Antioxidants Attenuate Oxidative Stress-induced Hidden Blood Loss In Rats

BackgroundHidden blood loss(HBL),commonly seen post total knee or hip arthroplasty,causes postoperative anemia even after reinfusion or blood transfusion based on the visible blood loss volume.Recent studies demonstrated oxidative stress might be involved in HBL.However,whether antioxidants proanthocyanidin(PA)or hydrogen water(HW)can ameliorate HBL remains poorly understood.The aim of the study was to evaluate the effect of PA and HW on HBL.Rat HBL model was established through administration of linoleic acid with or without treatment of PA or HW.The levels of hemoglobin,red blood cell count,superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),Malondialdehyde(MDA),and ferryl hemoglobin(Hb)were measured.RBC and Hb values as well as the activity of SOD and GSH-PX were reduced after administration of linoleic acid,which were ameliorated by treatment with PA or HW.In addition,the quantity of MDA was significantly decreased along with the administration of PA or HW.In conclusion,PA or HW could ameliorate HBL in rat model through reducing oxidative stress,suggesting they might be used as novel therapeutic approach in the prophylaxis or treatment of HBL in clinic.IntroductionArtificial joint replacements are widely employed to alleviate pain,and improve the quality of patients’ life.the quantity for primary and total hip arthroplasty and total knee arthroplasties was estimated to increase by174%-673%by 2030 as the population ages,However,hidden blood loss(HBL)dominantly occurs after artificial joint replacement,such as total knee arthroplasty(TKA)and total hip arthroplasty(THA).The consequential acute anemia and transfusion are the major concerns for joint surgeons.The pathogenesis of HBL is very complicated involving several factors.A recent study demonstrated that free fatty acids(FFA)generated from fatty emboli in blood circulation are responsible for the hidden blood loss through peroxidating injury of membrane molecules of RBC and hemoglobin.In addition,antioxidants administered intra-or post-operatively are predicted to play a protective role in erythrocytes oxidation and potentially reduce the volume of hidden blood loss after arthroplasty,suggesting oxidation might be involved in the pathogensis of HBL.Consistent with this,our previous study also demonstrated that FFA can induced red bloods cells and hemoglobin damage via ROS toxicity in vivo.As a natural antioxidant extract from grape seeds,proanthocyanidin(PA),possesses a wide scope of bioavailability.PA exhibits higher protective effect against DNA damage and lipid peroxidation induced by ROS compared with b-carotene,vitamins C and E.PA is a safe and effective bioavailable antioxidant and ROS scavenger,which is used for the treatment of ischemia-reperfusion injury of multiple organs,malignant tumor progression,carcinogenesis,gastrointestinal disorders and Parkinson’s and Alzheimer’s illnesses.As a new antioxidant,hydrogen water(HW)has been also applied to prevent and treat oxidative stress-associated illnesses using establishment of animal models.HW has been proven to selectively remove strong oxidants including peroxynitrite and hydroxyl radical.Alternative ROS play a physiological role in preventing cells from oxidative stress.Considering the role of oxidative stress in the pathogenesis of HBL,whether PA and/or HW(as an antioxidant)ameliorate HBL remains poorly understood.The objective of this study was to evaluate the effect of PA and HW on HBL as well as compare their protective effects through measuring the levels of.hemoglobin,red blood cell count,superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),Malondialdehyde(MDA),and ferryl hemoglobin(Hb).Materials and methodsAnimalsForty 10-week male Sprague-Dawley rats weighed 250±20g were obtained from Nanjing University Unit.All animals were fed daily with diet,potable water or HW under appropriate laboratory conditions at 24℃ with 12-h light/dark cycle.The animals were randomly assigned into four groups(n=10).Experimental procedures were performed strictly according to the Care and Use of Laboratory Animals proposed by National Research Council in 1996.All animals were properly regulated.Animal ethics approval was obtained for this research.All experimental procedures were conducted comply with the guidelines by the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee of Nanjing University.Preoperatively,all animals were anesthetized via ether inhalation.Instruments and reagentsInstruments:Hematology analyzer(SYSMEX XE-5000,Japan),centrifuge(Hermle Universal Centrifuge Z323,German),microplate reader(BIO-RAD680,USA),polarizing microscope(NIKON ECLIPSE 501,Japan),spectrophotometer(Hewlett Packard 8453 UV-visible diodearray spectrophotometer,USA).HW generating apparatus(Bio Coke Laboratory,Tokyo,Japan).hydrogensensor(DHS-001,ABLE,Tokyo,Japan).Reagents:The concentration of MDA and the activity of SOD and GSH-PX were measured by with commercially available assay kits(Nanjing JianchengBioengineering Institute,Nanjing,China).Linoleic acid was purchased from Sigma-Aldrich(St.Louis,USA).PA was purchased from Shanghai Aladdin Bio-chem Technology Institute(Shanghai,China).HW preparation:HW was prepared by dissolving H2 gas into drinking water under high pressure of 0.4 MPa using a HW-generating apparatus.SD rats were supplied with HW(0.7 mM)through a closed glass vessel(300 ml)equipped with an outlet line containing 2 ball bearings to prevent water degassing.The H2 concentration of HW was detected by a hydrogensensor(Unisense,Aarhus,Denmark).Experimental protocol and drugThe procedures below were performed on the rats in all four groups and the dose use was referred as previously described.The control group(CON)rates were fed with portable water and injected with ethanol alone(0.5 ml,20%)via intravenous administration into the tail vein after 2-week feeding.The LIN(receiving linoleic acid,LIN)group animals were fed with portable water and injected with a 0.5ml of 60 mmol/L linoleic acid diluted in 20%ethanol by intravenous administration into the tail vein after 2-week feeding.The LIN+PA group was fed with a 100 mg/kg dose of PA diluted with portable water daily,and injected with 0.5 ml of 60 mmol/L linoleic acid diluted in 20%ethanol by intravenous administration into the tail vein after 2-week feeding ·The LIN+HW group was fed with HW daily.And injected with 0.5ml of 60 mmol/L linoleic acid diluted in 20%ethanol by intravenous administration into the tail vein following 2-week feeding.During all treatments,rats were daily monitored and weighted one to six times per day until the end of the experiment.All rats had no special discomfort throughout the experiment.Routine and biochemical Analysis of bloodThe blood samples were taken from the caudal vein under anesthesia(0.5 ml each time)at the beginning of the injection and 24-,48-,and 72-h following administration.RBC,hematocrit and Hb levels were detected by a hematology analyzer immediately after sampling collection.Morphological changes of blood cells were observed following Wright’s staining under polarizing microscope.The remnant blood samples were centrifuged and restored at 80℃ for subsequent biochemical analysis.MDA,T-SOD and GSH-PX activities were measured by a spectrophotometer.The absorbance values were detected at 532 nm,550 nm and 412 nm wavelength.Spectral changes of Hb in the LIN and LIN+PA groups were quantitatively measured by a spectrophotometer.Hb at a concentration of 10 mM was mingled with 0.1 M sodium phosphate buffer containing 100 mM DTPA.All experimental procedures were conducted at 25℃.Statistical analysisStatistical analysis was performed using SPSS 19.0 statistical software.All data were expressed as mean ±standard deviation(SD).One-way analysis of variance(ANOVA)was performed followed by Dunnett’s t-test.A P value of less than 0.05 was considered as statistically significant.ResultsDaily consumption of water and body weight among all groups was monitored.Rats in the CON group consumed(20.0±3.5)ml of portable water daily,(21.0±2.7)ml of portable water daily in the LIN group.In the LIN+PA group,daily consumption of PA solution was(22.0±2.4)ml,and(24.0±3.4)ml of HW daily in the LIN+HW group.Water consumption and body weight did not significantly differ among four groups.Blood Routine TestBefore linoleic acid administration,no significant differences were observed inRBC and Hb levels among the four groups.After administration of adose of 0.5 ml(60 mmol/L)linoleic acid,RBC and Hb level significantly changed compared with the control group,which showed that an in vivo hidden blood loss model was established successfully.We further analyzed the RBC and Hb levels of LIN+PA group and LIN+HW group compared to that of LIN group.After 24 hours of administration,the Hb and RBC had reduced to different extents in the three groups.In LIN group,the RBC and Hb values reduced by0.66±0.34×1012/L,16.3±8.25g/L,and in LIN+PA group,those decreased by0.35±0.1 ×101/L,9.1±4.01g/L.Significant difference was noted in the changes between the LIN and LIN+PA groups.After 48 hours of administration,the changes of RBC and Hb levels of the LIN group and LIN+PA group was still significantly different.Besides,in LIN+HW group,we found the RBC and Hb value decreased by 0.45±0.22×1012/L,10.7±3.56g/L after 24 hours,with a tendency to alleviate the reduction of RBC and Hb level.After 48 hours,the decrease of RBC and Hb(0.72±0.23×1012/L,18.2±5.85 g/L)of LIN+HW group was signicantly different to that of LIN group(1.15±0.48 × 1012/L,25.7±8.38 g/L).Oxidative Stress MarkersThe activity of SOD and GSH-PX in LIN group significantly declined after 24 hours of administration,and reached the lowest level after 48 hours,and had a mild increase after 72 hours.Both the LIN+PA and LIN+HW groups showed a similar variation tendency in these two markers.However,both SOD and GSH-PX activity in group of LIN+PA and LIN+HW were obviously higher than that of group LIN at each time point.In terms of MDA,its concentration in LIN group reached peak after 24 hours,and then started to decrease slowly.The LIN+PA and LIN+HW groups also displayed a similar changing pattern in MDA level.However,both SOD and GSH-PX activity in group of LIN+PA and LIN+HW were consistenly lower than that of group LIN.The ferryl Hb was present and formed by reacting with H2O2,which was confirmed by characteristic absorbance band around 620 nm via the reaction with sulphide ions.The effect of linoleic acid upon the hemolysis of RBCs,either itself or in conjunction with ROS,can be utilized to assess the severity of oxidative injury of erythrocytes(1).Blood samples were collected from three groups before administration and every 24 hours thereafter.Absorbance peak values were detected at approximately a wavelength of 425 nm,consistent with the Soret peak of ferryl Hb.Histologic investigationIn LIN group,a number of shrunken,deformed and ruptured blood cells were seen compared with the control group and the morphological changes werre the most distinct after 24 hours of administration.In contrast,we could also identfy some shrunken and deformed RBC in group LIN+PA and LIN+HW.But,ruptured blood cell could be hardly found in those two groups.