Zusso Morena, Mercanti Giulia, Belluti Federica, Di Martino Rita Maria Concetta, Pagetta Andrea, Marinelli Carla, Brun Paola, Ragazzi Eugenio, Lo Rita, Stifani Stefano, Giusti Pietro, Moro Stefano
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy.
Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Bologna, Italy.
Br J Pharmacol. 2017 May;174(10):1090-1103. doi: 10.1111/bph.13746. Epub 2017 Mar 31.
Toll-like receptor 4 (TLR4) plays a key role in the induction of inflammatory responses both in peripheral organs and the CNS. Curcumin exerts anti-inflammatory functions by interfering with LPS-induced dimerization of TLR4-myeloid differentiation protein-2 (MD-2) complex and suppressing pro-inflammatory mediator release. However, the inhibitory mechanism of curcumin remains to be defined.
Binding of bis-demethoxycurcumin (GG6) and its cyclized pyrazole analogue (GG9), lacking the 1,3-dicarbonyl function, to TLR4-MD-2 was determined using molecular docking simulations. The effects of these compounds on cytokine release and NF-κB activation were examined by ELISA and fluorescence staining in LPS-stimulated primary microglia. Interference with TLR4 dimerization was assessed by immunoprecipitation in Ba/F3 cells.
Both curcumin analogues bound to the hydrophobic region of the MD-2 pocket. However, only curcumin and GG6, both possessing the 1,3-diketone moiety, inhibited LPS-induced TLR4 dimerization, activation of NF-κB and secretion of pro-inflammatory cytokines in primary microglia. Consistent with the ability of 1,3-diketones to coordinate divalent metal ions, LPS stimulation in a low magnesium environment decreased pro-inflammatory cytokine release and NF-κB p65 nuclear translocation in microglia and decreased TLR4-MD-2 dimerization in Ba/F3 cells. Curcumin and GG6 also significantly reduced cytokine output in contrast to the pyrazole analogue GG9.
These results indicate that phenolic 1,3-diketones, with a structural motif able to coordinate magnesium ions, can modulate LPS-mediated TLR4-MD-2 signalling. Taken together, these studies identify a previously uncharacterized mechanism involving magnesium, underlying the inflammatory responses to LPS.
Toll样受体4（TLR4）在外周器官和中枢神经系统的炎症反应诱导中起关键作用。姜黄素通过干扰脂多糖（LPS）诱导的TLR4-髓样分化蛋白2（MD-2）复合物二聚化以及抑制促炎介质释放发挥抗炎功能。然而，姜黄素的抑制机制仍有待明确。
使用分子对接模拟确定双去甲氧基姜黄素（GG6）及其缺乏1,3-二羰基功能的环化吡唑类似物（GG9）与TLR4-MD-2的结合情况。通过酶联免疫吸附测定（ELISA）和荧光染色检测这些化合物对LPS刺激的原代小胶质细胞中细胞因子释放和核因子κB（NF-κB）激活的影响。通过在Ba/F3细胞中的免疫沉淀评估对TLR4二聚化的干扰。
两种姜黄素类似物均与MD-2口袋的疏水区域结合。然而，只有姜黄素和GG6都具有1,3-二酮部分，能够抑制LPS诱导的原代小胶质细胞中TLR4二聚化、NF-κB激活和促炎细胞因子分泌。与1,3-二酮配位二价金属离子的能力一致，在低镁环境中LPS刺激可减少小胶质细胞中促炎细胞因子释放和NF-κB p65核转位，并减少Ba/F3细胞中TLR4-MD-2二聚化。与吡唑类似物GG9相比，姜黄素和GG6也显著降低了细胞因子产量。
这些结果表明，具有能够配位镁离子结构基序的酚类1,3-二酮可调节LPS介导的TLR4-MD-2信号传导。综上所述，这些研究确定了一种以前未被表征的涉及镁的机制，该机制是LPS炎症反应的基础。
