Efficacy And Mechanism Of Hypothermic Self-made Liver Solution On Liver Via Portal Vein In Situ Irrigation During Portal Vein Occlusion In Rats With Obstructive Jaundice

BackgroundJaundice is always the most common symptom in patients with malignant tumors such as pancreatic carcinoma,cholaniocarcinoma and so on.The character of special location and early portal vein invasion always lead to low radical resection rate.In recent years,the application of pancreaticcoduodenectomy with portal vein resection in managing these cancers has been improved.But acute portal vein occlusion(PVO) can cause progressive damage to liver especially in patients with obstructive jaundice(OJ).Until now,there is no report about hepatic ischemical reperfusion injury caused by PVO in patients with OJ.Therefore ,profound research of hepatic pathophisiology changes after PVO and safe time limit to PVO in rats with OJ has important influence on promoting the safety and toleration of operation and the following recovery.Previous studies showed that apoptosis was the main character of hepatic ischemical reperfusion injury.Orthotopic hypothermical perfusion was widely used to prevent ischemical reperfusion injury that can promote hepatic tolerance of ischemia.At present,mainly used perfusion solution included Lactated Ringer'S Solution,HTK solution,UW solution and so on.These solutions can lessen ischemical reperfusion injury to some extent.But whole hepatic blood flow blockage with continuous orthotopic hypothermic perfusion also had some disadvantages:First,this technique has big difficulty and multiplicity;Second,it can cause complication such as severe hypotension, edema of lung,depression of potassium,acidosis,DIC and so on.3,HTK and UW solutions can't exist in systemic circulation.Then we used a hepatic preservation solution(self-made liver solution,SMLS) and assessed its effect and mechanism in protecting hepatocytes .ObjectiveThe mRNA level and expression of A20 along with the relation between the activation of MAPKs and the expression of related proteins in mitochondria mediated apoptosis pathway were observed in rats with OJ and hepatic continuous hypothermic perfusion with SMLS after PVO to elucidate the mechanisms of hepatic damage by PVO in rats with OJ and protection by SMLS.Methods and results1,Animal model and effect of PVO on hepatic energy metabolism:normal rats were randomly divided into four groups:(1)group SO:shame operation;(2)group A:biliary tract recanalization;(3)group B:biliary tract recanalization with PVO 30m;(3)group C:biliary tract recanalization with PVO 60m;(3)group D: biliary tract recanalization with PVO 90m.Summary mortality of group D was 60 percent which was obviously higher than that of other groups at 1 post operation week. ALT,AST and TBIL of group D were marked higher than those of other groups and the tendency of decreasing was not showed at 24h post operation.RCR,P/O and ATP of group D were also lower than those of other groups.Hepatic tissue disorder,extensive vacuolization and necrosis of hepatocyte,infiltration of inflammatory cells and cholestasis were still obvious in group D.2,Protective effect of SMLS on hepatic function in rats with OJ and PVO:rats with OJ were randomly divided into four groups:group E(biliary tract recanalization,liberation of portal vein,resection of caudate lobe of liver and closure of abdomen at 90m ).group F(biliary tract recanalization,portal vein bypass in caudate lobe of liver,PVO of left hepatic tissue 90m,then recovery of portal vein flow and resection of caudate lobe of liver).group G(group F with perfusion of 4℃Lactated Ringer'S Solution).group H(group F with portal vein perfusion of 4C SMLS).The level of ALT,AST and TBIL of all groups were heightened post operation and got to peak at 6h post operation,and then declined after 24h post operation.The increasing value of ALT,AST and TBIL in group F,group G and group H were obviously higher than that of group E.Because of no PVO,the level of ETX of group E gradually descended which means obstruction of biliary tract is the main reason for increasing of ETX. and biliray tract recanalization is of advantage to decrease ETX.In group F,the level of ETX markedly increased post operation and no tendency of decreasing was showed even at 72h post operation.In group G and group H,the level of ETX also increased post operation but amplitude of increasing was lower than that of group F(p<0.05).The level of ETX in group H was lower than that of group G at every time point post operation(p<0.05).All groups showed hepatic apoptosis post operation by TUNEL.Group E showed a tendency of decreaing but group F,groupG and group H had the adeverse tendency.3,MAPK signal transduction pathway mediating protection of hepatocyte by SMLS: reverse transcription PCR,Western blotting and TUNEL were used respectively to determine A20 mRNA,A20,P-JNK,P-ERK,P-p38,bcl-2,Bax, caspase-3 and hepatocyte apoptotic rate.Specific inhibitor of MAPK signal transduction pathway SP600125(specific inhibitor of JNK), PD98059(specific inhibitor of ERK)and SB203580(specific inhibitor of p38)were also used in the experiments.The results showed that A20 mRNA and protein level of A20,P-ERK and bcl-2 were obviously higher than those of other groups(p<0.05).But the apoptotic index showed an adverse tendency in which the apoptotic index of group G was marked higher than that of group F(p<0.05).P-JNK,Bax,caspase-3 and apoptotic index of group F were different from those of other groups(p<0.05).Conclusion1,Acute occlusion of portal venous flow in rats with obstructive jaundice can cause mitochondrial energy metabolic block in liver cells.2,The safe tolerant time limit to portal vein ligation in rats with obstructive jaundice and portal flow bypass is sixty minutes.3,Apoptosis of hepatic cells is the mainly representation of ischemia reperfusion injury to liver by portal vein ligation in rats with obstructive jaundice.4,Hepatic apoptosis caused by portal vein ligation in rats with obstructive jaundice is decreased by portal vein perfusion in low temperature with SMLS.5,Acute portal vein ligation can promote hepatic apoptosis by activating JNK signal transduction pathway,mediating the offset balance of bcl-2/bax protein and activating downstream apoptosis-related protein caspase-3.6,The expression of anti-apoptosis protein A20 is activated by hepatic continuous hypothermical perfusion with SMLS,then ERK signal transduction pathway is activated to regulate the balance of bcl-2/bax protein and decrease the expression of apoptosis executive protein caspase-3.