p38MAPK-eNOS-N0信号通路在人脐静脉内皮细胞凋亡中的保护作用

目的探讨p38MAPK信号通路在胰高血糖素样肽1(GLP-1)拮抗人脐静脉内皮细胞凋亡中的作用。方法实验分为对照组、糖基化终末产物(AGE)组、GLP-1组、AGE+GLP-1组、AGE+SB203580组、AGE+GLP-1+SB203580组及AGE+GLP-1+L-NAME组,Western blot检测p-p38MAPK/p38MAPK、磷酸化内皮型一氧化氮合酶/内皮型一氧化氮合酶(p-eNOS/eNOS)蛋白表达情况,NO检测试剂盒(一步法)检测NO含量,DCFH-DA荧光探针检测细胞活性氧(ROS)含量,Annexin V/PI流式检测细胞凋亡率。结果与AGE组相比,GLP-1预处理可诱导p-p38MAPK蛋白表达下降(P=0.000);与对照组比较,GLP-1或p38 MAPK抑制剂(SB203580)预处理后,受AGE抑制的eNOS蛋白表达或诱导的ROS水平分别显著升高(P=0.004)或下降(P=0.000);GLP-1预处理后,因AGE诱导的细胞凋亡率显著降低(P=0.000),而加入L-NAME后,GLP-1的抗凋亡作用显著减弱(P=0.002);GLP-1预处理后,细胞NO含量较单纯AGE组明显升高(P=0.000),而予以L-NAME后,细胞NO含量显著降低(P=0.011)。结论GLP-1可抑制p38 MAPK信号通路的活化,拮抗AGE对血管内皮细胞的氧化损伤;上调eNOS蛋白的表达,拮抗AGE诱导的内皮细胞NO生成障碍及细胞凋亡,从而延缓糖尿病合并动脉粥样硬化的发生发展。

The Role of p38MAPK-eNOS-NO Signaling Pathway in the Protection Mechanisms of Apoptosis of Endothelial Cells

Aim To investigate whether p38MAPK signaling pathway is involved in the protective mechanism of glucagon like peptide-1 (GLP-1) on endothelial cell damage induced by advanced glycation endproducts (AGE). Methods The experiment was divided into control group, AGE group, GLP-1 group, AGE+GLP-1 group, AGE+SB203580 group, AGE+GLP-1+SB203580 group, AGE+GLP-1+L-NAME group. Expression of p-p38MAPK/p38MAPK, phospho endothelial nitric oxide synthase/endothelial nitric oxide synthase (p-eNOS/eNOS) protein were examined by Western blot. Nitric oxide (NO) generation was detected by NO detection kit. Reactive oxygen species (ROS) generation was examined by fluorescent probe technique. The apoptosis rate was tested by Annexin V/PI flow cytometry. Results Compared with AGE group, GLP-1 inhibited the expression of p-p38MAPK protein(P=0.000). Compared with control group, after adding GLP-1 or p38MAPK inhibitor (SB203580), the expression of eNOS protein inhibited and ROS generation induced by AGE was significantly increased(P=0.004)or decreased(P=0.000). After adding GLP-1, the increased apoptosis rate induced by AGE was inhibited significantly(P=0.000). While after adding L-NAME, the anti-apoptosis effect was significantly weakened(P=0.002). After adding GLP-1, NO generation increased significantly(P=0.000) compared with AGE group. While after adding L-NAME, NO generation decreased significantly(P=0.011). Conclusion GLP-1 can inhibit the oxidative damage of vascular endothelial cells by inhibiting the activation of p38MAPK signaling pathway, and increase the expression of eNOS protein to increase NO generation and antagonize apoptosis induced by AGE.