Cytokines And Inflammatory Mediators In Mice Liver Ischemia-reperfusion Injury

Background and AimsFor the effective control of bleeding,liver surgery is often necessary to block the hepatic artery and portal vein.But as the liver hepatic inflow occlusion time extends, with the restoration of blood flow,it results in the inevitable cause of liver I/R injury.At home and abroad many studies on hepatic ischemia-reperfusion injury in a basic understanding of the mechanism consider the mechanism of their occurrence mainly in the following aspects of the combined effect:liver microcirculation disfunction;oxygen free radicals;calcium overload;cytokines and inflammatory mediators and so on,While cytokines and inflammatory mediators how to lead to hepatic ischemia-reperfusion injury mechanism is unclear.In this experiment,We created hepatic ischemia-reperfusion model in mice to study the cytokines and inflammatory mediators in the hepatic ischemia-reperfusion injury.Materials and methods24 male C57BL/6 mice,6-8 weeks of age and weighing 16-20 g were randomly divided into 4 groups:A group,B group,C group and D group,all animals were fasted for 12h before the experiment,but with water ad libitu.0.2%pentobarbital sodium solution were administered intraperitoneally in animals with the dosage of 60mg/kg. And then the abdomen was opened in the midline to expose the liver.In group B,C,D, the mice were subjected to 75 minutes of focal hepatic ischaemia by placing an atraumatic microvascular clamp on the left hepatic artery and portal vein carefully,then with the reperfusion of 2 hours,6hours,24hours.When the hepatic ischemia was over, the abdomen would be closed,then the mice eated freely.Group A was sham-operated group with laparotomy only,without any other treatment.In each group after reperfusion,the blood was obtained from retrobulbar venous plexus,then the part of hepatic ischemia-reperfusion liver was collected.After holding for 30min,then followed 3000rpm/min centrifuge for 10min,sera were preserved at 4°C. Part of liver tissue was kept in 10%formaldehyde fixation,the other was preserved in -70℃.Hepatic function assay:Blood samples were measured ALT and AST using a clinical biochemistry machine(Hitachi,Japan).Pathology:The specimens of liver were embedded in the paraffine.The sample was dyed with HE stain and observed through optics microscope,the changes are graded.MPO assay:MPO activity of the liver was analyzed by chromatometry using the kit according to the manufacturer's instructions(Nanjing Jiancheng Bioengineering Institute, China).liver tissue TNF-αand IL-1βmRNA expression levels:we used the Real time PCR to determine the TNF-αand IL-1βmRNA level.ResultsThe ALT and AST of B,C,D groups were significantly elevated,but with the reperfusion time increased gradually reduced(group B,C,D VS group A;group B,C VS group D,P＜0.01).The performance under light microscope of 2 hours of reperfusion was that the hepatic cell was significantly edema degeneration,with partial liver cell vacuolization Obviously,there was some patchy necrosis,sinusoidal congestion and narrower. Histopathological score(group B,C,D VS group A;group B VS group C,D,A,P ＜0.05).The liver tissue MPO content of B,C,D groups increased with the peak of 2 hours of reperfusion(group B VS group A,C,D,P＜0.01).The IL-1βmRNA expression in B,C,D groups increased with the peak of 2 hours of reperfusion(group B VS group A,C,D,P＜0.01);TNF-αmRNA expression in B,C, D groups was higher than A group(VS group A,P＜0.05).ConclusionThe release of inflammatory mediators can damage the vascular endothelial cells, resulting in microcirculation obstacles.And cytokines can stimulate and maintain the inflammatory response,also leading to reperfusion damage.Tumor necrosis factor (TNF)-αand(IL)IL-1βare two important cytokines involved in hepatic I/R.