The Role Of G6PD During Oxidative Stress Induced By Ang Ⅱ In HUVEC

Objective: Glucose-6phosphate dehydrogenase（G6PD）is one of themost important housekeeping enzyme in mammals. It is the first andrate-limiting enzyme of the pentose-phosphate pathway, which widelydistributed in all body tissues and cells. G6PD is an enzyme essential forbody’s normal metabolism and growth essential. It is indispensable tomaintain the cytoplasmic pool of NADPH and cell oxidation reductionequilibrium.G6PD gene mutation resulted in loss of function of the G6PDprotein, can lead to neonatal jaundice, drugs or infection mediated hemolyticcrisis, horsebean disease. Recent studies suggest that G6PD plays an importantrole in the cell and organism physiological function etc. G6PD deficiency willdisturb the oxidation-reduction balance, can lead to cell growth disorders andabnormal signal transduction, there will be abnormal embryonic development,increase the degenerative disease susceptibility.But so far, under oxidative stress the activity changes of G6PD and therelation with the pentose-phosphate pathway is unclear in the cell？Studyactivity of G6PD to learn how to maintain cellular NADPH and cellularoxidation-reduction balance and related diseases will be of very greatsignificance.G6PD human gene is an X-linked gene that maps to the Xq28region.The gene consists of13exons and12introns, about21kb. The first exon is nocoding sequence, the intron that located in the middle of the2nd and3exon islength about9KB.Angiotensin II (AngⅡ) generated by the conversion of angiotensin I arepeptides (octapeptide) substances; is the most important active substance ofrenin-angiotensin system (RAS).Ras played an important role in maintainingbody’s blood pressure stability and regulating water and electrolyte balance. Changes of Ras activity are closely associated with cardiovascular disease anddevelopment. AngⅡthrough a variety of effects take part in the process of theoccurrence, development of arteriosclerosis. Ang II of the stimulus leads tovascular excessive free radicals, further causing vascular endothelial cell injury,inflammation and dysfunction, that become atherosclerosis based. But invascular endothelial cells under oxidative stress, there will be what kind ofchanges within the cell?This activity of G6PD will be how to change and withthe pentose-phosphate pathway is not clear. Thus, we use Ang II to stimulateHUVEC, observe the cellular reactive oxygen species (ROS), nitric oxidesynthase (NOS), nitric oxide (NO), changes of NADP/NADPH and G6PDprotein, to Study on oxidative stress of HUVEC induced by Ang II and itsmechanism，To observe the changes of the intracellular nitric oxide synthase(NOS), nitric oxide (NO), NADP/NADPH and G6PD proteins. Under oxidativestress how is G6PD to change.Methods:1To culture cellHUVEC were cultured to use DMEM medium containing10%newbornfetal serum at37℃,5%CO2and saturated humidity. Cells were seeded in6well plates or75mL flask in1×105/cm2density, and to culture for24hours.After switching to serum-free DMEM medium and then to culture for24hours. Changing containing10%FBS DMEM medium, while adding thestimulus. Receiving the cells at the point in time.2To observe the changes of ROS(1) After incubated for1h with different concentrations of AngⅡ(0，10-6，10-7，10-8mmol/L) in6well plates, active oxygen ROS detection kitmeasure different concentrations of samples.(2) Cells were incubated with10-6mmol/L Ang Ⅱ in6-well plates for0h，0.5h，1h，3h，6h respectively. Active oxygen ROS detection kit measuredifferent times of samples.3To observe the changes of NADP/NADPH.Cells were incubated with10-6mmol/L AngⅡ in6-well plates for0min, 5min,15min,30min,60min,90min,120min respectively, according to the kitsmelting cells2times, were measured.4To observe the changes of the activity of G6PD.Cells were incubated with10-6mmol/L AngⅡ for0min,5min,15min,30min,60min,90min,120min respectively, the cells are collected forextraction of protein, frozen at-70℃. According to the kit for determination.5To observe the changes of G6PD protein.Cells were incubated with10-6mmol/L AngⅡ for0min,5min,15min,30min,60min,90min,120min respectively, the cells are collected forextraction of protein to determine the amount of G6PD by Western Blotting.6To observe the expression of NOS and NO(1) Cells were incubated with10-6mmol/L AngⅡ in6-well plates for0min,5min,15min,30min,60min,90min,120min respectively. To charge thecell supernatant, were detected by NO kit.(2) Cells were incubated with10-6mmol/L AngⅡ in6-well plates for0min,5min,15min,30min,60min,90min,120min respectively. To charge thecell supernatant, were detected by NOS kit.7Data analysisAll date were expressed as X S.The results were analyzed by SPSS16.0software. The mean variance compared with single factor analysis ofvariance (one way ANOVA), P <0.05for the difference has statisticalsignificance.Results:1Ros of HUVEC is enhancing by Ang II stimulated.(1) After incubated for1h with different concentrations of Ang Ⅱ (0,10-6,10-7,10-8mmol/L), under the fluorescence microscope, fluorescence intensitywas the strongest at10-6mmol/L.(2) Cells were incubated with10-6mmol/L Ang Ⅱ for0h,0.5h,1h,3h,6hrespectively, under the fluorescence microscope, fluorescence intensity was thestrongest in1h. 2NADP/NADPH is decline.Cells were incubated with10-6mmol/L AngⅡ for0min,5min,15min,30min,60min,90min,120min respectively.NADP/NADPH changes suggestive the occurrence, development,of inoxidative stress, metabolic activity of the pentose-phosphate pathwaysuppressed transiently, gradually increase.3Changes of G6PD activity and content, in the development process ofendothelial cells oxidative.Cells were incubated with10-6mmol/L AngⅡ for0min,5min,15min,30min,60min,90min,120min respectively. The results show that: the activityof G6PD decreased firstly then increased. In the initial period of oxidative stressG6PD was inhibited, with the extension of time its activity recovered gradually.A parallel relationship between changes of G6PD activity and expressionchanges of G6PD protein.4The expression of NO and the activity of NOS is decline.Cells were incubated with10-6mmol/L AngⅡ for0min,5min,15min,30min,60min,90min,120min respectively.a On the5min the expression level of NO reflection raising, With theincrease time showed a time-dependent decrease, to reach the minimum peakat1h.b On the5min the expression level of NOS reflection raising, With theincrease time showed a time-dependent decrease, to reach the minimum peakat1h.Decrease of NO and reduction of NOS activity in the cell, the function ofendothelial cells were damaged.Conclusions:110-6mmol/L AngⅡ HUVECwith1h is the best conditions of incubationin vitro model for oxidative stress in endothelial cells.2Oxidative stress disrupts the pentose phosphate pathway, metabolichomeostasis, and stimulating endothelial dysfunction through changing theactivity of G6PD.