The Effects Of Antibiotic-induced Alteration Of The Intestinal Flora On Intestine And Immunity System In Mince

Purpose:To study the effects of various antibiotic(Antibiotic,ABx)on the immune system after changing the intestinal flora of mice.Method:1.We used a mixture of four different antibiotics(ampicillin,metronidazole,vancomycin,and neomycin)to feed male Balb/c mice at the same time.The processing time nodes were 7 days and 21 days.After 7 days of ABx treatment(ABx7 group),the mice were divided into two groups,one continued to be administered(ABx21 group),and the other one was given sterile water for natural recovery(RE group),then fed to 21 day.By measuring the body weight,water consumption and feed volume of mice,analyzing the changes in the body affected by the administration.2.Take the ileum,proximal colon and spleen tissue of mice,by measuring the length and weight of the colon,the weight of ileum and spleen,investigating the effects of antibiotics on intestinal tissues and immune organs of the body.Each group of mice was randomly divided into two parts.One part of the intestinal tissue samples of the mice was stained with hematoxylin-eosin(HE).The other part of the sample was frozen in liquid nitrogen,total RNA was extracting for RNA-seq and which was used to analyze the changes of colonic transcription level for bioinformatics analysis.3.Genomic DNA was extracted from fresh stools of mice,through 16 S rDNA V3-V4 hypervariable regions sequence was carried out to analyze the differences of gut microbiota in each group.Analyz the differences between group and effect of antibiotics on the intestinal flora through the species ? and ? diversity.4.Immunofluorescence staining technology was used to observe the expression level and secretion position of regenerating islet-derived protein(Reg3b).5.To clarify whether the above-mentioned gene expression changes were caused by changes in the bacterial flora,bacterial suspension was prepared using mouse stool samples;the mouse colon cancer cells(MC38)were co-cultured with bacterial suspensions in vitro,and the RNA was extracted from the cells at different time points,then RT-q PCR was used to verify the genes with significant expression in the transcriptome.Results:1.The mice were fed with a mixture of four different antibiotics cocktails(ABx).Compared with the control group,the mice in the ABx7 group had a sharp decrease in body weight,water consumption and feed volume;colon weight decreased and length increased;ileum weight increased,reduced spleen weight.By observing the HE pathological sections,the length of the colonic crypts after ABx treatment became shorter,and there were inflammation.After the 7th day,the body weight,water and feed of the mice in the ABx21 group and the RE group gradually increased;the weight of the colon increased but there was no significant difference in length;the weight of the ileum and the spleen increased.The pathological section of HE showed a slight increase in the length of the crypts of the colon,and the edema and inflammation improved.2.Based on the results of the 16 SrDNA V3-V4 hypervariable region of microbial diversity,most of the intestinal microorganisms in normal mice were composed of Bacteroidetes and Firmicutes.Compared with the control group,after 7 days of ABx treatment,the diversity of intestinal microorganisms decreased sharply,the abundance of Bacteroides and Pachyphyta was significantly reduced,and the abundance of Proteobacteria significantly increased.After 21 days of treatment,the biodiversity of the RE group increased but could not restore to the control group level,and the flora composition of the ABx21 group was similar to that of the ABx7 group,and the Proteobacteria were still the main group.3.In colonic transcriptome sequencing results,colonic gene expression levels in ABx7 changed significantly,and most differential gene expressions were down-regulated.Through GO functional enrichment and KEGG pathway analysis of which found that the main genes focus on adaptive and innate immune functions.It was further found that Nfkbia?Tnf?Il6?Reg3b?Reg3g?Stat3 and other genes related to immunity have significant expression differences.Among them,the gene and protein expression of Reg3 b and Reg3 g was significantly increased in ABx7,but decreased to the control level after 21 days of ABx.Conclusion:After ABx treatment,the composition of the intestinal flora changed significantly,but continued to extend the treatment time without increasing the further changes in the flora.Host immune status can change in different ABx treatment times,the body adapted to the changed intestinal flora gradually and tried to revert to a certain extent after long-term ABx treatment.Reg3 b may affect the changes in host immune homeostasis during continuous ABx treatment and which correlated with the growth of Escherichia-Shigella.