The Protective Role Of Rapamycin For I/R Injury In A Cirrhotic Rat Small-graft Liver Transplantation Model And Study Of The Activation Of Hepatic Stellate Cells And Rho-ROCK Signaling Pathway

Objectives: On the basis of cirrhotic rat small-graft liver transplantation model, investigating the effect of rapamycin on graft injury after small-graft liver transplantation and the role of Rho-ROCK signaling pathway and activation of HSC in the graft injury after small-graft liver transplantation, investigating the effect and mechanism of rapamycin and providing primary data for further basic science study and possible clinical application.Methods: 1. Establishment of cirrhotic rat small-graft liver transplantation model well-health male Sprague-Dawley rat(330-380g) as donor; male cirrhotic Sprague-Dawley rat(330-380g) as recipient. The method of inducing cirrhosis: male Sprague-Dawley rat were injected CCL4 (dosage: 1ml/kg/time) subcutaneously since 6-week old and continued by 8 weeks. 2. Group planning two groups were set up: small-graft control group(n=30), small-graft treatment group(n=30). 6 pares of rat liver transplantation were included at every time point. 12 pares of rat liver transplantation were used for 7-day survival. The method for treatment: injecting rapamycin solution v.p. 0.2mg/kg/time. Recipient received injection 24h before operation, post-anesthesia, and post-reperfusion, respectively. Donor received only one injection after anesthesia. 3. Laboratory study 3.1 Masson's staining graft were fixed by 10% formalin, slice making, using Masson's staining to stain collagen to green to show the extent of cirrhosis of liver. 3.2 Intragraft expression of a-SMA by immunostaining the paraffin sections of the graft were immunochemically stained for a-SMA. After de-paraffinization, endogeneous peroxidase activity was quenched by 1%H₂O₂ the section were then treated with 30% normal bovine serum for 30min to reduce the background staining, followed by treatment of primary antibodies of a-SMA at 4℃ overnight. After washing, the sections were incubated with secondary antibody,developing. PBS was used instead of primary antibody as negative control. 3.3 Intragraft expression of ROCK I , Rho and VEGF by western-blot the whole protein of the graft was extracted, fifty micrograms of protein were size-separated in 12% SDS-PAGE and transferred to nitrocellulose membrane. The immunoreactive signals were visualized and analyzed to determine the expression of protein. 3.4 Determination of liver function analyzing blood sample of 6, 24, 48h time point after operation to determine AST and ALT level.Results: 1. successfully establishment of cirrhosis rat liver transplantation model. Masson's staining show typical cirrhotic characters in the section: fibrous connective tissue components in Glisson's sheath, formation of a pseudolobules and fibrotic septa. These characters show that the cirrhosis of liver of recipient was successfully induced (figure 1). 2. significant difference between the two groups on 7-day survival the 7-day survival of the small-graft control group and small-graft treatment group were 8.33%(1/12) and 66.67% (8/12), respectively.(P=0.027). 3. significant difference between the two groups on liver function at all time points the ALT and AST of control group were significantly higher than treatment group at 6,24,48h after reperfusion.(figure 2). 4. significant difference between the two groups on expression of a-SMA at all time points, the expression of a-SMA of control group was significantly higher than treatment group, showing that the extent of activation of HSCs of control group is much higher than that of the treatment group.(figure 3) 5.significant difference between the two groups on expression of Rho, ROCK and VEGF. At all time points, the expression of Rho, ROCK and VEGF of control group were significantly higher than that of treatment group, (figure 4). Conclusions: 1. our study successfully established cirrhotic rat liver transplantation model for the first time. This model has great value in the future study about cirrhosis liver transplantation. 2. the liver function and 7-day survival of control group were significantly lower than that of the treatment group, showing that rapamycin indeed has protective effect on the graft injury after small-graft liver transplantation. 3. Laboratory studies showed: through inhibition of Rho-ROCK signaling pathway and inhibition of...