FTY 720 attenuate ischemia-reperfusion injury in normal and cirrhotic rat liver

Abstract

Objective: The aim of this study is to probe into the protective mechanism of FTY720 in ischemia reperfusion injury in normal and cirrhotic rat liver models by the investigation of intragraft gene expression pattern related to acute phase inflammatory responses and cell survival signaling pathway. Materials and Methods: A 70% rat liver ischemia model was used. The male lewis rats underwent lobar warm ischemia for 60 minutes followed by reperfusion. The cirrhotic rat liver model was induced by CCl4 subcutaneous injection. Treated rats (n=6 for each time point) received FTY720 by iv injection at 20 mins before ischemia and 10 mins before reperfusion (1mg/Kg). Rats were sacrificed at 60 mins, 90 mins, 6 hours and 24 hours after reperfusion for sample collection for morphological examination, intragraft gene detection for inflammatory response, vasoconstriction and cell survival signaling pathway by real time RT-PCR and Western blot. Blood samples were analyzed for liver function and cytokine levels. Results: FTY significantly reduced serum ALT and AST levels in normal and cirrhotic rats (normal liver: ALT – 6hr : 736 vs 1810 U/L, p=0.028, 24 hr: 310 vs 414 U/L, p=0.025; AST – 24 hr: 470 vs 880 U/L, p=0.037; cirrhotic liver: ALT – 24 hr: 616 vs 1041U/L, p=0.043). FTY also significantly down-regulated mRNA levels of Egr-1, which is a master switch of ischemia-reperfuson injury as well as ET-1, a vasoconstriction gene related to sinusoidal damage both in normal and cirrhotic liver. Although there was no difference in intragraft expression of caspase 3, the anti-apoptotic gene A20 and HO-1 was up-regulated in FTY groups. Less apoptotic hepatocytes were found in the FTY groups. IP-10 was over-expressed accompanied with CXCR-2 (IP-10: 90 min: 3816 vs 997% relative to basal level, p=0.043, 24 hrs: 113 vs –87%, p=0.049; CXCR- 2: 90 min: 1121 vs 186%, p=0.046, 24 hr: 45 vs –73%, p=0.027). MIP-2 was downregulated accompanied with the less intracellular expression of iNOS in the FTY groups. The cell survival signaling Akt pathway was activated in the FTY groups reflected by over-expression of GSK and Akt. The hepatic ultrastructure was well maintained in FTY group compared with the disruption of integrity of sinusoids in control group in cirrhotic model. Conclusions: FTY 720 attenuates hepatic ischemia-reperfusion both in normal and cirrhotic rats by amelioration of acute inflammatory response by downregulation of Egr-1 and prior induction of anti-inflammatory and anti-apoptotic genes together with activation of Akt pathway.