Tan Ji Wei, Israf Daud Ahmad, Md Hashim Nur Fariesha, Cheah Yoke Kqueen, Harith Hanis Hazeera, Shaari Khozirah, Tham Chau Ling
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43300, Malaysia.
Faculty of Science, Universiti Putra Malaysia, Serdang 43300, Malaysia.
Biochem Pharmacol. 2017 Nov 15;144:132-148. doi: 10.1016/j.bcp.2017.08.010. Epub 2017 Aug 13.
Mast cells play a central role in the pathogenesis of allergic reaction. Activation of mast cells by antigens is strictly dependent on the influx of extracellular calcium that involves a complex interaction between signalling molecules located within the cells. We have previously reported that tHGA, an active compound originally isolated from a local shrub known as Melicope ptelefolia, prevented IgE-mediated mast cell activation and passive systemic anaphylaxis by suppressing the release of interleukin-4 (IL-4) and tumour necrosis factor (TNF)-α from activated rat basophilic leukaemia (RBL)-2H3 cells. However, the mechanism of action (MOA) as well as the molecular target underlying the mast cell stabilising effect of tHGA has not been previously investigated. In this study, DNP-IgE-sensitised RBL-2H3 cells were pre-treated with tHGA before challenged with DNP-BSA. To dissect the MOA of tHGA in IgE-mediated mast cell activation, the effect of tHGA on the transcription of IL-4 and TNF-α mRNA was determined using Real Time-Polymerase Chain Reaction (qPCR) followed by Calcium Influx Assay to confirm the involvement of calcium in the activation of mast cells. The protein lysates were analysed by using Western Blot to determine the effect of tHGA on various important signalling molecules in the LAT-PLCγ-MAPK and PI3K-NFκB pathways. In order to identify the molecular target of tHGA in IgE-mediated mast cell activation, the LAT and LAT2 genes in RBL-2H3 cells were knocked-down by using RNA interference to establish a LAT/LAT2 competition model. The results showed that tHGA inhibited the transcription of IL-4 and TNF-α as a result of the suppression of calcium influx in activated RBL-2H3 cells. The results from Western Blot revealed that tHGA primarily inhibited the LAT-PLCγ-MAPK pathway with partial inhibition on the PI3K-p65 pathway without affecting Syk. The results from RNAi further demonstrated that tHGA failed to inhibit the release of mediators associated with mast cell degranulation under the LAT/LAT2 competition model in the absence of LAT. Collectively, this study concluded that the molecular target of tHGA could be LAT and may provide a basis for the development of a mast cell stabiliser which targets LAT.
肥大细胞在过敏反应的发病机制中起核心作用。抗原激活肥大细胞严格依赖于细胞外钙的内流，这涉及细胞内信号分子之间的复杂相互作用。我们之前报道过，tHGA是一种最初从当地一种名为阔叶蜜茱萸的灌木中分离出的活性化合物，它通过抑制激活的大鼠嗜碱性白血病（RBL）-2H3细胞释放白细胞介素-4（IL-4）和肿瘤坏死因子（TNF）-α，预防了IgE介导的肥大细胞激活和被动全身性过敏反应。然而，tHGA肥大细胞稳定作用的作用机制（MOA）以及分子靶点此前尚未得到研究。在本研究中，用DNP-IgE致敏的RBL-2H3细胞在受到DNP-BSA攻击前先用tHGA进行预处理。为了剖析tHGA在IgE介导的肥大细胞激活中的作用机制，使用实时聚合酶链反应（qPCR）测定tHGA对IL-4和TNF-α mRNA转录的影响，随后进行钙内流测定以确认钙在肥大细胞激活中的作用。通过蛋白质免疫印迹分析蛋白质裂解物，以确定tHGA对LAT-PLCγ-MAPK和PI3K-NFκB途径中各种重要信号分子的影响。为了确定tHGA在IgE介导的肥大细胞激活中的分子靶点，使用RNA干扰敲低RBL-2H3细胞中的LAT和LAT2基因，以建立LAT/LAT2竞争模型。结果表明，tHGA抑制了IL-4和TNF-α的转录，这是由于抑制了激活的RBL-2H3细胞中的钙内流。蛋白质免疫印迹结果显示，tHGA主要抑制LAT-PLCγ-MAPK途径，对PI3K-p65途径有部分抑制作用，而不影响Syk。RNA干扰结果进一步表明，在不存在LAT的LAT/LAT2竞争模型下，tHGA未能抑制与肥大细胞脱颗粒相关的介质释放。总的来说，本研究得出结论，tHGA的分子靶点可能是LAT，并可能为开发靶向LAT的肥大细胞稳定剂提供基础。
