The Effects Of Exosomes And Their Cargo On Cow’s Milk Allergy And Lipid Metabolism

Food allergies have become a widely concerned food safety and health issue among the public.It is a complex immune process involving multiple cells and molecular.As a mediator of cell-to-cell communication,the role of exosomes in food allergy is not yet known.In addition,previous studies have shown that lipid metabolism disorders increase the risk of food allergies.Gastrointestinal symptoms caused by cow’s milk allergy reactions can cause changes in the intestinal environment,which will directly affect the digestion and absorption of nutrients,as well as interfere with intestinal glucose and lipid metabolism.Exosomes are related to lipid synthesis,transport,and decomposition.Whether cow’s milk allergies cause lipid metabolism disorders,and the role of exosomes play in them are still unknown.This study will focus on the exosomes derived from cow’s milk allergic mice.By comparing the changes in quantity,the differences of carrying proteins and small RNA,the study aims to identify the patterns of changes in exosomes in cow’s milk allergies;At the same time,small animal imaging is used to explore the target organs and cells of exosomes derived from intestinal tissue.Furthermore,the expression levels of genes or proteins related to cholesterol biosynthesis,cholesterol transport,and cholesterol reabsorption in the liver and small intestine of mice with cow’s milk allergy were detected to evaluate the effect of exosomes on lipid metabolism in milk allergy.The main methods,results,and conclusions of the study are as follows:(1)A cow’s milk allergy model was constructed byβ-lactoglobulin(BLG),and extracellular vesicle release was inhibited by N,N′-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3′-p-phenylene-bis-acrylamide dihydrochloride(GW4869).The changes in rectal temperature and elevated levels of histamine and Ig E in serum of mice indicate the successful construction of cow’s milk allergy mouse model,and the inhibition of exosome release alleviated allergic symptoms in mice;Through principal component analysis(PCA),it was found that inhibiting the release of exosomes changed the proportion of Th cell population in the spleen.After inhibiting the release of exosomes,T lymphocytes in peripheral blood mononuclear cells(PBMCs)of mice were upregulated by 7%,while B lymphocytes were downregulated by 34%.In addition,mouse serum derived exosomes were isolated by size exclusion chromatography(SEC).The serum exosomes of the sensitized group were 16.6%higher than those of the control group,and the proteins carried by the serum exosomes of the sensitized group were mainly enriched in adaptive immune response pathways such as B cell receptors.These data indicate that exosomes are involved in the process of cow’s milk allergic reactions and are mainly affected by changes in B cell function.(2)A separation and purification method for intestinal tissue-derived exosomes(Ti-Exo)was successfully established through enzymatic hydrolysis and ultra-high speed freeze centrifugation.The isolated Ti-Exo were identified by nanoparticle tracking analysis(NTA),transmission electron microscopy(TEM),and immunoblotting(WB),indicating that the purity of the extracted exosomes meets the requirements of subsequent experiments.Through proteomic analysis,it was found that the proteins carried by Ti-Exo-s are mainly enriched in pathways such as tight junctions,antigen processing and presentation,and cholesterol metabolism;Through small RNA sequencing,it was found that there were 9 differentially expressed mi RNAs in the Ti-Exo,among which 6 were significantly upregulated:mmu-mi R-370-3p,mmu-mi R-125b-1-3p,mmu-mi R-194-2-3p,mmu-mi R-184-3p,mmu-mi R-486a-3p,and mmu-mi R-665-3p;Significantly downregulated three genes:mmu-mi R-29b-2-5p,mmu-mi R-221-3p,and mmu-mi R-379-5p.Through small animal imaging technology,the targeted organs of fluorescent labeled Ti-Exo were the liver,spleen,and femur,and the targeted cells were CD4~+cells and CD11b~+cells;The simultaneous transcriptome sequencing results indicate that Ti-Exo-s are involved in regulating liver lipid metabolism and spleen T cell differentiation.(3)By detecting total cholesterol(TC),triglycerides(TG),low-density lipoprotein(LDL-c),and high-density lipoprotein(HDL-c)in serum,as well as TC and total bile acids(TBA)in feces,the results showed that serum TC and LDL-c were significantly increased in cow’s milk allergic mice,while TC and TBA in feces were significantly reduced,indicating that cow’s milk allergic mice experienced cholesterol metabolism disorders.The expression of key regulatory genes or proteins in cholesterol synthesis,cholesterol transport,bile acid synthesis,and cholesterol reabsorption were detected by real-time fluorescence quantitative PCR(q PCR)and WB of the liver and small intestine tissues.The results showed that cow’s milk allergic mice increased cholesterol biosynthesis by upregulating the expression of HMGCR and SREBP2 genes/proteins;The PCSK9 gene level in sensitized mice significantly increased,leading to a decrease in LDLR protein level.At the same time,the SR-B1 gene expression level in the sensitized group was significantly reduced.These results indicate that cow’s milk allergy led to impaired cholesterol transport to the liver;The levels of CYP7A1 and CYP27A1 genes/proteins in the liver were downregulated in milk allergic mice,leading to milk allergy and inhibition of bile acid synthesis in the liver of mice;The significant increase in NPC1L1 protein expression in the small intestine tissue of sensitized mice lead to milk allergy and increased intestinal cholesterol reabsorption.The changes in the expression levels of the above key regulatory factors indicate that cholesterol metabolism is disrupted in sensitized mice,and the levels of TC and LDL-c in the blood were abnormally elevated.In addition,inhibiting the release of exosomes reduced the levels of TC and LDL-c in serum,as well as the levels of TC and TBA in feces.And the expression of key enzyme genes and proteins in liver cholesterol synthesis,cholesterol reverse transport,and bile acid conversion/transport,as well as cholesterol reabsorption was reversed,indicating that inhibiting exosome secretion improved lipid metabolism disorders caused by cow’s milk allergies.(4)In order to verify the involvement of Ti-Exo in immune regulation and lipid metabolism processes,an in vitro cell experiment was constructed.In this line,differential mi RNA mimetics(mi RNA-mimics)were synthesized and transfected into Caco-2 cells.The results showed that Ti-Exo-s promote the polarization of spleen cells towards Th2 cells and promote the secretion of Th2 cytokines(IL-4 and IL-13).After24 hours of stimulation of Caco-2/HT-29 co-culture transport model with Ti-Exo-s,the transmembrane electrical resistance(TEER)was significantly reduced when compared to the blank group,and the protein expression levels of Claudin1 and ZO-1 were significantly downregulated.After transfection of Caco-2 cells using mi RNA-mimic revealed that mi R-125b-1-3p,mi R-486a-3p,and mi R-184-3p downregulated the levels of claudins family proteins,while mi R-29b-2-5p,mi R-221-3p,mi R-379-5p,and mi R-665-3p upregulated the levels of claudins family proteins;Mi R-221-3p upregulates the level of occludin protein.In addition,by simulating cholesterol transport and absorption through the fluorescence analogue of cholesterol(NBD-TC),it was found that compared to Ti-Exo-c,Ti-Exo-s increased cell uptake and absorption of cholesterol.In summary,an increase in the number of circulating and intestinal tissue derived exosomes in cow’s milk allergy mouse were founded.And their carried proteins and mi RNAs were related to the allergic reaction process,which providing a theoretical basis for subsequent research on biomarkers of cow’s milk allergy.At the same time,this study firstly found that cow’s milk allergy reactions cause lipid metabolism disorders via exosomes,providing a new perspective for the health management of allergic patients.