Effects Of TAT And Heme Oxygenase-1 Fusion Protein On Liver Cell Apoptosis During Orthotopic Liver Transplantation In Rats

Objective: In clinical liver transplantation, primary graft nonfunction or dysfunction caused by hepatic ischemia-reperfusion (I/R) injury was a serious complication after orthotopic liver transplantation.Research shows that hepatocyte apoptosis is one of the important mechanisms of hepatic I/R injury. There were several reports in which hepatic I/R injury could be reduced by inducing the overexpression of heme oxygenase-1(HO-1,heat shock protein 32) through graft preconditioning with drugs or organ ischemia or heat shock in various organs in animal models. HO-1 is the rate-limiting enzyme in the catabolism of heme, a process that leads to formation of equimolar amounts of carbon monoxide (CO), the bile pigment biliverdin, and free iron, HO-1 has the following characters:anti-apoptosis, anti-oxidation, and anti-inflammation, which cause an extensive interesting of researchers. Protein transduction domains (PTDs) are short peptides which can freely pass through cell membrane independent of classical receptors or endocytosis. Proteins or peptides can be directly transferred into cells when covalently linked to small peptide domains. Among PTDs, the PTD containing 11 amino acids (peptide sequence: YGRKKRRQRRR) from HIV trans-activator (TAT) protein is the most extensively studied peptide.Research has shown that TAT and HO-1 fusion protein (TAT-HO-1) was transduced into myocardial cells successfully during cold preservation and its biological activity was maintained, which extended the time of cold preservation and reduced I/R injury of the transplanted heart. But there are few reports about its role during hepatic I/R injury. The previous experiments of this study confirmed that TAT-HO-1 fusion protein could be effectively transduced into cultured liver cells in vitro and hepatocytes in vivo and rat liver at the cold storage period which reduced apoptosis in hepatic parenchymal cells and sinusoidal endothelial cells (SECs) and protected the liver function. The present study was designed to investigate whether TAT-HO-1 fusion protein would be transduced into liver cells of orthotopic liver transplantation and attenuate apoptosis in hepatic parenchymal cells and sinusoidal endothelial cells (SECs).Methods:TAT-HO-1 was prepared as described previously. HO-1 cDNA fragment and synthesized TAT were cloned into fusion expression vector pET32a by molecular cloning techniques, and the TAT-HO-1-pET32a plasmid confirmed by DNA-sequencing was transfected into E.coli BL-21(DE3). The TAT-HO-1 protein was induced by IPTG and purified by Ni-NTA-agarose, then identified by Western blot.Male adult Sprague-Dawley rats weighing 250-280g each were randomly divided into two groups: donor group and receptor group. The basic techniques of liver harvesting and orthotopic transplantation without hepatic artery reconstruction were performed according to the method previously described by Kamada. The recipient rats were randomly divided into two groups (n=24 each).In control group (group C), normal saline 10 ml/kg was given via the penile vein after reperfusion.In TAT-HO-1 group (group P), TAT-HO-1 fusion protein 10ml/kg(10mg/kg) was given via the penis vein after reperfusion.Six rats in each group were selected before isolating liver (T0), and at 1, 6 and 12h after liver transplantation (T1-3).The rats were anesthetized with ether.Blood samples were taken from the abdominal aorta and Serum was separated.Liver tissues were isolated,and immediately frozened in liquid nitrogen. Serum and liver tissues were placed in -80℃refrigerator.Immunohistochemical staining method was used to detect the HO-1 content in liver tissues and analyze the transduction of TAT-HO-1.Serum levels of ALT were analyzed using automatic biochemistry analyzer. Radioimmunoassay was performed to determine the level of serum HA.Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) method was used to detect the apoptosis in hepatic parenchymal cells and SECs, Fluorescent real-time quantitative PCR method (RT-PCR) was used to detect the caspase-3 mRNA expression. After hematoxylin-eosin (HE) staining, pathological changes in liver tissues were observed by light-microscopy.Results:1 The HO-1 content of liver tissue There was obvious liver HO-1-positive brown staining distribution at T1-3 in both groups. The HO-1c content in the liver tissues were significantly higher at each time point in group P than in group C.After liver transplantation, intravenous TAT-HO-1 fusion protein can be transduced into rat liver.2 ALT and HA level determination The levels of serum ALT and HA were significantly higher at T1-3 than at T₀ in two groups (P<0.05). Compared with group C, there was no significant difference in ALT and HA levels at T₀ in group P(P>0.05),ALT and HA levels were significantly lower at T1-3 in both groups (P<0.05).3 Apoptosis index of hepatic parenchymal cells and SECs and expression of Caspase-3mRNA in liver tissues The AI of hepatic parenchymal cells and SECs and expression of Caspase-3 mRNA in liver tissues were significantly higher at T_1-3 than at T₀, and at T₂ than at the other time points in the two groups (P <0.05). Compared with group C, there was no significant difference in AI of hepatic parenchymal cells and SECs and expression of Caspase-3 mRNA in liver tissues at T₀ (P > 0.05), but the parameters mentioned above were significantly decreased at T1-3 in group P (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.Conclusion:TAT-HO-1 fusion protein injected intravenously can be transduced into graft liver after graft reperfusion of orthotopic liver transplantation.The liver cell protection of TAT-HO-1 fusion protein after liver transplantation are possibly through down-regulating the caspase-3 mRNA expression and reducing apoptosis of hepatocytes and SECs.Thus protein transduction technology will be the new measure to reduce hepatic transplantation I/R injury .