Proteomic Approach To The Mechanism And Biomarkers Research Of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease(NAFLD) is an increasingly recognized cause of liver-related morbidity and mortality.The histologic spectrum of NAFLD ranges from steatosis liver alone to nonalcoholic steatohepatitis(NASH),fibrosis,and cirrhosis. Steatosis is generally a benign disease,wheras NASH may be complicated by progressive end-stage liver disease.However,the pathogenesis of NAFLD is still unclear.It appears that oxidative stress,mitochondrion disfuntion and adipocytokines involve in the patheogenesis of NAFLD.It still needs further elucidate the factors that lead to progressive hepatocellular damage.The proteomic is a new approach which is used to analyze the complements and functions of proteins at the particular period of the body.It is closely associated with the physiology and pathology of body and can provide a theoratical basis in disease research.Meanwhile,the liver biopsy is an invasive examination and the liver tissue sample is too small to be applied to proteomic research.Therefore,we based on the NAFLD model which is induced by methionine and choline deficient diet in mice and combined proteomic approaches and bioinformatics analysis to systematically elucidate the liver proteomes during the development of MCD-induced NAFLD which may help clarify the pathogenesis of NAFLD.By using serological proteome analysis technology,we identidied a variety of autoantigens which were closely associated with the pathogenesis of NAFLD.It will attribute to discover novel biomarkers for noninvasive diagnosis NAFLD.We detected differently expressed proteins participating in the progression of NAFLD by using difference in gel electrophoresis(DIGE) and MALDI-TOF/TOF technology.Male C57/BL6 mice were fed a methionine/choline deficient diet for 2,5, and 8 weeks respectively,at which times steatosis(SS),NASH and then NASH combined early fibrosis(NCEF) were evident.The mice receiving the normal diet for the corresponding durations served as controls.There were 58,82,and 162 different proteins in the stage of SS,NASH and NCEF respectively.These differently expressed proteins were mainly located in the mithochodria(52.2%),following by excellular region(13.4%),endoplasmic reticulum(10.4%),plasma(9.0%),nucleus(9.0%), peroxisome(4.5%) and unkonwn location(1.5%).Biological functions and network analysis of these proteins exhibited phase-specific characteristics during evolution of the disease.The metabolic enzymes involved in free radical scavenging,fatty acidβ-oxidation and gluconeogenesis were down-regulated during the pathogenesis of NAFLD,wheras those proteins involved in the lipogenesis and cytoskeletal proteins were up-regulated as well as stellate cells marked proteins including desmin and glial fibrillary acidic protein.More importantly,lipid metabolic disorder runs through the pathogenesis of NAFLD.The pathway analysis of these proteins identified that Akt/PKB pathyway and p38MAPK pathyway were activated in the stage of NCEF.Firstly,we validated the reliability of the DIGE data both at protein level and mRNA level by using Western blot and RT-PCR.Secondly,we verified that pho-Akt and p38MAPK were up-regulated in the stage of NCEF.As is a new adipocytokine,visfatin has been paied more and more attention by its mimicking insulin functions and promoting the inflammation. Furthermore,we identified that visfatin was up-regulated in NCEF not only in the liver tissue but also in the serum by using immunohistochemistry and ELISA.We supposed that visfatin may involve in the pathogenesis of NAFLD by increasing hepatic ligenesis enzymes and decreasing gluconeogenesis enzymes.We also identified 18 autoantigens which were associated with NAFLD by using SERPA and found that there is no correlation between the autoantigens with their expressions in the pathogenesis of NAFLD.We further confirmed the antigenicity of alpha-enolase by using ELISA with recombinant protein in the serum of NAFLD patients.The results showed that the frequency of the autoantibodies to alpha-enolase was positively related with the level of liver injury.Therefore,we screened a series of autoantigens relating to the pathogenesis of NAFLD.It will attribute to discover noninvasive biomarkers of NAFLD in the future.