Pyridoxine Ameliorates Uncoupled Endothelial Nitric Oxide Synthase Induced by ox-LDL Via PKCαSignal Pathway in Human Umbilical Vein Endothelial Cells

【作者】 季勇；

【Author】 LP Xie,Z Liu,H Lu,W Zhang,QY Mi,XZ Li,Qi Chen,Yong Ji (Key Laboratory of Cardiovascular Disease and Molecular Intervention, Key Laboratory of Human Functional Genomics,Atherosclerosis Research Centre, Nanjing Medical University,Nanjing 210029,China)

【机构】 Key Laboratory of Cardiovascular Disease and Molecular Intervention,Key Laboratory of Human Functional Genomics,Atherosclerosis Research Centre,Nanjing Medical University；

【摘要】 <正>Objective One key mechanism for endothelial dysfunction is endothelial nitric oxide synthase （eNOS）uncoupling,whereby eNOS generates superoxide（O₂.） rather than nitric oxide（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.Methods HUVECs were incubated with ox-LDL with or without pyridoxine,L-NAME,Chelerythrine chloride （CHCI）,apocynin.Endothelial O₂.was measured using lucigenin chemiluminescence,as well as the O₂. -sensitive fluorescent dye Dihydroethidium（DHE）.NO levels measured by Chemiluminescence,PepTag Assay for Non-radioactive Detection of PKC Activity,depletion of PKCαand p47phox by siRNA silencing and the states of phosphorylation of eNOS thr495,total-eNOS,phosphorylation of PKCα/ β Ⅱ ,total-PKC, phosphorylation of PKCα,total-PKCα,p47phox were measured by Western Blot.Results Ox-LDL significantly increased O₂.production and reduced NO levels released from HUVECs,which could be reversed by eNOS inhibitor L-NAME.Pretreatment with pyridoxine significantly ameliorated ox-LDL-induced O₂.generation and preserved NO levels.Pyridoxine also improved the ox-LDL-reduced phosphorylation of eNOS Thr495 and activation of PKC.After using PKC inhibitor CHCI or siRNA silencing of PKCα,Pyridoxine failed to reduce O₂ production,elevate NO level and activate the PKC in response to ox -LDL.Furthermore, depletion of p47phox or using the NADPH oxidase inhibitor apocynin had no influence on effect of Pyridoxine,meanwhile,p47phox expression was no difference between all treatments.Conclusions Pyridoxine mitigated eNOS uncoupling induced by ox-LDL.The protectant effect was related with phosphorylation of eNOS Thr495 stimulated by PKC,not with NADPH oxidase.These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.更多还原

【Abstract】 Objective One key mechanism for endothelial dysfunction is endothelial nitric oxide synthase （eNOS）uncoupling,whereby eNOS generates superoxide（O₂.） rather than nitric oxide（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.Methods HUVECs were incubated with ox-LDL with or without pyridoxine,L-NAME,Chelerythrine chloride （CHCI）,apocynin.Endothelial O₂.was measured using lucigenin chemiluminescence,as well as the O₂. -sensitive fluorescent dye Dihydroethidium（DHE）.NO levels measured by Chemiluminescence,PepTag Assay for Non-radioactive Detection of PKC Activity,depletion of PKCαand p47phox by siRNA silencing and the states of phosphorylation of eNOS thr495,total-eNOS,phosphorylation of PKCα/ β Ⅱ ,total-PKC, phosphorylation of PKCα,total-PKCα,p47phox were measured by Western Blot.Results Ox-LDL significantly increased O₂.production and reduced NO levels released from HUVECs,which could be reversed by eNOS inhibitor L-NAME.Pretreatment with pyridoxine significantly ameliorated ox-LDL-induced O₂.generation and preserved NO levels.Pyridoxine also improved the ox-LDL-reduced phosphorylation of eNOS Thr495 and activation of PKC.After using PKC inhibitor CHCI or siRNA silencing of PKCα,Pyridoxine failed to reduce O₂ production,elevate NO level and activate the PKC in response to ox -LDL.Furthermore, depletion of p47phox or using the NADPH oxidase inhibitor apocynin had no influence on effect of Pyridoxine,meanwhile,p47phox expression was no difference between all treatments.Conclusions Pyridoxine mitigated eNOS uncoupling induced by ox-LDL.The protectant effect was related with phosphorylation of eNOS Thr495 stimulated by PKC,not with NADPH oxidase.These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.更多还原