The inhibitory mechanisms of Tectochrysin (MCL-1), a natural compound isolated from Muntingia calabura L., on fMLP-induced superoxide anion production in human neutrophils

• Main Authors: Yin Huan Chien, 簡吟桓
• Other Authors: C. H. Liao
• Format: Others
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/63880778572163355539

碩士 === 長庚大學 === 中醫學系天然藥物 === 98 === Human neutrophils are the important defense line in host defence mechanisms against external pathogens. The generation of reactive oxygen species (ROS) by neutrophils also holds an important status in host defence system. However, excessive neutrophil activation and inappropriate ROS production can also substantial injury to the host’s tissues and results inflammatory diseases, such as rheumatoid arthritis, asthma, myocardial infarction etc. Therefore, develop natural products to mediate neutrophils function and study of their molecular mechanisms would be useful for anti-inflammatory therapy advance. In this study, we investigated the inhibiting mechanism of Tectochrysin [5-hydroxy- 7-methoxy-2-phenylchromen-4-one] (MCL-1), a natural product from Muntingia calabura L. on superoxide anion production in human neutrophils. MCL-1 could inhibit the fMLP-induced superoxide anion production in a concentration-dependent manner with respective IC50 value of 0.16 ± 0.03 μM. However, MCL-1 did not inhibit superoxide anion production cause by phorbol 12-myristate 13-acetate (PMA, 100nM) even though high concentration has using. MCL-1 had no influence on fMLP and PMA induced cathepsin G release. It is important to clarify whether MCL-1 affect fMLP and GPCR interaction or not. In FLPEP experiment, MCL-1 has no inhibiting effect on FITC-conjugated fMLP (FLPEP) binding to human neutrophil’s receptors. And MCL-1 did not inhibit superoxide anion production cause by NaF (20 mM), a direct activator of G protein, but MCL-1 could delay the founction of superoxide anion production cause by NaF. The results demonstrated that the inhibition effect of MCL-1 was through intracellular signal transduction. MCL-1 did not have cellular toxicity was observed in neutrophils. Moreover, MCL-1 was also incapable of scavenging oxygen free radical by Xanthine-Xanthine oxidase system. In addition, MCL-1 inhibited fMLP- induced intracellar calcium mobilization and inhibited the calcium influx in calcium free system. It’s due to MCL-1 inhibited PLCγ2 phosphorylation, but it could not cause intracellular cAMP level increasing. Production of O2.– requires the assembly of an active NADPH oxidase. It is generally believed that the phosphorylation and subsequent translocation of p47phox to interact with flavocytochrome b558 are essential steps for the activation of NADPH oxidase upon neutrophil activation. The results indicated that MCL-1 could inhibit fMLP-stimulated membrane translocation of p47phox. But MCL-1 could not affect NADPH oxidase activity directly. Previous studies show that a number of kinases have been proposed to participate in p47phox phosphorylation events, including protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), p21-activated kinase (PAK) and AKT. MCL-1 could inhibit the phosphorylation of PKC pan induced by fMLP. Nevertheless, MCL-1 was not observed to inhibit the phosphorylation of ERK 1/2, p38, PAK and AKT induced by fMLP. Additionally, MCL-1 inhibited the Tyrosine kinase phosphorylation and Tec translocation to the membrane induced by fMLP. However, MCL-1 had no influence on phosphorylation of Src family tyrosine kinase and Hck phosphorylation. In another set of experiments, MCL-1 also had not observed to inhibit AKT translocation to the membrane, which reflect MCL-1 had no effect on the PH domain of Tec tyrosine kinase interacts with the products of PI3K. MCL-1 also had not observed to inhibit F-actin polymerization. These results suggested that MCL-1 could inhibit fMLP-induced superoxide anion production through inhibition of Tec tyrosine kinase translocation to the membrane, PLCγ2 phosphorylation and blockade its downstream signal.