Protective Effect And Mechanism Of Novel Erythrocyte Preservation Solution On Autologous Erythrocyte In Patients With T2DM

Background:In recent years,there has been rapid development in surgery,and many medical restricted areas gradually has been broken down.The number of surgeries involving elderly tumors,orthopedics,and other comorbidities such as hypertension and diabetes increased,and the shortage of blood supply and potential risks of blood transfusions were becoming increasingly prominent.The demand for autologous blood transfusion,especially storage-type autologous blood transfusion also increased.However,there is still controversy over the use of autologous blood transfusion in some special patients,such as type II diabetes mellitus（T2DM）surgical patients.Red blood cells in T2DM patients are affected by multiple factors during storage,which generally results in increased hemolysis,decreased oxygen carrying capacity,and changes in metabolic abilities,making them more susceptible to storage damage.According to this background,the hypothesis is proposed that"preparing a new type of red blood cell preservation solution may alleviate storage damage in red blood cells of T2DM patients."Objective:1.Comparing and observing the changes in the morphology and function of red blood cells（RBCs）stored in different new preservation solutions and the conventional preservation solution of preoperative autologous blood transfusion（PABD）in Type II diabetes mellitus（T2DM）patients,this study aims to explore the changes in RBC membrane stability and oxygen carrying capacity with storage time,investigate the mechanism by which new preservation solutions protect RBCs and reduce storage damage.This research provides theoretical basis for optimizing blood storage and developing rational blood transfusion schemes for T2DM patients,reducing the incidence of transfusion-related complications,and improving the clinical scientific level of blood transfusion.Method:20 non-diabetic patients and 20 T2DM patients scheduled orthopedic surgery for storage-type autologous transfusion were screened.Blood was collected after PABD evaluation,and plasma was separated and kept aside for experimental use.It was mixed with either conventional storage solution or a new storage solution.Based on previous literature,the contents of glucose,citric acid,and sodium chloride in the conventional storage solution were divided into three gradients for pre-experiment.The appropriate concentration was selected for the new storage solution 1 for subsequent experiments.Then,Trolox was added to the new storage solution 1 to prepare the new storage solution 2,and the morphological structure,FHB,LA,LDH,and ATP content of RBCs were tested in the conventional storage solution and the three types of storage solutions for non-diabetic patients,and T2DM patients at five time points（NC group,DO-c group,DO-1 group,DO-2 group）including 0d（T₀）,7d（T₁）,14d（T₂）,21d（T₃）,and 28d（T₄）.The effects of different storage solutions on the physicochemical properties,functional metabolism of RBCs in T2DM patients were observed.2.A food-induced plus STZ-induced T2DM SD rat model was construct,and after separating red blood cells from tail vein blood collection,added corresponding preservation solution.At T₀,T₁,T₂,T₃,and T₄,biochemical indicators of red blood cells from non-diabetic rats stored in conventional preservation solution（group A）,T2DM rats stored in conventional preservation solution（group C）,and T2DM rats stored in novel preservation solution 2（selected as a more suitable modified preservation solution for T2DM patients in the first part）were detected,including red blood cell morphology,2,3-DPG,ROS,GLU-YA,UA content,and non-targeted metabolomics mechanism was investigated.The PKH26 was used to label red blood cells stored in vitro for 28d in groups A,C,and Y and establish a related model to observe the survival rate of red blood cells after transfusion into rats stored in different preservation solutions.Then,after labeling,the stored red blood cells were transfused back into the corresponding rats,and the red blood cell labeling rate was detected by flow cytometry after 1h,7h,13h,and 18h of transfusion.Results:1.At T₀,T₁,T₂,T₃,and T₄,the non-diabetic NC group had the most complete RBC morphological structure,with lower hemolysis,FHB,LA,LDH,and ATP levels than the other three groups,which showed positive correlation with storage time.The RBCs of T2DM patients had the best morphology in the new preservation solution 2,with lower hemolysis,FHB,LA,LDH,and ATP levels than the DO-c group and DO-1 group,and the lightest degree of RBC storage damage.The DO-1 group had the second-best results,and the difference was statistically significant.2.At T₀,T₁,T₂,T₃,and T₄,group A had the best RBC morphology,with lower levels of ROS and uric acid（UA）than the other two groups,a lower glucose intake rate（GLU-YA）than the other two groups,and slower decline in RBC oxygen release ability（2,3-DPG）with increasing storage time with statistical significance.In T2DM rats,group C had more severe RBC oxidative reactions and faster glucose metabolism,with lower RBC oxygen release ability than group Y.Metabolomic screen results revealed that 11 major metabolic pathways were significantly affected in RBCs stored in vitro for 21d and 28d in both groups C and Y,including the biosynthesis and degradation of valine and isoleucine,the biosynthesis of aminoacyl-t RNA,α-Stearidonic acid metabolism,arachidonic acid metabolism,lipid metabolism,purine metabolism,Arbutin,and the selective inhibitor of UDP-glucuronic acid transferase metabolism.3.The RBC labeling rate gradually decreased with increasing transfusion time after transfusion with significant difference.After 7h,13h,and 18h of transfusion,there was no significant difference between groups A and Y.However,both groups had significantly higher RBC labeling rates than group C.Conclusion:1.There are significant differences,the morphological structure and functional metabolism of red blood cells（RBC）between type 2 diabetes（T2DM）patients and non-diabetic individuals,increase with longer storage time.Treatment with reduced glucose and chloride in the conventional preservation solution and increased p H can effectively reduce storage damage to T2DM patient RBC.Adding the antioxidant Trolox to the modified RBC preservation solution further reduces storage damage to T2DM patient RBC.2.The changes in RBC morphology,structure,and functional metabolism during 28 days of in vitro storage of RBC from different T2DM rat groups were investigated.Trolox was found to effectively reduce RBC oxidative stress,lower glucose metabolism,and maintain RBC oxygen release function.Metabolomics analysis was used to screen for differential metabolites and corresponding metabolic pathways affected by Trolox in protecting RBC of T2DM rats from storage damage.3.The experiment of stored RBC re-injected into the body,the new preservation solution 2added Trolox was found to improve the quality of stored RBC in T2DM rats and increase the survival rate of RBC in rats.