Experimental Study Of Protective Effect Of PGE1 In The Hepatic Ischemia Reperfusion Injury In Fat Liver Rats

PrefaceWith the development of economy and the elevation of living standard, the injury to the health, especially to liver caused by excessive drinking and high fat diet are more and more obvious, in the same time the morbidity of fat liver is higher and higher. Now the study of fat liver becomes one of the hot topics in hepatic researches.Hepatic ischemia reperfusion is a common problem in clinical surgery and cannot be avoided especially in the course of complicated hepatectomy and hepatic transplantation. Hepatic ischemia reperfusion may cause not only hepatic injury but also many organs' dysfunction even failure, the severe can lead to death. In order to prevent hepatic ischemia reperfusion, a lot of experiments and clinical researches have done recently. A series of ways have been produced, such as ischemia pretreatment, hypothermy pretreatment, heat shock pretreat-ment, pretreatment in gene level etc. and all kinds of protective drugs. But some effective drugs are too expensive and more drugs cannot be used in clinics. So it is so significant to find an ideal clinical drug to prevent fat liver ischemia reperfusion.Prostaglandin E1 (PGE1) can improve hepatic function, increase amino acid metabolism, glycogen metabolism and synthesis of DNA. PGE1 has the role of hemodynamics in liver and protect hepatic cells. Now PGE1 is used for the patients to improve hepatic function after hepatectomy and to treat primary non-funtion after hepatic transplantation, which get evident effect. 70% PGE1 is metabolized by lung quickly through peripheric intravenous injection and some isutilized by arteries and bowels, so the role of PGE1 to liver decreases. In order to increase selectively hepatic blood flow and affect directly hepatic cells, it is best to give drugs through hepatic circulation. In this experiment we try to study the influencing factor of hepatic ischemia reperfusion to fat liver and discuss potential mechanism to protect fat liver of PGE1, so that we can get according theory which is provided in the prevention of fat liver ischemia reperfusion.Materials1. Object: 42 Male Wistar rats weighing 230 - 300g were offered by animal experiment center of second affiliated hospital of China Medical University ( Shengjing Hospital). 42 rates were randomly divided into 2 groups, one is expe-riental group which gave 0.1 ml/kg/min PGE1 (5fig/ml);The other group gave 0.1 ml/kg/min saline. Each group was divided into 3 subgroup according to 1 hour, 6 hour, 24 hour after reperfusion, n = 7.2. Agent;PGE1: alprostadil(injection) , C^H^Os, lOOug/bottle, Beijing Saisheng medicine limited company.ICAM -1 antibody: Wuhan Boshide bioengineering limited company. SABC hit;Wuhan Boshide bioengineering limited company.3. Apparatus: Hitachi 7600 whole automatic biochemistry analyzer OLYMPUS optical microscopeBECKMAN - LE80 centrifuger OLYMPUS BX51 image pickerMethods1. Establishment of fat liver model;1% cholesterol, 20% hog fat and 79% common stoyer were mixed then fed to rats for 10 weeks, in the same time the rats were lavaged by 38 degree alcohol (Dayishan) according to lml/100g/ day.2. Hepatic ischemia reperfusion of rats;After rats were anesthetized by 10% chloral hydrate (3ml/kg) through intraperitoneal injection, their abdomenwere opened by median incision. Polyethylene catheter was inserted into the branch of superior mesenteric vein, the pointed end enter portal vein, micro pump was connected then PGE1 (0. 5|xg/kg/min) or saline was pumped which continued 15 minutes. According to Pringle's maneuver, hepatic hilum was blocked for 15 minutes. In each group 5ml artery blood was collected in lh, 6h and 24h after reperfusion respectively from abdominal aorta. After artery blood was put for 20 minutes, it was centrifugated under 2500 turn/min for 10 minutes. Acquired serum was kept in refrigerator at - 20T!. A part of hepatic tissue which size was 1.0 X 1. 0cm was obtained and fixed in 10% formalin solution which could be used for pathologic examination and immunohistochemical stain.3. Observation of index;3.1 Measurement of biochemical series in serum: The level of ALT, AST and LDH were measured by whole automatic biochemistry analyzer.3. 2 The observation and comparison of hepatic cells under light microscope: We took 3 different fields in every section which is HE stain under high power field (400 x ) and observed and counted the number of complete hepatic cells.3.3 The detection of the expression of ICAM - 1 in liver tissue;We took SABC immunohistochemistry to detect the exoression of ICAM - 1. In every section we took 3 different fields to observe ICAM - 1 expression and collected images under light microscope (400 X ) by image picker and integrate optical density of image was measured by MetaMorph software.4. Data processing: All results were expressed as mean standard deviation (x ± s) and analyzed by independent - sample t test. All data were analyzed u-sing SPSS 11.0 software and differences were considered significant at P <0. 05.Results1. Evaluation of fat liver;The livers of rats at 6w, 8w, lOw after special eating were selected and made pathological sections for HE stain. At 6 weeks, there was a little of hydropic degeneration around hepatic sinus. At 8 weeks,there was hydropic degeneration in hepatic tissue and a little of fatty degeneration around hepatic sinus. At 10 weeks, the fatty degeneration of liver of rats is evident and we could see obvious fatty vacuolus.2. the level of biochemical enzyme in the abdominal aorta: After 15 minutes of ischemia of fat liver and 1 hour, 6 hours and 24 hours of reperfusion, the ALT level and AST level of experimental group are lower than those of control group (p <0.05). After 1 hour of reperfusion, the LDH level of experimental group is lower than that of control group (p < 0.05) , but there was no significance after 6 hours and 24 hours of reperfusion (p > 0.05). After 1 hour reperfusion, the level of biochemical enzyme;the experimental group ALT 80. 71 ± 16.49U/L, AST 197. 29 ± 30. 165U/L, LDH 878. 43 ± 399. 558U/L;control group ALT 184. 14 ±65. 157U/L, AST 338. 71 ±42. 98U/L.LDH 2136. 14 ± 849. 743U/L. After 6 hour reperfusion,the level of biochemical enzyme: experimental group ALT 124. 57 ± 39. 424U/L, AST 345. 14 ± 100. 463U/L, LDH 1467.86 ±578. 201 U/L;control group ALT 241.57 ±85.747U/L, AST 512.29± 69. 276U/L, LDH 1773. 86 ± 728. 286U/L. After 24 hour reperfusion, the level of biochemical enzyme: experimental group ALT 91.57 ±10.907U/L, AST 315.71 ±73. 785U/L,LDH 647. 00 ±232. 571 U/L;control group ALT 187.43± 27.597U/L, AST 477.00 ± 58.304U/L, LDH 498.71 ± 209.093U/L.3. The observation and comparison of hepatic cells under light microscope: Under light microscope, the degree of cytoclasis of experimental group wasslighter than that of control group at each time point.The count of complete hepatic cells: The number of hepatic cells of experimental group was more than that of control group at each time point. After 1 hour and 6 hours of reperfusion, the compare between experimental group and control group has significance Statistically (p <0. 05). After 1 hour,6 hours, 24hours of reperfusion the number of hepatic cells of experimental group 144. 81 ± 14.44/hf,110.81 ±15.85/hf,107.24±9.54/hf;controlgroupl34.00± 12.57/hf, 101.48 ± 12.66hf, 105.90 ± 10.89/hf.4. The detection of the expression of ICAM - 1 in liver tissue;Brown yellow particles were seen as positive staining, which lied in cytoplasm or cellular membrane. In experimental group, the vessel wall of central veins and partof peripheral hepatic cells were stained slightly and there was weak expression in the endothelial cells of sinus and Kupffer cells. In control group, the vessel wall of central veins and part of peripheral hepatic cells were stained deeply and there was strong expression in the endothelial cells of sinus and Kupffer's cells. The results manifested that the ICAM - 1 expression of control group was significantly increased than that of experimental group after hepatic ischemia reperfusion (p <0.05). After 1 hour,6 hours, 24hours of reperfusion, the integrated OD average of experimental group 20.94 ±2.9,42.68 ±3.0,32.94 ±2.5;control group 26.44 ±2.3,47.72 ±2.9,37.50 ±2.4.DiscussionFat liver is caused by metabolic disorder which is due to the excessive free fatty acid inputted into liver, the increasing free fatty acid synthesized in liver or triglyceride synthesized by carbohydrate, the decreasing of (3 oxidation of fatty acid in hepatic mitochondria, the decreasing of the synthesis and secretion of very low density lipoprotein and the transporting disorder of triglyceride, etc. Fat is accumulated in hepatocytes which makes swellen of hepatocytes, widen of hepatic sinus and excursion of nucleus so that hepatic microtubule system is compressed that caused microcirculation disturbance. Microcirculation disturbance is also the basic pathophysiological change and the fundamental reason of hepatic failure after operation and primary nonfunction after transplantation. The disbalance of nitrogen monoxidum (NO) and endothelial factor results in capillary construction, white cell aggregation, which cannot obstruct lumens completely , but can damage hepatic microcirculation, prolong the time of ischemia. Thus Kupffer cells and neutrocytes are activated and produce inflammatory cyto-kines and oxygen free radicals which lead to further injury. The mechanism of oxygen free radicals impairment is lipid peroxidation reaction. Because the hepatocytes which have fatty degeneration are more sensitive to free radicals and inflammatory factors, we can discern that the injury of fat liver ischemia reperfusion is based on microcirculation disturbance.PGE1 can increase the blood flow and the level of ATP, accelerate the re-covery of mitochondria s respiratory function after reperfusion and enhance the a-bility to resist free radicals'injuries, protect the cellular membrane. In the experiment , we infused PGEl constantly from 15 minutes before ischemia to 1 hour after reperfusion. The results showed at 1 h, 6h and 24h after reperfusion the level of AST and ALT in experimental serum are lower than that of control serum, the expression of ICAM - 1 also has significant difference. All of those could indicate that the continuous infusion of PGEl before and during operation can obviously protect liver from ischemia reperfusion. The possible mechanism of protection: (1) PGEl can increase hepatic blood flow through acting on hepatic vascular smooth muscle. PGEl cannot dilate portal vein, that is to say, it cannot affect portal venous pressure, so it cannot decrease hepatic blood supply when the hepatic microcirculation is changed. First, PGEl can inhibit protein kinase C (PKC) , lighten platelet aggregation and thrombogenesis, enhance va-sodilatation, cut down the sensitivity of vascular smooth muscle to vasoconstrictor substance. Second, PGEl can step down microviscosity, improve fluidity, reduce neutrophils aggregation in hepatic sinus and decrease postsinus resistance. At last, PGEl can diminish ICAM - 1 expression in endothelial cells, thereby inhibit the adhesion of neutrophils, endothelial cells and Kupffer cells, ameliorate microcirculation. (2.) PGEl can bind with special receptor in hepatic cellular membrane, prohibit platelet aggregation, restrain white cell adhesion and reduce the injury mediated by cytokine;PGEl can inhibit the activation of xanthinoxidase, decrease production of oxygen free radical;PGEl can relieve the containment of intranuclear histone to DNA synthesis through protein kinase system, then promote hepatic cell regeneration.After ischemia reperfusion, massive neutrophils immigrate into hepatic cells by adhesion to endothelial cell. During the course, ICAM - 1 as one of important adhesion molecules play certain role. ICAM -1 belongs to immunoglobulin superfamily of cell adhesion molecules. In normal hepatic tissues, sinus endothelial cell, thymus dendritic cell and tonsil endothelial cell there is low - level expression of ICAM - 1. ICAM - 1 can promote the action of lymphocytes and monocytes with endothelial cell, thrombogenesis,inflammation course and tumor metastasis, etc. In the experiment, ICAM - 1 expression in hepatic tissues ofexperimental group is weaker than that of control group, which demonstrates PGE1 can protect hepatocytes through down regulation of ICAM - 1 expression, decreasing cell facto adhesion.ConclusionsThe results of the experiment show that the continuous infusion of PGE1 before and during operation has obviously protection to fat liver ischemia reperfu-sion. The mechanism of action may be that PGE1 inhibit ICAM - 1 expression, diminish inflammatory cell adhesion and reduce destruction of hepatocytes, thereby it can protect liver. We can conclude from the above that it is significant to protect liver through direct PGE1 infusion into hepatic circulation. In the same time, PGE1 is so safe and its side effect is so small that it can be hoped to be used in clinics.