Effect Of Four Flavonoids On The Allergenicity Of ω-5 Gliadin Peptide And The Barrier Damage Of Caco-2 Intestinal Epithelial Monolayers Induced By ω-5 Gliadin Peptide

Objective: wheat food allergy in children is a type I anaphylaxis mediated by Ig E.Because of its rapid and strong reaction,it is also called immediate hypersensitivity reaction,which affects the skin,respiratory tract,gastrointestinal tract and other parts.ω-5 gliadin is one of the main allergens of wheat food allergy in children.The current management of patients is dietary avoidance.However,wheat is widely used in food processing,so it is difficult to completely avoid the intake of wheat protein.Therefore,it is necessary to study other adjuvant therapies to alleviate allergic reactions.Flavonoids are secondary plant metabolites of polyphenols which are widely present in vegetables,fruits and plants.They have antioxidant,anti-inflammatory and immunomodulatory properties,and may alleviate food allergic reactions and intestinal inflammation induced by ω-5 gliadin.This study aims to investigate the effects of four flavonoids on the allergenicity of ω-5 gliadin peptides and ω-5 gliadin peptide-induced barrier damage in Caco-2 intestinal epithelial monolayers,which laid a foundation for the exploration of flavonoids as adjutant therapy for wheat allergy.Methods:Through in vitro simulated gliadin digestion in infants and young children’s gastrointestinal tract,and combined with reported ω-5 gliadin allergic epitope infromation,the digestion-resistant ω-5 gliadin allergic peptides were screened and synthesized.Subsequently,the degranulation model of KU812 cells was established,and the release of β-Hex,histamine,IL-6 and TNF-α of KU812 cells were detected after KU812 cells were incubated with ω-5 gliadin allergic peptides for 4 h.According to the results of determination,the allergic strongest ω-5 gliadin peptide was screened out.In order to explore the effect of four flavonoids on the allergenicity of ω-5 gliadin peptide,the degranulation model of KU812 cells was established by stimulation of 150 μg/m L the allergic stronger ω-5 gliadin peptide,and the release ofβ-Hex,hiamine,IL-6 and TNF-α of KU812 cells were detected after 0.5 h pretreatment with flavonoids baicalin,luteolin,isorhamnetin and naeringin.A monolayer model of Caco-2 intestinal epithelial cells was established.After incubation with four flavonoids in advance for 3 h,150 μg/m L ω-5 gliadin peptide with strong allergenicity were added for 24 h to establish barrier damage of Caco-2intestinal epithelial monolayers induced by ω-5 gliadin peptide.The levels of inflammatory factors IL-6,IL-8 and Zonulin were detected by ELISA,and the expression of tight junction proteins Occludin and ZO-1 were detected by Western blotting,aim to investigate the regulatory effects of four flavonoids on inflammatory response and barrier damage of Caco-2 cells induced by ω-5 gliadin peptides.Results: The digestion-resistant ω-5 gliadin allergic peptide was screened out,and four peptides were synthesized,namely P1: AA374-404,P2: AA160-189,P3:AA252-271,P4: AA253-279.Among them,ω-5 gliadin peptide P4 had the strongest ability to induce degranulation of KU812 cells.Compared with the blank group without synthetic peptide treatment,peptide P4 inhibited the viability of KU812 cells and promoted β-Hex,histamine,and IL-6 and TNF-α release in KU812 cells(P<0.05),indicating that the degranulation model of KU812 cells induced by ω-5 gliadin peptide was established successfully.The four flavonoids baicalein,luteolin,isorhamnetin and naringenin significantly inhibited peptide P4-induced KU812 cell viability loss;and decreased the release of β-Hex,histamine,IL-6 and TNF-α of KU812 cells stimulated by peptide P4.The inhibitory effect of isorhamnetin increased with the increase of the concentration(P<0.05).ω-5 gliadin peptide P4 promoted the release of inflammatory factors IL-6、IL-8and Zonulin in the Caco-2 cell monolayer,and reduced the expression of tight junction proteins Occludin and ZO-1(P<0.05).The Caco-2 cell inflammation and barrier damage model induced by ω-5 gliadin peptide was successfully established.The four flavonoids baicalein,luteolin,isorhamnetin and naringenin significantly inhibited the loss of Caco-2 cells viability induced by peptide P4;inhibited the peptide P4 to induce the release of IL-6,IL-8 in Caco-2 cells in a dose-dependent manner(P<0.05);inhibited the release of Zonulin and significantly increase the expression of tight junction proteins Occludin and ZO-1 in the Caco-2 cell monolayer.Conclusion: The four flavonoids baicalein,luteolin,isorhamnetin and naringenin can inhibit the degranulation of KU812 cells stimulated by ω-5 gliadin peptide,and have a certain anti-allergic effect.The four flavonoids also reduce inflammation reaction of Caco-2 cells caused by ω-5 gliadin peptide and protect intestinal epithelial barrier function.