| Objective: This study was conducted (1) to build stable experimental model of orthotopic liver transplantation in rat (OLT), summarize operative skills and techniques, so as to make adequate preparations for the next research. (2) to observe the changes of serum transaminases from rat liver graft which pretreatment with taurine, interleukin-1 receptor associated kinase-4 (IRAK-4) and NF-kappa B in Kupffer cells from rat liver grafts, supernatant fluid tumor necrosis factor-α(TNF-α) level. liver histology, graft and animal survival were also investigated. Data was used to explore the protective mechanisms of taurine pretreatment against hepatic ischemic/reperfusion injury(IRI) after liver transplantation.Methods: (1) The animal model of OLT was performed with two-cuff technique, in which suprahepatic vena cava (SHVC) was anastomosed with continuous suture, portal vein (PV) and infrahepatic vena cava (IHVC) were anastomosed with cuff method, and the bile duct was reconstructed by interior stent. General state of health of rat after transplantation was observed. The operative skills and techniques were summarized to hold skilled techniques of OLT and make adequate preparations for further research. (2)Male Sprague-Dawley rats, weighing 180-220g, were divided into two groups in a randomized, blinded fashion. Normal saline group (NS group) and taurine pretreated group(Tau group). NS group were given a single infusion with normal saline (1.5 mL) 10 minutes before harvesting. Tau group were given a single infusion with taurine (1.5 mL, 300mmol/L) 10 minutes before harvesting. After organ harvest, standardized liver transplantation was performed. At 0 min, 60 min and 180 min after reperfusion, Kupffer cells (KCs) were isolated and cultured, the expression of IRAK-4 gene and protein level in KCs were determined by real-time fluorogenetie quantitative PCR (RT-PCR) and Western blotting. The activity of nuclear factor-kappa B (NF-κB) in KCs were determined by electrophoretic mobility shift assay (EMSA). The supernatant fluid tumor necrosis factor-α(TNF-α) level were detected by ELISA. serum transaminases, liver histology were also investigated.Results: (1) Stable rat model of orthotopicd liver transplantation had been established successfully. There was no significant difference between NS group and Tau group in the duration of operation, cold-ischemia time, anhepatic phase time and loss of blood. (2) serum transaminases: In two groups, 0 min after transplantation ALT and AST did not have a significant difference on serum parameters measured in this study(P>0.05). In NS group, 60 min, 180 min after transplantation ALT and AST increased to 652.88±162.76 U/L, 986.25±173.59 U/L and 1578.0±369.64 U/L, 2125.3±303.46 U/L, respectively. In Tau group, 60 min, 180 min after transplantation ALT and AST increased to 434.25±112.27 U/L, 535.375±183.23 U/L and 1088.38±258.73 U/L, 1201.63±298.67 U/L, respectively. In 60 min and 180 min taurine significantly reduced serum levels of ALT and AST (P<0.05). (3) the change of mRNA and protein level in Kupffer cells: Real-Time fluorogenetie quantitative PCR and Western blotting showed that, the expression of IRAK-4 mRNA and protein in KCs did not have a significant difference in NS and Tau groups at 0 min after reperfusion(P>0.05). At 60 min after reperfusion, the expression of IRAK-4 mRNA and protein level in two groups both rised to a peak, relative levels was rised to 5.44±1.04, 2.39±1.11 and 3.52±0.81, 1.67±0.37 respectively. Tau group significantly lower than NS group(P<0.01). At 180 min after reperfusion, the expression of IRAK-4 mRNA and protein level in two groups began to descent, relative levels was 3.95±0.98, 1.53±0.48 and 3.26±0.62, 1.49±0.19 respectively. Tau Ugroup significantly lower than NS group(P<0.05). (4) KCs NF-κB DNA binding activity with EMSA: At 0 min after reperfusion, due to the cold ischemia, KCs NF-κB DNA binding activity had part of expression in two groups, but the two groups had no significant difference(P>0.05). At 60 and 180 min after reperfusion, NF-κB DNA binding activity has rised and obviously higher than 0 min(530.19±53.24 and 449.87±41.58 vs 249.97±34.67, P<0.01). Following tuarine pretreatment, the NF-κB activity was decreased significantly compared with NS group at 60 and 180 min(333.96±36.43 and 279.69±35.49 vs 530.19±53.24 and 449.87±41.58, P<0.05). (5) supernatant fluid TNF-αlevel: In two groups, 0 min after reperfusion TNF-αlevels did not have a significant difference (P>0.05). In NS group, the TNF-αlevels was increased to 1349.27±189.67 (μg/L) and 398.61±43.28 (μg/L) after 60 min and 180 min. In Tau group, the TNF-αlevels was 674.18±85.32 (μg/L) and 43.28±9.78 (μg/L) after 60 min and 180 min. In Tau group, Taurine (300 mmol/L) given to donors before organ harvest significantly reduced TNF-αvalues to the NS group (P<0.05).Conclusions: This data suggests that taurine can protect hepatic I/R injury after liver transplantation and the protective effects may be via down-regulation of IRAK4 and its downstream NF-κB, TNF-αexpression in KCs.
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