| Red blood cells(RBC) are abundant in vivo. The main function of RBC is transport of oxygen(O2) and carbon dioxide(CO2), which completed by hemoglobin(Hb). RBC are one of the most susceptible cells due to the following reasons:firstly, RBC are exposed to high oxygen tensions in the circulation; secondly, RBC are rich in iron, a transition metal which promotes the formation of oxygen free radicals; Thirdly, RBC contains approximately one-half of the fatty acids which are unsaturated. Reactive O xygen Species(ROS) react with Hb to become methemoglobin(met Hb) that lose its function to bind oxygen. The oxidation of Hb also causes the formation of disulfide between adjacent globin chains which deposited on the inside of cell membrane to influence membrane fluidity. RBC membrane attacked by ROS causes lipid peroxidation and membrane protein cross- links. Lipid peroxidation reduced the fluidity and increased rigidity, which reduced RBC elongation index.RBC oxidative damage ofter occurred under various physiological and pathological conditions. Exercise- induced oxidative stress cause damage of RBC which weaken oxygen delivery capacity and exacerbate tissue hypoxia, thus affect the capability of organisms. Besides, excessive oxidants such as aniline could oxidate hemoglobin into Met Hb which lose its oxygen delivery capacity. Another example is RBC are oxidated during storage. Changes such as increased free hemoglobin and decreased deformability lead to adverse effects of blood transfusion. All in all, oxidative damage of RBC are harmful to organism and it is imperative to prevent and treate its oxidative damage.It is often use antioxidants, including endogenous antioxidants(vitamin E and vitamin C) and exogenous substances(salidroside and mango peel extracts), to prevent and cure RBC oxidative damage. Salidroside is one of the most used in RBC. It has been showed the protective effect of salidroside in high altitude polycythemia, radiated- induced RBC oxidative damage and exercised- induced RBC oxidative damage. Salidroside exerts its effect through improving the bodily endurance ability, which require pretreatment in advance. Besides, the active ingredients are difficult to extract and it is hard to tell the mechanisms due to the complication of plants. Therefore, such antioxidants could be more effective if they are endogenous and simple.Pyruvate is a three-carbon molecule that is endogenously produced during glycolysis. It can be converted in to Acetyl-Co A, which is oxidized in the tricarboxylic acid(TCA) cycle to produce ATP. Pyruvate is also reduced into lactate by lactate dehydrogenase(LDH). Sodium pyruvate(SP) is the ionic type of pyruvate and has been demonstrated to have the effects of antioxidative, correct acidosis and organ protective. It has been showed that SP protect organ function in hemorrhagic shock, brain damage and ischemia-reperfusion injury models. SP has also been used in preventing cataract which may caused by diabetes and in pretecting islet cells from zinc poisoning which are associated with oxidative stress. However, there are few investigation concerning pyruvate effects against oxidative damage in RBC. Rejuvesol, a pyruvate-enriched rejuvenation solution of stored RBC, could dramatically restore ATP levels in RBC and reverse RBC mechanical properties. Since SP is the key intermediate in metabolism which also have antioxidative effect, we speculate that it may exert its effects via mediating glycolysis pathway. Part I Establis h and screen proper red blood cell oxidative stress modelsWe chose phenazine methosulfate(PMS), hydrogen peroxide(H2O2) and sodium nitrite(Na NO2) to establish and screen proper RBC oxidative stress model. Whole blood was obtained from the rat carotid artery. centrifuged the samples and removed the buffy coat. RBC suspensions were divided into four groups: Group control; Group PMS which final concentration were 25, 50 and100 μM respectively; Group H2O2 which final concentration were 0.5, 5 and 8 m M respectively; Group Na NO2 which final concentration were 0.5, 1 and 1.5 m M respectively. 0.4m M(final concentration) sodium azide(Na N3, as an inhibitor of catalase) was added into Group H2O2. All groups were then incubated for 1h at 37°C. The samples were centrifuged and the hematocrit of all samples was then adjusted to 40% with self- plasma before the measurements of methemoglobin(Met Hb), erythrocyte aggregation, deformability.The present results showed that after incubation with oxidants, the level of Met Hb is increased in all group. Aggregation Index(AI) in Group H2O2 decreased and it was significantly lower at 8m M compared with Group control(P<0.05); AI in Group PMS and Na NO2 were not significantly different(P>0.05). Top Time(TT) increased when H2O2 at 5m M and 8m M and show significantly different(P<0.05); PMS showed no effect on TT; TT decreased in Group Na NO2, while it increased as the concentration increased although still lower than Group control. Elongation Index(EI) decreased significantly at all shear rates(SHR) in Group H2O2 and Group PMS(P<0.05). EI in Group Na NO2 decreased(P<0.05) when SHR≤100 s-1. However, as shear rates increased, EI tended to increase and it showed significantly different when SHR≥600 s-1(P<0.05).In summary, all oxidants could raise Met Hb levels. However,effects of different oxidants on erythrocyte aggregation and deformability differ from one another, suggesting that we should choose proper oxidant according to the purpose in vitro models; Na NO2 may improve RBC EI as a nitric oxide(NO) donor so that it is not suitable for the following experiment. Beside, according to the Met Hb production, we chose the action concentration of PMS and H2O2 are 50μM and 8m M respectively. Part II Sodium pyruvate protection against oxidative damage in rat erythrocytesTo evaluated SP protection against oxidative damage in RBC in PMS/ H2O2-induced oxidative stress models. Whole blood was obtained from the rat carotid artery. The samples were centrifuged and the buffy coat was removed. First, we screen the concentration of SP through determining the level of Met Hb, MDA(malondialdehyde) and GSH. RBC suspensions were divided into two groups: Group PMS and Group H2O2. And there are different condition in these two group, including Group control, Group oxidative stress and Group SP. All groups were then incubated for 30 min at 37°C before the addition of H2O2. Then, oxidants were added into Group PMS/H2O2 and Group SP and all groups were incubated for another 1 h at 37°C. Alterations in(Met Hb), malondialdehyde(MDA), reduced glutathione(GSH), were determined after incubation. The GSH/GSSG ration and the activity of glutathione reductase(GR) were measured in group PMS.10 The presents results showed that the content of Met Hb increased slightly after incubated with different concentration of SP, but there was no significant difference and is still in the normal range. MDA decreased significantly, but it was rebounded when SP at 100 m M. GSH level increased slightly but it declined when SP at 100 m M. The results suggest that low concentrations of sodium pyruvate no significant side effects on red blood cells, 100 m M of SP may be high. Therefore, it is determined that the final SP concentrations were 5 m M, 10 m M, 20 m M and 50 m M.After incubated with PMS, the content of Met Hb, MDA increased and the level of GSH and the GSH/GSSG ratio decreased(P<0.05), the activity of GR show no difference. The SP pretreatment decreased MDA levels and increased GSH/GSSG ratio(P<0.05) in a dose-dependent manner, while show no effect on Met Hb and GSH. H2O2-incuced oxidative stress increased Met Hb, MDA levels and decreased GSH content(P<0.05). Pretreatment of RBC with SP significantly reduced Met Hb and MDA formation(P<0.05). It also increased GSH level in a dose-dependent manner and showed significantly difference when SP at 20 m M and 50 m M(P<0.05).In summary, we screen the concentrations of SP are 5,10 20 and 50 m M for the following experiments. Moreover, presents results first demonstrated that sodium pyruvate protected rat RBC against PMS and H2O2-induced damage and the protective effect of H2O2 showed more obvious. Thus we chose the H2O2-induced damage model to verify the potential mechanisms of SP protection.Part III Potential Mechanis ms of SP protection against oxidative damage in rat e rythrocytesTo test the potential mechanisms of SP protection in H2O2-induced damage model. RBC were divided into three groups: Group control, Group H2O2 and Group SP by pretreatments with various SP concentrations. All groups were incubated at 37°C for 30 min after adding SP in Group SP. The final SP concentrations were 5 m M, 10 m M, 20 m M and 50 m M, respectively. Then, H2O2 was added in Group SP and Group H2O2. All groups were then incubated at 37°C for another one hour. Alterations in reactive oxygen species(ROS), adenosine triphosphate(ATP), Na+-K+ATPase, lactate dehydrohenase(LDH), 2,3-diphosphoglycerate(2,3-DPG) and oxygen affinity(P50) were determined after incubation.Results showed that H2O2-incuced oxidative stress increased ROS levels and decreased ATP, LDH and Na+-K+ATPase activities(P<0.05). It also decreased 2,3-DPG and P50 levels(P<0.05). Pretreatment of RBC with SP significantly reduced the ROS production induced by H2O2 and pretreatment with SP at 50 m M showed significantly difference(P<0.05). Meanwhile, it profoundly restored concentrations of ATP(P<0.05) compared with Group H2O2 and markedly restored the activities of Na+-K+ATPase in a dose-dependent manner. Pretreatment with SP at 10 m M and 20 m M completely restored the LDH activities(P<0.05) and showed no difference compared with Group control. The value of P50 was also obviously enhanced in Group SP at various concentrations, compared to Group H2O2(P<0.05). Pretreatment with SP at various concentrations appeared to raise 2,3-DPG levels to a control value without differences among them.In summary, first, SP decrease ROS, which protect RBC directly; secondly, SP also increase LDH activity, promote glycolysis pathway to restore ATP level, which could reduce the extent of oxidative stress damage. Beside, NAD+/NADH ratio increases when pyruvate is transformed into lactate, and NAD+ is indispensable to produce 2,3-DPG which is a key factore for P50, so we speculate that SP could mediate 2,3-DPG pathway to increase 2,3-DPG, thus increase P50 to improve RBC function.In summary, this study indicated that SP protected RBC against oxidative stress and it exerts its antioxidant effects via mediating the Luebering-Rapoport pathway and the glycoltic pathway which alleviated RBC damage and improved RBC function, providing a new way to prevent and cure RBC oxidative damage-related diseases. |
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