Screening Matrix Metalloproteinase Inhibitor And Effects Of The Inhibitor In Rabbit Model Of Osteoarthritis

Extracellular matrix (ECM) is important as a scaffold for tissue structure and is also regarded as an environment regulating cell function in tissues. The extracellular matrix appears to be a dynamic structure, which has a prominent role in normal development as well as in a variety of disease processes. Enzymes that degrade ECM components can be classified into six families. They are matrix metalloproteinases, serine proteinase, proline, cysteine proteinase, aspartyl proteinase and glycosidase. One class that appears to play a dominant role is that of matrix metalloproteinases.MMPs are a family of zinc metalloendopeptidases which is secreted by cells, and are responsible for much of the prevention and turnover of matrix components. The zinc-binding motif HEXGHXXGXXH in the catalytic domain is the special feature that assign proteinases to this family. Based on their substrate specificity and primary structure, the MMP family can be subdivided into six groups: collagenases, gelatinases, stromelysins, Matrilysins, membrane type (MT) and heterogeneous subgroup. Matrix metalloproteinases (MMPs) are essential actors in this complex interplay between cells and the extracellular matrix. MMPs can degrade ECM components, other proteases, clotting factors, chemotactic molecules, latent growth factors, growth-factor binding proteins, adhesion molecules, cell surface receptors etc. Thereby, MMPs mediste the synthesis and secretion of cytokine, growth factor, hormone, cell-cell adhesion molecules receptors and control cell migration, proliferation, morphogenesis and apoptosis and regulate tumor expansion, angiogenesis and dissemination. The development of many diseases, such as tumor, rheumatoid arthritis, osteoarthritis and cardiovascular diseases, all relate to the abnormal of MMPs activities. Therefore, the regulation of MMPs activities is significant to the treatment of these diseases. In addition, MMPs as the target for the treatment to find the antagonistic drugs, especially selective inhibitors for MMPs, has become a hot topic of drug screening research area.Osteoarthritis(OA) is a degenerative joint disease that affects a large and growing population, which characterized as cartilage degradation, scleroisis of subchondral bone and osteophyte formation. The progressive cartilage destruction that occurs in OA is thought to result from the proteolytic activity of matrix degrading enzymes such as matrix metalloproteinases (MMPs), and the inhibition of MMPs is effective in blocking the cartilage damage.To study the inhibitory activities and related mechanism of myricetin to MMPs by fluorescent assays in vitro, and characterize the effects of MMP inhibitors on cartilage injury in rabbit osteoarthritis. We cloned and purified two kinds of MMPs, which were MMP-3 and MMP-13. Then, we used DQ-gelatin as substrate to study the inhibitory activities of myricetin to MMPs by fluorescent assays in vitro. The MMP inhibitions for these drugs were competitive revealed by enzyme kinetics analysis. Based on the analysis of the results of computer simulation, we found that there was no interaction between the myricetin and the catalytic zinc. But its binding site was in the S1'pocket. Meanwhile, the simulation combined with the free enzyme kinetics is in concordance to the experimental data, which suggests the two data was in the same order of magnitude with little errors. Therfore these results based on the structure of the matrix metalloproteinase inhibitors (MMPIs) will provide useful informations to the drug design. Finally we use an OA model in rabbit to test the therapeutic efficacy of myricetin. The results demonstrated myricetin can inhibit the enzymatic activities of MMPs, which was competitive revealed by enzyme kinetics analysis. There were obvious difference in the living conditions among the groups with or without drugs, and also we observed remarkable improvement in groups with drugs. As a conclusion that MMP inhibition of myricetin is effective in reducing the joint damage that occurs in the rabbit osteoarthritis model and supports a potential therapeutic role for MMP inhibition in the treatment of OA. In addition, We will continue to test these chemicals on MMP inhibition, biological activities and preclinical assays to develop a new drug with independent intellectual property rights.