新合成chalcone類化合物 TIH以濃度及時間依存性的方式抑制 formyl-Met-Leu-Phe (fMLP) 刺激大白鼠的嗜中性白血球生成超氧自由基，其 IC50 值為 1.4 ± 0.1 μM。TIH 抑制 fMLP 刺激嗜中性白血球產生超氧自由基的作用具有可逆性，並且對嗜中性白血球不產生細胞毒性。TIH 也會以濃度依存性的方式抑制 phorbol 12-myristate 13-acetate (PMA) 刺激嗜中性白血球生成超氧自由基，其 IC50 值為 2.8 ± 0.3 μM。TIH (1-10 μM) 不影響 dihydroxyfumaric acid (DHF) 自體氧化產生超氧自由基的現象。在非完整細胞系統的反應中，TIH (1-10 μM) 不會明顯抑制 arachidonic acid 活化細胞質與細胞膜分劃混合液產生超氧自由基，也不會抑制細胞經 PMA 活化NADPH oxidase 的細胞膜分劃產生超氧自由基。TIH 不會影響 fMLP 刺激嗜中性白血球所產生的 extracellular signal-regulated kinase (ERK) 及 p38 mitogen-activated protein kinase (MAPK) 磷酸化作用，但會抑制 AktSer473 及AktThr308 磷酸化作用，其 IC50 值分別為 3.9 ± 0.5 μM 和8.3 ± 1.4 μM。TIH 也會抑制 fMLP 刺激嗜中性白血球所產生的 phospholipase D (PLD) 活性，其 IC50 值 5.2 ± 2.0 μM。然而，對於 fMLP 刺激細胞所引起的 [Ca2+]i 增加及蛋白質酪氨酸磷酸化的現象，TIH 需在較高濃度下才有明顯的抑制效果。此外，TIH 也會抑制 fMLP 刺激嗜中性白血球所產生的 protein kinase Cα (PKCα)、PKCβI 及 PKCδ之膜轉位作用，其 IC50 值分別為 2.4 ± 1.5、4.8 ± 1.1 及 3.9 ± 1.1μM。TIH 在 5 μM 濃度下，也會明顯減少 RhoA 及 ADP-ribosylation factor (ARF) 之膜轉位作用。綜合上述的結果，顯示 TIH 抑制 fMLP 刺激大白鼠的嗜中性白血球生成超氧自由基的作用，可能經由抑制 PLD 及 PI3K/Akt 訊息傳遞路徑，以及減少 PKCα、PKCβI、PKCδ、ARF 及 RhoA 之膜轉位作用。 A synthetic chalcone derivative TIH inhibited formyl-Met-Leu-Phe (fMLP)-induced superoxide anion generation in a time- and concentration-dependent manner in rat neutrophils with an IC50 value of 1.4 ± 0.1 μM. Inhibition by TIH was reversible and not caused by cytotoxic effect. TIH also inhibited phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion generation in a concentration-dependent manner with an IC50 value of 2.8 ± 0.3 μM. TIH (1-10 μM) had no appreciable effect on the superoxide anion generation during dihydroxyfumaric acid (DHF) autoxidation, in arachidonic acid (AA)-activated cytosol and membrane fractions mixture, and in PMA-activated NADPH oxidase cell-free systems. TIH did not affect the fMLP-induced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation, while attenuated the AktSer473 and AktThr308 phosphorylation with IC50 values of 3.9 ± 0.5 and 8.3 ± 1.4 μM, respectively. In addition, TIH inhibited the phospholipase D (PLD) activity in neutrophils with an IC50 value of 5.2 ± 2.0 μM, however, had appreciable effect on [Ca2+]i and protein tyrosine phosphorylation only at higher concentrations of TIH. TIH also blocked the membrane translocation of protein kinase Cα (PKCα)、PKCβI and PKCδwith IC50 values of 2.4 ± 1.5, 4.8 ± 1.1, and 3.9 ± 1.1 μM, respectively, and significantly decreased the membrane associated RhoA and ADP-ribosylation factor (ARF) at 5 μM TIH. These results indicate that the inhibition of superoxide anion generation by TIH is probably attributed to the blockade of phospholipase D and PI3K/Akt signaling pathways, and to the attenuation of the membrane recruitment of PKCα, PKCβI, PKCδ, ARF, and RhoA in rat neutrophils.
