Rapamycin attenuates liver graft injury in a cirrhotic rat liver transplantation model-the significance of down-regulation of rho-rock pathway

Abstract

Objective: The aim of this study is to investigate whether rapamycin could attenuate hepatic I/R injury in a rat liver transplantation model using small-for-size grafts and cirrhotic recipients, and to explore the underlying mechanism. Materials and Methods: A rat orthotopic liver transplantation model using normal liver grafts (100% and 50% of recipient rat liver) was applied in cirrhotic rat recipients. The cirrhotic rat model was induced by subcutaneous injection of CCl4 for 8 weeks. In the treatment groups, rapamycin was given systematically (0.2 mg/kg, iv) 30 minutes before graft harvesting in the donor and 24 hours before liver transplantation, 30 minutes before total hepatectomy and immediately after reperfusion in the recipient. Recipients were sacrificed at 6, 24, 48, 96 hours and on day 7 after liver transplantation. The liver tissues were sampled for morphological examination, hepatic stellate cell (HSC) activation (a-SMA expression), VEGF expression, Rho and ROCK signaling pathways by immunostaining, Western blot and real time RT-PCR. Liver function and 7-day survival rates were also compared among the groups. Results: Rapamycin significantly improved the small-for-size graft survival from 8.3% (1/12) to 66.7% (8/12) (p=0.027). It also increased the 7-day survival rates of the whole grafts (58.3% [7/12] vs 83.3% [10/12], p=0.371). Liver function was improved both in whole graft and small-for-size graft groups after rapamycin treatment during the first 48 hours after liver transplantation. Significant activation of hepatic stellate cells was found mainly in small-for-size grafts during the first 7 days after liver transplantation. Rapamycin remarkably suppressed hepatic stellate cell activation during the first 7 days after liver transplantation, especially in small-for-size grafts. Intragraft protein expression and mRNA levels of VEGF were down-regulated by rapamycin at 6, 24 and 48 hours both in whole and small-for-size grafts. Consistently, mRNA levels and protein expression of Rho and ROCK I were also decreased in the treatment groups during the first 48 hours after liver transplantation. Conclusions: Rapamycin improved graft survival in a cirrhotic rat liver transplantation model using whole and small-for-size grafts by suppression of hepatic stellate cell activation through down-regulation of Rho-ROCK-VEGF pathway.