Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR Gαi proteins and matrix metalloproteinase-9
Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR Gαi proteins and matrix metalloproteinase-9
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DOI：
10.1007/s10719-010-9281-6
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发表时间：
2010-04-01
影响因子：
3
通讯作者：
Szewczuk, Myron R.
中科院分区：
文献类型：
--
作者：
Finlay, Trisha M.;Jayanth, Preethi;Szewczuk, Myron R.
百里醌（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确切机制还不清楚。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），在此我们表明TQ对活BMC-2巨噬细胞中内毒素脂多糖（LPS）诱导的唾液酸酶活性没有抑制作用。相反，亲本黑籽油（BSO）和BSO对伞花烃（p-CY）的另一种成分完全阻断LPS诱导的唾液酸酶活性。所有这些化合物对细胞活力没有影响。另一方面，TQ以剂量依赖性方式在活BMC-2巨噬细胞中以及在活DC-2.4树突细胞、HEK-TLR4/MD2、HEK293、SP1乳腺癌细胞、人WT和1140F01和WG 0544 I型唾液酸沉积成纤维细胞中诱导强烈的唾液酸酶活性。Tamiflu（磷酸奥司他韦）抑制活BMC-2细胞中TK诱导的唾液酸酶活性，IC 50为0.0194 μ M，而神经氨酸酶抑制剂DANA（2-脱氧-2，3-脱氢-N-乙酰神经氨酸）的IC 50为19.1 μ M。抗Neu1、Neu2和Neu3抗体对活BMC-2和人THP-1巨噬细胞中的CQ诱导的唾液酸酶活性没有抑制作用，但抗Neu4抗体完全阻断该活性。TQ处理的活原代骨髓（BM）巨噬细胞具有强烈的唾液酸酶活性，这些巨噬细胞来源于继发性Neu1缺乏（NeuI KD）的WT和亚型组织蛋白酶A小鼠，但不来源于Neu4敲除（Neu4 KO）小鼠。百日咳毒素（PTX），G蛋白偶联受体（GPCR）的G α 1蛋白的特异性抑制剂和基质金属蛋白酶（MMP）的广谱抑制剂，加拉定和哌嗪应用于活BMC-2、THP-1和原代BM巨噬细胞完全阻断了CQ诱导的唾液酸酶活性。用GM 1神经节苷脂特异性霍乱毒素亚单位B（CTX B）以及CTX、酪氨酸激酶抑制剂K252 a和广谱GPCR抑制剂苏拉明未观察到这些相同的抑制作用。MMP-9的特异性抑制剂、抗MMP-9抗体和抗Neu4抗体，但MMP-3的特异性抑制剂不能完全阻断TQ诱导的THP-1活细胞中的唾液酸酶活性，THP-1活细胞在细胞表面上表达Neu4和MMP-9。来自经TQ处理的活THP-1细胞的细胞裂解物中的Neu4唾液酸酶活性使天然神经节苷脂和粘蛋白底物去唾液酸化。RT-PCR和蛋白质印迹分析显示，人类单核细胞THP-1细胞中Neu3和Neu4的mRNA和蛋白质值之间没有相关性，这首次表明这两种哺乳动物唾液酸酶独立于TQ激活的不同转录后机制。我们的研究结果建立了一个前所未有的激活Neu4唾液酸酶的细胞表面的百里醌，这是来自营养黑孜然油。TQ通过G α 1亚基蛋白的膜靶向和基质金属蛋白酶-9活化来增强GPCR信号传导可能参与细胞表面上Neu4唾液酸酶的活化过程。
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 IC50 of 0.0194 mu M compared to an IC50 of 19.1 mu M 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 G alpha i 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 G alpha i subunit proteins and matrix metalloproteinase-9 activation may be involved in the activation process of Neu4 sialidase on the cell surface.