| Summary: | 碩士 === 中山醫學大學 === 醫學研究所 === 90 === Neutrophils are crucial of importance in host defense against bacterial infection, in the pathogenesis of inflammatory processes, and tissue destruction. Upon activation with soluble or particular stimuli, neutrophils generate superoxide anion and its toxic oxygen metabolites. These reactive oxygen species that are strongly anti-microbial but which may also cause damage by destructing surrounding tissue. The understanding of mechanisms of superoxide anion generation will be undoubtedly of great interest and could provide additional therapeutic targets for anti-inflammatory drugs. In the present study, effects of a synthetic compound, CYL-26Z, and a natural product, artocarpol A, were examined on superoxide anion generation in rat neutrophils.
CYL-26Z showed concentration- and time-dependent inhibition of formyl peptide-induced superoxide anion generation with IC50 value of 2.0 ± 0.6 µM and reached maximal inhibition after 10-min incubation. CYL-26Z also inhibited the phorbol ester-stimulated superoxide anion generation in a concentration-dependent manner with IC50 value of 2.7 ± 0.4 µM This inhibitory effect was partially reversed after washing. CYL-26Z had no cytotoxicity over the reaction period and did not scavenge the superoxide anion generation during dihydroxyfumaric acid autooxidation. CYL-26Z failed to alter the arachidonate-activated NADPH oxidase in a cell-free system. CYL-26Z suppressed the formyl peptide-stimulated [Ca2+]i elevation in the presence as well as the absence of external Ca2+ in a concentration-dependent manner. In addition, CYL-26Z concentration-dependently attenuated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) (IC50 about 30 µM). CYL-26Z also attenuated the phosphorylation of Ser473 and Thr308 on Akt with IC50 values of 19.2 ± 2.2 µM and 7.5 3.0 µM, respectively. CYL-26Z concentration-dependently inhibited the membrane association of Protein Kinase C (PKC)-α, -βI (IC50 5.0 ± 0.8 µM) and -βII. However, CYL-26Z had no effect on protein tyrosine phosphorylation.
Artocarpol A showed a Ca2+-dependent but cytochalasin B and pertussis toxin-independent stimulation of superoxide anion generation with a lag in rat neutrophils. Pretreatment of cells with formyl peptide or phorbol ester reduced the artocarpol A-induced response. Artocarpol A weakly activated NADPH oxidase in a cell-free system. Artocarpol A did not stimulate ERK phosphorylation and two MAPK/ERK kinase inhibitors, U0126 and PD98059, had no effect on artocarpol A-induced superoxide anion generation. Artocarpol A showed concentration- and time-dependent stimulation of p38 MAPK and Akt phosphorylation. The superoxide anion generation in response to artocarpol A was reduced by p38 MAPK inhibitor, SB203580, and phosphoinositide 3-kinase inhibitor, LY294002, in a concentration-dependent manner with IC50 values of 4.3 ± 0.3 and 4.9 ± 0.4 µM, respectively. Artocarpol A stimulated the recruitment of PKC-α, -βI, and -βII to membrane. Treatment with the PKC inhibitor, GF109203X, inhibited the artocarpol A-induced superoxide anion generation with IC50 value of 7.8 ± 1.0 nM. In addition, artocarpol A stimulation of superoxide anion generation was almost abolished by exposure of cells to a phospholipase C (PLC) inhibitor, U73122, and an antagonist of D-myo-inositol 1,4,5-trisphospahte receptors, 2-APB. Artocarpol A indeed stimulation of [Ca2+]i elevation in the presence and absence of external Ca2+. Lavendustin A inhibited but genistein enhanced the artocarpol A-stimulated superoxide anion generation. In addition, artocarpol A time dependently induced the tyrosine phosphorylation of 40, 45, 60, 90 kDa proteins. Ethanol reduced the artocarpol A-stimulated superoxide anion generation.
In summary, CYL-26Z inhibition of formyl peptide-induced superoxide anion generation probably involved the PKC-βI membrane association and the Akt signaling pathway. The exact site of action of CYL-26Z awaits further investigation. Artocarpol A stimulation of superoxide anion generation is probably attributable to the activation of PLC/Ca2+, PKC, p38 MAPK, tyrosine kinase, PLD and PI3K/Akt signaling pathways, and activated NADPH oxidase to evoke superoxide anion generation in rat neutrophils .
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