Ding Yuanyuan, Wang Na, Zhao Chenrui, Du Hongfen, Yuan Yujuan, Zhang Tao, An Hongli
Center for Translational Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China.
College of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China.
Mediators Inflamm. 2025 Aug 30;2025:9580121. doi: 10.1155/mi/9580121. eCollection 2025.
Mast cells (MCs) are effectors of anaphylactoid reactions. Mas-related G-protein-coupled receptor X2 (MRGPRX2) receptor mediates the direct activation of MCs in anaphylactoid disease. Siglec-6 negatively regulates MC activation and is a promising target in the development of antianaphylactoid reaction drugs. While caffeine exhibits an inhibitory effect against anaphylactic shock, the molecular mechanisms underlying these activities remain unknown. Our objective was to investigate the inhibitory effect of caffeine and its underlying molecular mechanism in MRPGRX2-induced MC activation and anaphylactoid reactions. Local and systemic anaphylactoid reactions in mice and in vitro MC activation experiments were conducted to investigate the effects of caffeine on anaphylactoid reactions. Molecular docking and surface plasmon resonance (SPR) experiments were used to predict and verify the molecular target of caffeine activity. siRNA silencing and western blot analyses were utilized to investigate the molecular mechanisms underlying caffeine activity. Caffeine inhibited local and systemic anaphylactoid reactions in mice and attenuated MRGPRX2-induced MC activation. Release of β-hexosaminidase, histamine, and Ca in siRNA-Siglec-6-laboratory allergic disease 2 (LAD2) cells was significantly higher than in NC-LAD2 cells. The binding affinity between caffeine and Siglec-6 protein is with a calculated of 1.76 × 10 mol/L. Caffeine increased Siglec-6 expression, phosphorylation of SHP-1, and dephosphorylation of PLC-γ1, IP3R, and ERK1/2 in the MRGPRX2 signaling pathway. Western blot demonstrated that phosphorylated SHP-1 (p-SHP-1) protein levels showed no increase, and MRGPRX2, phosphorylated PLCγ1 (p-PLCγ1), and phosphorylated ERK1/2 (p-ERK1/2) were abolished with caffeine treatment in Siglec-6-knockdown cells than in NC-knockdown cells. Caffeine suppressed the m-3M3FBS-induced upregulation of p-PLCγ1 and p-ERK1/2 levels. We have demonstrated that caffeine is an agonist of Siglec-6 and that subsequent activation of the ITIM motif of Siglec-6 phosphorylates SHP-1. This arrests MRGPRX2/PLC-γ1/IP3R signal transduction, thereby attenuating anaphylactoid reactions, including anaphylactic shock.
肥大细胞（MCs）是类过敏反应的效应细胞。Mas相关G蛋白偶联受体X2（MRGPRX2）介导类过敏疾病中MCs的直接激活。唾液酸结合免疫球蛋白样凝集素6（Siglec-6）负向调节MC激活，是抗类过敏反应药物研发中有前景的靶点。虽然咖啡因对过敏性休克有抑制作用，但其作用的分子机制尚不清楚。我们的目的是研究咖啡因在MRPGRX2诱导的MC激活和类过敏反应中的抑制作用及其潜在分子机制。通过小鼠局部和全身类过敏反应以及体外MC激活实验来研究咖啡因对类过敏反应的影响。利用分子对接和表面等离子体共振（SPR）实验预测并验证咖啡因活性的分子靶点。采用小干扰RNA（siRNA）沉默和蛋白质免疫印迹分析来研究咖啡因活性的分子机制。咖啡因抑制小鼠局部和全身类过敏反应，并减弱MRGPRX2诱导的MC激活。在siRNA-Siglec-6-实验室变应性疾病2（LAD2）细胞中，β-己糖胺酶、组胺和钙离子的释放显著高于阴性对照LAD2细胞（NC-LAD2细胞）。咖啡因与Siglec-6蛋白的结合亲和力计算得出的解离常数为1.76×10⁻⁹mol/L⁻¹。咖啡因增加MRGPRX2信号通路中Siglec-6的表达、SHP-1的磷酸化以及PLC-γ1、IP3R和ERK1/2的去磷酸化。蛋白质免疫印迹显示，在Siglec-6基因敲低细胞中，与阴性对照基因敲低细胞（NC-敲低细胞）相比，咖啡因处理后磷酸化SHP-1（p-SHP-1）蛋白水平未升高，且MRGPRX2、磷酸化PLCγ1（p-PLCγ1）和磷酸化ERK1/2（p-ERK1/2）均消失。咖啡因抑制m-3M3FBS诱导的p-PLCγ1和p-ERK1/2水平上调。我们证明了咖啡因是Siglec-6的激动剂，随后Siglec-6的免疫受体酪氨酸抑制基序（ITIM）激活使SHP-1磷酸化。这阻止了MRGPRX2/PLC-γ1/IP3R信号转导，从而减轻包括过敏性休克在内的类过敏反应。
