| Morbidity of liver diseases is comparatively high in China, including hepatitis, hepatocirrhosis, carcinoma, Congenital and metabolic liver disease, routine medications and surgreys could not get satisfied endpoint in therapy. Liver transplantation as the only method could cure liver diseases was in ever-growing demand. But owing to the complexity and difficulty in surgrey and perioperative management, donor liver more or less might endure injure in the transplantation procedures that leading to poor or delayed graft function and even primary graft non-function(PNF). Poor graft function was closely relevant to the poor preservation and ischemia-reperfusion (I/R) injure. Literatures indicated ischemia-reperfusion was one of the most important causes that may induce poor early graft function and affect long-time outcomes. The great disparity between the demand of potential transplant patients in need of liver donors and the availability of grafts urgently demand a satisfied method of graft preservation, and the mechanism of ischemia-reperfusion injure spontaneously became a important research point. As ischemia-reperfusion injure could induce damage and apoptosis to hepatocyte, avoiding ischemia-reperfusion injure was important to the therapy and prognosis.Ischemic preconditioning (IPC) was a procedure to decrease tissue ischemia-reperfusion injure in long-time ischemia by pretreatment of short-time ischemia. Murry raised the conception of pretreatment of ischemia for the first time in1986. The research by Laske and Szmagala revealed pretreatment of ischemia may prevent injure of myocardial ischemia in Percutaneous transluminal coronary angioplasty (PTCA) and cardiosurgery. Research from Tossy showed IPC also had a protection in ischemia-reperfusion injure in renal tissue. Literatures showed that IPC could apply to many other organs, such as brain, liver, small intestine, skeletal muscle, kidney and lung, and the existence of cross ischemic tolerance phenomenon between the various tissues and organs. For example, transient ischemic in skeletal muscle or kidney could effectively reduce the volume of myocardial infarction induced by coronary vascular obstruction. Protective effects and mechanism of IPC against I/R in liver autotransplantation in rats transplantation is not clear, it may reveal the way to decrease hepatocyte injure in liver transplantation.Interleukin-6(IL-6) is one of the core members of the cytokine, with many kinds of biological stimulating activity, widely expressed on activated B lymphocyte, resting T lymphocyte, myeloma cell, hepatocyte, natural killer cell and myeloid leukemia cells. Human IL-6is composed of213amino acids, mainly produced by mononuclear macrophage and Fibroblast, could be induced to produce by Tumor necrosis factor (TNF) and enhanced the adverse effect of TNF in return. IL-6can induce T, B lymphocyte differentiation, stimulate the synthesis of acute phase protein in hepatocyte, catalyse and amplify the inflammatory response and toxicity effects and cause damage to tissues.As a pleiotropic cytokine, IL-6is one of the important factors involved in the regulation of immunity and inflammatory reaction, is the main medium of the body reaction to infection and tissue injury, cooperating with colony stimulating factor (CSF), it can promote the growth and differentiation of marrow cells and enhance the cracking function of natural killer cells. Both viral and bacterial infection could induce the increased expression of IL-6, but this increased expression was also observed in a variety of autoimmune diseases and cancers. As part of the immune system, the raised level of serum IL-6play a important role in eliminating virus. Literatures also indicated the level of serum IL-6positively correlated with the degree of infection and inflammatory reaction, that make IL-6a sensitive index for infection and inflammatory reaction.This study firstly constructed the model of liver autotransplantation of ischemia reperfusion injury in rats. Histologic examination for liver tissue injure degree, laboratory tests for serum Alanine aminotransferase(ALT), Aspartate aminotransferase(AST), Superoxide Dismutase(SOD), Malondialdehydem(MDA) and IL-6, were both conducted in experiment and control group. Serum level of IL-6, TNF-a and IL-1β were compared between two groups, explored its correlation to histologic degree of liver tissue injure and the potential action mechanism. By exploring the mechanism of I/S injure and the injure protection method, this study aimed to supply a evidence for therapy and protection in I/S injure in liver transplantation.Objective To investigate the effect of ischemic preconditioning on ischemia-reperfusion (I/R) injury in orthotopic rat liver transplantation and the changes of Interleukin-6(IL-6), Tumor necrosis factor-a(TNF-a) and Interleukin-1β(IL-1β) levels in liver, we discussed the pathological changes and protection mechanisms of ischemic preconditioning on ischemia-reperfusion injury in orthotopic rat liver transplantation.Methods The portal vein infusion of autologous liver transplantation model was established in rats.72rats were randomly divided into three groups:(1) normal rats control group (group A);(2) the rats liver transplantation group (group B):derectly recept autogenous liver transplantation.(3) the ischemic preconditioning rats liver transplantation group (group C):clip rats liver hepatic artery, portal vein hepatic vein and portal vein in15min, reply liver blood flow, autogenous liver transplantation was performed1h later. Cotton with ether was used for open inhalation anesthesia for rats. Conventional skin preparation and sterilization were conducted before making the median incision. Bowel was moved inside out with a cotton swab to get a better surgrey field. Isolated and cut falciform ligament with tissue shear, raised the left and middle lobe of liver rightward, identified, cut and ligatured the esophageal ligament of liver, isolated liver anticlockwise from here. Firstly separated upward along the esophagus, cut the left triangle ligament, suture ligated and cut the left phrenic vessels. Then raised the right lobe of liver and cut the right triangle ligament. Isolated upward along Suprahepatic vena cava(SVC) to phrenic vein hiatus, then separated downward along cava to the level of left renal vein after ligaturing and cutting the lumbar vein. Separated the ligamentum hepatorenale, ligtured and cut right suprarenal vein. Reseted the liver and isolated the Infrahepatic vena cava(IVC) to the above level of left renal vein. Open the hepatoduodenal ligament and indentified the hepatic artery(HA), isolated it and embeded the silk prepared to block the artery. Continue to free portal vein(PV) from confluence of inferior mesenteric vein and splenic venous to the hilar, pylorus vein was ligatured meanwhile. Finally, hepatogastric ligament was bluntly separated to free each lobe of liver, and the procesure of free of liver accomplished. The diluted heparin(35U/L)3mL were irrigatted into portal vein by puncture needle to sustain fully heparinization. Puncture abdominal aortic proximally between common iliac artery and the left renal artery, vascular clamp was used to fix the needle, then blocked the artery above celiac trunk and below the puncture site. with vascular clamp. Then blocked the SVC and IVC at the level of right renal vein with vascular clamp, cut the vein wall about lmm on the IVC in the longitudinal direction as the outflow tract. Infused4℃heparin (12.5U/mL) in Ringer’s lactate20mL through the PV and abdominal aortic puncture site, at the same time,4℃Ringer’s lactate was pouring the liver surface to get rapid cooling. When the liver turned into the color of yellow soil, ended the perfusion and pull out the needle, repaired PV and abdominal aortic puncture point. The outflow tract on IVC was sutured with8-0Prolene. Blocked the hepatic artery on the lighted line and release the vascular clamps on PV, SVC, IVC and abdominal aortic to regain the blood supply to liver and finish the anhepatic phase. At the same time,38℃saline was pouring the liver surface for rapid rewarming. After opening the hepatic artry, continuous abdominal closure was conducted if there was no intra-abdominal hemorrhage. When the surgrey accomplished, lights were used to rewarm the rats until they got through anesthesia recovery period and arisesed.Anaesthesia was conducted in rats of each group2h,6h,12h and24h after reperfusion,5ml blood was collected through the inferior vena cava. Blood was centrifuged and serum was preserved in-80℃refrigerator for later test of ALT and AST. Cut off liver tissue and wash away the blood with Ringer’s solution, and tissue was sliced into a thickness less than0.5cm, partly fixed in10%neutral formalin, the others preserved in-80℃refrigerator.HE staining was performed with paraffin section of the rats liver12h after operation to observe the pathology changes of liver and changes in the microstructure of the hepatocyte mitochondria by electron microscopy.2h,6h,12h and24h after ischemia reperfusion in each groups, the level of serum Alanine aminotransferase (ALT), aspertate aminotransferase (AST) and liver tissue superoxide dismutase (SOD) activity and Malondialdehyde, Malondialdehyde (MDA) content in rats were detected. The level of interleukin-6expression after ischemia-reperfusion Injury in orthotopic rat liver transplantation was detected by polymerase chain reaction. The levels of TNF-a and IL-1β following I/R injury in rat serum were measured by enzyme linked immunosorbent assay.Data was analyzed using IBM SPSS20.0for Windows software. Data were expressed as mean and standard deviation. The statistical significance of differences between groups was analyzed using factorial design ANOVA. P value<0.05was considered to be statistically significant.Results1. The rat serum ALT and AST levels after reperfusion. We tested ALT and AST level of the rats serum in each groups.The results show that the content of ALT and AST level in group B and group C reach the top at2h after reperfusion.The ALT and AST levels of rat serum were gradually reducing in24h after operation.In each period,the levels of serum ALT and AST in group B and group C is obviously higher than that in group A. AST and AST levels in group C were lower compared thanaaaa that in group B at each time point, whose differences were statistically significant.(P<0.05).2. The liver pathological tissue changes stain in HE. The result show that both the arrangement of liver cell line and liver sinus endothelial cells are normal.In12h after reperfusion,the liver cells around the central vein become markedly swollen,liver blood sinus become narrow,and the structure of hepatic lobule appears unclear,which is company with red blood cells and blood clots and infiltrated by inflammatory cells in group B.In the roup C,the structure of hepatic lobule is essentially normal,the liver cells appears slight swell,the liver tissue has no obvious change.3. The injury in ultrastructure of liver cell12h after ischemia reperfuson in group B,the mitochondria volume appears to be different size and markedly swollen state,which is round like with degeneration in vacuolar.It shows that crest reduction,fault and disappearance can be found in some serious ones,endoplasmic reticulum around structures were not clear.The mitochiondria volume is a little swollen, which appears to be oval and rupture in internal crest,which is most normal arrangement.The structure of endoplasmic reticulum is still exsit.4. Ischemic preconditioning reduce the expression of IL-6in liver after ischemia-reperfusion. The result of RT-PCR shows that,a small amount of expression of IL-6could be found in group A,little fluctuation appears in each time. Expression of IL-6in group B and group C were higher than that in group A,whose differences have statistically significant (P<0.05).In group B, the expression of IL-6reachs the highest level in12h after reperfusion,and a downward trend appears after that. Expression of IL-6in all period of group A were lower than group B,whose difference at2h,6h,12h were statistically significant (P<0.05).5. The changes of rats serum TNF-α and IL-10level in each group The result of ELISA shows that,compared with normal control group,there is a different degree of rise in the rats serum TNF-α and IL-1β in group B and group C after ischemia-reperfusion injury,whose differences have statistically significant,the level reach the peak in12h after I/R, the level of rats serum TNF-α and IL-1β at6h、12h and14h in group C is lower than that in group B.6.The experiment conclusion of change in expression of SOD and MDA in liver tissue in each groups shows that,SOD activity of liver tissue in group B and group C were lower than that of group A.Each time,the expression of SOD in group C is slightly higher than that of group B,the differences of expression at6h and12h after operation were statistically significant (P<0.05).The expression of MDA in liver tissue in group B and group C were higher than that in group A.In each period, the expression of MDA in group C is slightliy lower than group B,whose difference was statistically significant (P<0.05).compared with group B, the expression of malondialdehyde is in the low lever in all period of liver tissue in group C,the activity of superoxide dismutase (sod) is higher,which prompt the level of free radical in group C is lower than that in group C.ConclusionsIschemic preconditioning has certain protection effect on ischemia-reperfusion injury of rat liver transplantation. Ischemic preconditioning can significantly reduce the expression of il-6, alleviate mitochondria damage in liver cell and has important potential clinical effect. |
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