The Protective Effects Of Na～+/H～+ Exchanger-1 Inhibitor On Endothelial Impairment Caused By Advanced Glycation End-products And Its Mechanisms

Partâ… . Protective Effects of Cariporide on EndothelialDysfunction Caused by Advanced Glycation End products(AGEs) in Rat Isolated AortaObjective: To observe the effects of cariporide, a selective Na⁺/H⁺exchanger inhibitor, on endothelial dysfunction induced by AGEs-BSA inrat isolated aorta.Methods: Isolated aortic rings of rat were exposed to AGEs-BSAfor 120 min. The endothelium-dependent relaxation (EDR) induced byAcetylcholine (ACh) and endothelium-independent relaxation induced bysodium nitroprusside (SNP), malondialdehyde (MDA) were thendetermined. The superoxide dismutase (SOD), and nitric oxide (NO) inrat aorta were also measured using a spectrophotometry.Results:â‘ Incubated with AGEs-BSA for 120 min, rat aorticisolated rings showed a significant inhibition of EDR induced by ACh,but AGEs-BSA had no significant effects on endothelium-independentrelaxation elicited by SNP.â‘¡Cariporide (0.01, 0.1, 1μM) attenuated theinhibition of EDR caused by AGEs-BSA in a concentration-dependentmanner.â‘¢AGEs-BSA decreased SOD activity and contents of NO, andincreased MDA concentration in vascular tissues. Cariporide significantlyresisted the decrease of NO content and SOD activity, and the elevationof MDA concentration caused by AGEs-BSA.â‘£Unmodified albumin(BSA) had no significant effect on ACh-mediated vasorelaxation. Conclusion: Cariporide has protective effect on endothelialdysfunction induced by AGEs-BSA. The mechanisms may be involved inactivation of Na+/H⁺ exchange and increase of oxidative stress.Partâ…¡. Protective Effects of Cariporide on EndothelialImpairment Caused by Exogenous AGEs in RatObjective: To observe the effects of cariporide on endothelialfunction impaired by exogenous AGEs in rats.Methods: Rats were given exogenous AGEs by tail veininjections of AGEs-BSA to induce vascular dysfunction and were treatedwith cariporide (0.1, 1 mg/kg/d)simultaneously for 4 weeks. The aorticarches were used for histopathological examination and immunohisto-chemical study of NF-κB-p65. The thoracic aortas were used fordetermination of the ACh-induced endothelium-dependent relaxationresponse and SNP-induced endothelium-independent relaxation response.Serum NO and MDA content were also measured.Results:â‘ The EDR response induced by ACh was inhibited withdecreasing serum NO content and increasing MDA level, but endothe-lium-independent relaxation were not affected.â‘¡Cariporide improvedthe EDR response and maintain serum NO and MDA content in adose-dependent manner.â‘¢There was no visible histopathological change found under microscope. The activation of NF-κB in ratendothelium was significantly increased during the administration ofexogenous AGEs. Cariporide significantly reduced the activation ofNF-κB in AGEs-treated rat.Conclusion: Cariporide has protective effect on endothelialimpairment caused by AGEs-BSA. The mechanisms may be involved ininhibition of the activation of NF-κB and oxidative stress.Partâ…¢. The effect of AGEs on NHE-1 activity inendothelium and its mechanismObjective: To investigate the effects of AGEs on the activity andgene expression of NHE-1 in HUVECs and the relationship between theactivation of NHE-1 and oxidative stress.Methods:â‘ HUVECs were exposed to AGEs-BSA for 0, 1, 3, 6,12 h to study the time-effect of AGEs-BSA on NHE-1 activity inendothelium.â‘¡HUVECs were exposed to AGEs-BSA of differentconcentrations (10, 50 and 100μg/ml) to study the dose-effect ofAGEs-BSA on NHE-1 activity in endothelium and to investigate theeffects of different interventions (PKC inhibitor, anti-RAGE antibody andinhibitor of NADPH oxidase).â‘¢The [Ca²⁺]_i and ROS generation ofHUVECs were determined after incubating with Cariporide of different concentrations (0.1, 1 and 10μM) or with different inhibitors (PKCinhibitor, NADPH oxidase inhibitor).â‘£HUVECs were exposed toAGEs-BSA for 0, 3, 6, 12, 24 h to study the effect of AGEs-BSA onNHE-1 gene expression. The activity of NHE-1, pHi, [Ca²⁺]_i and ROSwere assayed by spectrofluorometer and the NHE-1 mRNA expressionwas assayed by reverse-transcription polymerase chain reaction.Results:â‘ AGEs-BSA increased NHE-1 and pH_i of endothelium ina time-and concentration-dependent manner. PKC inhibitor(GF-109203X, 10μg/ml) and anti-RAGE antibody abolished the effect ofAGEs-BSA, while NADPH oxidase inhibitor (Apocynine, 100μM) hadno significant effect on AGEs-BSA induced NHE-1 activation.â‘¡Cariporide (0.1, 1 and 10μM) reduced the elevation of [Ca²⁺]_i and ROSgeneration of endothelium caused by AGEs-BSA in a concentration-dependent manner. GF-109203X also remarkably decreased the [Ca²⁺]_iand ROS generation of endothelial cells exposed to AGEs-BSA.Apocynine remarkably decreased the ROS generation but had no effecton [Ca²⁺]_i of endothelial cells exposed to AGEs-BSA.â‘¢AGEs-BSA hadsignificant effect on NHE-1 gene expression in endothelial cells after 3, 6,12 and 24 h incubation.Conclusion:â‘ AGEs-BSA can increase the NHE-1 activity inendothelial cells and this activation may be mediated by PKC pathway.â‘¡The oxidative stress of endothelial cells induced by AGEs-BSA ispossibly associated with the increase of NHE-1 activity. Partâ…£. The effect of cariporide on AGEs induced NF-κBactivation and its downstream targetsObjective: To study the effect of cariporide on AGEs inducedNF-κB activation and its downstream target genes in HUVECs.Methods:â‘ To observe the time-effect of nuclear translocationof NF-κB-p65 in endothelial cells induced by AGEs-BSA usingimmunofluorescence assay. Then the effect of Cariporide (10μM) ordifferent inhibitors (NF-κB inhibitor, PKC inhibitor and NADPH oxidaseinhibitor) on nuclear translocation of NF-κB-p65 induced by AGEs-BSAwere investigated.â‘¡IκB-αlevels were measured simultaneously withNF-κB-p65 by Western-blot analysis.â‘¢The time-effect of AGEs-BSAon TNF-αand slCAM-1 levels in supematant of endothelial cells weredetermined by sandwich ELISA. Then the effect of Cariporide (0.1, 1 and10μM) or different inhibitors (NF-κB inhibitor, PKC inhibitor andNADPH oxidase inhibitor) on TNF-αand sICAM-1 levels of endothelialcells exposed to AGEs-BSA were investigated.Results:â‘ AGEs-BSA (100μg/ml) increased NF-κB activation(nuclear translocation of NF-κB-p65) and decreased IκB-αlevel ofendothelial cells in a time-dependent manner. Cariporide significantlyinhibited the NF-κB activation and IκB-αdecrease. PDTC (100μM),GF-109203X (10μg/ml) and Apocynine (100μM) also remarkablyinhibited the NF-κB activation and IκB-αdecrease.â‘¡The levels ofTNF-αand sICAM-1 in supernatant were increased time-dependentlyafter the incubation with AGEs-BSA (100μg/ml). Cariporidesignificantly inhibited the release of TNF-αand slCAM-1. PDTC (100 μM), GF-109203X (10μg/ml) and Apocynine (100μM)also remarkablyinhibited the elevation of TNF-αand sICAM-1.Conclusion: The NF-κB activation in endothelial cells caused byAGEs-BSA is related to oxidative stress. Cariporide reduce NF-κBactivation and the expression of its target genes possibly via inhibition ofoxidative stress.