| Objective:To explore the protection effect of Radix Isatidis polysaccharide on hepatic ischemia-reperfusion injury (IRI), this empirical study in the rat autologous orthotopic liver transplantation (AOLT) model, based on the rats through the preoperative been continue intragastric administrated of Radix Isatidis polysaccharide (30mg/kg/d) of2weeks.To investigate the protection effect on the transplanted liver mediated by Radix Isatidis polysaccharide through research the changes of liver cell structure and function, the expression of correlated inflammatory factors and their interrelationship of different period, observe pathological changes and the mitochondrial structure changes of rat liver cell after the operation, detecting the expression of hepar earlier pathological changes、 biochemical indicator TNF-α and so on. Explore the possible protection mechanism of IRPS on liver of AOLT, which may provide an experiment basis for liver transplantation.Methods:2.172pure clean SD rats are includede in the study,which are divided into3groups randomly,autologous orthotopic liver transplantation model group (AOLT group n=24.);Radix Isatidis polysaccharide+autologous orthotopic liver transplantation model group (RIPT group n=24); Normal rats in control group (Sham group n=24). Two groups of liver transplantation in rats with portal vein are70%autologous transplantation model, and make technical improvements implemented since the orthotopic rat liver transplantation. Sham group are given the same treatment except the infusion.2.2Comparison of autologous orthotopic liver transplantation in rats within48h after index change.6rats were killed in1h,12h,24h,48h, after liver transplantation in each group. Collect blood samples from inferior vena cava,separate serum, detect ALT, AST and other biochemical markers with an automatic biochemical analyzer, test the expression of TNF-α by ELISA, slice of median liver lobe as a sample preparation, observe changs of liver cells’ pathological and the damage of mitochondrial and organelles in liver cells through the light microscope and transmission electron microscope. Results:3.1The criteria for successful operation: after surgery, rats immediately regained consciousness, stood up, and to maintain effective breathing, heartbeat. Portal vein perfusion, the liver warm ischemia time, generally<5s, is negligible. After the end of anhepatic phase, RIPT group of liver re-color uniform and ruddy,as the same time ALOT group then in the complex color.3.2The liver function test showed that1h after liver transplantation ALT and AST of serum both in AOLT group and RIPT group had a rapid raise.In time periods as1h12h and24h, serum ALT and AST levels of both AOLT group and RIPT group were significantly higher than that from the Sham group, there were significant differences (P all<0.05). Serum ALT and AST of the ALOT group were continuity on a high level during the time within24h after the transplantation. Serum ALT and AST of the RIPT group returned to normal as the time went on. In time periods as12h and24h, serum ALT and AST levels of the RIPT group were significantly lower than that from the AOLT group, there were significant differences (P all<0.05).3.3In each group,pathological changes of liver cells under light microscope after operation:1h after transplantation,the organizational structure of the liver changes in rats both with hepatocellular swelling, while lobular architecture is normal, the gap between the liver cells wided,hepatic sinusoid narrowed,having siltation akaryocytes and thromb,around with in the ALOT group, the organizational structure of the sham group are normal,there are no severe changes with hepatic tissue.12h after transplantation, the RIPT group has slight swelling of the liver cells, no significant changes in liver tissue; In the ALOT group, hepatocyte swelling, hepatic sinusoid narrowing and inflammatory cell infiltration is obvious.24h after transplantation, in the RIPT group,hepatocyte swelling improved. hepatic sinusoid broaden. In the ALOT group, hepatocyte swelling aggravated, hepatic sinusoid narrowing as hepatic lobules structure is not obvious, there are red blood cells and thrombus deposition, inflammatory cell infiltration appeared around;3.4Transmission electron microscopy in each group under the mitochondria of liver cells can be seen:1h after transplantation in rats from the RIPT group,the swelling of mitochondria in rats is lighter, be oval-shaped, round the remaining structure of the; in rats from the ALOT group,the mitochondria are swelling severely,be similar cyclo-shaped;after12h, in rats from the RIPT group, the swelling of mitochondria become more serious with the crest part of the fault, but still a small amount of order.the endoplasmic reticulum around are still remained, in rats from the ALOT group,the swelling of mitochondria are much more seriously,with the mitochondrial cristae decreased broken or disappeared.24h after transplantation in rats from the RIPT group,the swelling of mitochondria improved,round with normol endoplasmic reticulum;in rats from the ALOT group,vacuolar degeneration was found,severe reduction of visible ridges, broken or disappeared;48h after transplantation in rats from the RIPT group,the structure of liver cell was almost recover,a number of cells with nuclear fragmentation can be found.in rats from the ALOT group,there were much more cells with nucleolus broken solidily and dissolved,even apoptotic body can be found.3.5the level of serum TNF-α in ELISA results showed that the sham group, see a small amount of liver tissue expression of TNF-α, no significant fluctuation of the time;1h after transplantation,TNF-α expression can be seen between the sham group and the ALOT group is obvious (P<0.05),Conclusion:The autologous orthotopic liver transplantation (AOLT) model, mer-portal vein perfusion, in this empirical study, have a well simulation with the pathophysiological process of hepatic ischemia-reperfusion injury in clinically,with less interaction from immunologic injury and have less influence to the downstream organs of ischemia-reperfusion injury.can reflx independently the process of hepatic ischemia-reperfusion injury. Radix isatidis polysaccharide can protect the hepatocyte after liver transplantation by being intragastric administrated before opertion.the probable mechanism is adjusting the activation of mononuclear phagocytic system during ischemical reperfusion injury, especially the Kupffer’s cells, to refrain the dilivery of mediators of inflammation such as TNF-α, to lighten soakage of inflammatory cell and damage of blood vessel endothelium, lessen hepatic ischemia-reperfusion injury finally. |
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