS对照组(Con)、糖尿病AS胰岛素干预组(Insulin)和糖尿病AS达格列净干预组(Dapa),每组10只,定期监测血糖、体质量、血脂等指标。腹腔注射胰岛素和达格列净进行干预,8周后处死小鼠,取主动脉行大体油红O染色,取主动脉根部切片行油红O染色、HE染色和免疫组化染色,观察动脉粥样硬化病变和巨噬细胞斑块内浸润情况。培养小鼠巨噬细胞RAW264.7,通过CCK-8毒性实验筛选达格列净合适的干预浓度。连续培养72 h,CCK-8增殖实验检测达格列净对巨噬细胞增殖的影响,Western blot检测凋亡相关蛋白裂解半胱天冬酶3(cleaved caspase 3,CL Caspase3)表达情况。脂多糖(lipopolysaccharide,LPS)诱导小鼠巨噬细胞RAW264.7构建炎症模型,分别设置空白对照组、炎症对照组和达格列净干预组,ELISA检测巨噬细胞上清液中白细胞介素6(interleukin 6,IL-6)、肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)、单核细胞趋化因子1(monocyte chemoattractant protein-1,MCP-1)和粒细胞巨噬细胞集落刺激因子(granulocyte macrophage colony stimulating factor,GM-CSF)分泌水平。结果 与Insulin组相比,Dapa组小鼠血糖、体质量和血脂均无显著差异。大体油红O染色显示,Dapa组较Insulin组AS面积减少38%(P<0.05)。HE染色结果显示,Dapa组较Insulin组斑块负荷减少25%(P<0.05)。免疫组化染色显示,Dapa组较Insulin组斑块内F4/80阳性巨噬细胞明显减少(P<0.01)。CCK-8毒性实验结果提示,达格列净对体外巨噬细胞干预合适浓度为50μmol/L。CCK-8增殖实验表明达格列净显著抑制巨噬细胞增殖(P<0.05),Western blot�Background In recent years, several studies have shown that sodium-glucose cotransporter 2(SGLT2) inhibitors may reduce the incidence of cardiovascular events in patients with type 2 diabetes, while the related mechanisms remain to be further studied. Objective To investigate the effect and mechanism of dapagliflozin, one of SGLT2 inhibitors, on diabetic atherosclerotic(AS) plaque. Methods Apo E-/-mice were used to construct the models of AS. Then the mice were divided into AS control group(Con), diabetic AS insulin intervention group(Insulin) and diabetic AS dapagliflozin intervention group(Dapa) with 10 mice in each group, and the levels of blood glucose, body weight and blood lipid were monitored regularly. At 8 weeks after intra-abdominal injection of insulin and dapagliflozin, the mice were sacrificed. Aortae were taken for gross oil red O staining, and aortic root sections were taken for oil red O staining, HE staining and immunohistochemical staining, to evaluate atherosclerotic lesion and infiltration of macrophages in plaque. Mouse macrophages line RAW264.7 were cultured, and appropriate intervention concentration of dapagliflozin was determined by CCK-8 toxicity test. After cultured for 72 hours, the effect of dapagliflozin on macrophage proliferation was detected by CCK-8 proliferation assay, and apoptosis-related protein Cleaved Caspase 3(CL Caspase 3)was detected by western blot. RAW264.7 was induced by lipopolysaccharide(LPS) to construct an inflammatory model, and blank control group, inflammation control group and dapagliflozin intervention group were set up, respectively. The secretion levels of interleukin-6(IL-6), tumor necrosis factor-alpha(TNF-α), monocyte chemoattractant protein-1(MCP-1) and granulocyte macrophage colony-stimulating factor(GM-CSF) in macrophages supernatant were determined by enzyme-linked immunosorbent assay(ELISA). Results There was no significant difference between Insulin group and Dapa group in blood glucose, body weight or blood lipid. Gross oil red O staining showed

关 键 词：钠-葡萄糖转运体2抑制剂 动脉粥样硬化 糖尿病 巨噬细胞 粒细胞巨噬细胞集落刺激因子 

分 类 号：R587.1[医药卫生—内分泌]