Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCa signalling pathway in human umbilical vein endothelial cells
Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCa signalling pathway in human umbilical vein endothelial cells
复制标题
吡哆醇通过 PKCa 信号通路抑制人脐静脉内皮细胞氧化低密度脂蛋白诱导的内皮 NOS 解偶联
DOI：
10.1111/j.1476-5381.2011.01607.x
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发表时间：
2012-02-01
影响因子：
7.3
通讯作者：
Ji, Yong
中科院分区：
文献类型：
--
作者：
Xie, Liping;Liu, Zhen;Ji, Yong
背景和目的内皮功能障碍的一个关键机制是内皮NOS（eNOS）解偶联，本研究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（HUVECs）eNOS解偶联的影响及其可能的分子机制。不含吡哆醇、N（G）-硝基-L-精氨酸甲酯（L-NAME）、氯化白屈菜红碱（CHCI）或夹竹桃素。采用光泽精化学发光法和O（2）中心点（-）敏感荧光染料二氢乙锭（DHE）测定内皮细胞O（2）中心点（-）。通过化学发光、用于非放射性检测PKC活性的PepTag测定、通过SiRNA沉默消除PKC和p47 phox以及磷酸化eNOS Thr 495、总eNOS、磷酸化PKC α/β II、总PKC的状态来测量NO水平PKC、磷酸化PKC α、结果Ox-LDL可显著增加O2中心点（-）生产和减少NO水平从HUVEC释放; eNOS抑制剂，L-NAME逆转的效果。吡哆醇预处理显著抑制ox-LDL诱导的O（2）中心点（-）的产生，并保持NO水平。吡哆醇也阻止ox-LDL诱导的磷酸eNOS Thr 495和PKC活性的降低。吡哆醇的这些保护作用被PKC抑制剂CHCI或PKC α的siRNA沉默所消除。然而，耗尽p47 phox或治疗与NADPH氧化酶抑制剂，夹竹桃苷，对这些效果没有影响。此外，细胞质p47 phox的表达是不变的不同treatment.CONCLUSIONS和含义吡哆醇减轻eNOS解偶联诱导的ox-LDL。这种保护作用与PKC α刺激的eNOS Thr 495磷酸化有关，而不是通过NADPH氧化酶。这些结果为吡哆醇在ox-LDL相关的血管内皮功能障碍中的应用提供了支持。
BACKGROUND AND PURPOSE One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O(2)center dot(-)) 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.EXPERIMENTAL APPROACH 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)center dot(-) was measured using lucigenin chemiluminescence, and O(2)center dot(-)-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKC and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKC alpha/beta II, total PKC, phospho-PKC alpha, total PKC alpha and p47phox were measured by Western blot.KEY RESULTS Ox-LDL significantly increased O(2)center dot(-) 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)center dot(-) 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 PKC alpha. 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.CONCLUSIONS AND IMPLICATIONS Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKC alpha, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.