桂皮醛激活血管内皮TRPA1防止高糖诱导的氧化应激保护血管内皮功能

Activation of TRPA1 by Cinnamaldehyde Protects Endothelial Function by Preventing High Glucose Induced Oxidative Stress

目的:探索桂皮醛对高糖诱导的内皮细胞氧化应激的影响及其对相关血管内皮功能损害的作用,初步分析其作用机制。方法:(1)制作高糖(30 m M)致人脐静脉内皮细胞(HUVEC)损伤和血管组织损伤模型,并以低糖(5.5 m M)作为对照,高糖干预的HUVECs给予桂皮醛(10μM)或桂皮醛+TRPA1特异性拮抗剂(HC030031,10μM)进行干预,以DHE染色和DAF-2DA染色观察各组细胞超氧阴离子和一氧化氮(NO)水平;western blotting分析核因子E2相关因子2(Nrf2),内皮一氧化氮合酶(eNOS),磷酸化eNOS及P22^(phox)水平。(2)以TRPA1敲除(TRPA1^(-/-))及相应的野生型(Wild type,WT)小鼠分离胸主动脉进行体外培养,设低糖(5.5 m M D-葡萄糖)组、高糖(30 m M D-葡萄糖)组、高糖+桂皮醛(10μM)组,以微血管张力测定仪测定血管内皮依赖性舒张功能和非内皮依赖性舒张功能。结果:(1)桂皮醛可显著减少高糖介导的内皮细胞超氧阴离子的产生,防止NO水平下降,但上述作用可被HC030031所阻断。(2)桂皮醛可显著防止WT小鼠胸主动脉血管内皮依赖性舒张功能减退,但对TRPA1^(-/-)小鼠无上述作用。(3)桂皮醛剂量依赖性地上调Nrf2的表达,还可促进eNOS磷酸化,减少P22^(phox),上述作用均可被HC030031阻断。结论:桂皮醛激活TRPA1通过Nrf2信号通路可改善高糖介导的血管内皮细胞氧化应激水平,防止NO水平下降,改善血管内皮依赖性舒张功能。

Objective： To explore the effects and mechanism of cinnamaldehyde on oxidative stress induced by high glucose in endothelial cells and related endothelial dysfunction. Methods： （~） Human umbilical vein endothelial ceils （blUVECs）and arteries tissue models induced by high glucose（30 raM） were erected. Cells and arteries tissue cultured under low glucose levels（5 raM） condition were used as control. Cells under HG condition were treated with cirmamaldehyde （CA, 10 μM） or CA＋HC030031 （A selective TRPA1 inhibitor, 10 μM）. DHE and DAF-2DA staining were used to analysis the superoxide anion and NO level in HUVECs respectively. Nrf2, eNOS, p-eNOS and P22^phox were analysis by western blotting. （2） Thoracic aorta separated from TRPA1 knockout or the wild type mice were cultured under HG or NG. The experiment was divided into three groups, low glucose group, HG group and HG＋CA group. Wire myograph was used to analysis the endothelium dependent relaxation and endothelium independent relaxation. Results： （1） CA decreased superoxide anion generation induced by HG in endothelial ceils, prevented NO decreased, these effects were blocked by TRPA1 inhibitor, HC030031.（2） CA prevented endothelium dependent relaxation in WT mice but not in TRPA14- mice.（3） CA up-regulated Nrf2 in a dose dependent manner, and also promoted eNOS phosphorylation, decreased P22^phox, these effects were reversed by HC030031. Conclusion： Activation of TRPA1 by CA decreased oxidative stress in HUVECs under HG condition, preserved NO level hence improved the endothelium dependent relaxation.