Effects Of Fusion Protein TAT And Heme Oxygenase-1 On Oxidative Stress And Inflammatory Response In Rat Liver Transplantation

Objective: Improvements in surgical technique and transplantation immunology have greatly improved the outcome following liver transplantation, and this is now well established as a method for the treatment of many incurable liver diseases. But many elements including ischemia-reperfusion injury, rejection will effect survival. At present more and more end-stage liver diseases patients need liver transplantation, the shortage of donor-liver becomes more seriously. So, how to more effectively protect the donor-liver and decrease the primary non-function (PNF) incidence abstract more and more attentions. Heme oxygenase-1(HO-1) is the rate-limiting enzyme in the catabolism of heme, a process that leads to formation of equimolar amounts of the bile pigment biliverdin, free iron, and carbon monoxide (CO), it has the following characters: anti-oxidation, anti-inflammation, anti-apoptosis, down regulation the expression of blood vessel endothelium adhesion molecule and decreasing the injury of blood vessel and increasing transplanted organs blood, it causes the extensive interestings of researchers. Protein transduction domains (PTDs) are peptides that can cross over the cell membrane, proteins or peptides can enter cells to exert their functions if they are linked to PTDs. HIV-1 trans-activator(TAT)is one of the most widely studied PTD. Some researches have confirmed that the fusion protein TAT-HO-1 can successively enter the pancreaticβTC-3 cells and their vitalities were strength. Our prior researches show that the fusion protein TAT-HO-1 can enter the liver cell during cold preservation or after ischemia-reperfusion injury. The fusion protein TAT-HO-1 could reduce injury to liver after ischemia-reperfusion and to attenuate hepatocytes and SECs apoptosis in cold preservation injury. There are no relevant studies whether TAT-HO-1 can transduce efficiently into liver and show protective characters after liver transplantation. On the basis of prior study, we go on to make a research to investigate above issue.Methods: TAT-HO-1 was prepared as described .The E.coli BL-21(DE3) containing TAT-HO-1- pET32a plasmid was cultured. The TAT-HO-1 protein was induced by IPTG then purified by Ni-NTA-agarosed and identified by Western blot analysis.Healthy male adult SD rats weighing 250~280g were randomly divided into equal numbers of donors and recipients . As described by Kamada'two cuff technique rat liver transplantation was established. After transplantation recipients were randomly divided into 2 groups (n=24): control group(group C) and TAT-HO-1 group(group P). In group P fusion protein TAT and heme oxygenase-1 10 ml/kg was injected via dorsal veins of penis after transplant. In group C, 10 ml/kg saline was administered in the same manner. After operation the animals were randomly sacrificed at 0h, 1h, 6h and 12h(6 animals at each time point from each group),blood samples and the liver specimens were placed in -80℃refrigerator. .Immunohistochemical analysis was performed to determine the expression of TAT-HO-1 in liver after transplantation. Selectra-E automatic biochemistry analyzer was employed to detect the level of ALT in blood. Tht content of MDA was detected by thiobarbituric-acid and SOD activity was detected by xanthine oxidase assay and tht content of NO was detected by nitrate reductase technique. The level of TNF-a was tested in ELISA.The expression of NF-κB was also detected by immunohistochemistry technique. Morphological changes were examined by light microscope.Results:1 A strong accumulation of HO-1 staining was observed at 1h, 6 h and 12 h after liver transplantation in group P compared with that in group C(P<0.05).2 The level of ALT and TNF-αin liver blood: at the time of 0h after liver transplantation, there were no significant differences among the levels of ALT and TNF-αbetween both groups (P >0.05), however at the time of 1h, 6h and 12h there were significant differences between both groups,the levels of ALT, HA and TNF-αin the group protein were significantly lower than in the group C(P < 0.05).3 The results of SOD activity, MDA and NO contents and the expression of NF-κB in liver tissue :At the time of 0h after liver transplantation,there were no significant differences among SOD activity, MDA and NO contents and the expression of NF-κB in liver tissue between both groups (P>0.05). However at the time of 1h, 6h and 12h, there were significant differences between both groups, the contents of MDA and NO in the group protein were significantly lower than the group C, but the activity of SOD was significantly higher than the group C (P<0.05) , the expression of NF-κB in the group protein were significantly lower than the group C (P<0.05) .4 Histopathological observation Swollen liver cells, slightly widened liver cables, and infiltrated neutrophils in portal area were observed in group P; swollen, empty liver cells, and even necrosis, significantly widened liver cables, and infiltrated neutrophils in portal area were observed in group C after liver transplantation 6h.Conclusions: TAT-HO-1 injected via dorsal veins of penis after transplant can be transduced efficiently into rat livers, and shows better protective effect on liver against ischemia-reperfusion injury.The mechanism is related to the anti-oxidation and anti-inflammation characters of HO-1.