Effects And Mechanisms Of VDR Agonists On High-glucose-induced Oxidative Stress In Human Endothelial Cells

Objective: Low levels of vitamin D are associated with increased risk ofcardiovascular disease and mortality. Vitamin D may inhibit cytokine-mediatedendothelial cell activation and adhesion molecule expression. However,themechanism by which vitamin D receptor （VDR） ligand acts in endothelial cellremains unclear．In this study,the effects of VDR agonists（1,25（OH）₂D₃） and RXRαagonists （9-cis-RA） on high-glucose—induced oxidative stress in human umbilicalvein endothelial cells（HUVECs） have been investigated．Our study may help us toseek and screen beneficial drugs preventing diabetic complications and a novel agentsfor preventing cardiovascular diseases.Methods: HUVECs were isolated from healthy umbilical cords and cultured．Reversetranscription real-time PCR and immunoblotting were performed to determine theexpression of the components of NADPH oxidase(gp91^phox and p22^phox) andcaspase-3．ROS production and HUVECs apoptosis rate were detected by flowcytometry and confocal microscopy．Activatioan and expression of PKC weredetected by immunoblotting．The interrelation between VDR signaling pathway andRXR signaling pathway was demonstrated with transfection of small interferingRNA（siRNA）．Results: Compared to control, the ROS production of HUVECs and HUVECsapoptosis rate were significantly higher in high glucose group. Treatment ofendothelial cells with1,25（OH）₂D₃（10-8M）or9-cis-RA（10-7M） resulted insignificant inhibition of high glucose induced oxidative stress, PKCactivation,expression of gp91^phox, p22^phoxand caspase-3, and HUVECs apoptosis.Furthermore,1,25（OH）₂D₃and9-cis-RA had a synergistic inhibitory effect by joint intervention. Our results also showed that transfection of VDR siRNA largelyabrogated the effects of VDR agonists，suggesting the inhibition of high glucoseinduced oxidative stress by VDR mediation. But we found that9-cis-RA on highglucose-induced HUVECs apoptosis, ROS generation, activation of PKC, mRNA andprotein expression of gp91^phoxand p22^phoxwas weakened after VDR siRNAtransfection. While the pre-transfected RXRα siRNA,1,25（OH）₂D₃inhibition had nosignificant change.Conclusions:（1）VDR agonist1,25（OH）₂D₃inhibited HUVECs apoptosis, theexpression of NADPH oxdase subunit gp91^phox,p22^phoxand ROS production ofHUVECs in induced with high glucose by VDR-PKC-NADPH oxidase-ROSsignaling pathway.（2）1,25（OH）₂D₃or9-cis-RA inhibited high glucose-inducedHUVECs apoptosis and oxidative stress, and both had a synergistic inhibitory effectby joint intervention.（3）9-cis-RA inhibited high glucose-induced oxidative stress ofvascular endothelial cells which may partly depend on VDR signaling pathwaymediation.