A Preliminary Study Of The Injury Mechanism During Transplanted Liver Cold Storage

BackgroundFor patients with end-stage liver disease,the most effective treatment at this stage is liver transplantation.Although the continuous improvement of surgical techniques and replacement of immunosuppressive agents have led to the improvement of the long-term survival rate of liver transplant patients,there are still a lot of patients who will have perioperative risks and primary liver dysfunction after liver transplantation,which is an urgent problem to be solved in the field of liver transplantation.Cold ischemia injury is unavoidable injury of liver graft during transport at low temperature,and its pathophysiological process is closely related to postoperative recovery of liver function and long-term prognosis.At present,basic research on liver transplantation focuses more on the ischemia-reperfusion injury of the liver after transplantation,which lacks a more comprehensive and comprehensive study on the changes and mechanisms of injury during the cold preservation of the liver graft.ObjectiveIn this study,we observed the pathological changes and ultrastructural changes in the liver of mice transplanted liver at different time points.Testing indicators include the indexes of liver function and injury:ALT,AST,MDA,GS,HIF-1?,AQP8 and KLF2,and its downstream genes eNOS.The effects of cold storage on the liver function and the molecular mechanisms of cold-storage damage are initially described.It has important basic research significance and clinical application for further understanding of the process of liver cold-storage injury changes,the development of related liver transplantation measures and clinical value.Method(1)The cold-preserved mice animal model of liver transplantation is established.Twenty healthy male C57 BL / 6 mice were randomly divided into 4 groups according to their age: 8 weeks.After in situ perfusion,the liver was separately preserved in cold for 3 time points:(1)Normal control group(Normal): not cold preservation,not perfusion;(2)Cold preservation 4h group(CS4h): After perfusion,4? for 4 hours;(3)Cold storage 12 h group(CS12h): After perfusion,4? for 12 hours;(4)Cold storage 24 h group(CS24h): After perfusion,4? for 24 hours,(2)Liver histopathology was taken.HE staining,PAS staining and transmission electron microscope photographs are used to observe the morphological and ultrastructure changes of the liver and glycogen breakdown.The reperfusion fluid is collected at various time points to detect the content of ALT and AST in the reperfusion fluid.Real-time PCR is used to detect the mRNA expression of MDA,GS,HIF-1?,KLF-2 and its downstream eNOS in liver.Western blot is used to detect the expression of AQP8 in liver tissue.Results(1)Changes of HE and ultrastructure in mouse liver during cold storageMice liver HE changes:After cold preservation 4 hours,no significant damage is observed in hepatocytes.After cold preservation 12 hours,part of the hepatocyte cytoplasm edema,manifested as loosely arranged,stained light,increased liver sinusoidal cleft.After cold preservation 24 hours,hepatocytes appeared cytoplasmic edema more severe,disordered loses its original shape,part of the nucleus dissolved and broken,liver and sinusoidal crevices increased more serious.Mouse liver ultrastructure changes:(1)Changes in the nucleus: Compared with the normal control group,the nuclei of the hepatocytes in different cold storage time points showed unclear nuclear membrane boundaries,irregular shape,heterochromatin edge sets.The final nuclei were pyknotic and nucleolus disappeared.(2)Mitochondrial changes: After cold storage 4 hours,mitochondria were mild swelling and the matrix fades.After cold storage 12 hours,mitochondria were moderately swollen,the inner chamber was enlarged,the volume was increased,the electron density of the matrix was decreased,the vacuolization occurred partially,the mitochondrial cristae were partially broken and dissolved.After cold storage 24 hours,the mitochondria were swollen,the mitochondria became larger and longer,the rod shape was diminished,the matrix became fainter or vacuolar degeneration,and the crest obviously shortened or even disappeared and the part of the outer membrane ruptured.(3)Changes in endothelial cells: After cold storage 4 hours and 12 hours,the endothelial cells did not show obvious damage.When the time was prolonged to 24 hours,the endothelial cells were obviously edema.There were many vacuoles in the cytoplasm and the appearance of autophagosomes.Some endothelial cell membranes appeared.(2)Changes of ALT,AST in liver reperfusion fluid and MDA content in liver tissue in mice: After 24 hours of cold storage,the ALT activity in the perfusate of mice increased significantly.After 12 hours of cold storage,AST activity in the perfusate of mice gradually increased.The MDA content in mouse liver also gradually increased with the extension of cold preservation time,indicating that the liver of mice in each group had different degrees of damage.(3)Mouse liver tissue sections glycogen staining results: The positive rate of glycogen staining in normal group was high.With the prolongation of cold storage time,the positive rate of glycogen staining decreased,the density decreased,and the distribution was uneven.(4)Cold preservation of liver tissue mRNA levels of GS changes: With the prolongation of cold storage,the expression of GS mRNA decreased in mouse liver.(5)Cold preservation of hepatic tissue KLF2 and eNOS mRNA levels: With the prolongation of cold storage,the expression of KLF2 mRNA in the liver of mice gradually decreased.After cold preservation 4 hours and 12 hours,the expression of eNOS mRNA did not change obviously.But the expression of eNOS mRNA decreased at after cold preservation 24 hours.(6)Cold preservation of liver tissue HIF-1? mRNA levels: After cold preservation 4 hours,HIF-1? mRNA expression in mouse liver did not change significantly.HIF-1? mRNA expression increased significantly after 12 hours of cold storage.After 24 hours of cold storage,HIF-1? mRNA expression has come down.(7)Cold preservation of liver tissue AQP8 protein levels: After cold preservation 12 hours and 24 hours,the mouse liver AQP8 protein expression was significantly reduced.Conclusions1.With the prolongation of cold preservation,the damage of hepatocyte,nucleus,mitochondria,endothelial cell appeared one after another and gradually increased.Impaired liver cells lead to the release of AST and ALT.Mass production of free radicals leads to increased MDA content in lipid peroxidation,impaired synthesis of glycogen and enhanced decomposition.2.Cold storage injury in liver transplantation may begin with mitochondria.The down-regulation of transcription factor KLF2 aggravates endothelial injury.Up-regulation of hypoxia inducible factor HIF-1? may play a potential protective role in cold ischemia injury of liver in mice.At the same time,the expression of AQP8 is down-regulated,suggesting that it may lead to the appearance of hepatocyte edema,which may be related to the expression of HIF-1?.