The Protective Effects And Potential Mechanisms Of Malus Toringoides Extracts In High Glucose-induced Huvec

Malus toringoides(MT),the dried leaves of genus Malus toringoides(Rehd.)Hughes.and Malus transitoria(Balal.Schneid.),which has long history of use as drinking and medicine,has shown biological activities of hypoglycemic,hypolipidemic and hepatoprotection.The vascular protection of MT has not been studied.In the present study,we examined the protective effects of ethanol extracts of MT in high glucose(HG)-induced human umbilical vein endothelial cells(HUVECs)and then simply investigated the underlying protective molecular mechanisms,which could provided the theory basis for the application in the diabetic complication.First,we analyzed the components of MT by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS).In HG-induced HUVECs experiments,the cells was grouped and treated as follow: control group(5.5 mM normal glucose),HG-induced model group(25 mM high glucose),50 μg/mL MT group(50 μg/mL MT ethanol extracts(MTE)+ 25 mM high glucose),100 μg/mL MT group(100 μg/mL MTE + 25 mM high glucose),200 μg/mL MT group(200μg/mL MTE + 25 mM high glucose),400 μg/mL MT group(400 μg/mL MTE + 25 mM high glucose).To evaluate the protective effects of MT,the HUVEC was pretreated with MTE for 2 h,and then was induced with HG for 48 h.After induction,the level of Relative Oxygen Species(ROS),the activities of superoxide dismutase(SOD)and Glutathione peroxidase(GSH-px)were measured bycorresponding assay kit to evaluate the anti-oxidative activities of MT.The levels of intracellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1)and E-selectin both in the supernatant and cells lysate were measured by ELISA and Western Blot,respectively,to study the anti-vascular inflammatory effects of MT.Finally,the protein expressions of Heme oxygenase-1(HO-1),Nuclear factor(erythroid-derived 2)-like 2(Nrf2)and nuclear factor’kappa-light-chain-enhancer’ of activated B-cells(NF-κB)relevant signal protein was evaluated by Western Blot to investigate the vascular protection molecular mechanism of MT in HG-induced HUVECs.In the MTE,there are primarily 3 components,including phlorhizin,isoquercitin,quercitrin by LC-MS/MS analysis.As for the cells experiments,after treatment for 2 h with MTE,the elevated ROS induced by HG was lowered(P<0.05),and the decreased activities of SOD and GSH-px induced by HG was increased(P<0.05)in concentration-dependent manners.In addition,MTE reduced the levels of ICAM-1,VCAM-1 and E-selectin(P<0.05)in the supernatant and inhibited the expressions of ICAM-1,VCAM-1 and E-selectin(P<0.05)in the cells lysate in a concentration-dependent manner.In the mechanism studies,it was shown that MTE significantly induced HO-1 expression in concentration-dependent and induced HO-1 expression time-dependently in certain time scale,and arrived the peak of HO-1expression at 12 h.At the same time,MTE promoted the nuclear translocation of Nrf2,i.e.decreased the Nrf2 expression of the cytoplasm and increased the Nrf2 expression of the nucleus.Finally,pretreatment with MTE for 12 h,the HG-induced IκB-α phosphorylation in the cytoplasm and elevated p65 expression of nucleus were markedly suppressed,and it was indicated that MTE inhibited NF-κB activition.Taken together,MTE could protect endothelial cells from HG-induced oxidative stress and vascular inflammation,which might achieved through inducing Nrf2/HO-1 and inactivating NF-κB signal pathways in certain extent.