PKCβ/p66Shc Pathway Attributed To The Production Of Reactive Oxygen Species And Inflammation In High Dose Urea Treated Human Umbilical Vein Endothelial Cells

Objective: To investigate the role of PKCβ/p66 Shc pathway in production of inflammation and reactive oxygen species(ROS)in human umbilical vein endothelial cells(HUVECs)treated with urea.Methods: HUVECs were randomly divided into normal control group,mannitol group,urea group,urea + LY333531(PKCβ inhibitor)group.The activities and the morphological changes of the cells were detected by the CCK 8 assay and observed under an inverted microscope.ROS content was measured through DCFH-DA method.Immunofluorescence assay was used to observe the expression of p-p66 Shc in the HUVECs.Expression levels of p66 Shc,p-p66 Shc,PKCβ,TNFα,ICAM-1,i NOS and e NOS were detected by Western blot.Results: 1.The viabilities of HUVECs were greatly inhibited by urea with a dose-dependent manner starting at 25 mmol/L.The morphologicchanges of HUVECs treated by 25 mmol/L urea were observed including disordered arrangement,increased cell gap,destructive cobblestone appearance.2.Compared with the control group,the fluorescence intensity of ROS in cytoplasm was significantly enhanced in HUVECs treated with 25 mmol/L urea and reached peak at 6 h urea-treated with decreased fluorescence intensity at 12 h urea-treated.The fluorescence intensity of ROS was significantly decreased by addition PKCβ inhibitor after 6 h urea loading.3.Urea could induce the protein expression of PKCβ,p-PKCβ,p66 Shc and p-p66 Shc protein increased in a time dependent manner;compared with the control group,the 6 h group,p-p66 Shc increased about 76%(P <0.01),24 h group increased about 2 times(P <0.01);And p-PKCβ increased about 64% at 24 h(P <0.05).TNFα,ICAM-1 and i NOS protein expression in a time dependent increase;TNFα at 6 h increased about 40% and at 24 h increased about two times compared with the control group(P <0.05);ICAM-1 i at 6 h increased about 80% and at 24 h increased about two times compared with the control group(P <0.05);i NOS increased about 100% at 24 h(P <0.05);the expression of e NOS decreased,It reduced about 34% at 12 h(P <0.05),and reduced about 56% at 12 h(P <0.01).4.The immunoreactivity of p-p66 Shc,TNFα and ICAM-1 in the cytoplasm was also obviously increased significantly after 6 h of 25 mmol/L urea treatment,and LY333531 could block this effect;western blot result also showed that increased expression of p-p66 Shc,TNFα and ICAM-1 induced by high concentration of urea was inhibited by LY333531.The expression level of p-p66 Shc protein,compared with the control group,reduced about 40%(P < 0.05);Compared with 6 h,TNFα decrease of about 61%(P <0.05);ICAM-1 was reduced about 94%(P <0.01);and compared with 6 h group,the e NOS protein expression level increased about 50%(P <0.01).Conclusion: PKCβ/p66 Shc signaling pathway plays an important role in the excessive production of ROS and inflammation in HUVECs treated by high concentration urea.