利拉鲁肽对瘦素受体缺陷大鼠主动脉内皮细胞的保护作用及机制

Protective effect and mechanism of liraglutide on aorta endothelial cells in Zucker diabetic fatty rats

目的观察利拉鲁肽对瘦素受体缺陷所致糖尿病大鼠主动脉内皮细胞的作用并探讨其作用机制。方法选取8只10周龄Zucker瘦型大鼠作为正常组（A组），另选16只10周龄无特定病原体级Zucker肥胖糖尿病大鼠，空腹血糖均〉11.1 mmol/L，按照随机数字表法分为糖尿病模型组（B组）和利拉鲁肽治疗组（C组）。A、B组均给予等量的生理盐水，C组予皮下注射利拉鲁肽150 μg/kg，2次/d，共干预6周。实验结束后下腔静脉取血，留取主动脉。检测血清脂质氧化代谢产物丙二醛（MDA）及抗氧化酶谷胱甘肽过氧化物酶（GSH-Px）、超氧化物歧化酶（SOD）；炎症因子白细胞介素6（IL-6）、血管细胞黏附分子1（VCAM-1）；检测主动脉内皮细胞凋亡并观察主动脉形态学变化；Western blotting法检测主动脉中表皮生长因子受体（EGFR）、蛋白激酶B（Akt）、磷酸化蛋白激酶B（p-Akt）和内皮型一氧化氮合酶（eNOS）的表达。两组间比较采用t检验，多组间比较采用单因素方差分析，数据不符合正态分布者使用秩和检验。结果（1）干预6周后血清MDA水平B组[（3.8±1.2）μg/L]高于A组[（3.3±0.7）μg/L]、C组[（1.2±0.5）μg/L]，差异有统计学意义（F=15.4，P〈0.05）；B组SOD、GSH-PX水平低于A、C组，差异有统计学意义（F=4.7、3.4，均P〈0.05）。（2）干预6周后B组血清IL-6水平高于A组（t=2.95,P〈0.05），但与C组比较差异不具有统计学意义（t=－0.60，P〉0.05）。B组血清VCAM-1的含量[51（48-70）μg/L]高于A组[29（19-30）μg/L]、C组[19（11-25）μg/L]（H=1.20，P〈0.05）。（3）干预6周后B组可见部分主动脉内皮细胞出现凋亡，而A、C组未见。（4）Western blotting显示EGFR、p-Akt、eNOS在B组的表达量最低，C组最高（t=2.09、2.10、13.75，均P〈0.05）。结论利拉鲁肽可降低瘦素受体缺陷大鼠的氧化应激水平，并通过EGFR-Akt-eNOS通路发挥对糖尿病大鼠主动脉内皮细胞的保护作用。

ObjectiveTo investigate the protective effect and mechanism of liraglutide on aorta endothelial cells in Zucker diabetic fatty （ZDF） rats.MethodsSixteen special pathogen free ZDF rats aged 10 weeks were adopted. The fasting plasma glucose （FPG） of them were greater than 11.1 mmol/L. They were divided into two groups by random number table and treated with subcutaneous injections liraglutide （group C） or saline （group B） 150 μg/kg, bid. Eight zucker lean rats were administered saline of the same volume （group A）. Glycemic control was assessed by measurements of blood glucose levels every week. At the end of the experiment （after 6 weeks）, the blood and the aorta were reserved. The oxidative stress marker malondialdehyde （MDA） and anti-oxidative enzyme glutathione peroxidase （GSH-Px）, superoxide dismutase （SOD） were measured. The inflammatory factor such as interleukin 6 （IL-6）, vascular cell adhesion molecule 1 （VCAM-1） were evaluated by enzyme-linked immunosorbent assay （ELISA）. The hematoxylin-eosin staining was used to observing the morphology of aorta. Western blotting was used for detecting the expression of epidermal growth factor receptor （EGFR）, protein kinase B （Akt）, phosphorylated Akt （p-Akt）, endothelial nitric oxide synthase （eNOS） in aorta. The t test was performed in comparison between the two groups, while one way variance analysis for multiple groups comparison. The rank sum test was used when the data did not meet the normal distribution.Results（1） After 6 weeks interventions, the level of MDA in group B [（3.8±1.2）μg/L] was increased compared with group A [（3.3±0.7）μg/L], which is decreased in group C[（1.2±0.5）μg/L, F=15.4, P〈0.01]; SOD and GSH-Px were decreased in group B, which were increased in group C （F=4.7, 3.4, both P〈0.05）. （2）The level of IL-6 was increased in group B compared with group A （t= 2.95, P〈0.05）, and it was decreased in group C compared with group B, but there was no statistical difference （t=-0.60, P〉0.05）. The VCAM-1 was increased in group B [51（48-70）μg/L] compared with group A [29 （19-30）μg/L], and it was decreased in group C [19（11-25）μg/L] compared with group B （H=1.20, P〈0.05）. （3） TdT-mediated dUTP nick-end labeling （TUNEL） staining showed that there were apoptotic endothelial cells in aortic sections in group B but apoptotic cell was not found in group A and group C. （4）The expression of EGFR, p-Akt and eNOS were decreased in group B compared with group A, however, they were increased in group C （t=2.09, 2.10, 13.75, all P〈0.05）.ConclusionLiraglutide can alleviate oxidative stress in ZDF rats independent of the blood glucose and body weight, liraglutide plays a protective role on macrovascular endothelial cell by EGFR-PI3K/Akt-eNOS pathway.