Lee Jin Young, Lee Byung Ho, Lee Joo Young
Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon, Korea.
Pharmacology Research Center, Korea Research Institute of Chemical Technology, Daejeon, Korea.
Toxicol Res. 2015 Mar;31(1):11-6. doi: 10.5487/TR.2015.31.1.011.
Our body's immune system has defense mechanisms against pathogens such as viruses and bacteria. Immune responses are primarily initiated by the activation of toll-like receptors (TLRs). In particular, TLR4 is well-characterized and is known to be activated by gram-negative bacteria and tissue damage signals. TLR4 requires myeloid differentiation factor 2 (MD2) as a co-receptor to recognize its ligand, lipopolysaccharides (LPS), which is an extracellular membrane component of gram-negative bacteria. Gambogic acid is a xanthonoid isolated from brownish or orange resin extracted from Garcinia hanburyi. Its primary effect is tumor suppression. Since inflammatory responses are related to the development of cancer, we hypothesized that gambogic acid may regulate TLR4 activation. Our results demonstrated that gambogic acid decreased the expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-12, and IL-1β) in both mRNA and protein levels in bone marrow-derived primary macrophages after stimulation with LPS. Gambogic acid did not inhibit the activation of Interferon regulatory factor 3 (IRF3) induced by TBK1 overexpression in a luciferase reporter gene assay using IFN-β-PRD III-I-luc. An in vitro kinase assay using recombinant TBK1 revealed that gambogic acid did not directly inhibit TBK1 kinase activity, and instead suppressed the binding of LPS to MD2, as determined by an in vitro binding assay and confocal microscopy analysis. Together, our results demonstrate that gambogic acid disrupts LPS interaction with the TLR4/MD2 complex, the novel mechanism by which it suppresses TLR4 activation.
我们身体的免疫系统具有针对病毒和细菌等病原体的防御机制。免疫反应主要由Toll样受体（TLR）的激活引发。特别是，TLR4已得到充分表征，已知可被革兰氏阴性菌和组织损伤信号激活。TLR4需要髓样分化因子2（MD2）作为共受体来识别其配体脂多糖（LPS），脂多糖是革兰氏阴性菌的一种细胞外膜成分。藤黄酸是从藤黄中提取的棕褐色或橙色树脂中分离出的一种呫吨酮类化合物。其主要作用是抑制肿瘤。由于炎症反应与癌症的发展相关，我们推测藤黄酸可能调节TLR4的激活。我们的结果表明，在用LPS刺激后，藤黄酸在骨髓来源的原代巨噬细胞中，使促炎细胞因子（TNF-α、IL-6、IL-12和IL-1β）的mRNA和蛋白质水平表达均降低。在使用IFN-β-PRD III-I-luc的荧光素酶报告基因检测中，藤黄酸并未抑制由TBK1过表达诱导的干扰素调节因子3（IRF3）的激活。使用重组TBK1进行的体外激酶检测表明，藤黄酸不会直接抑制TBK1激酶活性，相反，通过体外结合检测和共聚焦显微镜分析确定，它会抑制LPS与MD2的结合。总之，我们的结果表明，藤黄酸破坏LPS与TLR4/MD2复合物的相互作用，这是其抑制TLR4激活的新机制。
