Mechanism Of Lactobacillus Paracasei PC-01 Alleviating Amino Acidmediated Food Allergy

Food allergies are immune-mediated adverse reactions to certain foods,usually triggered by harmless food protein antigens.It is an Ig E-mediated immediate hypersensitivity reaction,which can cause gastrointestinal,skin,respiratory tract,and cardiovascular abnormalities,and even life-threatening anaphylactic shock.The gut microbiota is critical for healthy immune regulation and the development of gut barrier function,promoting immune responses to pathogens and systemic hyporesponsiveness to innocuous antigens such as food.Probiotics can induce oral immune tolerance and alleviate food allergies by regulating intestinal flora and metabolites.In addition,previous metagenomic studies found that the amino acid metabolism pathways were different between allergic and non-allergic people,especially with probiotic intervention,and preliminarily found the correlation between probiotics,amino acid metabolism,and food allergy.However,whether probiotics alleviate food allergy through intestinal amino acid metabolism and the immune regulation mechanism are still unclear.The relationship between probiotics,intestinal amino acid metabolism and changes in host food allergy sensitivity needs to be further studied.In view of this,firstly,this study conducted oral intervention with PC-01 for two months in allergic population(A)and non-allergic population(NA).It was found that PC-01 has an obvious relieving effect on allergy.In allergic people,PC-01 can down-regulate Th2-related cytokines(IL-4,IL-13,Eotaxin,etc.)and inflammation-related cytokines(IL-6,IL-17,IL-8,IL-1β,etc.),down-regulate the proportion of eosinophils,up-regulate the content of Ig G2 and Ig G4 in the serum,induce and maintain the high s Ig A response in the intestine.And by measuring the content of amino acids in fecal samples,to explore the relationship between intestinal amino acids and allergies.The level of amino acids in feces is positively correlated with the content of Ig E,and there are characteristic allergic populations with high Ile,Leu and low s Ig A,and the intervention of PC-01 significantly downgrades the content of Ile and Leu.It indicated that Lactobacillus paracasei PC-01 could alleviate allergic reaction by regulating intestinal amino acid metabolism;Secondly,by using in vitro cell models(CD2.4,CMT93,and co-culture cell model),we in-depth explored the immunomodulatory mechanism of amino acids(Ile,Leu).We found that Ile and Leu upregulated the expression of pro-allergic genes,such as OX40 L and IL-6 in DC2.4,TSLP in CMT93.In mouse spleen,amino acids upregulated the expression of Th2 cell-related cytokines IL-4 and IL-13 m RNA,which promoted the Th1/Th2 immune balance biasing towards Th2.Moreover,mTOR signaling pathway inhibition and SESN2 gene knocking-down significantly inhibited the pro-allergic activity of amino acids(Ile,Leu)in DC2.4 and CMT93 cells,and there was no significant difference with the Blank group.The above results indicated that SESN2 and mTOR signaling pathway mediates the pro-allergic activity of amino acids;Finally,an in vivo study in mice was carried out,and the food allergic(FA)mice were treated with the combination of Ile,Leu and PC-01 to verify the anti-allergic activity of PC-01 and the allergy-promoting activity of amino acids(Ile,Le),and to deeply explore the immunoregulatory mechanism of PC-01 alleviating food allergy through amino acid metabolism.The study found that Ile,Leu can induce the up-regulation of Th2 cell proportion and the downregulation of Th1 and Treg proportion in mouse spleen.When treated by high Ile and Leu,the mice would show stronger food allergy responses,which verifies the conclusion of the above in vitro cell experiments.When the body is in a high Ile or Leu environment,PC-01 inhibited the Th2-type immune response by up-regulating the ratio of Th1 and Treg cells in the spleen,thereby alleviating the allergic response induced by amino acids.Through the analysis of intestinal microbiota,it was found that PC-01 can significantly improve the microbiota structure of FA mice,reduce the relative abundance of Proteobacteria and up-regulate the ratio of Firmicutes/Bacteroidetes(F/B)to induce intestinal immune regulation,change the amino acid level in mouse feces,and down-regulate the relative abundance of amino acid metabolism-related genes in the intestinal microbiota of FA mice.At the same time,under high Ile condition,PC-01 could significantly up-regulate the species richness of gut microbiota to exceed the level in healthy mice;under high Leu condition,PC-01 up-regulated species diversity.The above results indicated that PC-01 changed the amino acid environment of the body by regulating the structure of intestinal microbiota and amino acid metabolism,and then induced positive immune regulation effect on FA mice;The expression of genes related to the mTOR signaling pathway in mouse tissues and the immunofluorescence analysis of the small intestine showed that PC-01 inhibited the regulatory effect of amino acids on the expression of SESN2 and mTOR signaling pathway downstream gene Cyclin D1 the in the spleen and small intestine of mice.At the same time,it inhibited the activation of mTOR signaling pathway.This indicated that PC-01 could regulate the amino acid homeostasis by regulating the amino acid metabolism of intestinal microbiota,interfere with the regulation of SESN2 on the immune activity of amino acids,and inhibit the activation of amino acids on the mTOR signaling pathway in intestinal epithelial cells,so as to alleviate the food allergy-promoting response of amino acids,thereby relieving or preventing food allergies.