| Summary: | 碩士 === 國立臺灣大學 === 分子醫學研究所 === 92 === Rosiglitazone (RSG) is a synthetic PPARγ agonist, possessing the effect of lowering blood sugar. It is used clinically to treat type 2 diabetes. PPARγ has a restricted pattern of expression, mainly in the white and brown adipose tissues, whereas other tissues such as skeletal muscle and heart contain very limited amounts. Furthermore, PPARγ is expressed in vascular cells including endothelial and smooth muscle cells and macrophages/foam cells. The role of PPARγ in the immune system has been a focus of recent research. For instance, it has been recognized that during inflammatory processes, PPARγ inhibits the activation of macrophages and the production of pro-inflammatory cytokines (TNF-α and IL-6), but not the anti-inflammatory IL-10. These effects are through the restrain of the p42/44 MAPK signaling pathway. Both p38 MAPK and p42/44 MAPK signaling pathways have been show to be involved in LPS-induced TNF-α and IL-6 expression in macrophage. However, IL-10 is regulated by the p38 MAPK and PI3K and Akt signaling pathways. Interestingly, activated Akt could inhibit p42/44 MAPK and p38 MAPK activation. Therefore, the objective of thesis is to study the signal transduction by which RSG regulated LPS-stimulated cytokine release in macrophage.
IL-4 was used to induce the differentiation of macrophage from human monocyte, monitored by the expressions of PPARγ, CD36, and the LPL. Then we titrated the optimal concentrations at which LPS and RSG did not cause apoptosis of the cultured macrophage. We found that at a concentration lower than 4.5μM of RSG did not initiate apoptosis of cells. However, at this concentration, RSG decreased LPS-induced secretion of IL-6 and TNF-α, but not IL-10.
We then studied the signaling effects of p38 MAPK on cytokine secretion from macrophage. In the presence of a p38 inhibitor（SB203580）, LPS-induced TNF-α, IL-6 and IL-10 secretion was suppressed. Addition of RSG further suppressed the secretion of these cytokines, implying that some signaling pathway other than p38 MARK might involve this effect. Interestingly, addition of P13K inhibitor（LY294002）, caused an increase in TNF-α and IL-6 secretion, but a decrease in IL-10 secretion from macrophages stimulated by LPS. RSG decreased the response of secretion of TNF-α, IL-6, and IL-10, indicating that different signaling pathways might be responsible for TNF-α, IL-6, and IL-10 secretion. More interestingly, AMP-dependent protein Kinase （AMPK）was acutely phosphorylated by RSG. To study the role of AMPK，we added an AMPK activator（AICAR） to the macrophage cells to study cytokines release. We found that the secretion of IL-6 and TNF-α was suppressed, but there was no such an effect on IL-10 secretion. These data suggest that RSG is able to inhibit p38 MAPK and activate AMPK in a PI3K-dependent manner.
To further study the signaling pathways but which RSG mediates cytokine release p38 MAPK and PI3K and Akt and AMPK were studied We found that RSG suppressed the phosphorylation of the p38 MAPK but promotes the phosphorylation of the Akt.
To sum up, RSG inhibits the macrophage cells in their production of cytokines via signal transmission through the suppression of the p38 MAPK and activation of the PI3K，Akt and AMPK kinases. These results in the reduction in the pro-inflammatory cytokines, such as TNF-α and IL-6, while no diminish of anti-inflammatory IL-10 was observed.
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