Investigation of the Inhibitory Mechanisms of Cinnamophilin on Matrix Metalloprotinase-9 Activation in THP-1 Cells

• Main Authors: Chiao-Chi Tsuei, 崔巧琪
• Other Authors: George Hsiao
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/14390908727174045005

碩士 === 臺北醫學大學 === 醫學研究所 === 91 === Matrix metalloporteinases (MMPs) are a family of over 20 enzymes that cleave the various components of the extracellular matrix (ECM). The ECM serves many roles in addition to its structural and barrier functions, including ground substrates and connecting fibers, which have their function to maintain tissue structure. MMPs contribute to connective tissue development and proteolysis of ECM components by MMPs can alter these functions and are associated with a variety of normal and pathological conditions that involve matrix degradation, remodeling, tumor invasion, arthritis and atherosclerotic plaque rupture. Many different factors have been shown to influence the transcription of MMPs, including hormones, growth factors, oncogene, and cytokines. In general, inflammatory cytokines such as tumor necrosis factor-a, (TNF- a), lipopolysaccharides (LPS), interleukin 1b (IL-1b) and chemokines such as CC chemokine monocyte chemotactic protein-1 (MCP-1) could stimulate inflammatory cells to express MMPs genes and protein, and the secretion of specific tissue inhibitors of metalloproteinases (TIMP), of which there are four to date, tightly control MMP activity. Each TIMP differs in affinity for MMP, with TIMP-1 being the major inhibitor of MMP-9. According to previous experiments, we found that cinnamophilin (MA-1), a natural compound isolated from Cinnamomum philippinense, presented significant inhibitory effect on MMP-9 activation. We also found that histone deacetylase inhibitor II (HDI II), as a potent inducer of growth arrest, differentiation, or apoptotic cell death in a variety of transformed cells in culture and in tumor bearing animals, also showed obvious inhibitory effect on MMP-9. We used human monocyte THP-1 cells in our preliminary experiments and by using different concentration of LPS or MCP-1 treatment for 24 hours. By using gelatine zymography analysis, we found that MCP-1- or LPS-stimulated human monocyte THP-1 cells could significantly induce MMPs activities, especially MMP-9. The gelatin effect is most appropriate and significant at the condition when LPS stimulation concentration at 10 ng/ml and MCP-1 at 240 ng/ml with 1×106 cell/ml. Then, based on this experimental condition, we observed that cinnamophilin (1-50 mM) and HDI II (0.01-5 mM) both concentration-dependently inhibit MMP-9 activation induced by LPS and MCP-1significantly in zymography method. We also found that the inhibitory effect of cinnamophilin and HDI II were not due to impairment of cellular viability by MTT assays. According to Western blot method, we found that the inhibition on expression of MMP-9 protein is concentration-dependently by cinnamophilin and HDI II in various stimulations. By using RT-PCR method, we observed that cinnamophilin and HDI II can inhibit the expression of MMP-9 mRNA, thus they could have deeper influences on the level of MMP-9 transcription. At the same time, we investigated the mechanism of action of cinnamophilin and HDI II in various signaling pathways. We found that cinnamophilin could significantly inhibit the degradation of IkB-a induced by LPS, therefore nuclear factor-kB (NF- kB) may not translocate for transcription. However, the inhibitory effect on degradation of IkB-a of HDI II is not obvious. Furthermore, in MAPKs aspect, cinnamophilin and HDI II could inhibit JNK expression, and no direct inhibitory influence on ERKs expression was observed. In the cell migration assay, we observed that MCP-1 produced induction of THP-1 cell migration. The presence of cinnamophilin and HDI II would reduce MCP-1-induced migrating effect. By flow cytometry method, we found neither cinnamophilin nor HDI II has significant effect on THP-1 cell surface CD11b expression. In summary, we found that HDI II Chinese herbal compound, cinnamophilin, with inhibitory effect on MMP-9 expression, and cinnamophilin with its main mechanism of action might through NF-kB signal pathway on LPS stimulation. It will be interesting to do further studies and investigation on cinnamophilin and HDI II as therapeutic targets on inflammatory research in vivo.