Intermedin Reduced ROS Production To Protect Endothelial Cells Via Inhibiting ENOS Decoupling

Objective:Intermedin?IMD?,a biological active peptide,is expressed in renal endothelial cell.It reduced the renal interstitial fibrosis via inhibiting oxidative stress to delay the onset of chronic kidney disease.Studies have shown that IMD can activate adenosine monophosphate activated protein kinase?AMPK?,inhibit 26S proteasome and reduce guanosine triphosphate cyclohydrolase-I?GTPCH-I?degradation.GTPCH-I can prevent endothelial nitric oxide synthase?eNOS?decoupling to reduce reactive oxygen species?ROS?production.Therefore,we hypothesized that IMD might protect endothelial cells via inhibiting eNOS decoupling.This study investigated whether the AMPK/26S proteasome-GTPCH-I signaling pathway is involved in the process of IMD inhibited eNOS uncoupling,moreover,it indicated the molecular mechanism by which IMD reduced ROS production to protect endothelial cells via inhibiting eNOS decoupling.Methods:1.The model of cell hypoxic reoxygenation was established.Human umbilical vein endothelial cells?HUVECs?were cultured in a three-gas incubator containing92%N₂+5%CO₂+3%O₂ for 18 hours hypoxia culture,then put into a normal incubator for6 hours under reoxygenation.2.HUVECs in good condition were randomly divided into the normal control group,anoxic reoxygenation model group,IMD intervention group,IMD+compound C?AMPK inhibitor?intervention group,and the cells in the intervention group were pretreated in the medium with drugs for 1 hour before anoxic reoxygenation.3.The expression of eNOS dimer/monomer was determined by Western blot,to observe the effects of IMD on eNOS uncoupling.4.The expression of GTPCH-1 was detected by Western blot to observe the effect of IMD on the expression of GTPCH-I.5.The activity of 26S proteasome was measured by Elisa to observe the effects of IMD on 26S proteasome activity.6.Western blot analysis was used to determine the expression of p-AMPK and total AMPK to observe the effects of IMD on AMPK activity.Results:1.Compared with the control group,the expression of eNOS dimer/monomer in the anoxic reoxygenation model group was significantly reduced,indicating that eNOS uncoupling occured.After IMD intervention,this ratio was significantly increased,indicating that IMD could inhibit eNOS decoupling.The ratio was significantly decreased in IMD and compound C therapy group,indicating that IMD inhibited eNOS decoupling by compound C.2.Compared with the control group,the expression of GTPCH-I in the hypoxic reoxygenation model group was decreased,indicating that hypoxia led to the degradation of GTPCH-I.After IMD intervention,the expression of GTPCH-I was significantly increased,indicating that IMD could inhibit its degradation.The expression of GTPCH-I was significantly decreased after the intervention of IMD and compound C,indicating that IMD inhibits GTPCH-I degradation by compound C.3.Compared with the control group,the activity of 26S proteasome was increased in the anoxic reoxygenation model group,indicating that anoxia can induce the increase of the activity of 26S proteasome.After IMD intervention,its activity was significantly decreased,indicating that IMD could inhibit its activity.Compound C was added on the basis of IMD,its activity was significantly increased,indicating that IMD inhibited the activity of 26S proteasome by compound C.4.Compared with the control group,p-AMPK/AMPK expression was decreased in the anoxic reoxygenation model group,indicating that anoxia inhibited AMPK phosphorylation.After IMD intervention,this ratio was significantly increased,indicating that IMD could activate AMPK.The ratio was significantly reduced in IMD and compound C intervention group,suggesting that IMD inhibited eNOS decoupling by reducing the expression of GTPCH-I by inhibiting the activity of 26S proteasome by activating AMPK.Conclusion:IMD inhibits eNOS decoupling and protects against endothelial cell injury induced by oxidative stress via activating the AMPK/26S proteasome-GTPCH-I signaling pathway.