| Summary: | 碩士 === 長庚大學 === 天然藥物研究所 === 97 === The generation of reactive oxygen species (ROS) from neutrophils can act against pathogen invasion in host. However, ROS overproduction by neutrophils may also damage host tissues and result in some diseases, such as asthma and ischemia-reperfusion injury. Therefore, we aim to study the inhibiting mechanism of ZIL-25 and CMH-1001 on superoxide anion production in human neutrophils.
PART I : 2,2,6-trimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one (N-methylflindersine (ZIL-25)) is a natural product which is isolated from the leaves of Zanthoxylum integrifoliolum (Merr.). We found that ZIL-25 could inhibit superoxide anion production and Cathepsin G release in neutrophils by N-formyl-methionyl-leucyl- phenylalanine (fMLP, 1 μM) in a dose-dependent manner. The 50% inhibitory concentration (IC50) values of ZIL-25 for superoxide anion production and Cathepsin G release were 12.3 ± 2.8 μM and 20.4 ± 5.0 μM, respectively. In addition, ZIL-25 could not inhibit phorbol 12-myristate 13-acetate (PMA, 100 nM)-induced superoxide anion production and Cathepsin G release. ZIL-25 had no effects on scavenging oxygen free radicals by Xanthine-Xanthine oxidase and on cellular toxicity of neutrophils. Based on some studies, fMLP has been considered to induce superoxide production of neutrophils through activation of ERK1/2, p38 and AKT pathways. However, ZIL-25 had no influence on phosphorylation of ERK1/2, p38 and AKT by fMLP. The intracellular calcium mobilization is also well known as one of the crucial elements of NADPH oxidase regulation. In the presence of fMLP stimulation, the intracellular calcium concentration was elevated to 604.5 ± 26.3 nM in neutrophils. Howevwe in the condition that pretreatmented ZIL-25 (50 μM) to cells resulted in a decrease of fMLP-stimulated calcium concentration at 288.5 ± 22.2 nM. ZIL-25 could inhibit fMLP-induced intracellular calcium mobilization. Intracellular calcium mobilization is majorly influenced by cAMP and PLC pathways. Increasing cAMP levels inhibit the raise of calcium concentration in neutrophils. In our studies, ZIL-25 did not alter cAMP levels in neutrophils, and we suggested that the effect of ZIL-25 on intracellular calcium concentration did not via cAMP pathway. We further examined the membrane translocation of p47phox, which is one of NADPH oxidase protein components, and then found that ZIL-25 inhibited fMLP-stimulated membrane translocation of p47phox. Afterwards, we examined whether ZIL-25 would target on PLC pathway to regulate intracellular calcium mobilization. The results indicated ZIL-25 could inhibit PLC-γ2 phosphorylation by fMLP. These results suggested that ZIL-25 may inhibit fMLP-induced intracellular Ca2+ increase and p47phox translocation to membrane via inhibiting PLC-γ2 phosphorylation, and further prevent ROS production in human neutrophils.
PART II : CMH-1001 is a chemical compound which could inhibit superoxide anion production and cathepsin G release in human neutrophils by fMLP (1 μM). The 50% inhibitory concentration (IC50) values of CMH-1001 for superoxide anion production and Cathepsin G release were 1.4 ± 0.3 μM and 9.4 ± 4.7 μM, respectively. However, CMH-1001 did not inhibit PMA (100 nM) -induced superoxide anion production and Cathepsin G release. Moreover, CHM-1001 was incapable of scavenging oxygen free radicals by Xanthine-Xanthine oxidase and had no cellular toxicity of neutrophils. Then we examined the regulation of calcium levels, which is the messenger, in neutrophils. The results suggested that CMH-1001 could partially inhibit fMLP-induced intracellular calcium mobilization. Furthermore, we focused on the ERK1/2, p38 and AKT signaling pathways which contributed to stimulation of NADPH oxidase activity by fMLP. The results showed that CMH-1001 could inhibit ERK1/2 phosphorylation by fMLP. However, CMH-1001 did not affect either fMLP-induced p38 and AKT phosphorylation or PMA-induced ERK phosphorylation in human neutrophils. We further found that CMH-1001 could inhibit fMLP-induced p47phox membrane translocation to regulate activity of NDAPH oxidase. These results suggest that the inhibition of fMLP-induced superoxide anion production by CMH-1001 may act through inhibition of ERK phosphorylation and p47phox translocation to membrane in neutrophils.
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