| Milk is an important food source for infants and young children.However,according to domestic and international epidemiological survey,3%-6% of children are allergic to β-lactoglobulin(β-Lg).In this study,we selected a strain of Lactobacillus acidophilus KLDS 1.0738 with anti-allergic properties,and found that the strain can reverse the allergic inflammation in the Treg/Th17 imbalance,but the concrete mechanism is not clear.Recent studies suggest that a class of non-protein-encoded RNA-micro RNA(miRNA)is an important regulator of intestinal mucosal immunity,plays a key role in the differentiation of Th cells in the development of inflammation.But miRNA expression on the regulation of milk induced by Th17 imbalance is not yet known;whether the probiotic interaction with the host intestinal tract is regulated by a particular miRNA is also unclear.Therefore,based on the previous work,by constructing cell and animal models,first of all,to explore the role of β-Lg induced allergic cells to express miRNA,Secondly,the relationship between miRNA expression,Th17 imbalance and allergic inflammation was analyzed in the organs of β-Lg allergy mice;Further studies on the role of miRNAs in the inhibition of Th17 differentiation by Lactobacillus acidophilus.Further studies on the role of miRNAs in the inhibition of Th17 differentiation by Lactobacillus acidophilus.For the first time,this study revealed the immune mechanism of probiotics from miRNA levels,to provide a new theoretical basis for the prevention and treatment of milk allergies.In this study,we first analyzed the effect of β-Lg on miRNA expression from the cell level.The effect of β-Lg on the proliferation of splenic lymphocytes in mice was analyzed by MTT assay.β-Lg with the final concentration of 0.1,0.5 and 1 mg/mL was incubated with mouse spleen lymphocytes for 24 h,It was found that β-Lg could significantly promote the proliferation of spleen lymphocytes(P<0.05),and showed a dose-dependent relationship.Preferably 1 mg/mL of β-Lg incubated with spleen lymphocytes for 24 h.The expression of miR-146 a,miR-155,miR-21 and miR-9 was detected by RT-PCR.The results showed that β-Lg could induce the expression of miR-21,miR-155,miR-146 a and miR-9(P<0.05),in particular,miR-155,miR-146 a expression was 7 times and 5 times the blank group,respectively.Animal model was used to investigate the role of miRNA and Th17 cells in β-Lg allergy.Female KM mice were randomly divided into blank group and β-Lg allergy group(n=6).β-Lg allergic mice were given 1 mg/m L of allergen(1 m L Freund’s adjuvant + 1 mL 1 mg/mL β-Lg)once a week,a total of 3 weeks.Day 21 of the experiment,mice were given 1 mg/mL β-Lg after oral challenge with cervical dislocation.The expression of miR-146 a,miR-155,miR-21 and miR-9 in spleen and colon tissues were detected by RT-PCR.The expression of Th17-related cytokines and transcription factors in serum and spleen of allergy mice was detected by RT-PCR and enzyme-linked immunosorbent assay(ELISE).The results showed that the sensitization of β-Lg sensitized mice was significant,Manifested as allergic mice with diarrhea,the average weight loss phenomenon,colon length and weight were decreased,pathological results showed that allogeneic mouse colon tissue had significant inflammatory cell infiltration,and the number of eosinophils and serum Ig E levels were significantly increased,respectively(6.28±0.87)×109/L and(134.78±9.51)ng/m L(P<0.05).Compared with the blank group,the levels of serum Th17 cytokines IL-17,IL-6,TNF-α and IL-21 was significantly increased(P<0.05).Correspondingly,the expression levels of RORγt and IL-17 mRNA in spleen of allergic mice were also significantly higher than those in blank group(P<0.05).The expression of miRNAs in the spleen and colon tissues of each group showed that,the expression of miR-146 a,miR-155,miR-21 and miR-9 in β-Lg allergy group was significantly higher than that in blank group(P<0.05).Consistent with the results of the cell model,β-Lg allergen-induced mice overexpress miR-146 a and miR-155.Further analysis of the correlation between Th17 cells and miRNA expression in β-Lg allergy mice.The expression levels of miR-21,miR-155,miR-146 a and miR-9 mRNA in spleen tissue of β-Lg allergic mice were significantly correlated with IL-17 mRNA(r=0.997,0.996,0.998,0.995,P<0.05)and RORγt mRNA(r=0.983,0.974,0.983,0.983,P<0.05)were positively correlated.Effect of Lactobacillus acidophilus on the Expression of miRNA in β-Lg Allergic Lymphocytes.Compared with Con A positive control group,Lactobacillus acidophilus can inhibit β-Lg allergy lymphocyte proliferation,especially in the high concentration of Lactobacillus acidophilus inhibitory effect is higher.The expression of miRNAs in spleen lymphocytes of β-Lg allergy was detected by RT-PCR.Results miR-146 a,miR-155,miR-21 and miR-9 expression were significantly decreased(P<0.05),and the higher the concentration,the more the decline.The mice were treated with different concentrations of Lactobacillus acidophilus(low concentration of 107 cfu/m L;medium concentration of 108 cfu/m L;high concentration of 109 cfu/mL).The morphology of each group of mice was observed,take the mouse colon tissue to measure the weight and length as well as tissue sections,the therapeutic effect of Lactobacillus acidophilus on β-Lg allergic mice was evaluated by detecting serum Ig E content and eosinophil count.The results showed that after 4 weeks of continuous administration of Lactobacillus acidophilus,the symptoms of acute attack in mice were significantly reduced;with the increase of Lactobacillus acidophilus concentration,The mean body weight of mice also increased significantly(respectively,4.028 ± 0.74 g;4.59 ± 0.52 g;5.34 ± 0.54 g).The length of the colon to shorten the phenomenon are not the same degree of relief,Inflammatory cell infiltration,histopathological damage score was significantly reduced;and effectively relieve allergic symptoms to reduce the number of allergic mice eosinophils and serum levels of antibodies,with the increase of Lactobacillus acidophilus concentration,the effect is remarkable,and dose dependent(P<0.05).Lactobacillus acidophilus can significantly reduce the levels of inflammatory cytokines such as IL-17,IL-6,TNF-α and IL-21 in serum of allergic mice,the expression of IL-17 and RORγt m RNA in spleen tissue of allergic mice was inhibited,and the inhibitory effect of Lactobacillus acidophilus was significantly better than that of low dose group(P<0.05).The expression of miR-21,miR-155,miR-146 a and miR-9 in colonic and spleen tissues of allergic mice was significantly lower than that of Lactobacillus acidophilus(P<0.05),and dose dependent.In summary,miRNA gene expression and IL-17 and RORγt m RNA expression were closely related to the severity of β-Lg allergy symptoms,can be used as one of the biological indicators to evaluate allergic reactions.Lactobacillus acidophilus to alleviate allergy effect is significant,it may play a role in regulating the expression levels of miR-155,miR-146 a and Th17. |
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