背景:维生素K3(VK3),维生素K家族的脂溶性合成类似物,有凝结剂,抗炎,抗菌,和抗癌特性。假性变态反应是与肥大细胞相关的非IgE依赖性免疫应答。这项研究调查了VK3在非IgE依赖性肥大细胞活化中的作用。
方法:使用P物质(SP)诱导LAD2细胞活化,以分析VK3的体外作用。使用皮肤过敏和全身过敏小鼠模型来分析VK3的抗假性过敏作用。使用蛋白质组微阵列测定来分析VK3结合蛋白。生物层干涉法和免疫沉淀法用于验证VK3与其关键靶标之间的相互作用。RNA干扰用于确定GAB1在LAD2细胞活化中的作用。
结果:VK3抑制SP诱导的LAD2细胞活化,并导致β-己糖胺酶的释放,组胺和细胞因子;VK3抑制SP诱导的小鼠假性过敏反应,血清组胺和TNF-α水平降低。皮肤肥大细胞的脱颗粒减少;肥大细胞中的GAB1与VK3稳定结合。GAB1参与SP诱导的LAD2细胞活化。LAD2细胞中GAB1敲低可阻止SP诱导的β-己糖胺酶释放,钙动员和细胞骨架重塑。VK3直接结合GAB1并降低其表达以抑制SP诱导的LAD2细胞活化。
结论:在体外和体内均证实了VK3的抗假性过敏活性。VK3可以通过直接靶向GAB1来抑制SP诱导的肥大细胞活化。这项研究为VK3的活性和假性过敏反应的机制提供了新的见解。
BACKGROUND: Vitamin K3 (VK3), a fat-soluble synthetic analog of the vitamin K family, has coagulant, anti-inflammatory, antibacterial, and anticancer properties. Pseudo allergy is a IgE-independent immune response associated with mast cells. This study investigated the role of VK3 in IgE-independent mast cell activation.
METHODS: Substance P (SP) was used to induce LAD2-cell activation in order to analyze the effects of VK3 in vitro. Cutaneous allergy and systemic allergy mouse models were used to analyze the anti-pseudo-allergic effects of VK3. Proteome microarray assays were used to analyze VK3-binding protein. Biolayer interferometry and immunoprecipitation were used to verify interaction between VK3 and its key targets. RNA interference was used to determine the role of GAB1 in LAD2cell activation.
RESULTS: VK3 inhibited SP-induced LAD2-cell activation, and resulted in the release of β-hexosaminidase, histamine and cytokines; VK3 inhibited SP-induced pseudo allergic reactions in mice, and serum histamine and TNF-α levels decreased. Degranulation of skin mast cells was reduced; GAB1 in mast cells was stably bound to VK3. GAB1 participated in SP-induced LAD2-cell activation. GAB1 knockdown in LAD2 cells prevented SP-induced β-hexosaminidase release, calcium mobilization and cell skeletal remodeling. VK3 directly binds to GAB1 and reduces its expression to inhibited SP-induced LAD2 cell activation.
CONCLUSIONS: The anti-pseudo-allergic activity of VK3 was confirmed in vitro and in vivo. VK3 can inhibit SP-induced mast cell activation by directly targeting GAB1. This study provides new insights on the activity of VK3 and the mechanism of pseudoallergic reaction.