Study On Changes Of Liver Protein Profiling During The Formation Process Of Nonalcoholic Fatty Liver

Object:The pathological change of non-alcoholic fatty liver disease(NAFLD) is caused by many factors and lead to the disease. However, in recent years, energy surplus draws more attention of researchers as one of the etiological factors besides the pathological change. Accumulation of high-energy substances in liver is a specific expression of metabolic syndrome, also a risk factor for the devolopment from non-alcoholic steatohepatitis to cirrhosis or cancer of liver. As the incidence of metabolic syndrome increased, NAFLD has aroused unprecedented attention. With the social development, environmental risk factors are clearly while the molecular mechanism and pattern of development of NAFLD is not clear yet, which brings some certain difficulties to the prevention and treatment. Therefore, in this study, we fed rats with high-energy material to form accumulation of high-energy substances and lead to metabolic syndrome and NAFLD. Two dimensional electrophoresis and mass spectrometry were used to detect the changes of proteins in different stages of NAFLD, expecting to find one or two kinds of functional protein molecules and pointing out some directions for further study.Methods:1. The establishment of animal model for metabolic syndrome:20 SD rats were randomly divided into two groups,one group was fed with normal diet and the other was fed with high-fat diet. The change trend of body weight,Lee's index,OGTT value,blood lipid level was observed. The pathological characteristics and subcellular damage under electron microscope were involved.2.Analysis of liver proteomics:The total liver protein was separated by two-dimensional gel eletrophoresis at indicated different timepoints. Protein expressed differently were selected.After in-gel tryptic digestion,the peptide mass footprints were drawn up by MALDI-TOF-MS and the protein database was searched to identify the proteins. Then,analyzed how the indicated proteins participated in the pathogenesis of the formation process of nonalcoholic fatty liver.Results:1.The body weights increased in group of high-fat diet in compared with the group of normal diet (P<0.05); TG level, TCH level and liver score were increased in group of high-fat diet in compared with the group of normal diet (P<0.05);the OGTT values were stable in group of normal diet while impaired in group of high-diet.2. Changes of pathology:The level of fatty deposition and fatty degeneration were much severe in liver which were fed with high-fat diet (P<0.05); At the subcellsular level, atrophy of hepatocyte microvillus, microbiliary dilatation,and the damage of hepatocyte mitochondria could be observed in group of high-fat diet under electron microscope.3. After the high-fat diet feeding, liver proteomics showed its regularities as follows:a. protein spots gradually decreased, and at the 18 weeks, the protein spots were no more than 80%. b. During the study, protein spots changes at different isoelectric point were the most obvious. Neuter protein spots showed a down trend, while acidic protein spots suddenly increased at 18 weeks. c.Four kinds of zymoprotein were effectively identified:methionine adenosyltransferase?, alpha?glutathione S-transferase, mu 2?NADH dehydrogenase and aldehyde dehydrogenase family 9, subfamily A1. The first three zymoproteins were gradually reduced, but aldehyde dehydrogenase increased along with the high fat diet feeding time.Conclusion:1. The SD rat model induced by high-fat diet can mimic the hepatic pathologic alterations of human NAFLD caused by metabolism syndrome. The alterations include:?Morphology:a. Body weight increased to obesity, as well as body fat and hepatic lipid. b. Hepatocyte microvesicular steatosis were formed. c. At the subcellsular level, microvillus of hepatocyte atrophied, followed by microtubules appeared in cholangiocyte, then the hepatocyte mitochondrial swelled.?Biochemics:Glucose tolerance showed abnormal. Blood triglyceride and total cholesterol also increased.2. After the high-fat diet feeding, liver proteomics showed its regularities as follows:?protein spots gradually decreased, and at the 18 weeks, the protein spots were no more than 80%.?During the study, protein spots changes at different isoelectric point were the most obvious. weeks, the protein spots were no more than 80%.?During the study, protein spots changes at different isoelectric point were the most obvious. Neuter protein spots showed a down trend, while acidic protein spots suddenly increased at 18 weeks.?Four kinds of zymoprotein were effectively identified:methionine adenosyltransferase I, alpha glutathione S-transferase, mu 2?NADH dehydrogenase and aldehyde dehydrogenase family 9, subfamily A1. The first three zymoproteins were gradually reduced, but aldehyde dehydrogenase increased along with the high fat diet feeding time. It indicated that with the fat depositing in liver, the anti-oxidizing ability came to diminished, while the ability to clear endogenous aldehyde group production were enhanced. Substance metabolism developed to anabolism.