Effect of fusion protein TAT and heme oxygenase-1 on liver sinusoidal endothelial cells apoptosis during preservation injury

Background Proteins or peptides can be directly transferred into cells when covalently linked to protein transduction domains （PTDs）. TAT is one of the most widely studied PTDs. The effect of fusion protein TAT and heme oxygenase-1 （HO-1） on liver sinusoidal endothelial cells （SECs） apoptosis during cold storage is unknown. The present study aimed to determine whether fusion protein TAT-HO-1 would transduce efficiently into liver during cold storage, and, if so, to determine whether TAT-HO-1 would attenuate SECs apoptosis during preservation injury in rat. Methods Livers of Sprague-Dawley rats were harvested and randomly assigned to group 1 （HTK solution） and group 2 （HTK solution containing TAT-HO-1 fusion protein） according to the type of the preservation solution. The transduction efficiency of TAT-HO-1 was examined and the impairment of SECs was assessed during the period of cold storage followed by 1 hour of reperfusion. Results TAT-HO-1 can transduce efficiently into liver during cold storage. A significantly lower apoptotic index of SECs was observed in group 2, at 6, 12 and 18 hours of cold storage after 1 hour reperfusion, when compared with group 1. TAT-HO-1 reduced HA and ET levels in liver at each time point. Both Bcl-2 and Bax protein were expressed in hepatocytes and SECs at the periphery of the sinusoidal space. Moreover, higher Bcl-2 expression and lower Bax expression were observed in group 2. Conclusions TAT-HO-1 can transduce efficiently into rat livers and shows a protective effect on SECs by attenuating apoptosis during cold ischemia/reperfusion injury. Protein transduction will be a novel therapeutic strategy to reduce the risk of preservation injury in liver transplantation.

Effect of ulinastatin donor-pretreatment on liver graft during cold preservation in rats

Background Donor-pretreatment with ulinastatin may influence the liver graft during cold preservation. The aim of this research was to determine whether pretreatment of donor liver with Ulinastatin can attenuate cold preservation injury,and to explore the mechanism by which Ulinastatin affects the donor liver graft.Methods One hundred and forty-four Wistar rats were divided into the Ulinastatin treatment group （T group） pretreated with Ulinastatin 50 000 U/kg and control group （C group） treated with 0.9% normal saline via peritoneal injection prior to the anesthetization. After the abdominal cavity was opened and perfused with cold Ringer＇s lactate solution, the liver was harvested. The harvested liver was preserved in cold Ringer＇s lactate solution for 0, 2, 6, 24 hours, at which time the liver tissue was sampled for determination of dry weight and wet weight, Na＋-K＋-ATPase and Ca2＋-ATPase activity, lactic acid dehydrogenase （LDH） activity, lactic acid and malondialdehyde levels. Light microscopy and electron microscopy were used to observe liver morphology. The liver cold-preservation solution was taken for measurement of aspartate aminotransferase （AST） and alanine transaminase （ALT） levels. Correlation between ATPase activity and lactic acid level was analyzed by SPSS 13.0 for Windows.Results The morphology in the T group had improved cell boundaries vs. The C group at each time point. Dry weight to wet weight in the T group was lower than in the C group at 6 hours （P 〈0.05）, but the difference was not significant at 24 hours. ALT levels in the T group were lower than that in the C group at 6 hours （P 〈0.05） and 24 hours （P 〈0.01）. AST levels in the T group were lower than those in the C group at 2 hours （P〈0.05）, 6 hours （P 〈0.01） and 24 hours （P 〈0.01）.Na＋-K＋-ATPase activity in the T group was higher than in the C group and the mean difference between two groups was significant at 0 hour （P 〈0.05） and 2 hours （P 〈0.05）. Ca2＋-ATPase activity in the T group was higher than in the C group with the mean difference between two groups significant at 2 hours （P 〈0.05）. The T group had increased lactic acid levels at 0 hour （P 〈0.01） and 2 hours （P 〈0.05） compared with the C group, but there was no influence on the LDH activity at the same time. There were no obvious differences in the levels of malondialdehyde between the two groups at any time point. A linear correlation between Na＋-K＋-ATPase activity and lactic acid levels （r=0.295, P 〈0.05） was found.Conclusions Donor-pretreatment with ulinastatin may protect the cells in a liver graft from ischemia injury during cold preservation; the mechanism may be due to its promotion for cell glycolysis and its preservation of ATPase activity.