储存红细胞上清液对血管内皮细胞损伤作用及机制研究OA

Objective To investigate the damaging effects of red blood cell supernatant(RBC-S)stored for different durations(7 d,14 d,and 28 d)on vascular endothelial cells,and to explore the underlying mechanisms using bioinformat-ics analysis,so as to provide references for optimizing red blood cell transfusion strategies.Methods Human umbilical vein endothelial cells(HUVECs)were co-cultured with RBC-S stored for 7,14 and 28 days,designated as the 7 d group,14 d group and 28 d group respectively,which were collectively defined as the experimental groups.Cell damage was evalu-ated by cell proliferation assay(Cell Counting Kit8,CCK8),lactate dehydrogenase(LDH)release assay,4',6diamidino2phenylindole(DAPI)staining,and flow cytometry for apoptosis and reactive oxygen species(ROS)levels.The damage degree of RBC-S on vascular endothelial cells was assessed by statistical analysis of damage data among different groups.Since the damage effect reached a plateau at all time points,the 28 d storage group was selected as the representa-tive for further mechanistic studies.Transcriptomic analysis was performed to explore the role of frizzled class receptor 1(FZD1)and Wnt signaling pathway in red blood cell storagerelated endothelial dysfunction.Results Compared with the control group,the storage groups treated with 7 d,14 d,and 28 d RBC-S showed significantly decreased cell proliferation rates[control group 100%,7 d group(69.51±2.30)%,14 d group(74.54±2.89)%,28 d group(73.59±2.36)%,P<0.05],significantly reduced numbers of DAPI-stained cell nuclei[control group(213±12.5)per field,7 d group(140.33±17.04)per field,14 d group(152.00±23.72)per field,28 d group(144.33±19.09)per field,P<0.05]and significantly increased LDH release[control group(1),7 d group(8.33±1.41),14 d group(9.23±0.83),28 d group(9.16±0.60),P<0.05].There was no significant difference in the degree of damage caused by RBC-S among dif-ferent storage groups(P>0.05).With the prolongation of storage time,free hemoglobin(FHb)gradually increased[con-trol group(not detected),7 d(16.57±6.38)mg/L,14 d(76.80±22.83)mg/L,28 d(286.97±29.02)mg/L,P<0.05].The apoptotic rate(20.53±2.94)%and ROS relative intensity(5.13±0.91)in the 28 d storage group were sig-nificantly higher than those in the control group(P<0.05).Transcriptomic analysis showed that FZD1 played a key role in vascular endothelial dysfunction induced by red blood cell storage and was closely related to the Wnt signaling regulatory net-work.Conclusion RBC-S stored for 7 d,14 d,or 28 d can all significantly damage vascular endothelial cells,and the damaging effect reaches a plateau at 7 d of storage.Mechanistic investigation of the 28 d group indicated that the downregu-lation of the FZD1/Wnt signaling pathway may play a critical role in vascular endothelial dysfunction induced by red blood cell storage,providing a theoretical basis for further optimizing red blood cell storage and transfusion strategies.