Bian Yun-Fei, Wang Dong-Xue, Yang Hui-Yu, Xiao Chuan-Shi
Department of Cardiology, the Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001, China.
Sheng Li Xue Bao. 2011 Aug 25;63(4):387-95.
The present study was to investigate the effect of glucagon like peptide-1 (GLP-1) on high glucose-induced oxidative stress of cardiomyocytes and the possible role of the PI3K-Akt signal path in this process in the neonatal SD rats. With enzymatic digestion and immunofluorescence identification, cardiomyocytes after 72-96 h of primary culture were used in experiment. The cells were divided into 5 groups: normal control group, high glucose group, high glucose + GLP-1 group, high glucose + GLP-1 + LY294002 group and high osmolarity control group. The content of MDA was detected by TBA colouration method. The content of SOD was detected by xanthine oxidase method. The change of NADPH P47phox subunit mRNA quantity was detected by PCR gel electrophoresis. The level of ROS was detected by flow cytometry, and was also observed by fluorescence microscope. The DNA ladder was examined by agarose gel electrophoresis, and the cell apoptosis was determined by Annexin-V-FITC/PI flow cytometry, and the phosphorylation of Akt was determined by Western blotting. Compared with those in the normal control group, in the high glucose group, the cells grew poorly, and the beating rate was significantly lower (P < 0.05); The apoptotic rate was significantly increased (P < 0.05); The MDA content was increased (P < 0.05); It showed the typical DNA ladder, which is the characteristic of apoptosis; The SOD activity was decreased (P < 0.05); The level of intracellular ROS increased (P < 0.05); And the expression of NADPH P47phox subunit mRNA was increased; However the phosphorylation level of Akt was decreased. Pretreatment with GLP-1 improved the above-mentioned parameters and decreased the expression of NADPH P47phox subunit mRNA (P < 0.05). However, compared with the high glucose + GLP-1 group, LY294002, an inhibitor of PI3K-Akt signal path, attenuated the protective effect of GLP-1 in the high glucose + GLP-1 + LY294002 group. It is suggested that GLP-1 plays a protective role in the high glucose-induced injury and apoptosis of cardiomyocytes, and the PI3K-Akt signal path is involved in this process.
本研究旨在探讨胰高血糖素样肽-1（GLP-1）对高糖诱导的新生SD大鼠心肌细胞氧化应激的影响以及PI3K-Akt信号通路在此过程中的可能作用。采用酶消化法和免疫荧光鉴定法，将原代培养72 - 96小时后的心肌细胞用于实验。细胞分为5组：正常对照组、高糖组、高糖+GLP-1组、高糖+GLP-1+LY294002组和高渗对照组。采用TBA比色法检测丙二醛（MDA）含量；采用黄嘌呤氧化酶法检测超氧化物歧化酶（SOD）含量；采用PCR凝胶电泳法检测NADPH氧化酶P47phox亚基mRNA量的变化；采用流式细胞术检测活性氧（ROS）水平，并通过荧光显微镜观察；采用琼脂糖凝胶电泳检测DNA梯形条带，采用Annexin-V-FITC/PI流式细胞术检测细胞凋亡，采用蛋白质免疫印迹法检测Akt的磷酸化水平。与正常对照组相比，高糖组细胞生长不良，搏动率显著降低（P<0.05）；凋亡率显著升高（P<0.05）；MDA含量增加（P<0.05）；呈现典型的DNA梯形条带，这是凋亡的特征；SOD活性降低（P<0.05）；细胞内ROS水平升高（P<0.05）；NADPH氧化酶P47phox亚基mRNA表达增加；然而Akt的磷酸化水平降低。GLP-1预处理可改善上述参数，并降低NADPH氧化酶P47phox亚基mRNA的表达（P<0.05）。然而，与高糖+GLP-1组相比，PI3K-Akt信号通路抑制剂LY294002在高糖+GLP-1+LY294002组中减弱了GLP-1的保护作用。提示GLP-1对高糖诱导的心肌细胞损伤和凋亡具有保护作用，且PI3K-Akt信号通路参与此过程。
