Effect Of Hyperglycemia On Antioxidase System And ATP Level In Brain Microvascular Endotheliar Cell & The Antioxidant Mechanism Of Resveratrol

To study the effect of hyperglycemia on antioxidase gene expression and ATP level in brain microvascular endothelial cells(BMESs). Mouse brain microvascular endothelial cells line bEnd.3 was used. Reactive oxygen species (ROS) producrion was measured using 2'7'-dichlorofluorescin diacetate (DCFH-DA); SOD1,SOD2,GPX,CAT,UCP1,UCP2,UCP3,UCP4 and UCP5 gene expression were determined in bEnd.3 under high glucose(25mM) or normal glucose(5.5mM) by reverse transcription PCR;We also determined the protein level of SOD2 and the effect of inhibitors of PI3K,NF-kB,JNK,PKC on protein level of SOD2.We found that hyperglycemia can significantly increased the mRNA levels of SOD1,SOD2,UCP4 and UCP5,while there were no increase in protein of SOD2 found under hyperglycemia condition.The inhibitor of NF-kB and PI3K can increase the protein level of SOD2 in hyperglycemia,while the inhibitors of JNK,PKC have little effect.At the same time,we found the inhibitor of NF-kB have no effect on the mRNA level of SOD2 and UCP5 under hyperglycemia.Hyperglycemia also can reduce the ATP level of bEnd.3 while the inhibitor of NF-kB could not significant increase the ATP level of bEnd.3 under hyperglyceia. These results sugget that hyperglycemia inhibit SOD2 protein synthesis maybe partial through NF-kB pathway in spite of increased mRNA level; The increased gene expression of UCP4 and UCP5 and the decreased ATP level have no significant relative with the protein level of SOD2. Elevated oxidative stress plays an important role in diabetes-associated micro vasuclar disease in which NADPH oxidases maybe a major source of ROS generation.In this study we tested the hypothesis that high glucose induced oxidative stress was associated with changes in the expression of NADPH oxidase subunits and resveratrol can counteract this change. Fluoresence labelling with dihydroethidum,lucigenin-enhenced chemiluminescence, polymerase chain reaction,western blotting were empbyed to determine oxidative stress,NADPH oxidase activity and NADPH oxidse subunits mRNA and protein expression in cell cultures of mouse brain microvessel endothelial cells(bEnd.3).High glucose enhenced NADPH oxidase activity and ROS production in bEnd.3.High glucose also increased protein and mRNA level of NADPH catalytic isoform Noxl but had little effect on mRNA level of NOX2 and p22phox. Inhibition of NADPH oxidase activity by apocynin can significant reduced high glucose induced ROS production. The increases of NADPH oxidase activity,Noxl protein and mRNA level were significant suppressed by resveratrol which pretect endothelial cells from injury.High glucose increased NOX1 protein level was decreased by the inhibitor of NF-kB sulfasalazine and Resveratrol can inhibit the NF-kB activity by suppressing high glucose increased phosphate-IkB-alpha.These results suggest that Resveratrol could inhibite the NADPH oxidase activity through NF-kB pathway which can protect brain microvessel endothelial cells from oxidative stress damage induced by high glucose.