| Sepsis is a life-threatening acute organ dysfunction syndrome caused by a dysfunctional host response to infection,and is one of the main causes of death in critically ill patients.Thrombocytopenia is an important manifestation of sepsis,closely related to an increase in patient mortality.Platelets have unique advantages in hemostatic function and bidirectional immune regulation,so platelet infusion is not only an important means to prevent hemorrhagic diseases caused by thrombocytopenia or dysfunction caused by sepsis,but also a potential tool for immunotherapy of sepsis.At present,the clinical platelet supply generally uses 22±2℃ oscillation storage.The stored platelets can be stored for 5 days in a dedicated blood bag,but due to their short shelf life and susceptibility to bacterial contamination,the long-term stable supply of platelets has become a major challenge in clinical treatment.Cold storage and freezing storage of platelets at 4℃ can effectively prolong the shelf life of platelets while reducing the possibility of bacterial contamination.Cryopreserved platelets(CPP)have outstanding storage advantages and hemostatic functions.Compared to liquid stored platelets(LSP)that oscillate at 22±2℃,CPP has stronger ability to induce thrombin production and faster hemostatic speed,and a lower infusion volume can achieve hemostatic effects comparable to LSP,which is expected to become an effective supplement to LSP.However,due to storage damage,CPP quickly accumulates in the liver and is cleared by the liver after internal infusion.Previous literature has shown that CPP infusion can cause a certain degree of liver damage;On the other hand,abnormal liver function is an independent risk factor for mortality in sepsis patients.Therefore,there are no relevant reports on the effects of CPP infusion on sepsis patients,whether it can lead to adverse transfusion reactions,whether it can play a bacterial clearance role like fresh platelets(FP),and what effects it can have on liver function.Therefore,elucidating the impact of CPP infusion on sepsis and its related mechanisms is particularly crucial,which is expected to provide a theoretical basis for preventing the occurrence of adverse reactions to CPP infusion.This study is conducted from the following sections:1.Establishment of preparation method and quality evaluation of mouse CPPEstablishing a stable mouse CPP preparation technology is an important prerequisite for exploring the effects of CPP infusion on sepsis mice.To obtain high-quality and stable mouse CPP,a stable mouse platelet extraction technique was successfully established through cardiac blood collection and one-time centrifugation,and the stable recovery rate of mouse platelets was optimized by changing the volume of whole blood centrifugation.Subsequently,mouse platelets were frozen and stored for more than 48 hours using Valeri’s "no cleaning" method.The quality of the thawed CPP was evaluated through blood routine analysis,transmission electron microscopy(TEM)observation,and flow cytometry detection.The blood routine results showed that compared with FP,there was no significant difference in platelet count(PLT)of CPP,while platelet hematocrit(PCT),mean platelet volume(MPV),and platelet distribution width(PDW)were significantly increased;The TEM observation results showed that the integrity of CPP cell membrane was poor,and a large amount of content was lost;The flow cytometry results showed that compared to FP,the apoptosis and activation levels of CPP increased,while the aggregation ability and membrane surface glycoprotein levels decreased.The changes in the above indicators are consistent with the characteristics and structure of CPP from human and mouse sources reported in current literature.2.Establishment of a visual sepsis mouse modelTo analyze the impact of CPP infusion on sepsis,this study fully utilized the advantages of in vivo fluorescence imaging technology in real-time tracking of the same animal disease process and effectively reducing the number of animals used.Pseudomonas aeruginosa labeled with lux CDABE reporter gene was injected through the tail vein to establish a visualized sepsis mouse model.Firstly,in vitro imaging of luminescent bacteria confirmed that bacteria mainly accumulate in the liver of mice after entering the body.Subsequently,Pseudomonas aeruginosa was injected through the tail vein into C57BL/6J mice to induce sepsis in a mouse model.In vivo imaging was used to monitor bacterial proliferation in the mice,and small animal blood routine analyzer was used to detect changes in the mice’s blood routine.Serum biochemical tests and Hematoxylin eosin(HE)staining were used to analyze liver damage in mice.The results showed that there was a positive correlation between the mortality rate of mice and the amount of Pseudomonas aeruginosa infusion,with infusion of 3 × After107 CFUs of bacteria were introduced into the mice,they exhibited symptoms such as diarrhea,lethargy,and decreased activity.The 24-hour mortality rate was 40%,and the number and proportion of white blood cells(WBC),lymphocytes(Lymph),PLT,and PCT were all lower than normal levels.The proportion of neutrophils(Gram),MPV,and PDW increased,and the levels of ALT,AST,LDH,and CK in the mouse serum were significantly increased,Multiple organs in mice,such as the heart,liver,spleen,lungs,and kidneys,exhibit pathological changes characterized by extensive watery degeneration.The physical signs,blood routine,biochemical indicators,and pathological changes of the model mice in this study are consistent with the clinical characteristics of sepsis,indicating the successful establishment of the sepsis mouse model.3.Preliminary exploration of the effect of CPP infusion on bacterial clearance and liver injury in sepsis and its mechanism of action in liver innate immunityBased on stable mouse CPP preparation technology and a visualized sepsis mouse model platform,the effects of CPP infusion on sepsis and related immune mechanisms were preliminarily explored.(1)The effect of CPP infusion on bacterial clearance and liver damage in sepsisFirstly,based on a visualized sepsis mouse model,the effects of FP and CPP infusion on bacterial clearance,survival rate,and blood routine were analyzed using sepsis mice infused with physiological saline as control.The results showed that there was no statistically significant difference in bacterial clearance between the FP infusion group and the saline infusion group in mice,while the CPP infusion group had significantly lower bacterial clearance ability than the FP and control groups;In terms of mouse survival rate,FP infusion group>control group>CPP infusion group,which is consistent with the results of in vivo bacterial clearance;The blood routine test results showed that the platelet levels in sepsis mice after 2 hours of FP infusion were significantly higher than those in the control group and CPP infusion group,which may be the reason for the high survival rate of FP infusion group mice.Due to the important role of liver function in the prognosis of sepsis,this study further analyzed the effects of CPP infusion on serum ALT/AST levels and liver pathology in septic mice.The results showed that compared with the control group mice treated with FP infusion,the serum ALT/AST levels in the CPP infusion group were significantly increased,and there were more obvious inflammatory lesion areas and extensive liver cell degeneration and water like degeneration in the liver.There was significant congestion in the liver sinuses,reduced gaps,and significantly increased liver damage.The above results indicate that CPP infusion inhibits bacterial clearance in sepsis mice and exacerbates liver damage.(2)The correlation between CPP infusion inhibiting bacterial clearance and exacerbating liver damage in septic mice and liver innate immunityThe natural immune system of the liver plays an important role in the dynamic balance of sepsis,liver injury,and liver regeneration.To further investigate the role of liver innate immunity in the aggravation of sepsis liver injury by CPP infusion,the number and activity of lymphocytes,the number and phenotype of Kupffer cells,and the number and release of neutrophil extracellular traps(NET)in the liver innate immune cells of sepsis mice after FP and CPP infusion were analyzed.The results are as follows: 1)Compared with the FP group,there was no statistical difference in the number and activity of lymphocytes,the proportion and activity of NK cells,and the activity of NK T cells in the CPP group.However,the proportion of NK T cells decreased,which is consistent with reports of bacterial infection leading to a decrease in the number of NK T cells and mediating liver damage in sepsis mouse models.This suggests that the decrease in the number of NK T cells may be one of the reasons why CPP infusion inhibits bacterial clearance and exacerbates liver damage.2)The analysis of the number and phenotype of Kupffer cells showed that compared with the FP group,the CPP group significantly reduced the number of Kupffer cells in the liver,while there was no significant difference in cell phenotype.This is consistent with the results reported in the literature that a decrease in the number of Kupffer cells leads to a decrease in liver bacterial clearance ability,which in turn leads to a decrease in bacterial survival ability in sepsis mice.3)The analysis of neutrophils and NET in the liver showed that compared with the FP group,there was no significant difference in the number of neutrophils and NET expression that chemotactic to the liver after 2 hours of CPP infusion.After 24 hours,the number of neutrophils significantly decreased,while the expression of NET significantly increased,consistent with the results reported in literature that excessive NET generation caused multiple organ dysfunction in the body.The above results suggest that the aggravation of liver damage in sepsis mice by infusion of CPP may be closely related to the dysfunction of NKT,Kupffer,and neutrophils in the liver,and the specific mechanism needs further in-depth research.In summary,infusion of CPP will inhibit bacterial clearance and exacerbate liver damage in septic mice,preliminarily confirming its correlation with liver innate immunity.This study preliminarily explored the impact of CPP infusion on sepsis and its related liver innate immune mechanism,which has certain reference significance for preventing the occurrence of adverse reactions after CPP infusion. |
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