| Type I hypersensitivity is Ig E-mediated allergic reaction.Currently,the main therapeutic drugs include antihistamines,leukotriene blockers,mast cell stabilizers and glucocorticoids,but these drugs have some side effects.Avenanthramides are bioactive compounds derived from edible grain oats that play an important role in anti-inflammatory,antioxidant,antipruritic and anticancer properties,and the chemical structure of avenanthramides is very similar to antihistamines Tranilast.Therefore,the RBL-2H3 cells model and animal passive skin sensitization model were used to evaluate the effect of avenanthramides on the degranulation of RBL-2H3 cells in type I hypersensitivity reaction.It aimed to find a food-source and healthy anti-alleric substance without side effects.The research in this dissertation include the extraction and purification of avenanthramides from oats,the effect of avenanthramides on degranulation of basophilic granulocyte RBL-2H3,and the effect of avenanthramides on Ig E-mediated passive skin.The main methods,results and conclusions are described as follows:1.The optimum conditions for the preparation of total avenanthramides from oats by ethanol extraction method are described as follows: ethanol volume fraction is80.88%,solid-liquid ratio(g/m L)is 1:20,extraction time is 2.05 h,extraction temperature is 61.86 °C.The average extraction rate of avenanthramides after crude extraction by solvent extraction is 15.85%,and the yield is 21.88% after purification by AB-8 macroporous resin.2.The CCK8 cell proliferation toxicity kit was used to detect the reproductive toxicity of 10-3-200?M avenanthramides on RBL-2H3 cells.The proliferation rate of RBL-2H3 cells treated with avenanthramides at each concentrationranged from 97%to 101%.There was no significant difference when compared to the blank group without avenanthramides(P>0.05),indicating that avenanthramides had no proliferative toxicity to RBL-2H3 cells.3.By measuring the amount of ?-HEX and histamine released from RBL-2H3 cells,it was shown that 100 ?M of total avenanthramides and 120 ?M of avenanthramide A,B,and C could active RBL-2H3 cells.The release rates of ?-HEX were 19.5%,26.2%,20%,and 19.8%,respectively,and the inhibition is siginificant when compared with the control group,where the release rate was 43.6%(P<0.05).The release rate of histamine was 21.2%,23.6%,35.1% and 34.3%,respectively,and there is also a significant inhibiting release when compared with the control group,where the release rate was 54.28%(P<0.05).However,the effect of 30 ?M of avena alkaloids on the release of ?-HEX and histamine was 40% and 55%,respectively,indicating the inhibition is not significant difference(P>0.05).4.By measuring the release of intracellular neo-synthetic substances TNF-? and IL-6,it was shown that after 100 ?M of total avenanthramides in oats and 120 ?M avenanthramide A,B,and C stimulated cells,release the amounts of 130.5 pg/ml,151.7 pg/ml,160.2 pg/ml,and 155.6 pg/ml of TNF-? were produced,respectively,which is significantly lower than the control group(243.6 pg/ml)(P<0.05).Moreover,the release of IL-6 was 376 pg/ml,380.6 pg/ml,441.3 pg/ml,and 387 pg/ml,respectively,which were significantly lower than 548.7 pg/ml in the control group(P<0.05).After 60 ?M and 30 ?M avenanthramides were applied to the cells,the producing of TNF-? and IL-6 was also decreased,but the release was lower than that of the high-concentration avenanthramides.It indicated that avenanthramides can significantly inhibit the release of TNF-? and IL-6,and the higher the concentration,the better the inhibitory effect.5.The detection of fluorescent calcium ion about the changes of intracellular calcium content in RBL-2H3 cells treated with different concentrations of avenanthramide B.Revealing that the higher the avenanthramides concentration,the weaker the fluorescence intensity and indicating that avenanthramide B can reduce the intracellular Ca2+content and inhibit the activation and degranulation of cells.6.The RBL-2H3 cells were treated with 120 ?M avenanthramide B and 100 ?M total avenanthramides,the phosphorylation of P38 phosphorylated by Lyn,Syk,NF-?B and MAPK family was detected by Western blot.The results showed that: compared with the control group,avenanthramides can reduce the expression of phosphorylated proteins P-Lyn,P-Syk,P-NF-?B and P-P38,while the expression of Lyn,Syk,NF-?B and P38 have no significant effect.7.Avenanthramide B can significantly improve capillary permeability induced by allergic reactions(P<0.05),when 30 ?M,60 ?M,and 120 ?M avenanthramide B were used in a passive skin anaphylaxis test in mouse,the degranulation inhibitory effects of avenanthramides on cells increased with the increasing content of avenanthramide B acting on the cells.In summary,based on the results of RBL-2H3 cells test and passive skin anaphylaxis in mouse,it was shown that avenanthramides can effectively inhibit the degranulation of mast cells mediated by Ig E and have a certain anti-allergic effect. |
PDF Full Text Request