A new method to evaluate anti-allergic effect of food component by measuring leukotriene B4 from a mouse mast cell line

• Published in: Cytotechnology (Dordrecht) 2018-02, Vol.70 (1), p.177-184
• Main Authors: Takasugi, Mikako, Muta, Emi, Yamada, Koji, Arai, Hirofumi
• Format: Article
• Language: English
• Subjects: Arachidonate 5-lipoxygenase; Arachidonic acid; Biochemistry; Biomedicine; Biotechnology; Calcimycin; Calcium ionophores; Chemistry; Chemistry and Materials Science; Cytotoxicity; Daidzein; Enzymes; Flavonoids; Food; Food allergies; Genistein; Hay fever; High-performance liquid chromatography; Histamine; Isoflavones; Kaempferol; Leukemia; Leukotriene B4; Lipoxygenase; Mast cells; Phospholipase A2; Quercetin; Signal transduction; Soybeans
• ISSN: 0920-9069; 1573-0778
• DOI: 10.1007/s10616-017-0129-9

Leukotrienes (LTs), chemical mediators produced by mast cells, play an important role in allergic symptoms such as food allergies and hay fever. We tried to construct an evaluation method for the anti-LTB 4 activity of chemical substances using a mast cell line, PB-3c. PB-3c pre-cultured with or without arachidonic acid (AA) was stimulated by calcium ionophore (A23187) for 20 min, and LTB 4 production by the cells was determined by HPLC with UV detection. LTB 4 was not detected when PB-3c was pre-cultured without AA. On the other hand, LTB 4 production by PB-3c pre-cultured with AA was detectable by HPLC, and the optimal conditions of PB-3c for LTB 4 detection were to utilize the cells pre-cultured with 50 µM AA for 48 h. MK-886 (5-lipoxygenase inhibitor) completely inhibited LTB 4 production, but AACOCF 3 (phospholipase A 2 inhibitor) slightly increased LTB 4 production, suggesting that LTB 4 was generated from exogenous free AA through 5-lipoxygenase pathway. We applied this technique to the evaluation of the anti-LTB 4 activity of food components. PB-3c pre-cultured with 50 µM AA for 48 h was stimulated with A23187 in the presence of 50 µM soybean isoflavones (daidzin, genistin, daidzein, and genistein), equol, quercetin, or kaempferol. Genistein, equol, quercetin, and kaempferol strongly inhibited LTB 4 production without cytotoxicity. These results suggest that a new assay system using PB-3c is convenient to evaluate LTB 4 inhibition activity by food components. This method could be utilized for elucidation of the mechanisms of LTB 4 release suppression by food components such as flavonoids and the structure–activity relationship.