| Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver diseases, which can cause a variety of liver damages, including steatosis, steatohepatitis, fibrosis, cirrhosis or other liver symptoms. However, the molecular mechanisms of NAFLD pathogenesis remains unclear. Since proteomics studies reveal the protein levels of cell or organism under certain conditions or functional status, the application of the technical studies can provide NAFLD theoretical and experimental basis. The purpose of this study is to identify differentially expressed proteins in lipid-induced steatosis model through proteomics technology, then study the molecular mechanism of the proteins. These results may provide potential targets for future mechanistic studies concerning the development and prevention of NAFLD. The details were as follows:(1) Establishment of NAFLD in vitro model, screening and validation of different expressed proteins:for lipid accumulation in NAFLD process, in vitro model of NAFLD was established.We selected exogenous fatty acid-OA to treat SMMC-7721 cell lines, determined the inducers suitable life time and concentration. We found the best results were the process for 24h in 1mM OA.Then by two-dimensional electrophoresis and mass spectrometry methods applied to the control and treatment groups, the proteins were verified by q-PCR and western blot techniques. Approximately 650 proteins were separated and fifteen were found to be significantly affected by oleic acid compared to the untreated control. Among them, seven were upregulated (CALR/Lamin B1/Glycine-tRNA ligase/EEF1G/TPI1/PGK1/EIF3I) and other eight were downregulated (CALU/TPM4/KCIP-1/Annexin A5/HSP90/HSP70/plastin-3/Actin-2). The results showed that these proteins are involved in transcription regulation (53.4%), metabolic process (13.3%), cytoskeleton (20%), and cellular signaling transduction (13.3%), respectively. It is indicated that pathogenesis of NAFLD relates to different functional proteins. Among multiple differently expressed proteins, CALR proteins were selected for futher study.(2) Researchs on CALR proteins in lipid metabolism research: we appliyed immunofluorescence techniques to detect CALR cellular localization, CALR proteins were expressed in the cytoplasm and cell nucleus; then we used Fluo-3-Am staining and flow cytometry techniques, it was showed that calcium ions had no difference changes.With siRNA interference on SMMC-7721 cells, we found CALR proteins and genes were significantly down-regulated in 24h and 48h. Meanwhile CD36 and PPARa mRNA and protein levels were increased. Next we constructed pcDNATM3.1 (+)-CALR plasmids, by cis-transfected 293T cells to overexpress the CALR gene, we found CALR were significantly up-regulated in proteins and genes level for 24h and 48h, which CD36 and PPARa mRNA and protein levels were down-regulated.When added OA, Oil red O staining found CALR promoted lipid accumulation.These results suggest that CALR could affect CD36 and PPARa to promote fat accumulation, which not invlove with the changes of calcium ion levels in this process.(3) The gemfibrozil reduced hepatocyte lipid accumulation:gemfibrozil, regarded as PPARa activator, is a cholesterol-lowering drug, but the cellular level of lipid-lowering mechanism is unclear. We employed oleate and drugs to treate the SMMC-7721 cells, the best effect of cell proliferation and lipid-lowering effect were found in 1mM OA and 50?M GEM when the cells were treated 24h. By oil red O staining and TLC experiments, we found the total lipids and triglyceride levels were decreased significantly.Then we checked the key genes and proteins on lipid metabolism pathway(CD36, SREBP1, PPARa), these changes partially mediated by PPARa and SREBP1 signaling. With verification of downstream genes of PPARa and SREBP1 signaling, like LIPIN1/DGAT2/CPT2, the experiments indicate that gemfibrozil increased lipid oxidation and lipid synthesis. |
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