| Objective:To study the effects of long-chain inulin(inulin-type fructans)on the intestinal flora and intestinal mucosal immune function of mice treated with antibiotics(ABx),and evaluate the recovery of long-chain inulin to the intestinal flora and intestinal mucosal immunity of mice after antibiotic-induced intestinal flora imbalance.Method:1.Feed the mice with a mixed solution of four antibiotics(ampicillin,neomycin,metronidazole,vancomycin)for 1 week(ABx group),and randomly divide the mice into a long-chain inulin recovery group and a spontaneous recovery group.The long-chain inulin recovery group was treated with 5%(W/W)ITFs for 2 weeks(ITFre2w group),the spontaneous recovery group was free to drink sterilized water for 2 weeks(Sre2w group),and the control group was free to drink sterile water(Ctrl group).Mice feces were aseptically collected at the end of ABx treatment for 1 week and at the end of 2 weeks of recovery,and 16S r RNA sequencing analysis was performed to observe the recovery of the intestinal flora of mice after short-term supplementation of ITFs after ABx treatment for 2 weeks.2.Feed the mice with ABx for 3 weeks,and randomly divide the mice into a long-chain inulin recovery group and a spontaneous recovery group.The long-chain inulin recovery group was treated with 5%(W/W)ITFs for 4 weeks(ITFre4w group),the spontaneous recovery group was free to drink sterilized water for 4 weeks(Sre4w group),and the control group was free to drink sterile water(Ctrl group).The weight and diet of the mice recorded during the experiment.The mice were dissected at two time points at the end of 3 weeks of ABx treatment and at the end of 4 weeks of recovery,and the changes in organ index of each group were analyzed;the proximal colon tissue sections were analyzed by HE staining;The mouse feces were collected aseptically for16S r RNA sequencing analysis.Observe the recovery of mouse intestinal tissue and flora,and compare with the effects of short-term ABx treatment and inulin recovery on the intestinal flora of mice.3.Using enzyme-linked immunosorbent assay(ELISA)to analyze the diamine oxidase(DAO)activity and lipopolysaccharide(LPS)content in mouse serum;real-time quantitative polymerase chain reaction(RT-q PCR)Analyze the relative expression levels of colonic tight junction genes Occludin,Zo-1 and Mucin 2(Mucin2,Muc2);observe the expression level of Occludin in the colon by immunofluorescence staining technique,and explore the repair of ITFs on antibiotic-induced intestinal epithelial barrier damage in mice influences.4.Flow cytometry was used to analyze the proportion of B220~+B cells and CD4~+T cells in the colonic lamina proprial(LP)and spleen of regulatory T cells(Regulatory cells,Tregs);ELISA analysis of serum TGF-β1,IL-10,IL-17A and colon secretory immunoglobulin A(Secretory Immunoglobulin A,SIg A)content;RT-q PCR detection of related genes in the colon and spleen(i NOS,Il-1β,Tnf-α,Il-10,Ifn-γ,Tgf-β1)relative expression changes,to explore the effect of ITFs on the immune regulation of the intestinal mucosa and spleen of mice after antibiotic treatment.Results:1.The 16S r RNA microbial analysis results show that the intestinal microbes of the mice treated with ABx for 1 week are almost completely exhausted.By spontaneous recovery or supplementation of ITFs for 2 weeks,the diversity and richness of the intestinal microbes of the mice did not return to normal levels.At the phylum level,the predominant bacteria(Firmicutes,Bacteriodetes)in healthy mice had greater recovery in the two recovery groups,and the recovery in the Sre2w group was closer to that of the control group.At the genus level,the Bacteroidales＿S24-7 group bacteria in the Sre2w group returned to the control level,while the ITFre2w group showed high relative abundance of Parabacteroides＿distasonis and Bacteroides＿acidifaciens.Although supplementing ITFs can promote the growth of beneficial bacteria(Bifidobacterium,Lactobacillus)in the intestine,it also stimulates the reproduction of pathogenic bacteria(Enterbacter,Klebsiella).Compared with spontaneous recovery,the intervention of long-chain inulin delayed the recovery of the diversity and composition of the intestinal flora.2.After 3 weeks of ABx treatment,the mice’s spleen became smaller and the cecum became enlarged.After 4 weeks of spontaneous recovery and supplementary ITFs,the spleens of the mice recovered to the same level as the control group.The cecum of the Sre4w group recovered to the same level as the control group,while the length of the colon in the ITFre4w group was shortened and the cecum was still enlarged.Colon HE sections showed that the ITFre4w group had a little inflammatory cell infiltration and the mucosal epithelium was discontinuous,while the Sre4w group had only a few discontinuous mucosal epithelium.The 16S r RNA sequencing results showed that the diversity and abundance of intestinal microbes in mice treated with ABx for 3 weeks were similar to that of ABx for one week,and both decreased dramatically.After stopping ABx treatment and recovering through spontaneous recovery and supplementation of ITFs for 4 weeks,the composition and structure of the intestinal flora basically recovered to be the same as the control group,and there was no significant difference between spontaneous recovery or ITFs supplementation,indicating that antibiotic treatment Later,long-term supplementation of ITFs is more conducive to the recovery of intestinal flora than short-term supplementation of ITFs.3.ELISA results showed that after ABx treatment for 3 weeks,DAO activity and LPS content in mouse serum increased.RT-q PCR results showed that the relative expression of Zo-1 and Muc2 genes increased,while the relative expression of Occludin gene remained unchanged.After 4 weeks of recovery,the levels of DAO and LPS in the two recovery groups decreased,but the ITFre4w group was higher than the Sre4w group.The relative expression of the Zo-1 gene in the Sre4w group returned to the control level,the relative expression of the Muc2 gene did not decrease,and the relative expression of the Occludin gene did not change;the relative expression of the Zo-1gene in the ITFre4w group returned to the control At the group level,the relative expression of Occludin gene decreased.Colon immunofluorescence showed that the expression of Occludin protein in ABx3w and Sre4w groups was not different from that in the control group,while the expression of Occludin protein in ITFre4w group was decreased.It shows that ABx treatment can cause the integrity of the intestinal epithelial barrier to be damaged.Compared with spontaneous recovery,the recovery of supplementary ITFs delays the repair of the intestinal epithelial barrier.4.The results of flow cytometry showed that the ratio of B220~+B cells and CD4~+Foxp3~+Tregs in mouse colonic LP after ABx treatment decreased;spontaneous recovery and supplementary ITFs recovered for 4 weeks,B220~+B cells and CD4~+Foxp3~+Tregs in the two recovery groups The proportion of Foxp3~+Tregs increased and the Sre4w group was higher than the ITFre4w group.However,in the spleen,the ratio of B220~+B cells and CD4~+Foxp3~+Tregs in ABx-treated mice increased;spontaneous recovery and supplementary ITFs recovered for 4 weeks,the two groups of recovery groups of B220~+B cells and CD4~+Foxp3~+Tregs The proportion drops.And the proportion of Tregs in the ITFre4w group was lower than that in the Sre4w group.ELISA results showed that after ABx treatment,SIg A and serum TGF-β1 in mouse colon decreased after ABx treatment,and IL-17A content in serum increased.After 4weeks of recovery after spontaneous recovery and supplementary ITFs,the two recovery groups The content of SIg A and TGF-β1 of Sre4w increased,and the content of IL-17A decreased.The SIg A and TGF-β1 of Sre4w group were higher than those of ITFre4w group,and IL-17A was lower than ITFre4w group.RT-q PCR results showed that after ABx treatment,the relative expression of i NOS and Tnf-αgenes in the colon and spleen of mice increased;the relative expression of Il-4 and Ifn-γgenes in the colon decreased,while the relative expression of spleen Ifn-γgene decreased,and the relative expression of Il-4 and Il-10 genes increased;After 4 weeks of recovery,compared with spontaneous recovery,supplementation of ITFs can reduce the relative expression of i NOS and Tnf-αgenes in colon and spleen,but has no effect on the expression of Il-4and Ifn-γgenes.It shows that ABx treatment affects the immune regulation status of mice.Compared with spontaneous recovery,supplementation of ITFs delays the recovery of colonic mucosal immune regulatory cells such as Tregs and B cells,but reduces the pro-inflammatory factor genes i NOS and Tnf-αin the colon.And the expression of the spleen.Conclusion:Both continuous treatment of ABx for 1 week and continuous treatment for 3weeks can cause serious imbalance of the intestinal flora of mice.After 1 week of ABx treatment,whether by supplementing ITFs or spontaneously recovering for 2 weeks,the intestinal microbial diversity and richness did not return to normal levels.Supplementing ITFs can promote the growth of beneficial bacteria,but it can also stimulate the reproduction of conditional pathogenic bacteria.After 3 weeks of ABx treatment,supplementation of ITFs for 4 weeks is more conducive to the restoration of the intestinal flora to the original structure,but compared with spontaneous recovery,supplementation of ITFs has no significant effect on the recovery of intestinal flora.After antibiotics induce serious imbalance of the intestinal flora of mice,the immune status is a complex and dynamic process.ABx treatment will affect the immune regulation of the mouse body.Regulatory immune cells such as B cells and Tregs are reduced.It is worth noting that B cells and Tregs may have compensatory"systemic accumulation"in the spleen.ABx treatment can damage the intestinal epithelial barrier,increase the expression of pro-inflammatory related genes such as i NOS and Tnf-α,and decrease the expression of Il-4 and Ifn-γgenes.Compared with spontaneous recovery,supplementation of ITFs reduces the relative expression of i NOS and Tnf-αgenes,but delays the repair of the intestinal epithelial barrier and the recovery of immune regulatory cells B cells and Tregs. |
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