Hayashi Masahiko, Rho Mun-Chual, Fukami Akiko, Enomoto Akiko, Nonaka Shinobu, Sekiguchi Yoshino, Yanagisawa Tadashi, Yamashita Ayano, Nogawa Toshihiko, Kamano Yoshiaki, Komiyama Kanki
The Kitasato Institute, Tokyo, Japan.
J Pharmacol Exp Ther. 2002 Oct;303(1):104-9. doi: 10.1124/jpet.102.036137.
Interleukin (IL)-6 is a key mediator in the regulation and coordination of the immune response and participates in pathogenesis of cancer cachexia, autoimmune disease, and postmenopausal osteoporosis. In the course of a screening program aimed at IL-6 inhibitor from natural products, we isolated 20S,21-epoxy-resibufogenin-3-formate (ERBF) from bufadienolide and examined the effect of ERBF on activities of various cytokines. ERBF dose dependently suppressed IL-6 activity and caused a parallel rightward shift of dose-response curves to IL-6 at concentrations of 0.03 to 10 ng/ml. Analysis of data yields a pA(2) of 5.12 and a slope of 0.99. Selectivity of ERBF on activity of cytokines was examined using cytokine-dependent cell lines. ERBF did not affect IL-2-dependent growth of CTLL-2 cells, IL-3-dependent growth of Baf3 cells, or tumor necrosis factor (TNF)alpha-induced growth suppression in TNFalpha-sensitive L929 cells. ERBF also did not affect IL-4-stimulated expression of FcepsilonR II receptor (CD23) in U-937 cells, the IL-8-induced chemotaxis of human neutrophils, or nerve growth factor-stimulated neuronal differentiation in PC-12 cells. In contrast, ERBF dose dependently suppressed IL-6-induced neuronal differentiation in PC-12 cells. Furthermore, ERBF suppressed only IL-6-induced osteoclast formation without affecting osteoclast formation induced by IL-11, leukemia inhibitory factor, and 1alpha,25-dihydroxyvitamin D(3). In receptor binding assay, unbound (free) IL-6 was increased in a dose-dependent manner by pretreatment with ERBF on IL-6 receptor (IL-6R), suggesting that ERBF suppresses binding of IL-6 to IL-6R. These results clearly indicate that ERBF is a novel specific small molecule to show IL-6 receptor antagonist activity.
白细胞介素（IL）-6是免疫反应调节与协调过程中的关键介质，参与癌症恶病质、自身免疫性疾病和绝经后骨质疏松症的发病机制。在一项从天然产物中筛选IL-6抑制剂的研究过程中，我们从蟾蜍二烯羟酸内酯中分离出20S,21-环氧-脂蟾毒配基-3-甲酸酯（ERBF），并检测了ERBF对多种细胞因子活性的影响。ERBF以剂量依赖的方式抑制IL-6活性，在浓度为0.03至10 ng/ml时，使IL-6剂量反应曲线平行右移。数据分析得出pA(2)为5.12，斜率为0.99。使用依赖细胞因子的细胞系检测了ERBF对细胞因子活性的选择性。ERBF不影响CTLL-2细胞依赖IL-2的生长、Baf3细胞依赖IL-3的生长，也不影响肿瘤坏死因子（TNF）α敏感的L929细胞中TNFα诱导的生长抑制。ERBF也不影响U-937细胞中IL-4刺激的FcepsilonR II受体（CD23）表达、IL-8诱导的人中性粒细胞趋化作用，或PC-12细胞中神经生长因子刺激的神经元分化。相反，ERBF以剂量依赖的方式抑制PC-12细胞中IL-6诱导的神经元分化。此外，ERBF仅抑制IL-6诱导的破骨细胞形成，而不影响IL-11、白血病抑制因子和1α,25-二羟基维生素D(3)诱导的破骨细胞形成。在受体结合试验中，用ERBF预处理IL-6受体（IL-6R）后，未结合（游离）的IL-6以剂量依赖的方式增加，表明ERBF抑制IL-6与IL-6R的结合。这些结果清楚地表明，ERBF是一种新型的具有IL-6受体拮抗剂活性的特异性小分子。
