Comparative Proteomic Study Of Experimental Cirrhosis And Effect Of Fuzheng Huayu Decoction On Plasma Proteome

Part oneHepatic comparative proteomic study of cirrhotic rats induced by CCl₄Background and aims: Cirrhosis is a popular chronic progressive disease in China,and how to cure it is a major task of the Chinese medical specialists. Liver fibrosis isan indispensable pathological status during the development of cirrhosis. Proteomicsis a novel method to determine the constitution of proteins and to analyze the dynamicchanges of the expression and modification of proteins from an integrative level, bywhich the interaction and relationships among proteins are studied and the functionsof proteins and the principles of cellular activities are discovered. Disease proteomicsaims to lay theoretic foundation for studying the mechanisms, prevention,pre-diagnosis and therapy of diseases. In the present study a rat model ofCCl₄-induced cirrhosis was constructed, a platform for proteome of liver tissue wasbuilt up, and the conditions for two-dimensional gel electrophoresis of rat liver tissuewere determined. By analyzing the proteome of the rat model of CCl₄-inducedcirrhosis the study laid a basis for the further research of liver fibrosis, and cirrhosis.Methods: Male S-D rats were randomly divided into three groups: the control group(n=6) was injected with olive oil into abdominal cavity, cirrhotic animal model group(n=10) was treated in the same way with CCl₄(CCl₄/olive oil:v/v=1/1) 1ml/kg twicea week and for 8 weeks. The degree of liver fibrosis and inflammation grade wasobserved using histopathology study, collagen were visualized by Masson-trichrome--staining.α-SMA positive cells in hepatic sinus were counted to observe the activationof hepatic stellate cell. After two-dimensional gel electrophoresis the gels fromcirrhosis group and control group were analyzed by image analysis softwarePDQUST to match same points. Twice or more different content were considereddifferent points. Those points were digested, analyzed with MALDI-TOF-TOF-MSmass spectrograph and searched in protein database. The expression of protein wasidentified by immunoblotting test.Results: Typical fibrotic septa and pseudo-lobule were observed in cirrhotic animal model. The expression ofα-SMA positive cell increased. The reproducibility wassatisfying, proteins had approximately the same distribution and background. Afterprotein digestion in situ and mass spectrometric analysis, 33 points corresponding to28 proteins were identified. In those different proteins, 5 proteins only appear in livercirrhotic animal model group, 2 proteins only appear in control group. 12 proteinsexpression was promoted and 9 depressed in cirrhotic animal model group. Thoseproteins function in cell proliferation, energy metabolization, substancemetabolizetion and transport of acting enzyme, cytoskeleton and protein movement,oxidation stress, endoplasmic reticulum stress and hot shock stress respectively. Thechange trend of alpha crystalline B chain was further approved by immunoblottingtest.Conclusions: We successfully established a cirrhotic rat model. The change of thoseproteins was considered integratively, from which we obtained the characteristicchange in the liver tissue of cirrhotic animal induced by CCl₄. On the progress ofcirrhosis, the expression of diverse proteins may participate in the occurring anddeveloping of cirrhosis in the ways of cell proliferation, energy metabolization,substance metabolization, cytoskeleton structure and protein movement, and stressrespond.Part twoPlasma comparative proteomic study of cirrhotic rats induced by CCl₄Background and aims: Many diseases may affect proteins in plasma. Thecharacteristic changes are important to diagnosing diseases and monitoring curativeeffect. So far, only few plasma proteins are used in clinic as markers of some diseases.With the development of plasma proteome, more plasma proteins are entering ourview. Studying the change of plasma proteins of hepatic fibrosis and cirrhosis helps tounderstand the pathologic process, diagnoses and curative effect. In this research, weestablished a plasma proteomic technologic platform, on which we tried to establishthe methods of eliminating rat plasma albumin and globulin, and to optimize theconditions forplasma two-dimensional gel electrophoresis. It can be served as a basisfor further researches in cirrhosis by studying the change of plasma proteome of CCl₄-induced rat cirrhostic model.Methods: Cirrhotic rats were induced by CCl₄ injection to abdominal cavity, whileolive oil was administrated in the control group. Eight weeks after injection, bloodwas collected and plasma from the same group was mixed. Albumin andimmunoglobulin were wiped off. After two-dimensional gel electrophoresis, gel mapswere analyzed with PDQUST. Points from model group and control group werematched, and twice or more different content were considered different points. Thosepoints were digested, analyzed with MALDI-TOF-TOF-MS mass spectrograph andsearched from protein database. The expression of protein was identified byimmunoblotting test.Results: Albumin/IgG Removal Kit can wipe off most of albumin andimmunoglobulin from plasma. After the wiping, the number of gel map protein pointsincreases from 260±15 to 598±42. The repeatability was satisfying, and proteins hadapproximately same distribution and background. After protein digestion in situ andmass spectrometric analysis, 20 points are identified which belongs to 16 kinds ofproteins: plasma glutathione peroxidase, glutathione peroxidase precursor,apolipoprotein A-â…£precursor, apolipoprotein E, haptoglobin, prealbumin,retinol-binding protein, alpha-1-antitrypsin, serine/threonine-protein kinase MARK1,alpha-1-Macroglobulin, complement component 4, inter-alpha inhibitor H4 heavychain, transferring, liver regeneration-related protein LRRG03, vimentin, RIKENcDNA1700025B16. The change trend of haptoglobin was further approved byimmunoblotting test.Conclusions: Wiping off albumin and immunoglobulin from plasma can increase theprotein points display obviously, and optimize the plasma 2-DE map. The platformestablished in our study can be used in studying disease mechanism. In the liver ofcirrhotic rat model induced by CCl₄, those protein participate in cell defense andimmunity, protein transport and cytoskeleton and transformation, and acute phasereactive proteins. This hint the change of protein may be induced by the pathogenesis ofcirrhosis, those protein may take part in diverse ways during the occurring anddeveloping of cirrhosis.Part threeThe effect of Fuzheng Huayu Decoction on plasma proteome of cirrhotic rats Background and aims: Using proteomics we studied the role of Chinese traditionalmedicine compound on the differential expression of proteins in liver fibrosis andcirrhosis induced by CCl₄. The aim is to find the proteins involved in the certainphysiological and pathological progress, and to lay foundation for the early diagnoseand therapy for liver fibrosis and cirrhosis. Presently, Chinese traditional medicine hasshown obviously advantage in treatment of liver fibrosis. Fuzheng Huayu Decoctionhas nice clinic result in treating post-hepatitis cirrhosis and chronic hepatitis B liverfibrosis. The antifibrotic mechanism is various, and the compound works in amulti-component, multi-step, multi-arrangement, multi-target way. Proteomics opensup a new way in researching Fuzheng Huayu Decoction from an integrative level. Westudied the change of plasma proteome in the progress of cirrhosis in rat modelinduced by CCl₄, and tried to find how the multi-way function mechanism works intreating liver fibrosis.Methods: Male S-D rats were randomly divided into three groups: the control group(n=6) was injected with olive oil into abdominal cavity, cirrhotic animal model group(n=10) was treated in the same way with CCl₄(CCl₄/olive oil:v/v=1/1) 1ml/kg.Animals in Fuzheng Huayu group received compound intragastric administration inaddition of CCl₄ abdominal cavity injection. Eight weeks later, collagen was stainedby Masson-trichrome-staining and analyzed by image analysis software. Collagenfiber was half quantitively analyzed with image analysis software. After twodimensional gel electrophoresis, silver staining and analyzing gel image by softwarePDQUST 7.3, the diversity of protein points was analyzed with mass spectrometricanalysis.Results: The degree of fibrosis of Fuzheng Huayu group was much slighter than thatof model group. The collagen deposited in liver decreased (8.9%±3.7% vs. 12.4%±4.7%, P＜0.05). Wiping off most albumin and immunoglobulin from plasma can getmore satisfying repeatability. In this research, we got 10 evidently diversityexpression proteins. In those proteins, plasma glutathione peroxidase and itsprecursor, prealbumin, haptoglobin, precusor of ApoA-â…£, complement component4, inter-alpha-inhibitor H4 heavy chain and serine/threonine-protein kinase MARK1exhibited less expression in cirrhosis group and more expression in Fuzheng Huayugroup. The expression of proteins related to regeneration of liver and vimentinincreased in cirrhosis group and decreased in Fuzheng Huayu group. Conclusions: Animal trial confirmed that Fuzheng Huayu Decoction had the functionof anti-fibrosis and anti-cirrhosis. The plasma proteomic study on the pharmaceuticalintervention of Fuzheng Huayu decoction explained the mechanism of its anti-fibrosisand anti-cirrhosis function. It is discovered that Fuzheng Huayu decoction mayfunction as anti-fibrosis agent through accelerating the protein composing,anti-oxidation, adjusting the cell proliferation and transformation. Proteomicsexplores a new way in the research of inspecting the curative effect and mechanism ofChinese traditional medicine compound.