The Protective Effects Of Trypsin Inhibitor On Hepatic Ischemia / Reperfusion Injury （HIRI） And Liver Graft Survival

Background: Liver ischemia reperfusion injury(HIRI) is a common cause of liver graft nonfunction or function failure post transplantation. It is also a main reason which restricts the use of marginal liver donors. The mechanism of HIRI is very complicated. So far as we have known that several factors/pathways have been involved in the mechanisms of HIRI, including anaerobic metabolism, acidosis, intracellular calcium overload, and oxidative stress, etc. Trypsin inhibitor Ulinastatin(UTI) has been reported that it can inhibit the proinflammatory cytokine release through multiple pathways in which protects the cell membrane integrity, protects the hepatocytes from apoptotic or necrotic death, and may further reduce the HIRI.Aims: In this study, we designed to further explore the optimal concentration and timing of the UTI on cold preservation of liver graft and the underlying mechanisms. Furthermore, to examine the liver graft function post transplantation by using the liver donors from extended cold preservation time with or without addition of UTI in the preservation solution, and to explore the potential applications of UTI in marginal organ donors.Methods: In bred C57BL/6 mouse was employed. The liver cold ischemia reperfusion injury and orthotopic liver transplantation(OLTx) models were established. Different concentrations of UTI were added to the lactose ringer’s(LR) solution for liver perfusion and preservation in vitro and the timings of preservation were set. The liver injury score and functions were evaluated by liver ALT, AST, LDH enzyme release and histological examination. The apoptosis of hepatocyte was detected by TUNEL staining and the proinflammatory cytokines were detected by western blot and immunohistochemistry. In addition, the liver grafts were challenged by extended cold preservation time in vitro to further examine the protective effects of UTI on liver graft IRI and function.Results: 1. UTI supplementation to the perfusion and preservative LR solutions significantly protects the liver from cold ischemia injury in a dose dependent manners, the optimal concentration is 1000 U/ml. The ALT,AST,and LDH levels from the preserving solution were significantly reduced in the UTI treated group. 2.Liver histological examination revealed better preserved morphology and architecture with decreased hepatocyte swelling, cytoplasmic vacuolization, nuclear pyknosis, sinusoidal dilatation, and focal necrosis in the UTI treated group. 3. The apoptosis of hepatocyte, Caspase-3 and Bax gene expression on liver tissue were reduced, but Bcl-2 was increased significantly in the UTI treated group. 4. In addition, the productions of proinflammatory cytokines IL-6, TNF-α, and IFN-γ in the liver tissues of UTI supplemented LR solution with extended cold preservation time were reduced and IL-10 was increased significantly. 5. Moreover, the liver grafts with extended cold preservation time of 1 hour in the UTI supplemented LR solution demonstrated improved graft survival time post transplantation. The liver graft survival rate at day 3 and day 7 post transplantation were 80% and 50%, respectively, and were significantly increased compare to 40% and 20% in the LR only group(P<0.05). 6. The liver histological structure and serum enzyme release were significantly reduced in both UTI supplemented LR preservation(UTI group) and combined with UTI treatment to the liver graft recipients(UTI+RT) at 1, 6, and 18 hours post transplantation, respectively, compared to the LR only control. 7. At the meanwhile, the amount of hepatocyte apoptosis and liver graft Caspase-3 and Bax gene expression were reduced and Bcl-2 was increased significantly in both UTI and UTI+RT groups compared to the LR control group. 8. Furthermore, the proinflammatory cytokine IL-6, TNF-α, and IFN-γ expression were reduced, but IL-10 expression was increased significantly in the liver grafts of both UTI and UTI+RT groups. 9. Finally, the restoration of liver injury started at 18 hours posttransplantation among those 3 groups. However, the UTI+RT showed a better protective effect than UTI preservation only.Conclusions: UTI treatment affords significant protection from cold ischemic and reperfusion injury to donor livers. UTI supplementation to the preservation solution improves liver graft survival and acute function post-transplantation with the donors from extended cold preservation time. The underlying mechanisms of UTI on HIRI may be mediated by inhibition of proinflammatory cytokine release, increasing anti-apoptotic gene Bcl-2 and decreasing pro-apoptosis genes of Caspase-3 and Bax expression, and further protects hepatocytes from apoptotic death and improves the liver function.