| Proteomics analysis methods including two-dimensional electrophoresis, mass spectrometry and bioinformatics technology, can describe the changes of the type and quantity of proteins in host, integrally and dynamically. They are often applied to research the disease mechanism or action mechanism of some drugs. In order to explore the possible mechanism of curative effect on rheumatoid arthritis (RA) of bee venom, we studied the proteomics on type II collagen-induced arthritis (CIA) mice model. The main contents and academic contributions of this thesis are summarized as follows:1. The study was designed to evaluate the anti-arthritis effect of bee venom on a CIA mice model. Firstly, the emulsion of bovine type-II collagen and complete Freund’s adjuvant (CFA) was used to immunize male mice to establish CIA model. Then bee venom treatment groups were administered with three therapeutic doses of bee venom (2 mg·kg-1,1 mg·kg-1 and 0.5 mg·kg-1; respectively), model group and control group were administrated with normal saline (NS) for 3 weeks. The levels of serum TNF-a, IL-1/1β, IL-6, IL-18 and MMP-3 in experimental groups were detected by enzyme-linked immunosorbent assay. The results have shown that the serum cytokines in model group were significantly higher than those of normal control group, while there was no significant difference between BV treatment group and normal control. These results demonstrated that bee venom can inhibit production of proinflammatory cytokines and has a certain therapeutic effect on the CIA model.2. A two-dimensional electrophoresis (2-DE) based method for the analysis of mice serum proteome was established. Prior to proteomics research, serum samples were depleted from high-abundant protein, to enrich the indetectable protein of small molecule weight. Then, the critical steps in protein separation by 2-DE technology were optimized, including the choices of IPG strip, sample amount and the condition of isoelectric focusing (IEF). According to the results of this study, TCA/acetone precipitation was used to collect the serum proteins. The IPG strips with 17 cm and pH 4-7 were applied on IEF, and the sampling amount was 800μg. The gels were stained by Coomassie blue staining after the finish of 2-DE.3. To identify disease-related proteins and study action mechanism of BV, proteomic profiling of serum was performed in CIA mice, disease mice with BV (2 mg·kg-1) treated as well as control mice. As the results of images analysis, there were 165 proteins significantly different between model and control before BV treatment. After 3 weeks with BV therapy, the expressions of 36 proteins were found to be up-regulated and 33 were down-regulated in model group, while the expressions of 28 proteins were found to be up-regulated and 23 were down-regulated in BV treatment group compared to normal control. The proteins with significantly different expressions were identified by MALDI-TOF/TOF-MS, and 22 proteins were successfully determined, including cytoskeletal proteins (e.g. gamma-Actin), transport related proteins (e.g. Serotransferrin, Vitamin D-binding protein), inflammatory proteins (e.g. Haptoglobin, isoform CRA-b, Haptoglobin alpha chain, Kininogen-1 isoform 2), metabolism related proteins (Apolipoprotein A-I preproprotein) and blood proteins (serum albumin precursor). The results suggested that the mechanism of action of B V curing RA was related to the regulation of interactions between proteins and metabolic, immunity and the inhibition of cytokines. |
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