Department of Microbiology & Immunology, Queen's University, Kingston, ON, K7L3N6, Canada.
Glycoconj J. 2010 Apr;27(3):329-48. doi: 10.1007/s10719-010-9281-6. Epub 2010 Mar 6.
Anti-inflammatory activities of thymoquinone (TQ) have been demonstrated in in vitro and in vivo studies. However, the precise mechanism(s) of TQ in these anti-inflammatory activities is not well understood. Using a newly developed assay to detect sialidase activity in live macrophage cells (Glycoconj J doi: 10.1007/s10719-009-9239-8 ), here we show that TQ has no inhibitory effect on endotoxin lipopolysaccharide (LPS) induced sialidase activity in live BMC-2 macrophage cells. In contrast, the parent black seed oil (BSO) and another constituent of BSO para-cymene (p-CY) completely block LPS induced sialidase activity. All of these compounds had no effect on cell viability. On the other hand, TQ induces a vigorous sialidase activity in live BMC-2 macrophage cells in a dose dependent manner as well in live DC-2.4 dendritic cells, HEK-TLR4/MD2, HEK293, SP1 mammary adenocarcinoma cells, human WT and 1140F01 and WG0544 type I sialidosis fibroblast cells. Tamiflu (oseltamivir phosphate) inhibits TQ-induced sialidase activity in live BMC-2 cells with an IC(50) of 0.0194 microM compared to an IC(50) of 19.1 microM for neuraminidase inhibitor DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid). Anti-Neu1, -2 and -3 antibodies have no inhibition of TQ-induced sialidase activity in live BMC-2 and human THP-1 macrophage cells but anti-Neu4 antibodies completely block this activity. There is a vigorous sialidase activity associated with TQ treated live primary bone marrow (BM) macrophage cells derived from WT and hypomorphic cathepsin A mice with a secondary Neu1 deficiency (NeuI KD), but not from Neu4 knockout (Neu4 KO) mice. Pertussis toxin (PTX), a specific inhibitor of Galphai proteins of G-protein coupled receptor (GPCR) and the broad range inhibitors of matrix metalloproteinase (MMP) galardin and piperazine applied to live BMC-2, THP-1 and primary BM macrophage cells completely block TQ-induced sialidase activity. These same inhibitory effects are not observed with the GM1 ganglioside specific cholera toxin subunit B (CTXB) as well as with CTX, tyrosine kinase inhibitor K252a, and the broad range GPCR inhibitor suramin. The specific inhibitor of MMP-9, anti-MMP-9 antibody and anti-Neu4 antibody, but not the specific inhibitor of MMP-3 completely block TQ-induced sialidase activity in live THP-1 cells, which express Neu4 and MMP-9 on the cell surface. Neu4 sialidase activity in cell lysates from TQ-treated live THP-1 cells desialylates natural gangliosides and mucin substrates. RT-PCR and western blot analyses reveal no correlation between mRNA and protein values for Neu3 and Neu4 in human monocytic THP-1 cells, suggesting for the first time a varied post-transcriptional mechanism for these two mammalian sialidases independent of TQ activation. Our findings establish an unprecedented activation of Neu4 sialidase on the cell surface by thymoquinone, which is derived from the nutraceutical black cumin oil. The potentiation of GPCR-signaling by TQ via membrane targeting of Galphai subunit proteins and matrix metalloproteinase-9 activation may be involved in the activation process of Neu4 sialidase on the cell surface.
姜黄色素（TQ）的抗炎活性已在体外和体内研究中得到证实。然而，TQ 在这些抗炎活性中的精确机制尚不清楚。使用新开发的检测活巨噬细胞中唾液酸酶活性的测定法（Glycoconj J doi：10.1007/s10719-009-9239-8），我们在这里表明，TQ 对活 BMC-2 巨噬细胞中的内毒素脂多糖（LPS）诱导的唾液酸酶活性没有抑制作用。相比之下，母体黑种草籽油（BSO）和 BSO 的另一种成分对-侧柏烯（p-CY）完全阻断 LPS 诱导的唾液酸酶活性。所有这些化合物对细胞活力均无影响。另一方面，TQ 以剂量依赖的方式在活 BMC-2 巨噬细胞以及活 DC-2.4 树突状细胞、HEK-TLR4/MD2、HEK293、SP1 乳腺腺癌细胞、人 WT 和 1140F01 和 WG0544 型唾液酸贮积症纤维母细胞中诱导强烈的唾液酸酶活性。Tamiflu（奥司他韦磷酸盐）抑制活 BMC-2 细胞中 TQ 诱导的唾液酸酶活性，IC50 为 0.0194 microM，而神经氨酸酶抑制剂 DANA（2-脱氧-2,3-去羟乙酰神经氨酸）的 IC50 为 19.1 microM。抗-Neu1、-2 和 -3 抗体对活 BMC-2 和人 THP-1 巨噬细胞中 TQ 诱导的唾液酸酶活性无抑制作用，但抗-Neu4 抗体可完全阻断该活性。与 TQ 处理的活原发性骨髓（BM）巨噬细胞相比，WT 和低功能 cathepsin A 小鼠来源的次级 Neu1 缺乏（NeuI KD）的次级 Neu1 缺乏（NeuI KD）具有强烈的唾液酸酶活性，但来自 Neu4 敲除（Neu4 KO）小鼠则没有。百日咳毒素（PTX）是 G 蛋白偶联受体（GPCR）Galphai 蛋白的特异性抑制剂，基质金属蛋白酶（MMP）的广谱抑制剂 galardin 和哌嗪完全阻断 TQ 诱导的唾液酸酶活性。这些相同的抑制作用在 GM1 神经节苷脂特异性霍乱毒素亚基 B（CTXB）以及 CTX、酪氨酸激酶抑制剂 K252a 和广谱 GPCR 抑制剂苏拉明中观察不到。MMP-9 的特异性抑制剂抗-MMP-9 抗体和抗-Neu4 抗体，但不是 MMP-3 的特异性抑制剂，可完全阻断活 THP-1 细胞中 TQ 诱导的唾液酸酶活性，该细胞在细胞表面表达 Neu4 和 MMP-9。TQ 处理的活 THP-1 细胞中 Neu4 唾液酸酶活性使天然神经节苷脂和粘蛋白底物脱唾液酸化。RT-PCR 和 Western blot 分析显示，人单核细胞 THP-1 细胞中 Neu3 和 Neu4 的 mRNA 和蛋白值之间没有相关性，这首次表明这两种哺乳动物唾液酸酶存在独立于 TQ 激活的不同转录后机制。我们的发现确立了前所未有的姜黄色素对细胞表面 Neu4 唾液酸酶的激活，该酶来自营养黑种草籽油。TQ 通过 Galphai 亚基蛋白的膜靶向和基质金属蛋白酶-9 的激活增强 GPCR 信号传导，可能参与了细胞表面 Neu4 唾液酸酶的激活过程。
