Computational and Mechanistic Study of the Therapeutic Potential of Compound Formononetin in Preventing Mast Cell Activation and IgE Production in Food Anaphylaxis

Abstract

The prevalence of allergic diseases such as Asthma, Rhinitis, Anaphylaxis, Eczema, Urticaria, and Angioedema have been rising dramatically in low‐ and middle‐income countries around the world. According to American College of Allergy, Asthma & Immunology, allergies are the 6th leading cause of chronic illness in the U.S. and more than 50 million Americans suffer from allergies each year. Extensive data suggest that food allergies affect up to 10% of the world population and have been increasing in the last two to three decades. Globally 300 million people have asthma and about 200 to 250 million people suffer from food allergies. Previously we have shown that a small molecule compound, formononetin have shown decreases IgE production from B cells. We hypothesize that formononetin will be a potential candidate for preventing food anaphylaxis in food allergy by regulating molecular pathways that involved in IgE production in B cells, mast cell activation and degranulation. The objective of this study is to reveal potential therapeutic mechanisms of formononetin in prevention and treatment of food anaphylaxis from food allergy, by using computational modeling, including target mining, gene ontology enrichment, pathway analyses, protein‐protein interaction analyses and in silico molecular docking to identify gene and protein targets that are regulated in food allergy and mast cells diseases. The targets identified were further validated using qRT‐PCR in B cell multiple myeloma cell line. U266 cells were cultured to 1.0 × 106 cells/mL and then incubated with formononetin at different concentrations of 20 μg/mL for 72 hours. Supernatants were collected for measuring IgE levels by ELISA and cell viability was determined using Trypan blue dye. mRNA expression was determined for the top targets identified and compared with GAPDH. Mining formononetin targets into obtained disease targets uncovers 25 targets for food allergy, 51 targets for IgE diseases and 19 targets for mast cell diseases. This targets where further used for gene ontology, KEGG pathway analysis, CTPD network and protein‐protein interaction. Top regulated KEGG pathways include the primary immunodeficiency pathway, Epstein Barr virus infection pathway, Th17 cell differentiation, PI3K‐Akt pathway. Regulated gene ontology biological processes include B cell proliferation, B cell differentiation, B cell activation and apoptotic process. Formononetin decreased IgE at after 72 hours, P < 0.05 without cytotoxicity. Formononetin decreased mRNA expression of P53, TYK2, CASP8 significantly (P < 0.05) and IgEH very significantly (P < 0.01) and increased the mRNA expression of BCL2, NFKBIA and BTK significantly (P < 0.05) after 72 hours in the culture group compared to the untreated group. These findings suggest that formononetin may be a novel therapeutic candidate for treatment and prevention of IgE/mast cell mediated food allergy and other allergic diseases.