| In liver surgeon, in order to reduce bleeding, often require the blocking of the hepatic blood flow, but a variety of vascular occlusion will definitely leads to liver ischemia-reperfusion injury. Ischemia-reperfusion injury not only will cause liver necrosis, but also affects the liver regeneration capacity, which is one of the important factors affect ing the success rate of the liver surgery and the survival rate of patients. With the continuous development of surgical techniques, there are a lot of patients with liver disease,like liver cancer, liver fibrosis, and fatty liver, receiving complex and larger range of liver resection surgery, even the liver transplantation surgery. In spite of by further familiarity with liver anatomy, especially through the Intrahepatic biliary and vessels, reducing venous pressure during anesthesia in surgery as well as the various cutting and coagulation surgical instruments,it is possible to make non surgical hepatic blood flow occlusion become a reality, however,nowadays reducing blood loss by blocking blood flow, is still a common means of liver surgery. The common methods to block are Pringle, total hepatic ischemia,selective hepatic inflow occlusion,and so on. No matter which way of a vascular occlusion is used,it can to some extent reduce the incidence of liver surgery blood loss. But ischemia-reperfusion injury is still a complicated problem which we must face with. In addition to the means of hepatic serum enzymes in detection, histopathology, and CT, is there possibility to find a new means of detection, which is a more comprehensive judgment of liver’s function after liver resection and liver transplantation in patients, even the effect of other related organs? So as to provide clues to better and further diagnosis and treatment.Hepatoduodenal ligament with oppressive control of hepatic arterial bleeding Pringle, is the most widely used clinical measure to control bleeding after cutting liver, and this method can be used for almost all liver surgery because it is simple, easy, and having precise effect. This study uses The Pringle maneuver to construct hepatic ischemia-reperfusion model,and it is a higher representative for the study of ischemia and reperfusion.The metabolomics was developed in the1990s, through in vitro factors to analyze quantitatively the changes in vivo metabolites, looking for the mutual relations and dynamic law between the changes in the type and quantity of metabolites and the changes in physiology and pathology. Metabolomics focuses on the pathway of endogenous metabolites in biological whole organ or tissue,whose effect of internal or external factors is paied the same attention.Besides,the law of change over time is also included. By analyzing the metabolic samples of metabolite and using software combined with models to analyze differences in metabolites to identify potential marker (biomarker), so as to provide the basis for the diagnosis of the disease. Metabolomics is a new subject following genomics, transcriptomics, and proteomics, and it can be used as a new clinical biochemical techniques in the detection, diagnosis and treatment of certain diseases. Urease pretreatment-gas chromatography-mass spectrometry (GC/MS) is an important method in metabolomics research, which has high sensitivity and it’s able to analyze highly complex biological fluids. This method not only has the sample separation, identification and quantification completed quickly once for all, to a large extent, it can also promotes the overall and dynamic analysis of the bulk material. GC/MS is capable of one-time determination of hundreds of molecular weight less than1000molecules, and can outline instantly the situation of synthetizing and using small molecule metabolites in the liver. Similar with routine used in clinical serum enzymes in liver function tests, pathology and CT means,GC/MS reflects the general situation of liver function in some stages, as well as the information of other organs, such as the kidney, heart and other organs. Furthermore, It can even be used for drug metabolism compared before and after treatment. Currently, there is temporarily no report on injured assessment of metabolomics applied to liver postoperative ischemic reperfusion.The liver is the body’s largest metabolic organ, known as "substance-generation metabolomics",which is able to metabolize sugar, protein, fat, vitamins and hormones. It play a very important role in the maintenance of environmental balance in the body.Now, there is lack of a way to be able to reflect the body’s metabolism in the overall level, and comprehensively test the dynamic distribution of metabolite in liver during every period, so as to assess the treatment of disease and prognosis. At present, the use of metabolomics for liver transplantation, cirrhosis, hepatitis, fatty liver, etc. has been gradually carried out. The purpose of the present study is that using the method of metabolomics makes comparison with the chang of metabolites before and after hepatic ischemia-reperfusion injury,in order to have a command of injury, outcome and the trend of the metabolism after liver surgeon.On other hand, this study is combined with the conventional method of serum enzymology, pathology and so on to get better detection of preoperative and postoperative liver conditions, and comprehensive detection of liver.Metabolomics, whose research objects are urine, blood, and tissue homogenates,has an easy access to specimens and is convenient to monitor. The metabolomics now have two theories:nuclear magnetic resonance (NMR) and mass spectrometry (mass spectrometry).GC/MS is currently the most widely used separation technique, and it has stronger analytical capabilities and higher sensitivity. This study uses metabolomics GC/MS to compare and analyze the rat liver before and after ischemia-reperfusion injury, in order to seek for the important markers of early detection and diagnostic. This study is of a certain value in clinical applications. Materials and Methods1. To construct models of I/R on rats.Take SPF grade pure line SD (Spargue-Dawley) male rats which are220-250g.1.1GroupThe rats were randomly divided into a control group (20) and the experimental group (60), the experimental group were randomly divided into three groups according to the design time points (postoperative days1,3,5)(n=20).1.2Blocking the Hepatic portalSD rats of the experimental group etherized, whichever abdominal incision into the abdominal cavity. Fully reveal the first hepatic portal, use vascular to clip running through the hepatic artery and portal vein, common bile duct, clipping30minutes after the release.2. The collection of urine samples2.1. Place the rats in metabolic cages to collect urine for12hours before laparotomy,each about4ml.2.2Store these urine samples at refrigerator at-20℃.After standardized treatment in one week, carry out detection of metabolomics GC/MS.3. The detection of Serum enzymes3.1Each group were laparotomy to take the portal venous blood, about3ml each at a specified time.3.2Blood samples were allowed to stand for10minutes, centrifuged, and serum samples frozen at-70℃refrigerator, together with inspection, were all detected to get the measurement of serum AST, ALT, TBIL4. HE staining4.1After venous blood samples were collected, completely remove the liver tissue, hepatic pick and cut the middle of specimens, and placed them in10%neutral buffered formalin solution to fix. 5. Statistical analysis5.1Data were presented as median (interquartile range) and tested using the Kruskal-Wallis (K-W) test. Once a significant result was revealed by the K-W test, the Mann-Whitney U test was used for post-hoc pair-wise comparisons.5.2Principle component analysis (PCA):(PCA) was implemented to verify the distribution of variables for study groups as well as confirm the time trend of variable changes. The significance level a was set at0.05. When multiple comparisons were needed, the significance level was adjusted to0.01. All analyses were carried out using SAS statistical software (version9.1.3, SAS13.Result1. Serum enzymes in indicators:The level of ALT, TBIL in1day after the group was significantly higher after3days decreased,and gradually returned to normal levels five days after surgery.2. Result of HE staining:under the rules of Suzuki’s,1day after surgery:Liver cell injury is more serious, more cell necrosis, significant congestion, congestion performance, we can see a small amount of vacuoles.3day after surgery:Better than1day, the cell recover gradually, restored hepatic sinusoid compensatory expansion, there is still a very small amount of vacuoles; even visible individual necrotic cells; very small amount congestive performance.5day after surgery:No vacuolation; no necrotic cells; still very little congestion, congestive performance. It has been restored to a normal level basically. Shame group:Clear lobular architecture, sinusoidal was the streak, closely arranged morphologically normal liver cells, nuclear stained uniform cytoplasmic.3. GC/MS:1day after surgery, lactic acid, glycerol-3-phosphate were significantly higher than the shame group. From3day to5day, the lever reduce to normal gradually.The level of Alanine and serine in one day after surgery are significantly reduced;gradually increased to near normal levels in3to5days after surgery.Pyruvic acid, threonine, oleic acid was not statistically significantly different.Conclusion1. After30min by pringle, Serum enzyme indicators reached a peak and the damage by HE tests were severe, the first day after I/R damaged. The third day, it become better. The fifth day, the level were normal practically.2. Using GC/MS to tests the urine samples by the rats which for I/R damaged:the trend of indexes of the lactic acid of glucose metabolism, the alanine, serine of protein metabolism and the glycerol-3-phosphate of lipid metabolism were similar with the trend were resemble with Serum and HE.3. Use GC/MS to test urine, can reflect the process which damaged by I/R really. Observed and contrasted the trend of urine indexes, was resemble with Serum and HE. Using GC/MS, not only can reach the sample easily, but also can observe the situation comprehensivly and Dynamically. GC/MS can become a new and Sensitive method to evaluate the liver. |
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