PKC?/p66Shc Mediates Cyanate-induced Oxidative Stress Injury In Huvecs

Objective: To explore the effect of the PKC?/p66Shc pathway with cyanate induced oxidative stress and dysfunction in human umbilical vein endothelial cells(HUVECs)and discuss its possible mechanism.Methods: HUVECs were cultured in vitro.The changes of cell morphology were observed by inverted microscope.The effect of cyanate on the activity of HUVECs was detected by CCK-8 method;the content of ROS in cells was measured by DCFH-DA method;NO,CAT and GSH-Px were detected by colorimetry.The immunofluorescence staining was conducted to observe the contents of p-p66 Shc,p-PKC?,eNOS and ICAM-1.Western blot analysis was used to assess the protein levels of p66 Shc,p-p66 Shc,PKC?,p-PKC?,ICAM-1 and eNOS.Results: 1 mM cyanate significantly inhibited the viability of HUVECs.Compared with the control group and mannitol group,the content of ROS in the cyanate group was greatly increased.The induced of cyanate increased the levels of NO,reduced the enzyme activity of CAT,GSH-Px and SOD in endothelial cells,however the content of MDA was decreased.The expression of p66 Shc,p-p66 Shc,PKC?,p-PKC? and ICAM-1 in endothelial cells was clearly up-regulated,while eNOS expression was down-regulated.After pretreatment with inhibitor LY333531,ROS levels were decreased,the levels of NO reduced,the enzyme activity of CAT,GSH-Px and SOD raised,the content of MDA decreased.The expression of p66 Shc,p-p66 Shc,PKC?,p-PKC? and ICAM-1 was remarkably down-regulated,while eNOS expression was up-regulated.Conclusion: Cyanate induces endothelial cell oxidative stress and dysfunction by activeting the PKC?/p66Shc signaling pathway,producing a large amount of ROS;inhibiting the enzymatic activity of CAT,SOD and GSH-Px;reducing the production of NO and the expression of eNOS and increasing the content of MDA and the expression of ICAM-1.