Effect Of Sinomenine On Ischemia-Reperfusion Injury In Orthotopic Rat Liver Transplantation

Effect of Sinomenine on ischemia-reperfusion injury in orthotopic rat liver transplantationThe damage to the liver caused by ischemia and reperfusion(I/R) represents a continuum of processes that culminate in hepatocellular injury. These processes are triggered when liver is transiently deprived of oxygen and reoxygenated, and can occur in a number of clinical settings, such as those associated with low flow states, diverse surgical procedures, or during the organ procurement for transplantation. In fact, I/R injury to the liver, an antigenindependent component of "harvesting" insult, represents an important problem affecting transplantation outcome. It causes up to 10% of early organ failure, and can lead to the higher incidence of both acute and chronic rejection. Moreover, I/R injury contributes to the acute shortage of livers available for transplantation because of the higher susceptibility of marginal livers to the ischemic insult. Indeed, minimizing the adverse effects of I/R injury could significantly increase the number of patients that may successfully undergo liver transplantation. However, at present there is no treatment available to prevent hepatic I/R injury. As intervention on more than one level is most likely needed to allow the recovery of cellular and organ function, extensive research efforts to better understand the mechanisms of hepatic I/R injury are warranted.The medicinal plant Sinomenium acutum Rehd.et Wils. has been used in China for more than 2000 years, for the treatment of various diseases. The main active constituent has been identified as the alkaloid Ssinomenine(7, 8-didehydro-4-hydroxy-3, 7-dimethoxy-17-methyl-9α, 13α, 14α-morphinan-6-one). Sinomenine is in clinical use in China and Japan due to its analgetic andanti-inflammatory activities. Its anti-rheumatic properties have been proved inclinical studies.Based on the conclusions mentioned above, in this study, we observe the effect of different dosage of Sinomenine on ischemia and reperfusion injury during orthotopic liver transplantation of rat and try to explore the possible mechanisms.PARTⅠProtective effects of Sinomenine on ischemia-reperfusioninjury during orthotopic liver transplantation in ratsObjective: To investigate the protective effect of Sinomenine on the cold ischemia and reperfusion(I/R) injury during orthotopic liver transplantation (OLT). Methods: We used syngeneic SD as animal model, OLT were performed in normal rats based on Kamada's two-cuff technique. 196 syngeneic SD rats were randomly divided into sham operation, NS control and two Sinomenine groups. The rats in sinomenine groups were treated with low(40mg/kg) and high dose(80mg/kg) of sinomenine respectively. After the donor liver was preserved in Ringer's(LR) solution for 5 h, the orthotopic implantation was performed. The serum and tissue samples of 2, 6, 12 and 24 h were collected for analysis after reperfusion of the portal vein, and the one-week survival rate was observed in each group. The apoptosis index(AI) of liver after OLT was detected by TUNEL; The level of TNF-α,IL-1βand ICAM-1mRNA in rat's liver were detected by RT-PCR; The level of IL-2 in serum was detected by ELISA. Results: In comparison with those in the NS control group, the levels of ALT and AST decreased significantly in low and high dose treatment groups at different time point post-transplantation, and their one-week survival rates also increased markedly (75%, 75% vs 12.5%, P<0.01). The AI was decreased markedly in Sinomenine treatment groups. At the 24h after liver transplantation, the AI of saline control group was 37.0±4.30, and in two Sinomenine treatment groups, the AI wre 23.8±3.27,18.6±3.03, respectively. Compared with saline control group, there was significant difference(P<0.01). Sinomenine treatment decreased the expression of tumor necrosis factor-a(TNF-α),interleukin-1β(IL-1β) and intercellular adhesion molecule-1(ICAM-1) in rat's livers, The elevation of serum IL-2 was suppressed by Sinomenine and Sinomenine treatment markedly ameliorated the focal necrosis of hepatocytes. Conclusion: Sinomenine can inhibit hepatocyte apoptosis by decreasing the discharge of TNF-α,IL-1βand ICAM-1, and decrease secretion of IL-2 correlating with T cell-mediated immunity. Sinomenine can prevent hepatic cells and SEC from cold ischemia and reperfusion injury during orthotopic liver transplantation in rats.PARTⅡEffect of Sinomenine on TLR4,MAPKs and NF-κB inrat's liver after ischemia-reperfusionObjective: To investigate the effect of Sinomenine on TLR4,MAPKs and NF-κBafter ischemia and reperfusion injury during orthotopic liver transplantation (OLT) and the mechanism of Sinomenine's protection of ischemia and reperfusion injury.Methods: We used syngeneic SD as animal model, OLT were performed in normal rats based on Kamada's two-cuff technique. 196 syngeneic SD rats were randomly divided into sham operation, NS control and two Sinomenine groups. The rats in Sinomenine groups were treated with low(40mg/kg) and high dose(80mg/kg) respectively. After the donor liver was preserved in Ringer's(LR) solution for 5 h, the orthotopic implantation was performed. We selected the sixth hour after ischemia and reperfusion injury as our observation points when the rat's liver function was the worst. The protein expression of TLR4,phosphorylated JNK,ERK,p38 MAPK and NF-kB were detected by Western Blot. Results: In the sham operation group, the TLR4 was low expression. High expression of TLR4 in saline control group, sinomenine decreased significantly the expression of TLR4 in liver tissue (P<0.01).The expression of phosphorylated JNK and ERK were no difference between four group, But the expression of phosphorylated p38 MAPK and NF-κB were low in sham operation group. The expression of phosphorylated p38 MAPK and NF-kB in saline control group were high, Sinomenine decreased significantly the expression of phosphorylated p38 MAPK and NF-kB in liver tissue (P<0.01).Conclusion: The mechanisms of sinomenine's protection of ischemia-reperfusion injury during liver transplantation in rats was related with its inhibition of innate immune responses mediated by TLR4. Sinomenine reduced the secretion of inflammatory cytokines such as TNF-α,IL-1βand ICAM-1 by inhibiting p38 MAPK and NF-κB signaling pathway. PARTⅢEffect of Sinomenine on the liver APC afterischemia-reperfusionObjective: To understand the Sinomenine's protective mechanism inischemia-reperfusion injury preferably, we investigated the effects of Sinomenine on maturation of liver derived dendritic cells (DC). Methods: We used syngeneic SD as animal model, OLT were performed in normal rats based on Kamada's two-cuff technique. 48 syngeneic SD rats were randomly divided into NS control and two Sinomenine groups. The rats in Sinomenine groups were treated with low(40mg/kg) and high dose(80mg/kg), respectively. After the donor liver was preserved in Ringer's(LR) solution for 2h, the orthotopic implantation was performed. Three days after operation, We Reseced the liver, separated and purificated liver DC, and observated the effects of Sinomenine on the maturion and function of DC. With fluorescent antibody markers and FACS, we analyzed the DC phenotypic expression of MHC-Ⅱ,OX62 and CD86. To investigate the effect of Sinomenine on the endocytosis of DC, we used FACS to analysis DCs endocytosis of protein antigen with OVA-FITC. Then detected the expressof IL-12, IL-1, TNF-αmRNA of liver DC by RT-PCR, and detected the expression of TLR4 on the liver DC of three groups. We collected the liver DC of three groups, and investigated allogeneic mixed lymphocyte reaction with ~3H-TdR. Results: The DC treated with sinomenine showed immature phenotype, and the liver DC of control group showed matured phenotype which MHC-Ⅱand CD86 were high expression. In Sinomenine treatment groups, the average intensity of fluorescence was stronger than that of the control group, and that showed the Sinomenine have the ability of inhibition on conversion of DC from immaturity to maturity. The test of endosome function found that Sinomenine can significantly decrease the antigen presenting function of liver DC. In addition, the liver DC treated with Sinomenine showed the expression of TLR4 and the IL-12,IL-1,TNF-αmRNA were low. At the same time, we found that the Sinomenine can significantly inhibit the liver DC mixed lymphocyte reaction result from MLR. Conclusion: Sinomenine can inhibit the maturity and function of liver DC. The effects of Sinomenine on ischemia-reperfusion injury during orthotopic liver transplantation in rats may be related to inhibition of the immune response mediated by T cell through inhibiting the maturation and function of liver DC.