Reseach Of Ischemia Reperfusion And Ischemic Preconditioning On The Regenerative Of Residual Liver After Hepatecytomy In Rats

PartlThe effects of ischemia reperfusion and ischemic preconditioning on the regenerative of residual liver after hepatecytomy in rat modelObjective:To explore the effects of hepatic ischemia reperfusion injury (IRI) and ischemic preconditioning (IPC) on regenerative of residual liver after major hepatectomy with the model we established, and set up a fundamental for further research.Methods:Female Sprague-Dawley(S-D) rats were randomly divided into3groups:①ham-operation control group (S group, n=6).②schemia Reperfusion Group (IR group, n=27):Reperfusion was administered after25minutes of portal triad clamping, and the major hepatectomy was performed as soon as hepatic inflow occlusion.③schemic Preconditioning Group (IPC group, n=27):Referring to10minutes hepatic ischemia and10minutes reperfusion before hepatic inflow occlusion, and then repeated the procedures of IR group. The liver regeneration tissues of6rats in each group were obtained at4hours,24hours, and48hours after operation, which were stained with Hematoxylinand Eosin (HE) for histologic study. The serum AST, ALT and TBil were measured with an automatic biochemistry analyzer.Results:The histopathology showed different damaged of the residual hepatocyte, with cell swelling and fatty degeneration, but without obvious necrosis. Comparing IR group and IPC group at24hours and48hours after operation, cell swelling and fatty degeneration were severer in IR group. Regeneration of hepatocyte was not significant at4hours after operation, which began to increase at24hours. Mitosis figures were more obvious in IPC group. With the time going, the serum ALT and AST increased gradually in both groups, and then began decreased24hours after operation. However, the detection value of serum ALT and AST were significantly higher in IR group than that in IPC group (P＜0.05). There was no death of rats24hours after operation with the improvement of liver function.Conclusion:With the regeneration of hepatocyte, the liver function of rats gradually recovered after major hepatectomy. IPC could relieve injury of hepatocyte in early stage, and mitosis figures could be found at24h after operation. The level of serum ALT and AST of IPC group were lower than that in IR group. IPC showed some protective effects on hepatocyte at early stage after operation. Part2The impact of ischemia reperfusion and ischemic preconditioning on the gene expression profile in rat liver regenerationHepatic ischemia-reperfusion injury (IRI) referred to exacerbation of hepatocyte dysfunction and structure damage after ischemia reperfusion caused by variant reasons of liver ischemia. Hepatic IRI is a common pathophysiological process in liver surgery, such as partial hepatectomy, liver transplantation, liver trauma and shock. Besides, IRI might induce liver failure and multiple organ dysfunctions, leading to higher morbidity and mortality, which became the main factor affecting the recovery and prognosis after operation. With a remarkable ability of regeneration, the liver function could recover through proliferation of residual liver tissue after variants of damage factors, such as operation, toxin and infection, etc. Therefore, reducing the ischemia reperfusion damage, improving the regeneration capacity had great significant for promoting the recovery of liver function. However, the mechanism of the impact of IRI on the hepatocyte regeneration capacity remained unclear.Hepatic ischemia-reperfusion injury (IRI) was inevitable during hepatic operation with hepatic vascular occlusion, as well as donor preservation and implantation period in liver transplantation. Hepatic IRI could lead to systemic inflammatory response syndrome and multiple organ failure, which increased the mortality of hepatectomy and liver transplantation. There were still no effective therapeutic approaches to prevent and avoid from hepatic IRI. However, recent research showed that ischemic preconditioning (IPC) might be an available technique in minimizing hepatic IRI. IPC was a brief ischemic reperfusion before the subsequent long-term ischemia-reperfusion injuries, which induced an endogenous protective mechanism, improved the tolerance of ischemia-reperfusion injury of the tissue and organ, and decreased the damage caused by hepatic IRI.Objective:To investigate the dynamic changes of the gene expression in the regeneration of residual liver of rats after the operation of ischemia reperfusion and IPC before major hepatectomy by using the gene chip, and to discuss the mechanism of liver regeneration capacity with IRI and IPC after major hepatectomy.Methods:Female Sprague-Dawley(S-D) rats were randomly divided into3groups:① Sham-operation control group (S group, n=6).(②Ischemia Reperfusion Group (IR group, n=27), Reperfusion was administered after25minutes of portal triad clamping, and the major hepatectomy was performed as soon as hepatic inflow occlusion.③Ischemic Preconditioning Group (IPC group, n=27), referring to10minutes hepatic ischemia and10minutes reperfusion before hepatic inflow occlusion, and then repeated the procedures of IR group. The liver regeneration tissues of6rats in each group were obtained at4hours,24hours, and48hours after operation. The total RNA was isolated from the liver tissue of rats. The differential expressed genes during different stages of liver regeneration were screened through Affmetrix RAT GeneArray1.OST gene chip technique, and their functions were analyzed and classified. mRNA of Socs3, Gadd45a, Ptgs2, Angl and Gck genes in the liver regeneration were confirmed by RT-PCR.Results:During the process of liver regeneration with ischemia reperfusion after major hepatectomy, there were1742differentially expressed genes, relating to cell cycle regulation genes, growth and differentiation-related genes, DNA repair genes, metabolism-related genes, biologic oxidation genes, inflammation-related genes, signal transduction-related genes, cytokine-related genes, apoptosis-related genes, and so on. Eight kinds of significant trends for differentially expression genes were found during the process of liver regeneration.During the process of liver regeneration with ischemic preconditioning after major hepatectomy,1103differentially expressed genes were selected, relating to cell cycle regulation genes, growth and differentiation-related genes, DNA repair genes, metabolism-related genes, inflammation-related genes, cytokine-related genes, biologic oxidation genes, signal transduction-related genes, apoptosis-related genes, and so on. There were7kinds of significant trends for differentially expression. In the process of liver regeneration after IPC and major hepatectomy, the expression of PK and GCK gene associated with glycolysis decreased and some genes related to amino acid metabolism changed obviously.Conclusion:The regeneration of residual liver of rats after the ischemia reperfusion and IPC was the process of dynamic changes with multiple genes regulation. The interactions of multiple genes together promoted liver regeneration. Genes related to growth and cell cycle up-regulated in4hours of liver regeneration after major hepatectomy, which promoted the liver regeneration. To adapt to the demand of substance metabolism and energy supplies, the expression of metabolism-related genes down-regulated at the begining, and then they up-regulated gradually24hours later. During the process of liver regeneration, the inflammation-related genes and the biologic oxidation genes also demonstrated different function. The changed in the expression of PK and GCK were beneficial to energy balance of liver cells in IPC groups. Part3The effect of ischemia reperfusion and ischemic preconditioning on the expression of protein COX-2and GADD45a in rat liver regeneration after hepatectomyObjective:To investigate the expression of protein COX-2and GADD45a in regeneration of residual rats liver after major hepatectomy with ischemia reperfusion and ischemic preconditioning.Methods:Female Sprague-Dawley(S-D) rats were randomly divided into3groups:①Sham-operation control group (S group, n=6).②Ischemia Reperfusion Group (IR group, n=27), Reperfusion was administered after25minutes of portal triad clamping, and the major hepatectomy was performed as soon as hepatic inflow occlusion.③schemic Preconditioning Group (IPC group, n=27), referring to10minutes hepatic ischemia and10minutes reperfusion before hepatic inflow occlusion, and then repeated the procedures of IR group. The liver regeneration tissues of6rats in each group were collected at4hours,24hours, and48hours after operation for detecting the expression of protein COX-2and GADD45a by Western blot respectively.Results:Compared with S group, the expression level of COX-2in IR group and IPC group were significantly increased at4hours,24hours and48hours (P<0.01). And the level at4h was higher than that at24h and48h (P<0.01). In IR group, the expression level of COX-2was significantly higher at4h than of24h and48h in IPC group (ρ<0.01).Compared with S group, the expression of GADD45a in IR group and IPC group were significantly higher at4h (P<0.01). But there was no significant change in the expression of GADD45α in group IR and group IPC at24h and48h compared to S group (P>0.05), while the expression of GADD45a in IPC group was significantly higher at4h than that in IR group (P<0.01).Conclusion:The COX-2expression of hepatocyte might be related to hepatic ischemia-reperfusion injury (HERI). Ischemic Preconditioning (IPC) could contribute to reduce HIRI in early stage through decreasing the expression of COX-2. Meanwhile, the activation of GADD45a by IPC would promote DNA repairing and contribute to the proliferation of hepatocyte through cell cycle regulation.