Department of Pathology Microbiology & Immunology, New York Medical College, New York, NY, United States.
Academy of Chinese Medical Science, Henan University of Chinese Medicine, Zhengzhou, China.
Front Immunol. 2024 Aug 15;15:1427563. doi: 10.3389/fimmu.2024.1427563. eCollection 2024.
Food allergy is a prevalent disease in the U.S., affecting nearly 30 million people. The primary management strategy for this condition is food avoidance, as limited treatment options are available. The elevation of pathologic IgE and over-reactive mast cells/basophils is a central factor in food allergy anaphylaxis. This study aims to comprehensively evaluate the potential therapeutic mechanisms of a small molecule compound called formononetin in regulating IgE and mast cell activation.
In this study, we determined the inhibitory effect of formononetin on the production of human IgE from peripheral blood mononuclear cells of food-allergic patients using ELISA. We also measured formononetin's effect on preventing mast cell degranulation in RBL-2H3 and KU812 cells using beta-hexosaminidase assay. To identify potential targets of formononetin in IgE-mediated diseases, mast cell disorders, and food allergies, we utilized computational modeling to analyze mechanistic targets of formononetin from various databases, including SEA, Swiss Target Prediction, PubChem, Gene Cards, and Mala Cards. We generated a KEGG pathway, Gene Ontology, and Compound Target Pathway Disease Network using these targets. Finally, we used qRT-PCR to measure the gene expression of selected targets in KU812 and U266 cell lines.
Formononetin significantly decreased IgE production in IgE-producing human myeloma cells and PBMCs from food-allergic patients in a dose-dependent manner without cytotoxicity. Formononetin decreased beta-hexosaminidase release in RBL-2H3 cells and KU812 cells. Formononetin regulates 25 targets in food allergy, 51 in IgE diseases, and 19 in mast cell diseases. KEGG pathway and gene ontology analysis of targets showed that formononetin regulated disease pathways, primary immunodeficiency, Epstein-Barr Virus, and pathways in cancer. The biological processes regulated by formononetin include B cell proliferation, differentiation, immune response, and activation processes. Compound target pathway disease network identified NFKB1, NFKBIA, STAT1, STAT3, CCND1, TP53, TYK2, and CASP8 as the top targets regulated at a high degree by formononetin. TP53, STAT3, PTPRC, IL2, and CD19 were identified as the proteins mostly targeted by formononetin. qPCR validated genes of Formononetin molecular targets of IgE regulation in U266 cells and KU812 cells. In U266 cells, formononetin was found to significantly increase the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. In basophils KU812 cells, formononetin significantly increased the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK, TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R.
These findings comprehensively present formononetin's mechanisms in regulating IgE production in plasma cells and degranulation in mast cells.
食物过敏是美国一种普遍存在的疾病，影响近 3000 万人。这种疾病的主要管理策略是避免食用食物，因为可用的治疗选择有限。病理性 IgE 和过度反应的肥大细胞/嗜碱性粒细胞是食物过敏过敏反应的核心因素。本研究旨在全面评估一种名为芒柄花素的小分子化合物在调节 IgE 和肥大细胞激活方面的潜在治疗机制。
在这项研究中，我们使用 ELISA 法确定芒柄花素对食物过敏患者外周血单核细胞中人类 IgE 产生的抑制作用。我们还使用β-己糖胺酶测定法测量了芒柄花素对 RBL-2H3 和 KU812 细胞中肥大细胞脱颗粒的预防作用。为了确定芒柄花素在 IgE 介导的疾病、肥大细胞疾病和食物过敏中的潜在靶标，我们利用计算建模分析了来自 SEA、Swiss Target Prediction、PubChem、Gene Cards 和 Mala Cards 等多个数据库的芒柄花素的机制靶标。我们使用这些靶标生成了 KEGG 途径、基因本体论和化合物靶标途径疾病网络。最后，我们使用 qRT-PCR 测量了 KU812 和 U266 细胞系中选定靶标的基因表达。
芒柄花素以剂量依赖的方式显著降低 IgE 产生的人类骨髓瘤细胞和食物过敏患者 PBMC 中的 IgE 产生，且无细胞毒性。芒柄花素降低了 RBL-2H3 细胞和 KU812 细胞中的β-己糖胺酶释放。芒柄花素调节食物过敏中的 25 个靶标、IgE 疾病中的 51 个靶标和肥大细胞疾病中的 19 个靶标。靶标 KEGG 途径和基因本体论分析表明，芒柄花素调节疾病途径、原发性免疫缺陷、EB 病毒和癌症途径。芒柄花素调节的生物学过程包括 B 细胞增殖、分化、免疫反应和激活过程。化合物靶标途径疾病网络确定 NFKB1、NFKBIA、STAT1、STAT3、CCND1、TP53、TYK2 和 CASP8 为芒柄花素高度调节的顶级靶标。TP53、STAT3、PTPRC、IL2 和 CD19 被确定为芒柄花素最主要的靶标蛋白。qPCR 验证了 U266 细胞和 KU812 细胞中 IgE 调节的芒柄花素分子靶标的基因。在 U266 细胞中，发现芒柄花素显著增加 NFKBIA、TP53 和 BCL-2 的基因表达，同时降低 BTK、TYK、CASP8、STAT3、CCND1、STAT1、NFKB1 和 IL7R 的基因表达。在 KU812 细胞中，芒柄花素显著增加 NFKBIA、TP53 和 BCL-2 的基因表达，同时降低 BTK、TYK、CASP8、STAT3、CCND1、STAT1、NFKB1 和 IL7R 的基因表达。
这些发现全面呈现了芒柄花素在调节浆细胞中 IgE 产生和肥大细胞脱颗粒中的作用机制。
