Luteoloside Protects Vascular Endothelium Against Iron Overload Damage Via ROS/ADMA/DDAH?/eNOS/NO Pathway

Objective:Endothelial dysfunction caused by iron overload is thought to be related to excessive production of reactive oxygen species(ROS)possibly,and antioxidants have been found to reduce ROS-induced damage.Luteoloside(Lut)is an isoflavonoid compounds with antioxidant properties that has the ability to scavenge free radicals.However,it has not been studied whether Lut can protects Human umbililical vein endothelial cells(HUVECs)against iron overload injury,and its underlying mechanisms remain unclear.Therefore,in this study,we investigated whether Lut protects HUVECs against iron overload damage via the ROS/ADMA/DDAHII/eNOS/NO pathway.Methods:In this study,HUVECs were treated with 50 ?M iron dextran for 48 h to construct the iron overload damage model.CCK-8 was used to determine cell viability,spectrophotometric was used to determine the degree of opening of mitochondrial permeability transition pores(mPTP),glutathione peroxide enzyme(GSH-Px),lactate dehydrogenase(LDH),superoxide dismutase(SOD)activities,and the level of NO(Nitric oxide)and malondialdehyde(MDA)content;Western blotting was used to determine the expression of DDAH?,eNOS,p-eNOS,cleaved-caspase3 and mitochondrial/cytoplasmic cyt c protein.Fluorescence-labeled flow cytometry was used to detect apoptosis,mitochondrial membrane potential(MMP),reactive oxygen species(ROS)in cells and mitochondria;TUNEL was used to detect apoptosis,and intracellular ADMA and DDAH? contents were determined by HPLC.Results:We found that 40 ?M Lut treatment increased cell viability,SOD and GSH-Px activities,and decreased LDH activity and MDA levels compared with 50 ?M iron treated HUVECs for 48 hours.In addition,treatment with 40 ?M Lut reduced ADMA content,up-regulated DDAH?expression and activity,and increased p-eNOS/eNOSratio and NO levels.Furthermore,40 ?M Lut significantly attenuated intracellular or mitochondrial ROS generation,maintained MMP,inhibited mPTP opening,prevented cyt c from mitochondria released into cytoplasm,reduced cleaved-caspase3 expression,and ultimately improved iron overload induced cell apoptosis.However,the addition of pAD/DDAH?-shRNA adenovirus reversed these beneficial effects of Lut.In addition,established L-arginine(L-Arg),a ADMA competitive substrate,cyclosporin A(CsA),a mPTP blocker agent,and edaravone(Eda),a free radical scavenger as positive control groups.The effects of Lut was similar to that of L-Arg,CsA and Eda treatment.Conclusion:Lut can protect HUVECs against iron overload damage via ROS/ADMA/D DAHII/eNOS/NO pathway,and it could be a potential drug for treating iron overload.