The Mechanism Of Wnt Agonist Promotes Liver Regeneration After Major Hepatectomy In Biliary-Obstructed Rats By Activating Wnt/β-Catenin Signaling Pathway

BackgroundObstructive jaundice (OJ) is one of the common clinical pathological state in Hepatobiliary and Pancreatic Surgery.To date, although the regenerative capacity of liver is extraordinary,major hepatectomy with OJ has still a high risk of morbidity and mortality associated with liver poor function.OJ has been shown to inhibit liver regeneration in previous studies. The mechanism of impaired hepatic regeneration in cholestatic liver has been studied extensively,but the molecular mechanism remains unclear.The current treatment strategy is biliary drainage before operation,damage control procedure in operation and protective liver function after operation.Although these methods have achieved certain positive effects,they were still therapeutic strategy of passive and there were some limitations.These methods did not improve the liver regeneration by the molecular mechanism.Thus,there is important clinical significance to explore the molecular mechanism of liver regeneration after major hepatectomy in OJ.With the development of proteomics technology and bioinformatics,some signaling pathway associated with liver regeneration have been identified by gene chip technology.After a pilot study,we found that wnt/p-catenin signaling pathway had still play an important role in liver regeneration after major hepatectomy in OJ.The wnt/β-catenin signaling pathway is critical for development, differentiation and cellular homeostasis.Previous study had demonstrated canonical wnt/β-catenin signaling pathway play a essential role in liver regeneration after major hepatectomy in non-jaundice rats by activating β-catenin and regulating expression of its downstream target gene,and loss of β-catenin results in delayed liver regeneration.However, whether the inhibitory effect of jaundice on liver regeneration associated with Wnt/β-catenin signaling pathway remains unclear.This study was conducted to observe the changes of wnt/β-catenin signal pathway during liver regeneration in rat model of major hepatectomy after OJ and to explore the potential molecular mechanisms ofthis pathophysiological process.Part IAn Improved Rat Model of Major Hepatectomy combined with Internal Drainage After Obstructive Jaundice Using Microsurgical TechniquesObjective:In order to better understand pathogenesis of jaundice,several rat models of major hepatectomy combined with internal drainage after OJ were established. However, complication rates such as biliary infection and blockage of bile duct were still high and models were not stable.We aim to establish an improved rat model of major hepatectomy combined with internal drainage after OJ using microsurgical techniques.Stability and safety of new rat model were evaluated.Methods:After a pilot study, an improved rat model was established.The first step of surgical procedure was induction of OJ.The common bile duct was isolated and resected.The second step was biliary drainage and hepatectomy after OJ.An new method for biliary drainage was restored in situ by reconnecting the proximal end and the distal end of the common bile duct by inserting an stent.And then a 70% hepatectomy was performed according to Higgins and Anderson method.All operations were carried out under a microscope. Male Sprague-Dawley rats were randomly divided into four groups.Group1 was the sham group,where rats were not treated with either 70% major hepatectomy after OJ or drugs;Group 2 is classical method group,where OJ was induced and choledochojejunostomy for internal drainage and major hetatectomy were performed.Group 3 was non-microsurgical techniques group,where OJ was induced and then internal drainage and major hetatectomy were performed using new method;Group 4 was new method group using microsurgical techniques. Stability and safety of new rat model were evaluated according to the complications and survival rate.The rats were sacrificed at 1,6,12,24,72,168,240 and 336 hour after hepatectomy.The liver biochemical indexes and histopathological analysis were examined.Results:The successful rate of three kinds of method inducing OJ were 86.3%(G2), 88.8%(G3),97.5%(G4) respectively (P<0.05).The omission ligation of caudate lobe bile duct and biliary fistula were the main reason for the failure model. The postoperative liver function of the three groups recovered rapidly.But liver function was most stable in G4 after 7days and the liver function was a rebound in G2 because of biliary complication.Two weeks after hepatectomy the total complications of the three groups were 20.0%(G2),11.8%(G3),6.25%(G4) respectively(P<0.05) and the survival rates were 60.0%(G2),68.8%(G3),92.0%(G4) respectively (P<0.05).The level of amylase was increased temporarily after operation.There was significant difference between G3 and G2(P<0.05),and the level between G4 and G2 had no significant difference (P<0.05)Conclusions:Stability and security of the improved rat model are superior to the traditional rat model because of small trauma,lesspostoperative complications and high survival. It is suitable for the study of long-term biliary internal drainage.Part ⅡInfluence of Obstructive Jaundice on Liver Regeneration aft er Major Hepatectomy in Rats:Roles of Wnt/β-catenin Sig-nalling PathwayObjective:This study was conducted to observe the changes of wnt/β-catenin signal pathway during liver regeneration in rat model of major hepatectomy after OJ and to explore the potential molecular mechanisms of this pathophysiological process.Methods:Male SD rats were randomly divided into three groups(n=30 in each group):sham,control and study groups.In the sham group,rats were not treated with either 70% major hepatectomy or OJ.In the control group,only 70% hepatectomy was performed.In the study group OJ was induced in the rats by bile duct ligation.After 7 days biliary internal drainage and 70% hepatectomy were performed.These rats were sacrificed after operation on 1,6,12,24,72 and 168 hours.Liver regeneration, liver damage and survival rates were assessed,and the protein and mRNA levels of wnt/β-catenin signal pathway in the liver tissue were determined by Western blot and qRT-PCR.ResultsrThe survival rates were 100% in three groups.The rats in the control group had lower ALT and AST levels compared to rats in the study group in 1,6,12 and 24h after operation (P<0.05).Moreover,the ALB level was significantly higher in the control group than the study group in 24,72 and 168h after operation (P<0.05).The liver body weight ratio and liver regeneration rate were significantly higher in the control group than the study group in 72 and 168h (P<0.05).The labelingindex of Ki-67 and PCNA were significantly higher in the control group than that in the study group in 12,24 and 72h (P<0.05).The protein expression of β-catenin began to increase at 6h after operation in control group and reached the peak at 24-72h, while the protein expression of β-catenin in study group were delayed and suppression compared with the control group.It began to increase at 12h after operation. The protein expression of cyclin D1 and cyclinE1 began to increase at 12h after operation in control group and reached the peak at 72h,while the protein expression of cyclin D1 and cyclinEl in study group were delayed compared with that in the control group.It began to increase at 24h after operation.The mRNA expression level of cyclin D1 and cyclinE1 began to increase at 6h after operation and it reached the peak at 24-72h while the mRNA began to increase at 24h in study group. The mRNA expression level of cyclin D1 and cyclinE1 were significantly higher at 12,24 and 72h in the control group than that in the study group (P<0.05)Conclusions:Compared with normal rat,liver regeneration in rat model of major hepatectomy after OJ was seriously inhibited and delayed.The mechanism was associated with the inhibition of wnt/β-catenin signal pathway,especially with the inhibition of expression ofβ-catenin.Part ⅢWnt Agonist Enhances Liver Regeneration After Major He-patectomy in Biliary-Obstructed RatsObjective:This study in the part Ⅱ had demonstrated that the delayed liver regeneration in rats with OJ had associated with the suppression of wnt/p-catenin signaling pathway.This study in this part was conducted to investigate the effect of wnt agonist on major hepatectomy after OJ and to explore the potential molecular mechanisms of this effect.Methods:Male Sprague-Dawley rats were randomly divided into three groups: sham, control and study groups.In the sham group,rats were not treated with e ither wnt agonist or saline.In the study group, OJ was induced by ligating and dividing the common bile duct, and a 70% hepatectomy was performe d with implementation of wnt agonist (2-amino-4-[3,4-(methylenedioxy)benzylam ino]-6-(3-methoxyphenyl)pyrimidine,5 mg/kg BW) treatment in the study group. In the control group, those rats in which OJ was induced received normal sal ine before the 70% hepatectomy. The rats were sacrificed after hepatectomy on lh,6h,12h 24h 72h 168h. The liver regeneration,liver damage and apoptosis 1 abeling index were assessed after hepatectomy.The protein and mRNA levels of wnt/β-catenin signal pathway and its downstream target gene in the liver tissu e were determined by Western blot and qRT-PCR.Results:The postoperative liver function and the indices of proliferation activities were better in the study group than that in the control group.The rats in the study group had lower ALT and AST levels compared to rats in the control group in 6h,12h and 24h after operation (P<0.05).The TBIL level was significantly lower in the study group than the control group in 12h,24h after operation (P<0.05).Moreover,the ALB level was significantly higher in the study group than that in the control group in 24h,72h and 168h after operation (P<0.05).The liver body weight ratio and liver regeneration rate were significantly higher in the study group than that in the control group in 72h and 168h (P<0.05).The labeling index of Ki-67 and PCNA were significantly higher in the study group than that in the control group in 24h and 72h (P<0.05).The protein expression of P-catenin began to increase at 6h after operation in study group and reached the peak at 24-72h, while the expression in study group were delayed and suppressed compared with the study group.It began to increase at 12h after operation. The protein expression of cyclin D1 and cyclinE1 began to increase at 12h after operation in study group and reached the peak at 72h, while the expression in control group were delayed compared with the study group. It began to increase at 24h after operation.The mRNA expression level of cyclin D1 and cyclinE1 in study group began to increase at 6h after operation and it reached the peak at 24-72h, while the mRNA began to increase at 24h in control group. The mRNA expression level of cyclin D1 and cyclinEl were significantly higher at 12h,24h and 72h in the study group than that in the control group (P<0.05)Conclusions:These results demonstrate that wnt agonist treatment is able to improve the liver regeneration in rats with OJ by activating the wnt/β-catenin signaling pathway directly.It may represent a novel therapeutic approach in the treatment of major hepatectomy with OJ.