The Effects And Mechanism Of Vitamin C Postconditioning On Arachidonic Acid-induced Platelet Activation That Had Undergone Anoxia-reoxygenation

Objective: Experimental and clinical studies provide evidence that platelet activation occurs upon ischemia?reperfusion,but the underlying mechanism is not very unclear.A lot of researches show that platelet activation is closely related with the burst of reactive oxygen species(ROS)which is induced in the development of ischemia-reperfusion injury.Vitamin C is a weak acidic vitamin,as well as a powerful antioxidant.Previous studies have found that VC could scavenge ROS,reduce platelet activation and attenuate the reperfusion injury.However,its effects on the platelets that had undergone anoxia-reoxygenation(A/R)in vitro remain unclear.Therefore,the study was designed to evaluate the effect of Vitamin C on the platelets in vitro by exposing to A-R and elucidate related mechanism.Methods: A total of 67 cases of fresh platelets puree were collected from the Blood Center of Hebei Province,which the platelet concentration is between 960×109/L and 1280×109/L.The inclusion and exclusion criteria are strictly in accordance with national standards for blood donation law.The platelets puree were diluted with saline to the concentration of 200×109/L.Aggregation and curve slope were evaluated in 4 hours with whole blood impedance analyzer(Model 590),made by Chrono-Log Corporation.At first,the aim was to find out the most appropriate experimental time.The platelets were treated with different hypoxia or reoxygenation time,and then the impedance aggregometry was carried out by measuring changes in electrical impedance,which induced by arachidonic acid(AA 0.5mmol/L),so the anoxia-reoxygenation(A/R)model in vitro was created.Three different antioxidatives(vitamin C,melatonin and probucol)were used to post treat the platelets puree that had gone A-R.The platelets puree were prepared into A/Rgroup,C1-3 group and concerning weak acidity of VC,HCl group(p H=3.24,that was the same with VC solution)was set up and impedance aggregometry reflected the effect of antioxidatives on platelet aggregation.The influence of VC on apoptosis-related indicators was detected.Reactive oxygen species(ROS)were obsereved by inverted fluorescence microscope,mitochondrial membrane potential(??m)was measured by flow cytometric analysis and Bax,Bcl-2,cytochrome-C,and Caspase-9 were detected by western blot analysis.SPSS 22.0 statistic software was used to analysis.When data was subjected to normal distribution,continuous variables were expressed as the mean value±standard deviation(SD),the overall comparison in each group was evaluated by single factor ANOVA analysis.If not,the variables were expressed as the median and quartile(QR),the overall comparison in each group was evaluated by Friedman's M test.P value of <0.05 was considered statistically significant.Results:1 The effect of different time of hypoxia or reoxygenation on the platelet aggregation of platelet puree1.1 The effect of different time of hypoxia on the platelet aggregation of platelet puree Compared with control platelets kept in atmospheric conditions,platelets exposed to A-R showed increase in platelet aggregation and curve slope,which was maximal after 20 minutes of anoxia.There were statistically differences among these groups(P<0.05)between the groups of 20 min and5min and no statistical difference was found among the groups of 5min,10 min and 30min(P>0.05);1.2 The effect of different time of reoxygenation on the platelet aggregation of platelet puree Compared with the group of 0min,platelet aggregation and curve slope in other groups increased,and the activation rate reached to maximum in group of 5min,but no statistical difference was found among them(P>0.05);2 The effect of three different antioxidatives to post treat the platelet puree that had gone A-R2.1 The effect of different concentrations of vitamin C on the platelet aggregation of platelet puree during A/R Compared with the A/R group,the platelet aggregation of VC group(1000?M)decreased significantly(P<0.05),while no statistical difference was found among the group of 10?M,100?M and HCl(P > 0.05).In comparison of 10?M and 100?M,the VC group(1000?M)showed the greatest inhibition rate(P<0.05);2.2 The effect of different concentrations of melatonin on the platelet aggregation of platelet puree during A/R Compared with the A/R group,the platelet aggregation of melatonin group(100?M)decreased significantly(P < 0.05),while no statistical difference was found between the group of 0.01?M and 1?M(P>0.05).In comparison of 0.01?M and 1?M,the 1000?M group showed the greatest inhibition rate(P<0.05),but the former activated the platelet not inhibited it;2.3 The effect of different concentrations of probucol on the platelet aggregation of platelet puree during A/R Compared to the A/R group,the platelet aggregation of probucol group(100?M)decreased significantly(P<0.05),while no statistical difference was found between the group of 1?M and 10?M(P>0.05).In comparison of1?M and 10?M,the 1000?M group showed the greatest inhibition rate(P <0.05);3 The effect of Vitamin C on platelet apoptosis-related indicators3.1 ROS Compared with the control group(platelet puree was kept in air),ROS in A/R group,VC group and HCl group increased significantly(P < 0.05).Compared with the A/R group,the ROS in VC group decreased(P < 0.05)while HCl group showed no change like that,which meant vitamin C could scavenge ROS powerfully;Compared with the control group,the low ??m rate in A/R group,VC group and HCl group increased significantly(P < 0.05).Compared with the A/R group,the rate in VC group decreased(P < 0.05),and there was no significant difference compared with HCl group(P>0.05),suggesting that the ability of alleviating mitochondrial injury was related with oxidizability,but not acidity;3.3 The apoptosis-related protein expression of Bax,Bcl-2,cyto-C and Caspase-9Compared with the control group,the expression of cyto-C,Bax,Bcl-2and Caspase-9 increased(P<0.05),suggesting that the cytoplasmic apoptotic events of mitochondrial pathway in platelets increased.Compared with the A/R group,the VC group and the expression of cyto-C,Bax and Caspase-9decreased and the expression of Bcl-2 increased significantly(P<0.05).This showed vitamin C abolished the hypoxia-induced events of intrinsic pathway of apoptosis and further,it hindered AA-induced platelet aggregation and adhesion.3.2 Mitochondrial membrane potential(??m)Conclusion:1 Different ROS scavengers dose-dependently inhibit platelet activation and aggregation.2 Vitamin C postconditioning scavenges ROS,which is related with down-regulation of cyto-C,Bax and Caspase-9 protein and up-regulation of Bcl-2 protein,reduces platelet apoptosis,and then inhibits platelet aggregation.