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胰高血糖素样肽-1抑制高糖诱导的新生大鼠心室肌细胞氧化应激效应

边云飞, 王冬雪, 杨慧宇, 肖传实^*

山西医科大学第二医院心内科，太原 030001

摘要

本文旨在观察胰高血糖素样肽-1 (glucagon like peptide-1, GLP-1)对高糖所致新生大鼠心室肌细胞氧化应激的影响，并探讨PI3K-Akt 通路在其中所起的作用。将酶消化法分离的经α-肌动蛋白免疫荧光法鉴定的原代培养72~96 h的新生大鼠心室肌细胞分为5组：正常对照组、高糖组、高糖 + GLP-1组、高糖 + GLP-1 + LY294002 (PI3K-Akt通路的抑制剂)组、高渗对照组。采用硫代巴比妥酸显色法测定细胞上清液中丙二醛(malonaldehyde, MDA)含量，用黄嘌呤氧化酶法测定细胞上清液中超氧化物歧化酶(superoxide dismutase, SOD)的活性，运用PCR凝胶电泳检测NADPH P47phox亚基mRNA的变化，运用荧光显微镜及流式细胞术检测心室肌细胞内的ROS含量，运用流式细胞术及DNA ladder法检测心室肌细胞的凋亡，Western blot检测各组心肌细胞Akt磷酸化水平。结果显示：(1)与正常对照组相比，高糖组细胞生长状态较差，搏动频率减慢甚至消失，细胞凋亡率显著增加，DNA ladder呈现凋亡独有的“梯状图谱”，胞浆MDA水平增高，上清液中SOD活性下降，心肌细胞内ROS活性明显增高，NADPH P47phox亚基mRNA表达增加，Akt 磷酸化水平降低，差异有统计学意义(P < 0.05)；(2)葡萄糖浓度为25 mmol/L的DMEM中加入终浓度10 nmol/L的GLP-1培养72 h后，与高糖组相比，细胞搏动频率较快，凋亡率明显减少，DNA梯状条带消失，接近正常基因组的DNA带形，MDA水平明显下降，SOD活性明显升高，心肌细胞NADPH P47phox亚基mRNA表达明显降低，Akt 磷酸化水平升高，差异有统计学意义(P < 0.05)；PI3K-Akt通路的抑制剂LY294002可拮抗GLP-1对高糖所致心室肌细胞氧化应激损伤的保护作用，差异有统计学意义(P < 0.05)。以上结果提示，GLP-1可以逆转高糖所致的心室肌细胞氧化应激损伤及凋亡，对心室肌细胞起保护作用；PI3K-Akt通路的抑制剂LY294002可以阻断GLP-1对心室肌的保护作用，提示PI3K-Akt通路是GLP-1发挥保护作用的途径之一。

关键词： 新生大鼠; 心室肌细胞; 高糖; 胰高血糖素样肽-1; PI3K信号转导途径; LY294002

分类号：R363.2

[Glucagon like peptide-1 inhibits high glucose-induced injury of oxidative stress in cardiomyocytes of neonatal rats.] [Article in Chinese]

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

Abstract

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.

Key words: neonatal rat; cardiomyocyte; high glucose; glucagon-like peptide-1; PI3K-Akt signal path; LY294002

收稿日期：2011-02-18　　录用日期：2011-05-30

通讯作者：肖传实　　E-mail: ganxibaozhongxin@sina.com

引用本文：

边云飞, 王冬雪, 杨慧宇, 肖传实. 胰高血糖素样肽-1抑制高糖诱导的新生大鼠心室肌细胞氧化应激效应[J]. 生理学报 2011; 63 (4): 387-395.

BIAN Yun-Fei, WANG Dong-Xue, YANG Hui-Yu, XIAO Chuan-Shi. [Glucagon like peptide-1 inhibits high glucose-induced injury of oxidative stress in cardiomyocytes of neonatal rats.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (4): 387-395 (in Chinese with English abstract).