Key Laboratory of Cardiovascular Disease and Molecular Intervention, Key Laboratory of Human Functional Genomics, Atherosclerosis Research Centre, Nanjing Medical University, Nanjing, China.
Br J Pharmacol. 2012 Feb;165(3):754-64. doi: 10.1111/j.1476-5381.2011.01607.x.
One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O(2) (•-) ) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism.
HUVECs were incubated with ox-LDL with/without pyridoxine, N(G) -nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O(2) (•-) was measured using lucigenin chemiluminescence, and O(2) (•-) -sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot.
Ox-LDL significantly increased O(2) (•-) production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O(2) (•-) generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments.
Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.
内皮型一氧化氮合酶（eNOS）解偶联是内皮功能障碍的一个关键机制，在此过程中，eNOS 产生超氧阴离子（O(2) (•-) ）而不是 NO。我们探讨了吡哆醇对氧化型低密度脂蛋白（ox-LDL）诱导的人脐静脉内皮细胞（HUVEC）中 eNOS 解偶联的影响及其潜在的分子机制。
将 HUVEC 与 ox-LDL 共同孵育/不共同孵育，加入吡哆醇、N(G) -硝基-L-精氨酸甲酯（L-NAME）、氯化Chelerythrine（CHCI）或 Apocynin。使用发光体化学发光法测量内皮细胞产生的 O(2) (•-) ，使用二氢乙锭（DHE）测量 O(2) (•-) 敏感荧光染料。通过化学发光法测量 NO 水平，通过 PepTag 测定法用于非放射性检测 PKC 活性，通过 siRNA 沉默耗竭 PKCα 和 p47phox，通过 Western blot 测量磷酸化 eNOS Thr495、总 eNOS、磷酸化 PKCα/βII、总 PKC、磷酸化 PKCα、总 PKCα 和 p47phox 的状态。
ox-LDL 显著增加了 HUVEC 中 O(2) (•-) 的产生并减少了释放的 NO 水平；此作用可被 eNOS 抑制剂 L-NAME 逆转。吡哆醇预处理可显著抑制 ox-LDL 诱导的 O(2) (•-) 的生成并保持 NO 水平。吡哆醇还可防止 ox-LDL 诱导的磷酸化 eNOS Thr495 和 PKC 活性降低。这些吡哆醇的保护作用可被 PKC 抑制剂 CHCI 或 PKCα 的 siRNA 沉默所消除。然而，耗竭 p47phox 或用 NADPH 氧化酶抑制剂 Apocynin 处理对这些作用无影响。此外，不同处理对胞浆 p47phox 表达无影响。
吡哆醇减轻了 ox-LDL 诱导的 eNOS 解偶联。这种保护作用与 PKCα 刺激的 eNOS Thr495 磷酸化有关，而与 NADPH 氧化酶无关。这些结果为在 ox-LDL 相关的血管内皮功能障碍中使用吡哆醇提供了支持。
