胰高血糖素样肽-1抑制高糖和高胰岛素诱导的大鼠主动脉内皮细胞氧化损伤

Glucagon Like Peptide-1 Inhibits High Glucose and Insulin Concentration Induced Oxidation of Endothelial Cells in Rat Aortic Endothelial Cells

目的 观察胰高血糖素样肽-1（GLP-1）对高糖高胰岛素诱导主动脉内皮细胞损伤的保护作用,并揭示其机理.方法 原代培养正常成年SD大鼠胸主动脉内皮细胞,将细胞分为五组,即（A）正常糖（5.5 mmol/L）组,（B）高糖（25 mmol/L）组、（C）高糖±GLP-1（10 nmol/L）组,（D）高糖＋高胰岛素（100 nmol/L）组和（E）高糖高胰岛素±GLP-1组.采用DAPI染液检测发生凋亡改变的细胞,各组细胞凋亡率=（凋亡改变的细胞数/总细胞数）×100％;采用比色法检测细胞内caspase-3和SOD1,SOD2的相对酶活性（A实验组/A对照组）;采用流式细胞术检测细胞中活性氧自由基（ROS）的相对含量（平均荧光强度）,并通过荧光定量PCR对细胞中NADPH氧化酶的亚基p22phox以及SOD1,SOD2和过氧化氢酶的mRNA转录水平进行检测（RQ值）.结果 ①B组和C组细胞的凋亡率分别为（36.9±5.90）％和（20.5±3.39）％,两者相比差异具有统计学意义（P=0.035）;D组和E组细胞的凋亡率分别为（52±8.14）％和（17.52±0.68）％,两者相比差异具有统计学意义（P=0.017）;②caspase-3相对酶活性在B组和C组中分别为1.50±0.07,1.06±0.03,两者相比差异具有统计学意义（P=0.000）,在D组和E组中分别为1.77±0.08和1.22±0.07,两者相比差异具有统计学意义（P=0.000）;③ROS的相对含量在B组和C组中分别为1.50±0.11,0.52±0.10,两者相比差异具有统计学意义（t=6.844,P=0.000）;④B组中SOD2和过氧化氢酶的mRNA转录水平（1.06±0.06,1.04±0.22）均低于C组（1.33±0.10,1.77±0.16）,差异具有统计学意义（P=0.002和0.018）,D组中SOD2和过氧化氢酶的mRNA转录水平（1.08±0.13,1.02±0.15）均低于E组（1.27±0.06,1.58±0.17）,差异具有统计学意义（P=0.022和0.020）;B组和D组分别与C组和E组相比,p22phox和SOD1的mRNA转录水平差异不具有统计学意义（P＞0.05）;⑤SOD2的相对酶活性在B和C组中分别为0.82±0.02和1.03±0.07,两者相比差异具有统计学意义（P=0.040）,在D和E组中分别为0.78±0.06和0.96±0.05,两者相比差异具有统计学意义（P=0.033）;SOD1的相对酶活力在各组中的差异不具有统计学意义（F=0.677,P=0.618）.结论 GLP-1能降低大鼠主动脉内皮细胞中ROS的含量,抑制细胞凋亡,能有效抑制高糖和高胰岛素对内皮细胞的损伤.

Objective To investigate the potential protection of GLP 1 on rat aortic endothelial cells,and elucidate the signal events in molecular level. Methotls Rat aortic endothelial ceils were isolated from healthy Sprague-Dawley rats,cells were grouped into 5 groups：（A） normal glucose （5.5 mmol/L）, （B） high glucose （25 mmol/L）, ？ high glucose plus GLP-1 （10 nmol/L）,（D） high glucose plus high insulin （100 nmol/L） and （E） high glucose plus high inulin plus GLPd. Cell apooptosis was detected with DAPI staining （Apoptotic rate -- number of apoptotic cells/number of total cells） and easpase- 3 activity assay Intraeellular ROS concentration was determined with FACS （fluorescence intensity）. Ex pression p22,SOD1 ,SOD2 and catalase were detected with real time PCR （RQ value）,and the activity of SOD1 and SOD2 were determined with colorimetry assay . Results @ The apoptotic rate in group B （36.9 ± 5.90） were significantly higher than that of in group C （20.5±3. 39） in endothelial cells （P=0. 0351 ,and the apoptotic rate in group D （52 ±8. 14）%were significantly higher than that of in high group E （17.52 ± 0.68）% in endothelial cells （19=0. 017 Caspase 3 relative activity in group 13 （1.5±0.07） were higher than that of in group C （1. 06±0.03） in en dothelial cells （P=0. 000）,and caspase-3 relative activity in group D （1.77±0.08） were higher than that of in group E（1.22±0.07） in endothelial cells （P= 0. 000） ;ROS relative concentration in group B （1.50 ± 0.11 ） were higher than that of in group C （0.52±0.1） in endothelial cells （t=6. 844,P=0. 000） Expression of SOD2 and catalase in group B were 1.06±0.06 and 1.04±0.22,which were lower than those of in group C （1. 33±0.10 and 1.77±0. 16） in endothelial cells （P〈0. 002 and 0. 018） ;Expression of SOD2 and catalase in group D （1.08±0.13 and 1.02±0.15） were lower than that of in group E （1.27±0.06 and 1. 58±0.17） in endothelial cells （P=0. 022 and 0. 020） ,and there were no significant difference between group B and C in expression of p22phx and SOD1 （P〈0.05） ,neither between group D and ESOD2 relative activity in group B （0.82±0.02） were lower than that of in group C （1.03±0.07） in endothelial cells （P=0. 040） ,and SOD2 relative activity in group D （0. 78±0.06） were lower than that of in group E （0. 96±0.05） in endothelial cells （P= 0. 033）. There were no significant difference among those groups in SOD1 relative activity （P〈0.05）. Conclusion GLP-1 decreased the concentration of ROS in rat aortic endothelial cells, and inhibited cells apoptosis. Moreover, GLP 1 inhibited injury of endothelial cells which were exposed in high glucose and insulin.