Nakamura Y, Murakami A, Ohto Y, Torikai K, Tanaka T, Ohigashi H
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Cancer Res. 1998 Nov 1;58(21):4832-9.
Double applications of phorbol esters trigger excessive reactive oxygen species (ROS) production in mouse skin. Previously reported data suggest that the two applications induce distinguishable biochemical events, namely, priming and activation. The former is characterized as a recruitment of inflammatory cells, such as neutrophils, by chemotactic factors to inflammatory regions and edema formation. The latter is responsible for ROS generation. Thus, inhibitory effects of 1'-acetoxychavicol acetate (ACA), previously reported to be a superoxide generation inhibitor in vitro, on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammatory responses in mouse skin model were examined using a double application of ACA. We demonstrated that two pretreatments and pretreatment with ACA (810 nmol) in the activation phase suppressed double TPA application-induced H2O2 formation in mouse skin. ACA exhibited no inhibitory effects on edema formation and the enhancement of myeloperoxidase activity during the first TPA treatment, whereas the anti-inflammatory agent genistein administered at the same dose inhibited both biomarkers. No inhibitory potential of ACA for TPA-induced H2O2 formation in the priming phase was confirmed. On the other hand, in the in vitro study, ACA inhibited ROS generation in differentiated HL-60 cells more strongly than did 1'-hydroxychavicol, which showed no inhibition by pretreatment in the activation phase. In addition, allopurinol did not inhibit double TPA application-induced H2O2 formation in mouse skin. These findings suggest that the NADPH oxidase system of neutrophils rather than the epithelial xanthine oxidase system is dominant for the O2--generating potential in double TPA-treated mouse skin. ACA significantly inhibited mouse epidermis thiobarbituric acid-reacting substance formation, known as an overall oxidative damage biomarker. Moreover, histological studies demonstrated that ACA inhibited double TPA treatment-induced morphological changes reflecting inflammatory response, such as edema formation, leukocyte infiltration, hyperplasia, and cell proliferation. Furthermore, pretreatment with ACA but not 1'-hydroxychavicol in the activation phase inhibits double TPA application-induced increases in both number of leukocytes and proliferating cell nuclear antigen index. These results suggested that ROS from leukocytes including O2- plays an important role for continuous and excessive production of chemotactic factors, leading to chronic inflammation and hyperplasia, which are inhibitable by ACA. Thus, we concluded that O2- generation inhibitors are agents that effectively inhibit oxidative stress and inflammatory responses in mouse skin.
佛波酯的双重应用会引发小鼠皮肤中过量的活性氧（ROS）生成。先前报道的数据表明，这两种应用会引发可区分的生化事件，即引发和激活。前者的特征是趋化因子将炎症细胞（如中性粒细胞）募集到炎症区域并形成水肿。后者则负责ROS的生成。因此，使用乙酸1'-乙酰氧基查维醇（ACA）的双重应用，研究了其对12-O-十四酰佛波醇-13-乙酸酯（TPA）诱导的小鼠皮肤氧化应激和炎症反应的抑制作用，此前已报道ACA在体外是一种超氧化物生成抑制剂。我们证明，在激活阶段进行两次预处理以及用ACA（810 nmol）预处理可抑制双重TPA应用诱导的小鼠皮肤中H2O2的形成。在第一次TPA处理期间，ACA对水肿形成和髓过氧化物酶活性的增强没有抑制作用，而相同剂量的抗炎剂染料木黄酮则抑制了这两种生物标志物。未证实ACA在引发阶段对TPA诱导的H2O2形成具有抑制潜力。另一方面，在体外研究中，ACA比1'-羟基查维醇更强烈地抑制分化的HL-60细胞中的ROS生成，而1'-羟基查维醇在激活阶段的预处理没有抑制作用。此外，别嘌呤醇不能抑制双重TPA应用诱导的小鼠皮肤中H2O2的形成。这些发现表明，在双重TPA处理的小鼠皮肤中，中性粒细胞的NADPH氧化酶系统而非上皮黄嘌呤氧化酶系统在产生O2-的潜力方面占主导地位。ACA显著抑制小鼠表皮中硫代巴比妥酸反应物质的形成，这是一种整体氧化损伤生物标志物。此外，组织学研究表明，ACA抑制了双重TPA处理诱导的反映炎症反应的形态学变化，如水肿形成、白细胞浸润、增生和细胞增殖。此外，在激活阶段用ACA而非1'-羟基查维醇预处理可抑制双重TPA应用诱导的白细胞数量和增殖细胞核抗原指数的增加。这些结果表明，包括O2-在内的白细胞产生的ROS在趋化因子的持续过量产生中起重要作用，导致慢性炎症和增生，而ACA可抑制这些反应。因此，我们得出结论，O2-生成抑制剂是有效抑制小鼠皮肤氧化应激和炎症反应的药物。
