| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Background : Ischemia/reperfusion (I/R) injury has been a major problem complicating liver transplantation. During hepatic I/R injury, nuclear transcriptional factor κB (NF-κB) is induced in liver grafts, resulting in a rapid activation of multiple genes, including cytokines, chemokines and inducible nitric oxide synthase (iNOS). To determine the role of NF-κB in cold I/R injury and expression of inflammatory genes, this study examined the effects of two methods of gene transfer to liver grafts with adenovirus encoding IκB (AdIκB), the inhibitory molecule of NF-κB.
Methods : Orthotopic syngeneic (LEW-LEW) rat liver transplantation was performed after 18 hr preservation in UW at 4℃. Three groups of liver grafts included:1) uninfected control, 2) infected ex-vivo with AdIκB (1x10^9 pfu) via dual perfusion of the portal vein and hepatic artery with vascular clamp technique (CT), and 3) pretreated donor with AdIκB 4 days before harvesting (PT). Adenoviral green fluorescent protein vector (A
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dGFP) was used as control. Animals were sacrificed 1,3,6,12,24,and 48 hrs after reperfusion. NF-κB activity, IκB and iNOS expression, serum AST level and histpathology were assessed by gel shift assay, Western blot, blood chemistry and tissue H&E staining, respectively.
Results : In untransfected control livers, NF-κB was activated biphasically at 1-3 hrs and 12 hrs after reperfusion with mean AST level of 4744 IU/L at 24 hrs. iNOS expression started to increase at 3 hrs, peaked at 6-12 hrs, and decreased by 24 hrs. Using CT with AdIκB, but not with AdGFP, there was a downregulation of 2^<nd> NFκB activation. This associated with more severe hepatic injuries ; AST increased to >9000 IU/L. In addition, there was a prolonged iNOS expression for 48 hrs. On the other hand, PT with AdIκB inhibited 1^<st> peak of NF-κB, resulting in significant ameliorations of AST (1711 IU/L). iNOS was weakly expressed in this group at only 3-6 hrs after reperfusion.
Conclusion : Results in this study demonstrate that the inhibition of 1^<st> peak of NFκB by PT With AdIκB improved I/R injury, probably via the downregulation of proinflammatry cytokine activation. On the other hand, 2^<nd> NF-κB peak is probably protective, since inhibition of 2^<nd> peak by CT with AdIκB increased I/R injury. Expression of iNOS well correlated with the severity of I/R injury. These results suggest that NF-κB activation during I/R injury has dual roles ; early activation at 1-3 hrs after reperfusion is deteriorative, whereas the late NF-κB activation at 12 hrs seems to be protective. Less
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