Study On Liver Warm Ischemia-reperfusion Injury Caused By Different Hemodynamic States And Its Intervention

BackgroundLiver transplantation has helped thousands of patients with end-stage liver disease. About300,000patients need liver transplantation in China. However, only2,000liver transplantations are done every year in China. The ratio of waiting candidates and donors is5versus1in the U.S.A.,3versus1in British and150versus1in China.Because of the scarcity of citizen voluntary donors in China, most organs came from executed prisoners in the past. The continuation of practices has caused controversy in the international transplantation community. Fewer death sentences are delivered in China with each passing year. Living donor transplantation might adversely affect the physical health of the donor. Huang Jiefu, Vice Minister of the Ministry of health, said recently that a voluntary donation system should be established in China to encourage citizen to donate their organs after death.Donation organs come both from donors of cardiac death(DCD) and donors of brain death(DBD). Legislation on the adoption of brain death as the standard was subsequently not endorsed. So donation after cardiac death will be the main source of organs.In1995Maastricht criteria of donation after cardiac death was established. There are five types of DCD. Type1:patient is dead on arrival. Type2:unsuccessful emergency resuscitation. Type3:imminent cardiac arrest in intensive-care unit after withdrawal of life support. Type4:cardiac arrest during or after brain death diagnostic procedure. Type5: unexpected cardiac arrest in intensive care. Our country promotes type3DCD in hospital.The characteristic of DCD liver is that it often suffered severe warm ischemia-reperfusion injury. From the withdrawal of life support to the declaration of death, the liver may suffer long time of warm ischemia. There are many different kinds of hemodynamic states which affect the liver such as low blood pressure, ischemia and extravasation. It is necessary to know how to estimate the warm ischemia-reperfusion injury, what is the mechanism and how to prevent it. This research was carried out through a rat liver warm ischemia-reperfusion model.The nature of ischemia is hypoxia. This research studied the hypoxia inducible factor-1(HIF-1) expression under hypoxia circumstances.The different expression of HIF-1 under different hemodynamic states was studied. HIF-1intervention was also studied.Part IDifferent hemodynamic states correlate with liver warm ischemia-reperfusion injury in ratsObjective:In a rat model of partial(50%) hepatic ischemia-reperfusion, different vessels(hepatic artery, portal vein, hepatic vein) were clamped separately to investigate the effect of different hemodynamic states on liver warm ischemia-reperfusion injury in rats.Methods:108SD(Sprague Dawley) rats were randomly divided into three groups:hepatic artery clamping group(HAC), portal vein clamping group(HPC), hepatic vein clamping group(HVC). In each group the vessels of the left part of the liver were clamped separately. A rat model of partial(50%) hepatic ischemia was used to determine the effect of15,30or60minutes of liver ischemia preceding reperfusion.①2,6,12,24and48hours later blood samples were collected to assess serum alanine aminotransferase(ALT);②6hours later blood and tissue samples were collected to assess serum alanine aminotransferase and total bilirubin(TB), hepatic myeloperoxidase(MPO) activity and tissue malondialdehyde(MDA) content.③After30minutes’ischemia and6hours’reperfusion tissue samples were collected to assess histological changes. The microcirculation state was observed by intravital fluorescence microscopy. TNF-a, IL-1and IL-6mRNA of hepatic tissue were measured by real-time PCR. Apoptosis of hepatic cell was detected in situ by TUNEL Expression of Caspase-3was determined by ELISA. Apoptosis associated proteins were determined by western blot.④All the data were shown in mean±standard deviation and analyzed by SPSS13.0.Results:A stable model of rat partial(50%) hepatic ischemia-reperfusion was successfully established, which was useful for studies on the effect of different hemodynamic states on liver warm ischemia-reperfusion injury in vivo. The model simulated three pathophysiologic states in the livers from the donors of cardiac death(DCD):venous congestion secondary to right ventricular failure, ischemia due to decreased hepatic blood flow, and hypoxemia due to decreased oxygen in the hepatic artery.①Usually6hours after reperfusion serum ALT reached its peak value.②The ischemia-reperfusion injury in the HVC group was much severer than that in the HAC and HPC groups.③Serum ALT and TB, histological changes, hepatic MPO activity and MDA content, microcirculation state, TNF-a, IL-1and IL-6mRNA of hepatic tissue, apoptosis of hepatic cell, expression of Caspase-3, and apoptosis associated proteins showed difference in three groups.Conclusions:The time of vessel clamping had very important influence on the hepatic I/R injury. When the warm ischemia time got longer, the hepatic ischemia-reperfusion injury became much severer.The injury was obvious in the early phase of the reperfusion. If the clamping time didn’t exceed30minutes, the injury was relatively slight and reversible. When the clamping time exceeded60minutes, the injury was much severer and became irreversible.The time of clamping should be controlled within30minutes. There was remarkable difference between the liver warm ischemia-reperfusion injury in the three groups. The ischemia-reperfusion injury in the HVC group was much severer than that in the other two groups.Part Ⅱ Effect of different hemodynamic states on HIF-1different expression in liverObjective:To observe the effect of different hemodynamic states on hepatic microcirculation and oxygen metabolism when different vessels were clamped. HIF-1α played a crucial role in hepatic I/R injury. The hepatic content of HIF-1α protein and HIF-1α mRNA were measured to investigate the difference between the three vessel clamping groups. And the expression characteristics of HIF-1α were further studied.Methods:216SD rats were divided randomly into3groups(similar to part Ⅰ).①Morphological changes and microcirculation were observed by pathological examination and fluorescence microscopy. The decrease in liver oxygenation was confirmed by HypoxyProbe-1staining. Tissue metabolism was assessed by monitoring the interstitial levels of glucose, glycerol and lactate/pyruvate ratio.②2hours after reperfusion HIF-1α protein and HIF-1α mRNA were measured by western blot and real-time PCR. HIF-1α positive cells were detected in situ by immunohistochemical method. The relationship between HIF-1α expression in the liver and hypoxia was analyzed.③6hours and24hours after reperfusion liver function, hepatic myeloperoxidase(MPO) activity and histological changes were assessed.④All the data were shown in mean±standard deviation and analyzed by SPSS13.0.Results:①Under different hemodynamic states the hepatic ischemia-reperfusion injury significantly increased with time. The I/R injury was severer in HPC group liver compared with HAC group. Furthermore, even much severer I/R injury was observed in HVC group liver compared with HPC group, confirmed by the histological and biochemical parameters. The distribution characteristics of HypoxyProbe-1staining were different in the three group. The HypoxyProbe-1positive hepatocytes were seen mainly in the pericentral zone in HAC and HVC group. In contrast, HypoxyProbe-1was strongly stained in the periportal area in HPC group.②These histological results were consistent with the HIF-1α immunoreactivity area in the three groups, suggesting that hypoxia was the main trigger for HIF-la expression. HIF-la protein content in the nuclear increased in the HAC and HPC group. But it was barely detectable in HVC group. The hepatic HIF-1α mRNA levels increased in the HVC group.③After60minutes’ischemia and24hours’ reperfusion, livers from the HVC group revealed severe lobular edema, congestion, ballooning, and hepatocellular necrosis. The Suzuki score was higher in HVC group than that in HAC and HPC group (the value in HAC, HPC, HVC group was0.83±0.04,2.21±0.59,3.89±0.08, P<0.01). After24hours’ reperfusion the liver function improved slowly.Conclusions:There was a crucial relationship between liver warm ischemia-reperfusion injury and hypoxia. Hypoxia resulted in microcirculation dysfunction and metabolic changes in the liver. Hypoxia led to nuclear HIF-la protein accumulation, but the expression characteristics of HIF-la were different in the three groups. HIF-la played a protective role in liver warm I/R injury, but the levels of the protection were different under different hepatic hemodynamic states.Part Ⅲ Effect of inhibition of HIF-1on liver warm ischemia-reperfusion injury in ratsObjective:To study the effect of inhibition of HIF-1α by2-Methoxyestradiol on liver warm ischemia-reperfusion injury in rats.Methods:324SD rats were divided randomly into3groups(similar to part I).Then these rats were divided further into two groups, intervention group and control group. Separate groups of rats were treated with HIF-1α inhibitor2-ME(2-Methoxyestradiol, intervention group) or0.9%normal saline(control group) followed by HA, HP or HV clamping for15,30or60minutes respectively.①After30minutes’ischemia and6or24hours’reperfusion serum ALT and TB were assessed.②After30minutes’ischemia and24hours’reperfusion histological changes were assessed.③After15,30or60minutes’ischemia and2hours’ reperfusion tissue HIF-la protein expression were assessed.④After15,30or60minutes’ ischemia and6hours’ reperfusion tissue MPO activity and MDA content were assessed.⑤After30minutes’ ischemia and24hours’ reperfusion HO-1, eNOS, iNOS and A2AR mRNA of hepatic tissue, were detected in all the groups.⑥All the data were shown in mean±standard deviation and analyzed by SPSS13.0.Results:①2-ME intervention slowed down the recovery of liver function after reperfusion. After30minutes’ ischemia and6hours’ reperfusion, in HAC group, the serum ALT was (218±10)U/L in control group and (560±17) U/L in2-ME intervention group. The serum TB was (8.38±1.03) umol/L in control group and (15.0±1.88)umol/L in2-ME intervention group. In HPC group, the serum ALT was (459±7) U/L in control group and (847±27) U/L in2-ME intervention group. The serum TB was (17.3±2.22) in control group and (23.1±1.37) in2-ME intervention group. P<0.05②Hepatic pathology performance got worse after2-ME intervention.③After2-ME intervention HIF-1α protein expressed less.④MDA, MPO content increased in HAC and HPC groups after2-ME intervention (P<0.05).⑤HO-1, A2AR and iNOS mRNA of hepatic tissue expressed differently especially in HPC and HVC groups.2-ME intervention had little effect on eNOS mRNA expression.Conclusions:Rats receiving inhibition of HIF-1α with2-ME suffered much severer liver warm ischemia-reperfusion injury, evidenced by the worse liver function, less preserved liver architecture by histology and higher content of MDA and MPO. Meanwhile, the expression of HIF-1α protein decreased.2-ME intervention had little effect on I/R injury in HVC group. HIF-1α played a crucial protective role in the I/R injury and the protection was correlated with the activation of target genes such as HO-1, A2AR and iNOS.