Zhu Minmin, Wen Meilin, Sun Xia, Chen Wankun, Chen Jiawei, Miao Changhong
From the Department of Anaesthesiology, Fudan University Shanghai Cancer Center, and Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China.
Anesth Analg. 2015 Apr;120(4):781-9. doi: 10.1213/ANE.0000000000000616.
Perioperative hyperglycemia is a common clinical metabolic disorder. Hyperglycemia could induce endothelial apoptosis and dysfunction. Propofol is a widely used IV anesthetic drug in clinical settings. In the present study, we examined whether and how propofol reduced high glucose-induced endothelial apoptosis and dysfunction in human umbilical vein endothelial cells (HUVECs).
HUVECs were cultured with different concentrations (5, 10, 15, and 25 mM) of glucose for different times (4, 8, 12, and 24 hours). To study the effect of propofol, cells were incubated with different concentrations (0.2, 1, 5, and 25 μM) of propofol for 2 hours. In parallel experiments, cells were incubated in 5 mM glucose as control. Nitric oxide (NO) production was measured with a nitrate reductase assay. Cell viability was determined with a Cell Counting Kit-8. Protein expression of active caspase 3, cytochrome c, endothelial NO synthase (eNOS), p-eNOS-Thr, p66, protein kinase C βII (PKCβII), and p-PKCβII-Ser was measured by Western blot analysis. Accumulation of superoxide anion (O2˙) was measured with the reduction of ferricytochrome c. Cell apoptosis was determined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining.
Compared with control, high glucose decreased NO production (P < 0.0001) and reduced cells viability (P < 0.0001) in HUVECs. Compared with high glucose treatment, pretreatment of cells with propofol (5 μM, 2 hours) reduced high glucose-induced inhibitory p-eNOS-Thr phosphorylation (P < 0.0001), increasing NO production (P = 0.0007), decreased high glucose-induced p66 expression (P < 0.0001) and p66 mitochondrial translocation (P < 0.0001), O2˙ accumulation (P < 0.0001), mitochondrial cytochrome c release (P < 0.0001), active caspase 3 expression (P < 0.0001), and enhancing endothelial viability (P < 0.0001). Furthermore, propofol inhibited high glucose-induced PKCβII expression (P = 0.0002) and p-PKCβII-Ser phosphorylation (P < 0.0001). Moreover, the observed protective effect of propofol was quite similar to that of PKCβII inhibitor.
Propofol, by a mechanism of decreasing high glucose-induced PKCβII expression and p-PKCβII-Ser phosphorylation, inhibits high glucose-induced p66 mitochondrial translocation, therefore protecting HUVECs from high glucose-induced endothelial dysfunction and apoptosis.
围手术期高血糖是一种常见的临床代谢紊乱。高血糖可诱导内皮细胞凋亡和功能障碍。丙泊酚是临床广泛使用的静脉麻醉药物。在本研究中，我们研究了丙泊酚是否以及如何减轻高糖诱导的人脐静脉内皮细胞（HUVECs）凋亡和功能障碍。
将HUVECs用不同浓度（5、10、15和25 mM）的葡萄糖培养不同时间（4、8、12和24小时）。为研究丙泊酚的作用，将细胞用不同浓度（0.2、1、5和25 μM）的丙泊酚孵育2小时。在平行实验中，将细胞在5 mM葡萄糖中孵育作为对照。用硝酸还原酶法测定一氧化氮（NO）生成量。用细胞计数试剂盒-8测定细胞活力。通过蛋白质印迹分析测量活性半胱天冬酶3、细胞色素c、内皮型一氧化氮合酶（eNOS）、p-eNOS-Thr、p66、蛋白激酶C βII（PKCβII）和p-PKCβII-Ser的蛋白表达。用高铁细胞色素c还原法测量超氧阴离子（O2˙）的积累。用末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记染色法测定细胞凋亡。
与对照相比，高糖降低了HUVECs中NO生成量（P < 0.0001）并降低了细胞活力（P < 0.0001）。与高糖处理相比，用丙泊酚（5 μM，2小时）预处理细胞可降低高糖诱导的抑制性p-eNOS-Thr磷酸化（P < 0.0001），增加NO生成量（P = 0.0007），降低高糖诱导的p66表达（P < 0.0001）和p66线粒体易位（P < 0.0001）、O2˙积累（P < 0.0001）、线粒体细胞色素c释放（P < 0.0001）、活性半胱天冬酶3表达（P < 0.0001），并提高内皮细胞活力（P < 0.0001）。此外，丙泊酚抑制高糖诱导的PKCβII表达（P = 0.0002）和p-PKCβII-Ser磷酸化（P < 0.0001）。此外，观察到的丙泊酚保护作用与PKCβII抑制剂的作用相当相似。
丙泊酚通过降低高糖诱导的PKCβII表达和p-PKCβII-Ser磷酸化的机制，抑制高糖诱导的p66线粒体易位，从而保护HUVECs免受高糖诱导的内皮功能障碍和凋亡。
