The Protective Effect Of Hyperoxia Liquid Preconditioning On Ischemia-Reperfusion Injury Of Intrahepatic Bile Duct In Rats

Liver ischemia-reperfusion(I/R) injury is one of the most important causes of organ dysfunction after liver transplantation, and the biliary complications after liver transplantation mainly caused by the I/R injury of the intrahepatic bile duct. Which caused series of biliary complications after operation, such as cholangeitis, intrahepatic cholestasis, bile duct stenosis, bile duct loss syndrome etc. Seriously influenced the function and survival of the graft. The most trouble complication is ischemic-type biliary lesions(ITBL), with an incidence varying between 5% and 15%. Finally up to 50% of the patients with ITBL need retransplantation or even die. Hperoxic liquid could decrease erythrocyte aggregation, lower the blood viscosity, quicken blood flow rate, improve the microcirculation of the ischemic tissue, It has been reported that the hperoxic liquid can prevent multiple organ cell damage during ischemia reperfusion, But the effect on intrahepatic bile duct was still uncertain. This study aimed on observing the effect of hyperoxic liquid on ischemia-reperfusion injury of intrahepatic bile duct in rats, and investigate its mechanism for clinical use.Methods40 SD rats were randomly divided into experimental and control group, which include 8 h and 24 h group respectively, 10 rats per group. The rats in experimental group were preconditioned by hyperoxia(20 mL/kg) 10min before ischamia, and the control group use physiological saline instead. Rats were anesthesiaed by intraperitoneal injection with pentobarbital sodium. The abdominal cavity was opened through a transverse incision, vessel clamps were used to occlude middle and left external liver lobe to produce the 70% part rat-hepatic I/R injury anima1 model in vivo, The occlusion were lasted for 90 minutes. Samples were taken for tests after reperfusion, liver samples were divided into three pieces, one fixed by polyoxymethylene and made into paraffin section for light microscope, another one fixed by glutaraldehyde and checked by transmission electron microscope, the last one fixed by glutaraldehyde for testing the catalase(CAT) activity by diaminobenzidine. Blood samples were taken from postcava for 4ml each, standing in freezing tube for 1 hour, centrifuged by 2000 r/min for 15min, the upper serum were taken and reserved under -20℃for analyzing alanine aminotransferase(ALT), asparate transaminase(AST) andγ-glutamyltransferase(GGT) activity.ResultsThe histological and ultramicrostructure lesions showed in experimental groups are significantly alleviated compared with those in control groups, intrahepatic bile duct endothelial cell shedding can be seen by light microscope, while hepatic tissues surrouded showed some lesions variously. Inflammatory cells infiltrated in the portal area,and the lesions showed in groups of 24 hours are higher than in 8 hours'. Cytoplasm rarefaction of the bile duct cells can be seen under the electron microscope, and the electron density decresed, nucleo-heterochromatin increased and side-gatherd, showing the degeneration change, And no significant ultramicrostructure lesions were detected in the hepatic cells of the experimental groups. The GGT activity in rats serum is significantly higher in the control groups compared to the experimental groups (P<0.05), and the difference is also significant between the group of 8h and 24h after reperfusion. The ALT and AST activity in rats serum is significantly higher in the control groups (P<0.05). The CAT activity showed significantly higher after preconditioned by the hyperoxia liquid in the experimental groups, and significantly higher in the group which 24 hours after reperfusion.DiscussionSince the blood flow blocked and the metabolin accumulated during the I/R procedure, inflammation cytokins will release generous oxygen free radical, especially the hydrogen dioxide and the hydroxy radical which are the most important mediate factors of I/R injuries, enhance the activity of antioxidase in the scavenging system of oxygen free radical can against such injuries. The hyperoxia liquid contains tittle ozone, ozone used in therapeutical dose can help against I/R injury in the rats'liver. Hyperoxia liquid preconditioning can stimulate endogenous defense system, help enduring ischemic and anoxic injury, so it can protect from I/R injury. In this study, we choose 2 time spot at 8 hours and 24 hours after reperfusion to observe the effect of hyperoxia liquid on I/R injury of intrahepatic bile duct.This experiment illustrated that the biliary lesions were controlled and alleviated after preconditioned by hyperoxia liquid. ALT and AST mainly reflect the lesions in the hepatic parenchymal cells, which increased with direct ratio to the hepatic tissue lesions, The hepatic tissue lesions partly showed injuries in the intrahepatic bile duct system. The results of this experiment showed that the lesions in the hepatic tissue were higher in the control groups, this indicats that the hyperoxia liquid can protect the hepatic tissue from I/R injury, which was the same as the documents had been reported before. Histo-pathomorphology and ultramicrostructure all showed that the lesions in the experimental groups were alleviated, conformed the biochemical indicator tested. Thus the biochemical indicator can properly reflect the I/R injury in the intrahepatic bile duct, the protective effect of hyperoxia liquid is definitely. The organism mainly depend on oxygen free radical scavenging system make up of antioxidases to prevent I/R injury. The catalase is one of the most important antioxidases, which mainly catalysis the hydrogen dioxide break into nontoxic water, in order to avoid the hydrogen dioxide change into free radical with high oxygenize activity, which will oxygenize the unsaturated fatty acid into hydroxy radical with hypertoxic, so the injury of free radical were alleviated. So testing the activity of the CAT can reflect the capability of the organism against the free radical injury. This experiment use the enzymohisto- chemistry method to determine the CAT activity, and the result showed that the CAT activity was significantly higher than the control group. and compared to the group of 8 hours, the activity of the CAT was even higher in the group of 24 hours, this result suggested that the antioxygen capability of rats in the experimental groups had been strengthened continuously. So we can conclude that the hyperoxia liquid can fairly protect the intrahepatic bile duct system of the rats from I/R injury.ConclusionThis experiment demonstrated that the hyperoxia liquid preconditioning can alleviate the I/R injury to the intrahepatic bile duct of the rats, which has a protective effect on the I/R injury to the intrahepatic bile duct. The probably mechanism may be: motivate endogenous protective system and enhance the capability of anti-oxidation; decrease the content of OFR and reperfusion injury induced by which; improve the blood rheology of the experimental animal, help improve the microcirculation and rectify the distortion of the blood rheology. It can provide useful information for clinical application.