Studies On Molecular Mechanism Of Rat Liver Regeneration Using The Techniques Of Proteomics And Metabonomics

Liver is one of the most important organs involved in physiological, pathological, and toxicological events. It is well known that the liver has the capacity to regenerate and restore its original size and function after partial hepatectomy (PHx), injury and hepatic diseases. Liver regeneration continues to be an important focus in mammalian liver research. Acceleration of liver regeneration could be of great clinical benefit in various liver-associated diseases. However, at present little is known about therapeutic interventions to enhance this regenerative process. Our limited understanding and the complexity of the mechanisms involved have prevented the identification of new targets for treatment. Previous studies have identified many hormones, growth factors, cytokines and specific genes involved in this process. However, the molecular mechanism of liver regeneration, especially the mechanism of termination and remodeling after partial hepatectomy was not clear yet. Previous works usually adopted the regular molecular biology method to study the expression difference of time and space course, signal transmission pathway, regulating mechanism that control the replication of hepatocytes, which make the works limit. As a result, the complexity of the regeneration mechanisms which is from priming, proliferation to termination is not of overall understanding.Proteins are central to our understanding of cellular function and disease processes, and without the participation of proteins, the substantial metabolism will not exist. Proteomics is the large-scale study of proteins, particularly their structures and functions, which provide a very effective approach to explore differentially expressed protein, expression level, modification state and even the interaction among proteins during the liver regeneration. Differential proteomics, the comparison of distinct proteomes (e.g. normal versus diseased cells, diseased versus treated cells, etc) is of paramount importance.Gene→Pr→Metabolite Life activities rely on metabolism including signal synthesis and release, cell communication, energy transfer, cell proliferation and differentiation etc. Metabonomics can reveal the discipline of metabolism, therefore it determines the final direction of'omics'research. Followings are the advantages of metabonomics: Magnify of slight change of gene and protein expression in metabolite; high throughput: testing interval lasts 5s to10s for single sample; huge information receive; reflection of the final results of metabolism events. Based on these facts, the research can be accomplished by means of omics method with high throughput. A comprehensive analysis of liver regeneration includes protein level and metabolite level can be approached through proteomics and metabonomics respectively: find and screen the expression chart and variation discipline of differential proteins by differential proteomics; find metabolism chart and variation discipline through relevant biomarker by means of mass spectrometry of the micromolecules during different phases in regeneration process. As a result of the combined analysis of differential proteins and metabolites, molecular mechanism of liver regeneration will be explained in different phases of liver regeneration.SectionⅠ: Differential proteomics analysis of the cytoplasmic proteins in rats during liver regeneration after partial hepatectomyIn this study, liver tissues were gained from partial hepatectomy (PHx), a classic experimental model of rapid liver cell proliferation. The proteomic changes in rat liver cytoplasm were analyzed in two groups, 70% PHx test group and sham-operation control group, at 3h,12h,3d,7d,11d and 14d after 70% PHx. By a modified two-dimensional gel electrophoresis, the differential protein spots were recognized using PDQuest7.4 software. Then the protein spots(12h,7d,11d) were identified by matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and/or tandem mass spectrometry reconfirmation. The data from MALDI-TOF/TOF MS were analyzed by using the program MASCOT v. 1.9 against SWISS-PROT database with GPS explorer software. To confirm these results, various important identified proteins (11d) were validated by Western-blot. The result shown that 15 differentially expressed proteins were identified based on a changing of threshold of 2-fold, and among them 13 proteins were identified by mass spectrometry successfully. We found the 13 proteins were associated with carbohydrate metabolism, lipid metabolism, amino acid metabolism, anti-oxidation, respiratory chain and oxidation-phosphorylation, biotransformation and other metabolic pathways. However, more attention was paid on two proteins (11d): peroxiredoxin 1 and peroxiredoxin 6 that both have the anti-oxidant capacity. The expressed level was shown in a contradistinctive manner. Compared with sham-operation control group, the former was up-regulated, the latter was down-regulated at 11d after 70% PHx. The two anti-oxidant proteins maybe play other important roles during the termination and remodeling progress. SectionⅡ: Metabonomics research in rats during liver regeneration after partial hepatectomyMetabolism is the total (bio) chemical changes in living organisms and metabolic activities are the basic features of living systems. Metabonome is the dynamic complement of the endogenous metabolites within the integrated biological systems. Metabonomics is the branch of science concerned with the quantitative understandings of the metabolite complement of integrated living systems and its dynamic responses to the changes of both endogenous factors (such as physiology and development) and exogenous factors (such as environmental factors). Metabonomics is a true multidisciplinary subject combining the physics-based analytical chemistry, mathematics-based chemometrics and biochemistry-based biological sciences. The life activities usually exist in the metabolism level in the cells, so metabonomics is thought the termination of the'omics'. The liver tissue and blood were treated with acetonitrile in two groups, 70% PHx test group and sham-operation control group, at 6h,12h and 7d,11d after 70% PHx. The data was obtained using Agilent LC/Q-TOF-MS and analyzed through PCA, PLS. Compared among test groups and with sham-operation control group, the biomarkers were identified. The biomarkers were associated with carbohydrate metabolism, lipid metabolism, amino acid metabolism and nucleic acid metabolism.Discussing about metabolic discipline and regulating mechanism of liver regeneration with'omics'data. Proteomic and metabonomic techniques are ideal for clarifying quantitative protein and metabolite changes in physiological and diseased conditions, respectively. In liver research, however, proteomics and metabonomics are still in their infancies and no studies have been performed so far comparing proteomic and metabonomic profiles in liver regeneration. The present study provides new insights into liver regeneration, which could not have been obtained by studying individual signaling pathways.The advent the two high-throughput experimental technologies transformed biological research from a relatively data poor discipline into one that is data rich. After the data analysis, we found the energy demanding increased during the early phase of liver regeneration. The level of D-Threitol was increased and the enolase-1 level was also increased, which indicated glycometabolism was strengthened. During the progress of liver regeneration, phospholipid as the main ingredient of cell membrane, its metabolic level experienced the course which is upward in the early phase of liver regeneration and downward in the late phase of liver regeneration. The level of Phytosphingosine,LysoPC(18:2(9Z,12Z)) is increased in the early phase and LysoPC(16:0),LysoPC (20:4(5Z,8Z,11Z,14Z)) is decreased in the late phase. The expression level of Prx-6 which has the PLA2 activity is also down-regulated after 70% PHx. An important challenge that is faced by us today lies in interpreting these large-scale data sets and thereby deriving fundamental and applied biological information about whole systems.