Mechanism Of Ischemic Preconditioning In Improving Insulin Resistance After Liver Transplantation

Objective: Linking hepatic ischemia-reperfusion injury during liver transplantation with inflammatory factors and insulin receptor substrates,exploring the mechanism of hepatic ischemia-reperfusion injury influence insulin resistance after liver transplantation,and the possible mechanism of how ischemic preconditioning improve insulin resistance after liver transplantation,and providing new ideas for the diagnosis and treatment of insulin resistance after liver transplantation.Methods: First part:(1)The data of patients who underwent liver transplantation in our hospital from 2016 to 2021 were collected,screened according to the inclusion and exclusion criteria,and divided into normal Normal group and insulin resistance group(including T-HG group and NODM group);(2)Univariate analysis was performed on the data between groups,and indexes with P<0.05 were included in multivariate Logistic regression analysis to analyze the independent risk factors of insulin resistance after liver transplantation,to construct a survival curve,and study whether the blood glucose status was related to the survival rate;(3)The data of T-HG group and NODM group were compared,and the factors of blood glucose reversal were discussed;(4)The blood glucose,TAC concentration,and inflammatory factors within 30 days after surgery were compared between the two groups,and the differences were analyzed.Second part:(1)60 rats were randomly divided into 3 groups,control group,liver warm ischemia-reperfusion group,and LT group,(2)SD rat orthotopic liver transplantation model was established according to the kamada two-cuff method.After operation 2h,1d,3d,5d,7d,samples were collected,and serum,liver tissue,pancreatic tissue were collected for analysis,IL-1β,IL-6,TNF-α,CRP,HMGB1,insulin,C-peptide,pancreatic amylase,lipase,AST,ALT levels in serum were detected by ELISA;xanthine redox method and colorimetric method to detect SOD and MDA content in liver and pancreas;liver tissue HE staining to observe the changes of liver injury degree at different time points in three groups;Immunohistochemical detection to observe the expression levels of HMGB1,NF-κB p65,IL-6 and IRS-2 in liver tissue;Western Blot to detect the protein levels of HMGB1,NF-κB p65,IL-6 and IRS-2 in liver tissue.Third part:(1)Exploration of the appropriate time for ischemic preconditioning:18 SD rats were randomly divided into 6 groups: A,warm ischemia-reperfusion,B,IPC3min+warm ischemia-reperfusion,C,IPC5min+warm ischemia-reperfusion,D IPC8min+warm ischemia-reperfusion,E,IPC10min+warm ischemia-reperfusion,F,IPC15min+warm ischemia-reperfusion group,samples were collected 2h after operation,and serum,liver tissue,and pancreatic tissue were collected for analysis,ELISA method was used to detect serum adenosine,protein kinase C,IL-1β,IL-6,TNF-α,CRP,HMGB1,insulin,C Peptide,pancreatic amylase,lipase levels;xanthine redox method and colorimetric method to detect SOD and MDA content in liver tissue and pancreatic tissue;nitric acid reduction method to detect NO content in liver tissue;liver tissue HE for changes of liver injury degree at different time points were;the expression level of HMGB1 in liver tissue was detected by immunohistochemistry;the protein concentration of HMGB1 in liver tissue was detected by Western Blot method.(2)Study on the mechanism of ischemic preconditioning in improving insulin resistance after liver transplantation:75 rats were randomly divided into3 groups,control group,LT group,and IPC+LT group.According to the kamada two-cuff method SD rat model of orthotopic liver transplantation was established.Specimens were collected at 2h,1d,3d,5d,and 7d after operation,and serum,liver tissue,and pancreatic tissue were collected for analysis,IL-1β,IL-6,TNF-α,CRP,HMGB1,insulin,C-peptide,pancreatic amylase,lipase,AST,ALT were detected by ELISA;xanthine redox method and colorimetric method to detect SOD and MOD content in liver tissue and pancreatic tissue;liver tissue HE staining was used to observe the changes in the degree of liver injury in the three groups at different time points;immunohistochemical detection was used to observe the expression levels of HMGB1,NF-κB p65,IL-6 and IRS-2 in liver tissue;Western Blot method was used to detect the protein levels of HMGB1,NF-κB p65,IL-6 and IRS-2 in liver tissue.Results:(1)Insulin resistance is common after liver transplantation,and some patients develop irreversible NODM.Insulin resistance is related to a variety of factors.In our study,age,warm ischemia time,preoperative hyperglycemia,Higher levels of IL-6 are risk factors for insulin resistance after liver transplantation,warm ischemia time is independent risk factors.In the comparison of T-HG and NODM groups,LDLT,lower age,and lower BMI receptor`insulin resistance status are more likely reversal,and insulin resistance has no significant effect on the survival rate of patients.(2)By detecting the postoperative indexes of the rats in the control group,the liver warm ischemia-reperfusion group and the liver transplantation group,it was found that the overall trend of blood glucose in the rats after LT was higher than that of the other groups,and the liver function damage and pancreatic damage were higher than other groups.The levels of inflammatory factors were higher than other groups,and the β-cell function was severely damaged.Compared with the other groups,the liver injury in the LT group showed severe liver damage,which gradually eased over time.The expression of HMGB1,NF-κB p65,and IL-6 in the liver tissue of the LT group was significantly up-regulated,and the expression of IRS-2 was significantly down-regulated.,Western Blot results are the same as immunohistochemistry.(3)Through the exploration of different ischemic preconditioning time,we found that ischemic preconditioning for 5 minutes and reperfusion for 10 minutes can make the liver produce the highest concentration of NO and adenosine,lower inflammatory factors and liver damage than other preconditioning groups,same to the liver glucose metabolism.Therefore,in the first part of the experimental section,we determined the optimal preconditioning time for LT donor livers.By comparing the postoperative specimens of the rats in the control group,the LT group and the IPC+LT group,it was found that the overall trend of blood glucose in the rats after IPC+LT was lower than that of the LT group,liver injury and pancreatic injury were significantly lower than those of the LT group,and the levels of inflammatory factors were significantly lower.Lower than the LT group,β cell function was better than the LT group.The damage degree of liver HE staining in the IPC+LT group was better than that in the LT group.At the same time,the expressions of HMGB1,NF-κB p65 and IL-6 were significantly down-regulated in the liver tissue.Western Blot results were the same as immunohistochemistry.Conclusion: In the analysis of clinical data,liver ischemia-reperfusion injury and the release of inflammatory factors are the causes of insulin resistance after liver transplantation.The degree of IR after liver transplantation is more severe than that of WIRI.The activation of HMGB1/NF-κB inflammatory pathway caused by HIRI influence the obstructed insulin signal transduction causes postoperative IR,and IPC,as an endogenous protective mechanism,not only alleviates HIRI,but also improves the occurrence and development of postoperative IR.