| BackgroundAt present,allogeneic liver transplantation has become the only recognized clinical strategy for the treatment of end-stage liver disease.With the remarkable development of liver transplantation technology and strategies,the incidence of postoperative complications has been greatly reduced over the years,and the long-term survival rate of patients has been significantly improved.However,due to the rapid increase in the demand for liver transplantation,the use of marginal donor livers has greatly increased in the past decade.This inevitably leads to a decline in the quality of the graft and a series of serious complications after transplantation.One of the serious complications is early allograft dysfunction(EAD).EAD describes the poor function of the graft in the initial period after liver transplantation.The survival time of EAD patients is poor,because EAD may develop into non-functioning primary grafts,leading to death or re-transplantation.Due to the lack of specific animal models,the underlying mechanism of EAD is poorly understood.In order to avoid the waste of donor organs caused by EAD,finding biomarkers and therapeutic targets with early warning significance for this fastdeveloping but life-threatening complication has important clinical value.AimsIt aims to explore the risk factors of EAD through the dynamic changes of the immune cell spectrum in the peripheral blood of liver transplant recipients;based on the existing animal model technology to verify the therapeutic effect of interference risk factors in ischemia-reperfusion injury and liver transplantation injury;according to the above results,we will explore the relevant signal pathways and cell damage mechanisms of EAD;find biomarkers with early warning significance for the clinical diagnosis of EAD and explore new therapeutic targets for the treatment of EAD.MethodsSection 1:This study retrospectively included data of 109 patients who underwent liver transplantation at the Hepatobiliary and Pancreatic Surgery Center of the First Affiliated Hospital of Zhejiang University from April 1,2017 to August 1,2019.We adopted the EAD definition proposed by Olthoff in 2010 as the diagnostic criteria,namely:(1)the highest serum ALT within 7 days after surgery> 2000 U/L;(2)the highest serum AST within 7 days after surgery> 2000 U/L;(3)Serum TB ≥10mg/d L on the 7th postoperative day;(4)INR ≥1.6 on the 7th postoperative day,only one of them should be satisfied.Perform dynamic analysis and comparison of the acquired clinical laboratory data(the latest laboratory test results before transplantation,the test results 2-4 days after transplantation,and the proportion of corresponding index changes)to screen out the risk factors for EAD;further analyze the impact on liver transplant recipients Independent risk factors for the prognosis and outcome of EAD.Based on Kaplan-Meier survival analysis,it is clear that the selected risk factors affect the overall survival(OS)of liver transplant recipients.Section 2:Based on mature animal models-mouse ischemia-reperfusion model and rat liver transplantation model,we explore the risk factors discovered in the first part-the role of natural killer cells(NK cells)in the process of ischemia-reperfusion.Use the medical database GEO for data analysis to explore molecules with abnormal changes in expression before and after liver ischemia-reperfusion injury.Exploring the shift of NK cells in mice after ischemia-reperfusion injury by flow cytometry,and detecting serum aminotransferase glutamate aminotransferase(ALT),alanine aminotransferase(AST),observe the severity of inflammation in histopathological sections,the degree of liver cell apoptosis,and the expression of pro-apoptosis-related protein molecules after interfering with NK cells and its upstream signaling molecule Toll-like Receptor 4(TLR4)to explore the therapeutic effect of ischemia-reperfusion injury.Section 3:Analyze IRI-related signal pathways and molecular mechanisms through new analytical techniques-RNA-sequencing(RNA-sequencing)and Mass Cytometry(Cy TOF).RNA sequencing technology was used to analyze the differentially expressed genes after liver ischemia-reperfusion,combined with pathway enrichment analysis technology to find the molecular mechanism and signal pathways associated with these genes,and then combined with Cy TOF analysis technology to find the subgroups of cells with different degrees of infiltration between the IRI group and the treatment group after liver IRI injury.Integrating the above analysis results,we have initially explored the possible mechanisms among TLR4,NK cells and liver IRI damage,and drawn a pattern map.ResultOf the 109 liver transplant recipients included,74 cases did not develop EAD after transplantation,and were defined as the non-EAD group,and 35 cases developed EAD,which was defined as the EAD group,and 9 of them whose EAD status was maintained for 7 days,which was defined as the 7d-EAD group.Comparing the components of the immune cell spectrum in the peripheral blood of transplant recipients between the nonEAD group and the EAD group,it was found that in the non-EAD group,the preoperative neutrophils to white blood cell ratio(NWR)was significantly lower than EAD group patients(0.226±0.035 vs 0.239±0.027,p=0.028),lymphocyte to white blood cell ratio(LWR)was significantly higher than EAD group patients(0.074±0.040 vs 0.058 ±0.032,p=0.034);After transplantation,the proportion of NK cells was significantly higher than that of patients in the EAD group(0.037±0.031 vs 0.027±0.015,p=0.008);in the dynamic comparison of various indicators,the NK of patients in the non-EAD group The dynamic change value of cell proportion(0.287±0.170 vs 0.222±0.081,p=0.008)and the dynamic change value of NWR(0.362±0.051 vs 0.344±0.039,p=0.042)were significantly higher than those in the EAD group.Further research found that after liver transplantation,regardless of whether EAD occurred,the proportion of NK cells in the peripheral blood of the recipient decreased significantly(0.106±0.070 vs 0.083±0.073,p<0.0001),and recovered about 1 week after the operation to the preoperative level.Combined with the data and information during the operation,we found that the dynamic change value of NK cell proportion was significantly negatively correlated with cold ischemia time,NK proportion shift =-0.013 * CIT + 0.375,R2 = 0.54,p = 0.016.Cox regression analysis found that the dynamic change of NK cell ratio(HR = 0.008,p = 0.027)and ABO blood type incompatibility(HR = 0.174,p = 0.003)were independent risk factors for the occurrence of 7d-EAD;survival by Kaplan-Meier.The analysis found that the dynamic change value of NK cell ratio may be related to the overall survival rate of transplant recipients.The above results suggest that NK cells may be a key factor in the occurrence of EAD in recipients after liver transplantation,and can affect their prognosis and outcome.It can be a potential indicator for predicting the occurrence of EAD and a new target for the prevention and treatment of EAD.Section 2:In the mouse liver ischemia-reperfusion model,we found that after IRI,the proportion of NK cells in peripheral blood of mice decreased significantly(p<0.01),while the proportion of NK cells in liver tissues increased significantly(p<0.001);the results of liver function test showed that IRI mice’s ALT(p=0.001)and AST(p=0.005)were significantly higher than those in the sham-operated group.Histopathological slices showed a higher degree of inflammatory cell infiltration in the liver tissue of IRI mice,and immunofluorescence results showed that IRI was small.The liver cell apoptosis of mice is more serious.WB results indicate that the expression of crude cell apoptosis molecules Bax and C-caspase 3 in IRI mice is significantly increased.This shows that NK cells from the peripheral blood are recruited to the liver and cause stronger liver damage.The use of the NK cell inhibitor α-ASGM1 can reduce the degree of recruitment,and through liver function,pathological sections,immunofluorescence and WB experiments,it can be found that they can alleviate the liver damage caused by IRI.Similar results were obtained in the rat liver transplantation model.The liver function of the transplanted liver in rats after the use of NK cell inhibitors was significantly better than that of unused rats.The pathological section results showed that the degree of liver cell damage was less.Through the analysis of the GEO public database,it was found that after liver ischemia-reperfusion,the expression of TLR4 increased significantly(p=0.007),which suggests that the role of NK cells in the liver IRI process may be related to TLR4.The use of TLR4 blocker TAK-242 can also have an effect similar to NK cell inhibitors,but the combination of the two drugs cannot bring greater benefits to the liver.Section 3: Analyze the differentially expressed genes in the liver tissues of mice in the IRI treatment group(n=3)and α-ASGM1 treatment group(n=3)by RNA sequencing technology,a total of 465 genes are screened out(p<0.05,fold change> 1.5).Pathway enrichment analysis showed that these genes were significantly related to immune-related pathways such as negative regulation of cytokine secretion,immune response,homeostasis,chemotaxis and platelet activation(p<0.05).Cy TOF analysis showed that in the immune cells infiltrated in the liver of the two groups of mice,the differences were CD49b-NK1.1+ NK,CD49b+ NK1.1+ NK and CD8+CD127+ T cells.This suggests that these cells play a key role in liver ischemia-reperfusion injury in mice and can be intervened by the NK cell inhibitor α-ASGM1.ConclusionPeripheral NK shift is associated with the incidence of EAD and its outcome after liver transplantation.The TLR4-NK-CD8+CD127+ T cell signaling pathway may be the key axis of graft injury after I/R treatment.Targeted NK cell inhibition can be a novel and effective strategy to protect liver tissue against EAD after liver transplantation. |
PDF Full Text Request