| The interaction of food components is an important scientific issue in the food field and has attracted widespread attention.Oat milk is becoming more and more popular because of its rich nutritional value.The components inevitably interact with each other.Their effects on the nutritional quality and safety of food have not been reported.Avenanthramide and β-lactoglobulin are the most important constituents in oat and milk,respectively.Whether the two components interact with each other and whether they affect each other’s physiological activities is the focus of this study.Avenanthramide B and β-lactoglobulin were selected as the research object in this article.The interaction between avenanthramide B and β-lactoglobulin was studied by UV absorption spectroscopy and fluorescence spectroscopy.The investigation of the effect of β-lactoglobulin on the inhibition of cell degranulation by avenanthramide B through the RBL-2H3 cell degranulation model.The explorationof the effect of avenanthramide B and β-lactoglobulin complex on cell degranulation by KU812 cell degranulation model.The main results and conclusions are as follows:(1)The results of ultraviolet absorption spectra and fluorescence absorption spectra showed that the fluorescence quenching of β-lactoglobulin was induced by the static quenching of the avenanthramide B;Under the condition of static quenching mechanism,the number of binding sites n was calculated to be around 0.5,indicating that the compound was formed in a ratio of 2:1 between avenanthramide B and β-lactoglobulin.(2)The CCK8 cell proliferation and cytotoxicity assay kits was used to detect the proliferation toxicity of 120μM avenanthramide B,60μM β-lactoglobulin,120μM β-lactoglobulin,240μM β-lactoglobulin and compounds formed with both β-lactoglobulin and avenanthramide B at concentration ratio of 0.5∶1,1∶1,2∶1 on RBL-2H3 cells.The results showed that the proliferation rate of RBL-2H3 cells in each group ranged from 93.1% to 97.1%,indicating that avenanthramide B,β-lactoglobulin of various concentrations and various complexes had no proliferative toxicity to RBL-2H3 cells.(3)IgE-mediated degranulation model of rat basophils RBL-2H3 was established to detect the release of bioactive substances β-HEX and histamine.The results showed that 120 μM avenanthramide B and three complexes significantly inhibited the release of β-HEX and histamine compared with the model group(P<0.05).Compared with avenanthramide B,there was no significant difference in the 0.5:1 complex group(P>0.05),while the β-HEX and histamine release in the 1:1 and 2:1 complex groups increased by 2.53% 、42.56% and 3.70%、26.31%,respectively,the difference was significant(P<0.05).The results showed that the inhibition of avenanthramide B on the release of β-HEX and histamine by basophils decreased after interaction with β-lactoglobulin.(4)After RBL-2H3 cells was treated with 120μM avenanthramide B and three groups of compounds,the release of inflammatory factors IL-6 and TNF-α was significantly reduced compared with the model group(P<0.05).Compared with avenanthramide B,the release of IL-6 and TNF-α in the 0.5:1 complex group was not significantly different from that in the avenanthramide B group(P>0.05),and the release of IL-6 and TNF-α in the 1:1 and 2-1 complex groups was significantly increased by 26.21%,19.95% and 9.61%,34.90%,respectively.The results showed that the inhibition of basophil release of IL-6 and TNF-α decreased after the interaction of avenanthramide B with β-lactoglobulin.(5)The phosphorylation levels of P38,Lyn,JNK and NF-κB were detected by western blotting in activated RBL-2H3 cells treated with avenanthramide B,β-lactoglobulin and their complexes.The results showed that,compared with the model group,avenanthramide B inhibited the phosphorylation of P38,Lyn,JNK,and NF-B proteins,and its inhibitory effect was weakened when it interacted with β-lactoglobulin to form 1:1,2:1 complexes.(6)The CCK8 assay showed that the proliferation rate of avenanthramide B(60μM,120μM and 240μM),120μM β-lactoglobulin and the complexes formed by them(concentration ratio of 0.5:1,1:1,2:1)in KU812 cells was between 93.1% and 97.1%,no significant difference compared with the blank group.Therefore,avenanthramide B,β-lactoglobulin in various concentrations and various complexes had no proliferative toxicity to KU812 cells.(7)IgE mediated degranulation model of human basophil KU812 cells was established,and the levels of β-HEX and histamine were measured.The results showed that compared with the β-lactoglobulin group,there was no significant difference in the release rate of the 0.5:1 and 1:1 complex group(P>0.05),while the release rate of the 2:1 complex group was significantly reduced(P<0.05)by 7.36% and 17.54%,respectively.The results indicated that when β-lactoglobulin formed a complex with twice concentration of avenanthramide B,it couldinhibit the release of β-HEX and histamine by basophildegranulation caused by antigen β-lactoglobulin.(8)The release of IL-6 and TNF-α in KU812 cells with 120μM β-lactoglobulin treatment were 212.82ng/L and 156.02 ng/L,respectively.Compared with the β-lactoglobulin group,there was no significant difference in the release of IL-6 and TNF-α in the 0.5:1 and 1:1 complex groups(P>0.05),while the release of IL-6 and TNF-α in the 2:1 complex group was significantly reduced(P<0.05)by 22.34% and 18.40%,respectively.The results showed that the combination of avenanthramide B and β-lactoglobulin in a ratio of 2:1 could effectively inhibit the release of inflammatory factors IL-6 and TNF-α by basophil degranulation induced by β-lactoglobulin.(9)The phosphorylation of P38,Lyn,JNK and NF-κB in KU812 cells treated with avenanthramide B,β-lactoglobulin and their complexes was detected by western blotting.The results showed that the phosphorylation levels of Lyn,P38,JNK and NF-κB proteins in the β-lactoglobulin group and the 0.5:1 and 1:1 complex group were higher than those in the 2:1 complex group.It is shown that when the compound of avenanthramide B and β-lactoglobulin was formed at a ratio of 2:1,it couldinhibit the phosphorylation of relevant proteins in the signal transduction pathway mediated by tyrosine kinase Lyn,and thus inhibited the activation and degranulation reaction of basophils stimulated by antigen β-lactoglobulin.(10)The above results indicated that avenanthramide B and β-lactoglobulin interacted to form a complex in a ratio of 2:1 and affected each other’s biological activities.β-lactoglobulin attenuated the inhibition of avenanthramide B on basophil degranulation.When the concentration of avenanthramide B was twice the concentration of β-lactoglobulin,the effect of β-lactoglobulin on basophil degranulation was reduced. |
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