The Addition Of Sodium Pyruvate During RBC Storage Can Attenuate The Storage Lesion And Liver Injury In A Murine Transfusion Model

Red blood cells(RBCs) are the main component of the blood, and the major function of RBCs is the transport of oxygen(O2) and carbon dioxide(CO2). In clinic, blood transfusion is a basic mean of treatment for patients with massive hemorrhage trauma or surgery. Hospitals in general adopt the “First Input First Output” principle in clinical blood transfusion, but it is mostly storage RBCs(SRBCs) that are used in the transfusion. It is inevitable that the physiological, biochemical, morphological structure and functions of RBCs go through a series of changes during storage, such as obstacle of energy metabolism, augment of oxidative stress level, decline of intracellular p H, decrease of erythrocyte deformation and oxygen carrying capacity. These are collectively referred to as "storage lesion". RBC storage lesion can lead to RBC transfusion adverse reactions. Animal experiments have shown that compared with fresh red blood cells(FRBCs), the deformability of RBCs stored for 7 days decreased significantly, which may aggravate acute liver injury after transfusion in patients experiencing hemorrhagic shock and resuscitation. In addition, the prolonged storage time may also contribute to the increased mortality in beagle dogs’ pneumonia model. Clinical studies have shown that the transfusion of SRBCs is closely related to acute lung injury caused by transfusion, the dysfunction of multiple organs, increased infection rate and mortality rate as well as other adverse reactions in trauma, critically ill or peri operative patients.Although a large number of studies have revealed that RBC storage lesion is closely related to the adverse reaction of transfusion, the mechanism of the adverse reaction after transfusion is still not clear. At present, there are many hypotheses, such as loss of Nitric oxide(NO) activity, energy depletion, oxidative damage of free hemoglobin, decreased deformation, iron overload can explain the adverse effects after RBC transfusion. It is reported that inhaled NO, washed RBCs or the treatment of SRBCs with rejuvenation and other methods can alleviate or reduce the occurrence of adverse reactions after transfusion. However, these methods are remedial measures after adverse effects have occurred irreversibly. If the storage lesion is inhibited during RBC storage, it may have greater therapeutic benefits.According to the standard of U.S. Food and Drug Administration(FDA) for SRBCs transfusion, the hemolysis rate of RBCs should be less than 1% after storage in vitro and survival rate in recipient should be at least 75% after transfusion 24 h. The hemolysis rate, survival rate and storage lesion are closely related with RBC storage solution. Since 1915 when Rous and Turner used citric acid and glucose mixed solution for RBC storage in vitro for the first time, the composition of RBC storage solution was improved progressively. At present, the maximum time of RBC storage is 42 days at 4 ℃ in clinic, with CPDA-1, sodium chloride-adenine-glucose and mannitol(SAGM) mainly used as RBC storage solution. The preservation solution contains glucose which provides energy for RBCs, citric acid/sodium citrate which plays the anticoagulant role, solution of phosphate buffer which maintains acid-base balance, adenine which promotes the generation of adenosine triphosphate(ATP), and mannitol which maintains membrane integrity of erythrocyte. Studies have found that by improving the composition and physicochemical property of RBC storage solution, it is possible to improve SRBC energy metabolism, reduce storage lesion, and prolong the storage time of RBCs. The addition of glucose as the energy metabolism substrate during RBC storage can effectively improve the RBC energy metabolism and reduce the hemolysis rate of SRBCs. However, the decreased antioxidant capacity and oxidized cell membrane and key proteins by reactive oxygen species result in irreversible oxidative damage during RBC storage, and the existing RBC storage solution is lack of antioxidant components. Stowell et al. found that the addition of vitamin C as the antioxidation during RBC storage can improve the survival rate of RBC and reduce the immunogenicity after transfusion, but it had no effect on inflammatory reaction. Despite the continuous study on RBC storage lesion and adverse reactions after transfusion, storage lesion has not been improved substantially and has still resulted in adverse reactions after transfusion. Under these circumstances, adding substances may improve RBC storage lesion and reduce the adverse reactions after transfusion.Sodium pyruvate(SP) is the sodium salt of pyruvate. Pyruvate is the key intermediate products in glycolysis pathway in Krebs cycle. It can produce carbon dioxide, water and energy in aerobic metabolism and generate lactic acid and energy in anaerobic glycolysis. Sodium pyruvate plays antioxidant and anti-inflammatory functions, protects organs and corrects acidosis. Previous studies have indicated that using the rejuvenation with sodium pyruvate to remodel the SRBCs could promote the regeneration of intracellular ATP and 2,3-DPG, thus improve the function of RBCs. However, the storage lesion cannot be reversed by the treatment with the rejuvenation solution containing sodium pyruvate before transfusion. At present, the effects of sodium pyruvate on RBCs storage lesion such as the decrease of oxygen-carrying capacity, the increase of oxidative stress level during RBC storage and adverse reactions after transfusion have not been reported. We hypothesized that the addition of sodium pyruvate during RBC storage could effectively lessen RBC storage lesion and reduce the adverse reactions after transfusion.The murine RBCs transfusion model is a commonly used animal model for the study of adverse reactions after SRBC transfusion. As has been reported, the murine RBCs stored for 14 days and the human RBCs stored for 42 days experience similar changes in morphology and function, and have similar states in physiology and biochemistry. Therefore, this study intends to use murine RBCs stored for 14 days to simulate human SRBCs and establish a murine RBCs’ storage platform to probe the effects of sodium pyruvate on RBCs during RBC storage, and to further explore the role of sodium pyruvate in reducing the adverse reactions after transfusion. Part I Establish the platform for the storage of RBCs in murine model in vitroThis study intends to explore the effects of sodium pyruvate on RBC storage lesion such as the decrease of oxygen-carrying capacity and the increase of oxidative stress level during RBC storage and adverse reactions after transfusion. The platform for the storage of RBCs in murine model in vitro was established. The mice were anesthetized with intraperitoneal injections of sodium pentobarbital(the injection volume was decided by 75mg/kg body weight), placed in a superclean bench with 75% alcohol disinfection animal surgery and surgical instruments, and mechanically ventilated. Mice were bled by cardiac puncture into CPDA-1 for anticoagulant and storage solution and its final concentration was 14%. The whole blood was leukoreduced and centrifuged at 400 g for 15 min and volume-reduced to a final hematocrit of 70% to 75%. The RBCs were stored at 4°C in blood storage refrigerator. RBCs were divided into two groups: RBCs in fresh RBC(FRBC) group were stored for less than 2 hours while the RBCs in stored RBC(SRBC) group were stored for 14 days. The blood gas, complete blood counts, the oxygen-carrying capacity, the hemolysis rate and the RBC 24-hour recovery were measured.The results showed that Na+ concentration was decreased significantly and K+ concentration was increased significantly in SRBC group compared with FRBC group(p(27)0.05). The hemolysis rate was increased significantly and the RBC 24-hour recovery was significantly decreased in SRBC group compared with FRBC group(p(27)0.05). The P50 value was decreased significantly in SRBC group compared with FRBC group(p(27)0.05). The results indicated that the RBC PO2 decreased, the acid base balance was destroyed, the distributions of Na+and K+ in the RBCs were abnormal and the oxygencarrying capacity decreased. In this part, we established an experimental platform for the storage of RBCs in vitro, and laid the foundation for the evaluation of the roles that sodium pyruvate played in RBC storage. Part II The effect on storage lesion of RBCs by adding sodium pyruvate during RBC storageThis part of study is based on the murine RBC storage platform established in Part I to verify the roles that sodium pyruvate played during RBC storage. As described in Part I, whole blood was leukoreduced and then equally divided into two groups(n=8):(1) SP group, concentrated solution of SP was added to RBC suspension to make the SP concentration of 2.5m M in RBC storage solution;(2) Control group, saline was added to RBC suspension at the same volume as SP group. The storage solutions were then centrifuged and volume reduced to a final hematocrit of 70% to 75%. The RBCs were stored at 4°C for 14 days. The blood gas, complete blood count, the oxygencarrying capacity, ATP content, oxidative stress levels, the hemolysis rate and the RBC 24-hour recovery were measured.The results showed that total hemoglobin concentration, RBC hematocrit, and mean corpuscular volume in SP group were not significantly differenct from those in control group(p(29)0.05). The ATP content and SOD activity were increased significantly in SP group compared with control group(p(27)0.05). The malondialdehyde(MDA) content was decreased significantly in SP group compared with control group(p(27)0.05). The P50 value was increased significantly in SP group compared with control group(p(27)0.05). Compared with control group, the hemolysis rate was on a downward trend and the RBC 24-hour recovery was on an upward trend in SP group, but there were no significant differences between the two groups(p(29)0.05). Results showed that effects on total hemoglobin concentration, RBC hematocrit, and mean corpuscular volume were not significantly different between the two groups. But adding sodium pyruvate during RBC storage can maintain the intracellular ATP concentration and oxygen-carrying capacity, improve the antioxidant capacity of SRBC, and reduce RBC oxidative stress injury. Part III The effect on liver injury after SRB transfusion by adding sodium pyruvate during RBC storageThis part of study is based on the murine RBC storage platform to explore the effects of adding sodium pyruvate to RBCs on liver injury in a murine transfusion model. As described in Part II, the SRBC added with sodium pyruvate or added with the same volume of normal saline were prepared. All recipient mice received by volume 20% of their total blood volume via the tail vein. Two hours later, the mice were anesthetized with intraperitoneal injections of sodium pentobarbital(the injection volume was decided by 75mg/kg body weight), then mechanically ventilated, aseptically bled by cardiac puncture. The whole blood was centrifuged at 4000 rpm for 90 s, the upper plasma was packaged and stored at-80°C. The liver tissue was taken into the liquid nitrogen and the other part was fixed with 4% formalin. Murine plasma biochemistry, including aspartate aminotransferase(AST), blood urea nitrogen(BUN) and lactate dehydrogenase(LDH) were measured. Murine hepatic MDA content, myeloperoxidase(MPO) activity, interleukin-6(IL-6) and tumor necrosis factor-a(TNF-a) levels were evaluated. Then murine pathological slices were observed through HE staining.The results showed that the AST activity, BUN content and LDH activity in murine plasma were decreased significantly in SP group compared with control group(p(27)0.05). The hepatic MDA content, MPO activity, IL-6 and TNF-a were decreased significantly in SP group compared with control group(p(27)0.05). The histopathologic results showed that SP group showed less cell necrosis and neutrophil cell infiltration in liver tissue and lighter liver injury in murine transfusion model compared with the control group. The results of this study showed that adding sodium pyruvate to SRBCs attenuated the oxidative stress and inflammatory response levels and lessened organ damage in a murine transfusion model.In summary, this study was based on the murine RBC storage platform to evaluate the effects of adding sodium pyruvate to SRBCs on RBC storage lesion, and furthermore to explore the effects of adding sodium pyruvate to SRBCs on liver injury in a murine transfusion model. The results of the study showed that adding sodium pyruvate to SRBCs during RBC storage can improve energy metabolism, oxidative stress damage and oxygen-carrying capacity, and can significantly reduce liver injury after SRBC transfusion. This study provides a new way of thinking and experimental evidence for the improvement of RBC storage solution and prevention of the adverse reaction after SRBC transfusion.