The Effect Of Ascorbic Acid Treatment On The PolyCHb Induced Oxidative Stress In The Kidney Of Guinea Pigs

Background:Hemoglobin based oxygen carrier is a hot topic and main stream in the field of developing red blood cell substitute.PolyCHb is polymerized human cord hemoglobin,its oxygen binding and loading property was well proved and its efficacy for resuscitating hemorrhagic shock rats is promising.As one kind of HBOC,PolyCHb is prone to be oxidized,while generating redical species,then cause oxidative stress and injury in organs.Ascorbic acid,one kind of reducing agent,maybe helpful to solve this problem.Our previous studies usually use rats,but rat can produce ascorbic acid while human can’t.Therefore it’s necessary and of great value to study the in vivo metabolic and oxidative stress aspects of PolyCHb using animals that can’t produce ascorbic acid,eg,guinea pig.The effect of ascorbic acid to the oxidative stress caused by PolyCHb is also worth to investigate,to support further research on PolyCHb.Purpose:To investigate the pharmacokinetic properties and renal oxidative effects of PolyCHb using exchange transfusion model in guinea pigs.Meanwhile,the effects of AA treatment were also researched.The findings will be helpful for further laboratory studies and clinical inveatigations.Ferrous and ferric PolyCHb was added to the culture medium of HK-2 cells,then the cell vitality,intracellular ROS,marker for oxidative stress,marker for heme metabolism were detected,to explan the mechanism at the cellular level.Methods:We used cord blood as the raw material,to produce PolyCHb using glutaraldehyde cross-linking method.The in vitro effects of ascorbic acid to the oxidation of PolyCHb were studied.For animal study,we made the exchange transfusion model,6%PolyCHb is infused,urine and blood samples were collected at defined time points.By plotting the drug concentration-time curve,pharmacokinetic parameters were calculated and the oxidative status of transfused PolyCHb was checked.AA treatment is also included in this group.In the other group,infused animals were divided into the following groups.sham,exchange transfusion(ET)and exchange transfusion plus AA injection(ET+AA).Animals were sacrificed at different time points,kidneies were collected to evaluate the oxidative status and histopathology changes.Cultured kidney proximal tubular epithelial cells were exposed to ferrous and ferric PolyCHb,then cell vitality,mitochondria functions and the express of markers on heme metabolism were investigated.Results:1.PolyCHb can’t inhabit the auto-oxidation of PolyCHb at 37℃,but it can reduce the ferric PolyCHb.2.We set up the artery&venous catheterization model using guinea pigs,and then carried out exchange transfusion.3.Pharmacokinetic parameters were calculated using 50%ET,T1/2 is 8.5-9.5 hours.AA treatment don’t change the parameters.There was hemoglobinuria in all animals in the ET group,the components in the urine are dimers and tetramers,while hemoglobinuria couldn’t be fund in the ET+AA group.4.In the 50%ET operation,there were no kidney histopathology changes in the sham group,there were kidney histopathology changes in the ET group,including abnormal glomerular structure,necrosis of primary tube epithelial cell.The kidney histopathology changes in ET+AA group were smaller than in the ET group.5.In the 50%ET operation,there were no differences between the three groups on CAT,GP activity;SOD activity decline in ET group;MDA and 8-OHdG contents increase after ET,while AA treatment can lower it;4-HNE content increased after ET from zero bacground,while AA treatment can lower it;HO-1 and ferritin contents increased after ET from,while AA treatment can lower it;Nrf2 expression elevated in the ET group.6.In-vitro cell culture showed that both ferrous and ferric PolyCHb release hemoglobin a subunit and heme,there is no changes on cell vitality,and mitochondrial respiration function.mitcochondrial membrance potential in the PolyCHb treated group decreased slightly,while AA can prevent it.ferrous and ferric PolyCHb caused the up-regulated expression of HO-1 and ferritin.Conclusion:In vitro studies showed AA can’t inhabit the auto-oxidation of PolyCHb at 37℃,but it can reduce the ferric PolyCHb.We obtained the pharmacokinetic parameters of PolyCHb,the half life is 8.5-9.5 hours.Infused PolyCHb can induce oxidative stress in kidney,cause histopathologic injury.AA treatment is beneficial to alleviate renal oxidative stress and lower the damages caused.AA treatment can improve the ability for motablizing the free hemoglobin and heme of kidney primary tube epithelial.Generally,this study demonstrated the potential value of using ascorbic acid to attenuate the oxidative stress caused by PolyCHb.