GLP-1对AGEs诱导H9C2心肌细胞凋亡的保护作用研究

Protective effect of GLP-1 against AGEs-induced H9C2 myocardial cell apoptosis

目的研究胰高血糖素样肽素-1(glucogon like pep tide-1,GLP-1)对晚期糖基化终产物(advanced glycation end products,AGEs)诱导H9C2心肌细胞凋亡的保护作用机制。方法体外培养H9C2细胞,实验分为4组:正常对照组、100 mg·L^(-1)AGEs试验组、AGEs(100 mg·L^(-1))+GLP-1(10 nmol·L^(-1))组、AGEs(100 mg·L^(-1))+NAC(5 mmol·L^(-1))组,处理24 h。通过CCK-8比色法检测细胞存活率,相差显微镜观察细胞形态,双氯荧光素(DCFH-DA)染色荧光显微镜摄片观察细胞内活性氧(ROS)水平;RT-PCR法测定Bax、Bcl-2 mRNA基因的表达;运用Hoechst 33258检测试剂盒及流式细胞术检测心肌细胞的凋亡率,Western blot检测凋亡相关蛋白Bax、Bcl-2的表达量。结果与正常组相比,100 mg·L^(-1)AGEs组降低H9C2细胞生存率,活性氧(ROS)生成量增加;与AGEs组相比,AGEs+GLP-1组细胞生成率提高,活性氧(ROS)生成量减少。与正常组相比,AGEs组促凋亡蛋白Bax表达增加,抑制凋亡蛋白Bcl-2减少,细胞凋亡率升高;AGEs+GLP-1组较AGEs组下调促凋亡蛋白Bax,上调凋亡抑制蛋白Bcl-2,细胞凋亡率减少。结论 GLP-1对AGEs诱导的心肌细胞凋亡具有保护作用,其保护机制与减少活性氧(ROS)有关。

Aim To investigate the protective effect of Glucogon like pep tide-1（ GLP-1） on H9C2 cardiomyocytes against AGEs-induced apoptosis and the po-tential molecular mechanisms. Methods H9C2 cardiomyocytes cells cultured in vitro were divided into the following groups ： normal control group,1 0 0 mg · L^（-1）AGEs group,100 mg·L^（-1）AGEs ＋ 10 nmol·L^（-1）GLP-1 group,100 mg·L^（-1）AGEs ＋ 5 mmol·L^（-1）N-acetylcysteine（ NAC） group. Cell viabillity rate was measured by CCK-8 assay,ROS production was measured by DCFH-DA fluorescent probe; Cells in different groups were stained with Annexin V-FITC / PI and then apoptotic rate was detected by flow cytometry; Nucleus morphology was observed under fluorescence microscope after being incubated with Honchest 33258; Bax,Bcl-2 mRNA gene expression was measured using RTPCR; Western blot was applied to assess the apoptotic components expression including Bax and Bcl-2. Result Compared with control group,cell viability ratein AGEs group was decreased in a dose-dependent manner; cell apoptosis and ROS production in H9C2 cells were remarkably increased in AGEs group. However,compared with AGEs group,GLP-1 reduced ROS production and ameliorated cell apoptosis caused by AGEs; the expression of pro-apototic proteins Bax was decreased,the expression of anti-apoptotic proteins like Bcl-2 was increased. Conclusion GLP-1 protects H9C2 cardiomyocytes against AGEs-induced apoptosis,which may be related to the reduction of the active oxygen（ROS）.