The Effects Of α-lipoic Acid On Indoxyl Sulfate-induced Oxidative Damage In Human Umbilical Vein Endothelial Cells

Objective：Compared to the general population，patients with chronickidney disease(CKD) have a much higher risk of cardiovascular diseases anddeath．Accelerated atherosclerosis is the key mechanism of cardiovascularcomplication in CKD，which is starting at endothelial damage．As renalfunction deteriorates，accumulated uremic toxins in patients with CKD caninduce oxidative stress reaction,which is an emerging key mechanism ofatherosclerosis in CKD．Indoxyl sulfate (IS)，a representative protein-bounduremic toxin，that accelerate the progression of chronic kidney disease，mayalso be responsible for vascular disease via an induction of oxidativestress．α-lipoic acid (LA) is a naturally antioxidant widely used in diabetes，diabetes complications and ischemia reperfusion injury etc，but rarely inCKD．This study aimed to determine if α-lipoic acid makes protective effectsagainst indoxyl sulfate-induced oxidative damage in HUVECs and thepossible mechanism．Methods：HUVECs were divided into four groups：control，LA，IS，IS+LA．Cell viability was analyzed using MTS method．To measure lipidperoxidation，malonaldehyde (MDA) was detected．Intracellular reactiveoxygen species (ROS) levels were measured by flow cytometry. Theexpression of NADPH oxidase4(NOX4) was evaluated using reversetranscription-polymerase chain reaction (RT-PCR).All the data were analyzed with GraphPad Prism5software. Data wereexpressed as mean±SD. Multiple comparisons were evaluated using one-wayANOVA and significant differences between two groups were analyzed byBonferroni test．P value<0.05was considered significant．Every experimentwas performed more than three times． Results：1Cell viability was decreased by IS（100、200、500μm/L）and reverselyincreased by LA (20、50、100、250、500μm/L) in a dose-dependentmanner．There were significant differences in survival rate at concentrations ofIS≥200μm/L and LA≥250μm/L compared with the control group．LApretreatment significantly increased survival of HUVECs （IS500μm/L+LA250μm/L vs IS500μm/L，P<0.01）. The concentrations of500μm/L IS and250μm/L LA were chosen to sebsequent experiments．2Compared to that in control group, the concentration of MDA washigher in IS group(2.87±0.66vs1.29±0.40nmol/ml)(P<0.001）; Pretreatmentwith LA, the concentration of MDA was decreased in IS+LAgroup(2.04±0.46nmol/ml) as compare to that in IS group(2.87±0.66nmol/ml)(P<0.05）.3Compared to that in control group，the percent of ROS positive cellswas higher in IS group（76.17±2.25vs62.07±1.60%)(P<0.001); Pretreatmentwith LA, the percent was decreased in IS+LA group(69.70±3.01%), ascompare to that in IS group（76.17±2.25%）(P<0.05)．4Compared to that in control group, the NOX4expression was higher inIS group(1.65±0.18vs1.00±0.24）(P<0.05); Pretreatment with LA，the NOX4expression was decreased in IS+LA group(1.11±0.41) as compared to that inIS group(1.65±0.18)(P<0.05）．Conclusion：1Indoxyl sulfate could induce oxidative damage in endothelial cells．2α-lipoic acid could restrain the oxidative damage induced by indoxylsulfate，which may be relied on down-regulating the expression of NADPHoxidase and ROS in HUVECs.