Preliminary Cytological Investigation On Food Allergy Susceptibility Induced By High Salt

The changes in dietary composition are thought to be a potential driver of food allergy being increased year by year.It was shown that the dietary models of high-fat and low-dietary fiber which are popular in Westerns can enhance susceptibility to food allergy.Some studies reported that high-salt diet could affect the the immune system,especially regulating the function of Th17 cells and Tregs.Therefore,The objective of this work was to target the two important cells in food allergies,KU812cells(basophils)and Caco-2 cells(an important intestinal epithelial cells).Effects of high salt exposured on KU812 cells were studied by triggering the cell degranulation induced by OVA,and the mechanism of the KU812 cells how to release IL-4 under high salt was clarified as well through the analysis of transcriptomics.Finally,effects of high salt on intestinal barrier function were studied in intestinal epithelial cell Caco-2 model in vitro and mouse animal test,respectively.The main methods,results and conclusions are presented as follows.1.The KU812 cells sensitized with OVA was used to study the effect of high salt on cell degranulation.There was no significant difference in the release of histamine and β-hexosaminidase in the high-salt group when compared with the positive control group(P>0.05),and there was a significant difference in the release of IL-6 and TNF-ɑ between the two groups(P<0.05).The release of IL-6 and TNF-ɑ in high-salt group increased by 239.78 pg/m L and 1.8 pg/m L,respectively.The m RNA levels of IL-6,TNF-ɑ and IL-4 in the high-salt group were also significantly increased(P<0.05).High salt could promote OVA-sensitized KU812 cells to secrete pro-inflammatory cytokines IL-6,TNF-ɑ and IL-4,but not histamine andβ-hexosaminidase,indicating that high salt could aggravate the inflammatory factor response of KU812 cells in food allergy stage.2.The molecular mechanism of the KU812 cells how to release IL-4 under high salt was explored through SGK1 and P38 inhibitor intervention experiment.The results showed that the production of IL-4 in KU812 cells was improved and the expression of NFAT5 and SGK1 genes were increased under the condition of high salt.It was also found that the expression of SGK1 and IL-4 genes in KU812 cells were significantly inhibited by the interfered of SGK1 inhibitor under high salt conditions,while P38 inhibitor could significantly inhibit the expression of NFAT5,SGK1 and IL-4 genes in KU812 cells.Therefore,high salt promoted the production of IL-4which was also a key cytokine in Th2-type cell immunity in KU812 cells through the P38-NFAT5-SGK1 signaling pathway,So high salt could also promote the appearance of food allergies.3.The mechanism of the KU812 cells how to release IL-4 under high salt was analyzed by transcriptome sequencing technology(RNA-seq).There were 437 differential genes being identified based on the DNBSEQ platform,including 293up-regulated genes and 144 down-regulated genes.According to the analysis results of KEGG and GSEA,the mechanism of the effect of high salt on KU812 cells included the participation of PI3K-Akt,MAPK,Rap1,m TOR,Fox O and Wnt signaling pathways.Among them,Wnt,MAPK and Rap1 signaling pathway which may play a leading role were significantly activated,and P38,NFAT5 and SGK1 genes were also involved.4.The expressions of ZO-1,Occludin and Claudin-1 were detected by Q-PCR and Western-Blot after Caco-2 cells were treated with high salt.High salt could reduce the expression of these three proteins to varying degrees.Among them,the expression levels of ZO-1(P<0.01)and Occludin genes(P<0.05)were significantly decreased by the result of Q-PCR,but the expression of Claudin-1 was not significantly decreased(P>0.05).The results of Western-Blot experiment showed that the expression of ZO-1 protein was significantly decreased(P<0.05),but the expression of Occludin and Claudin-1 proteins was not significantly decreased(P>0.05).High salt could destroy the function of tight junction protein in Caco-2 cells,resulting in increased permeability.5.Mice were fed a high salt diet(4.0% NaCl diet +1.0% NaCl drinking water)for 3 weeks,the expression levels of ZO-1,Occludin and Claudin-1 proteins in intestinal cells were decreased to varying degrees.The experimental results of FITC-dextran also showed that the concentration of FITC-dextran in serum of mice in high-salt diet group was significantly increased(P<0.01).A high-salt diet disrupted the intestinal barrier in mice and increased the intestinal permeability of mice.