化合物 TCH 抑制 fMLP 刺激大鼠嗜中性白血球生成超氧自由基的研究

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Title:	化合物 TCH 抑制 fMLP 刺激大鼠嗜中性白血球生成超氧自由基的研究 Inhibition of superoxide anion generation by TCH in rat neutrophils
Authors:	蔡雅如;Ya-Ru Tsai
Contributors:	藥學院藥物化學研究所碩士班
Keywords:	嗜中性白血球;超氧自由基;NADPH oxidase;PDK1;Akt;PKC;neutrophil;superoxide;NADPH oxidase;PDK1;Akt;PKC
Date:	2010
Issue Date:	2010-09-29 14:10:41 (UTC+8)
Abstract:	新合成 phenazine carboxylate 類的化合物 TCH 可以濃度依存性但並無明顯的時間依存性的抑制 formyl-Met-Leu-Phe (fMLP) 所誘導嗜中性白血球超氧自由基的生成(IC50 值為 4.5 ± 0.8 μM)。此抑制作用並非來自於影響細胞存活、超氧自由基清除或直接抑制 NADPH oxidase 的活性。在相同的 IC50 值下 TCH 抑制嗜中性白血球細胞中 p47phox 和 p22phox 的連結、Rac2 和 gp91phox 的連結、p47phox、p21-activated kinase 1 (PAK1) 及 Vav 的磷酸化。TCH 並不影響 p38 mitogen-activated protein kinase 與 MAPK-activated protein kinase-2 的磷酸化。TCH 可抑制細胞中 Akt 與 p47phox 的連結、Akt 的磷酸化及 Akt 酵素活性，但不抑制細胞溶解液中的 Akt 酵素活性。TCH 可抑制 active human recombinant PDK1 酵素活性，但不影響 Akt 與 PDK1 的細胞膜轉移。TCH 會抑制 PKC-α、-δ與-ζ和 p47phox 的連結作用，但不影響 PKC-β和 p47phox 的連結。TCH 會抑制 PKC-α，但不會抑制PKC-β、-δ與-ζ的細胞膜轉移。TCH 也不會抑制 fMLP 刺激細胞引起的 PKC 活性。TCH 不會增加細胞內 cyclic AMP 的含量。綜合以上結果，TCH 可能經由影響 PDK1/Akt、PKC 和PAK1 訊息傳遞途徑來抑制 p47phox 的磷酸化，經由影響 Vav/Rac2 訊息傳遞途徑來抑制 Rac2 的活化，進而抑制 NADPH oxidase 聚合及超氧自由基的生成。 A novel synthetic phenazine carboxylate derivative TCH inhibited formyl-Met-Leu-Phe (fMLP)-stimulated superoxide anion generation in rat neutrophils in a concentration- but not a time-dependent manner with IC50 value about 4.5 ± 0.8 μM. This inhibitory effect was not owing to the decrease in cell viability, scavenging of generated superoxide anion or direct blockade of NADPH oxidase activity. Under the same IC50 value, TCH inhibited the interaction of p47phox and Rac2 with p22phox and gp91phox, respectively, and the phosphorylation of p47phox, p21-activated kinase 1 (PAK1) and Vav in fMLP-stimulated cells. TCH had no effect on the phosphorylation of p38 mitogen-activated protein kinase and MAPK-activated protein kinase-2. Pretreatment of cells with TCH inhibited the interaction of Akt with p47phox, the phosphorylation of Akt and Akt activity, whereas, it did not affect the Akt activity in the cell-lysates of fMLP-stimulated cells. TCH inhibited the enzymatic activity of active human recombinant PDK1, whereas, it had no effect on the recruitment of Akt and PDK1 to membrane. TCH attenuated the interaction of PKC-α、-δ and -ζ with p47phox, whereas, it did not affect the association of PKC-β with p47phox. TCH decreased the membrane recruitment of PKC-α but not of PKC-β、-δ and -ζ. TCH had neither block the PKC activity nor increase the cellular cyclic AMP levels of fMLP-stimulated cells. Taken together, TCH probably attenuate PDK1/Akt, PKC and PAK1 signalings and Vav pathway leading to the inhibition of p47phox phosphorylation and Rac2 activation, respectively, which in turn blockade the assembly of active NADPH oxidase and then superoxide anion generation.
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