The Effects Of Fusion Protein TAT And Heme Oxygenase-1 On Liver Cells Apoptosis During Cold Storage In Rats

Objective: Liver transplantation is the therapy of choice for patients with end-stage liver disease. However, primary graft nonfunction caused by preservation injury remains a problem in orthotopic liver transplants. Although preservation injury likely is multifactorial, cold ischemia/reperfusion (I/R) injury of the liver contributes significantly to it. There is evidence that apoptosis of liver cells play a important role in cold I/R injury. Recently, it has been reported that in comparison with hepatocytes, sinusoidal endothelial cells (SECs) were more sensitive to cold I/R injury. Protein transduction is a novel technology by which proteins or peptides can be directly transferred into cells when covalently linked to small peptide domains, known as protein transduction domains (PTDs). The human immunodeficiency virus transactivator TAT protein is one of the most widely studied PTDs. It has been shown that TAT-fused proteins can be delivered to several tissues, including the liver, kidney, heart muscle, lung, spleen and brain, when injected into rats systemically. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme of heme degradation into its byproducts carbon monoxide (CO), iron, and biliverdin. The induction of HO-1 expression is considered a primary adaptive response of the cellular defense mechanism. Overexpression of HO-1 by gene therapy and chemical induction has been used to reduce the deleterious effects of apoptosis and oxidative stress in various cell types and animal models. It has been reported that TAT-HO-1, a fusion protein of the TAT and HO-1, can transduce efficiently into pancreaticβTC-3 cells and improve islet viability in culture.The present study was designed to determine whether TAT-HO-1 would transduce efficiently into liver during cold storage, and, if so, to determine whether TAT-HO-1 would attenuate hepatocytes and SECs apoptosis in cold preservation injury.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 analysis.Forty-eight male Sprague-Dawley rats weighing 250~300 g each were used for these studies. The rats were randomly divided into two groups according to the type of the preservation solution(n=24), Group C: livers were flushed and preserved with 4°C HTK solution; Group P: livers were flushed and preserved with 4°C HTK solution containing 50μg/ml TAT-HO-1. The rats were anesthetized by ether inhalation. Livers were harvested as described previously with some modifications.Liver specimens and preservation solution samples were collected at 0hr, 6 hr, 12 hr and 18 hr of cold storage. Immunohistochemical analysis was performed to determine whether TAT-HO-1 would transduce into liver during cold storage. Alanine aminotransferase (ALT) levels were analyzed using Selectra-E automatic biochemistry analyzer by the hospital laboratory. Radioimmunoassay was performed to determine the level of hyaluronic acid (HA) and endothelin (ET) in livers. The apoptotic index (AI) of hepatocytes and sinusoidal endothelial cells (SECs) was identified by TUNEL assay. The expression of Bcl-2 and Bax was evaluated by immunohistochemical analysis.Results:1 A strong accumulation of HO-1 staining was observed at 6 hr, 12 hr and 18 hr of cold storage in group P compared with that in group C(P<0.05).2 The levels of ALT, HA and ET all increased time-dependently during cold storage in both groups(P<0.05). The levels of ALT, HA and ET were lower in group P at 6 hr, 12 hr and 18 hr, when compared with that in group C (P<0.05).3 The AI of hepatocytes and SECs increased time-dependently during cold storage in both groups(P<0.05). The AI of hepatocytes and SECs was lower in group P at 6 hr, 12 hr and 18 hr, when compared with that in group C (P<0.05).4 The Bcl-2 and Bax protein was found to be expressed in both groups. The expression of Bcl-2 was higher in group 2 at each time point, throughout cold storage, when compared with that in group C. Whereas the expression of Bax was lower in group P at each time point, when compared with group C.Conclusion: TAT-HO-1 can transduce efficiently into rat livers and it shows a protective effect on both hepatocytes and SECs via upregulation of Bcl-2 expression and downgulation of Bax expression during cold storage. Protein transduction will be a novel therapeutic strategy to reduce the risk of preservation injury in liver transplantation.