Borges Perla Villani, Moret Katelim Hottz, Maya-Monteiro Clarissa Menezes, Souza-Silva Franklin, Alves Carlos Roberto, Batista Paulo Ricardo, Caffarena Ernesto Raúl, Pacheco Patrícia, Henriques Maria das Graças, Penido Carmen
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
Mol Pharmacol. 2015 Nov;88(5):949-61. doi: 10.1124/mol.115.098970. Epub 2015 Sep 1.
Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88- and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-β-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-β knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.
天然免疫系统对细菌脂多糖（LPS）的识别是由分化抗原簇14/ Toll样受体4/髓样分化蛋白2（MD-2）复合物介导的。在本研究中，我们研究了格杜宁（一种来自楝科植物的柠檬苦素，被描述为热休克蛋白Hsp90抑制剂）对永生化小鼠巨噬细胞中LPS诱导反应的调节作用。用格杜宁（0.01 - 100 μM，无细胞毒性浓度）预处理野生型（WT）巨噬细胞，以浓度依赖的方式抑制LPS（50 ng/ml）诱导的钙内流、肿瘤坏死因子-α和一氧化氮的产生。进一步研究了格杜宁对髓样分化因子88样衔接蛋白/髓样分化初级反应蛋白88和含TIR结构域衔接蛋白诱导干扰素-β相关衔接分子/TIR结构域衔接蛋白诱导干扰素-β依赖性信号通路的选择性作用。用格杜宁（10 μM）预处理WT、含TIR结构域衔接蛋白诱导干扰素-β基因敲除和髓样分化因子88样衔接蛋白基因敲除的巨噬细胞，在6小时和24小时时显著抑制LPS（50 ng/ml）诱导的肿瘤坏死因子-α和白细胞介素-6的产生，表明格杜宁调节这两种信号通路之间的共同事件。此外，格杜宁（10 μM）抑制LPS诱导的前列腺素E2产生、环氧合酶-2表达以及核因子κB向WT巨噬细胞核内的转位，证明了这种化合物的广泛作用。除了能够抑制LPS诱导的促炎介质外，格杜宁还能在LPS刺激或未刺激的巨噬细胞中触发抗炎因子白细胞介素-10、血红素加氧酶-1和Hsp70。计算机模拟研究表明，格杜宁能有效地对接至MD-2的LPS结合位点，表面等离子体共振进一步证实了这一现象。我们的结果表明，除了调节Hsp90外，格杜宁还作为LPS的竞争性抑制剂，阻断Toll样受体4/MD-2/LPS复合物的形成。
