| Abstract: | 嗜中性白血球在防禦細菌感染、產生發炎以及組織受損的過程中，扮演著相當重要的角色。嗜中性白血球受到可溶或不可溶的刺激物刺激時，會生成超氧自由基及其有毒的代謝產物。這些活性氧物質有非常強的抗菌效果，但如產生過多的話，則會造成周邊組織的傷害。如果對於嗜中性白血球生成超氧自由基的作用機轉能夠有更進一步的認識，即可研發出更有效的抗發炎藥物。於本實驗，主要是測試合成化合物 CYL-26Z 及天然化合物 artocarpol A 影響嗜中性白血球生成超氧自由基的作用機轉。
CYL-26Z 以濃度及時間依存性的方式抑制 formyl peptide 刺激嗜中性白血球生成超氧自由基，其 IC50 值為 2.0 ± 0.6 M，且於反應 10 分鐘時達到最大的抑制效果。CYL-26Z 也以濃度依存性的方式抑制 phorbol ester 刺激嗜中性白血球生成超氧自由基 (IC50 2.7 ± 0.4 M)。CYL-26Z 造成的抑制效果，可經由清洗後，產生部分可逆的現象。CYL-26Z 與細胞反應期間並不產生細胞毒性，也無明顯的抑制 dihydroxyfumaric acid 自體氧化產生超氧自由基的現象。在無細胞系統的反應中，CYL-26Z 並不會抑制 arachidonate 活化的NADPH oxidase 的活性。不論細胞外是否具有鈣離子，CYL-26Z 皆會以濃度依存性的方式抑制 formyl peptide 刺激細胞所引起的 [Ca2+]i 增加現象。CYL-26Z 以濃度依存性的方式抑制 formyl peptide 刺激嗜中白血球所產生的 p38 mitogen-activated protein kinase (MAPK) 及 extracellular signal-regulated kinase (ERK) (IC50 值大約 30 M)。CYL-26Z也抑制 Akt 上的Ser473 及Thr308磷酸化作用 (IC50值分別為19.2 ± 2.2 M和7.5 ± 3.0 M)。CYL-26Z有濃度依存性的抑制 fMLP 刺激 Protein Kinase C (PKC)-α、-βI (IC50 5.0 ± 0.8 M) 及 -βII 的轉位作用。CYL-26Z不影響蛋白質酪氨酸磷酸化的作用。
Artocarpol A 引起嗜中性白血球生成超氧自由基的作用是須要鈣離子存在下，但不需要 cytocalasin B。Pertussis toxin 不會影響 artocarpol A 的作用。嗜中性白血球先與 formyl peptide 或 phobor ester 反應後，會抑制再加入 artocarpol A 刺激嗜中性白血球生成超氧自由基的反應。Artocarpol A 對於無細胞反應系統中的 NADPH oxidase 具有微弱的活化作用。Artocarpol A 無法刺激 ERK 產生磷酸化的作用。利用 MEK/ERK kinase 的抑制劑 U0126 及 PD98059 對於 artocarpol A 刺激嗜中性白血球產生超氧自基也不會有影響。Artocarpol A 以濃度及時間依存性的方式促進 p38 MAPK 及 Akt 的磷酸化。利用 p38 MAPK 抑制劑 SB203580 以及 PI3K 抑制劑 LY294002 皆以濃度依存性的方式抑制 artocarpol A 所引起超氧自由基增加的現象，IC50 值分別為 4.3 ± 0.3 M 及 4.9 ± 0.4 M。Artocarpol A 可以使細胞質中的 PKC-α、-βI 及 -βII 轉位至細胞膜上。PKC 抑制劑 GF109203X 以濃度依存性的方式抑制 artocarpol A 所引起的超氧自由基生成作用。另一方面，phospholipase C (PLC) 的抑制劑 U73122 以及 D-myo-inositol 1,4,5-trisphosphate receptor 的抑制劑 2-APB 幾乎可以完全的抑制 artocarpol A 所引起的超氧自由基生成作用。不論細胞外是否有鈣離子存在下，artocarpol A 皆可以刺激細胞提高細胞內鈣離子的濃度。Artocarpol A 引起的超氧自由基生成作用可被lavendustin A抑制，而genistein反而有促進artocarpol A的作用。Artocarpol A有時間依存性的引起 40, 45, 60 及90 kDa 蛋白質酪氨酸磷酸化的作用。Ethanol可抑制artocarpol A 引起的超氧自由基生成作用。
綜合上述的結果顯示，CYL-26Z 抑制 formyl peptide 刺激嗜中性白血球生成超氧自由的機轉，可能是抑制PKC-βI 的轉位及 Akt 的訊息傳遞路徑，而降低 NADPH oxidase活性。但是 CYL-26Z 確實的作用位置，仍待更進一步的研究。Artocarpol A 可能經由活化 PLC/Ca2+、PKC、 p38 MAPK、 PLD、 tyrosine kinase、PI3K/Akt的訊息傳遞路徑以及活化NADPH oxidase來促使嗜中性白血球產生超氧自由基。
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 M 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 M 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 M). CYL-26Z also attenuated the phosphorylation of Ser473 and Thr308 on Akt with IC50 values of 19.2 ± 2.2 M and 7.5 3.0 M, respectively. CYL-26Z concentration-dependently inhibited the membrane association of Protein Kinase C (PKC)-α, -βI (IC50 5.0 ± 0.8 M) 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 M, 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 . |
