Effects Of Single,double And Triple Probiotics On Flora Recovery And Immunity In Mice With Intestinal Flora Imbalance

Objective:By intragastric administration of single,double and triple probiotics to mice with intestinal flora imbalance,the immune recovery and its possible mechanism of mice with intestinal flora imbalance were analyzed,and the effects of single,double and triple probiotics on the recovery of intestinal flora in mice with intestinal flora imbalance were elucidated.Method:1.Experimental grouping and cycleThirty 8-week-old SPF C57BL/6J mice were randomly divided into normal group(NC),antibiotic group(NR),single group(MT),double group(DT)and triple group(TT),with 6 mice in each group.NR group,MT group,DT group and TT group were given ceftriaxone sodium 8g/kg for one week.After successful modeling,the MT group was given Lactobacillus DM9811 0.2 ml/only/day.DT group was given Lactobacillus DM9811,Enterococcus faecalis and DM9112 bacteria liquid 0.2 ml/only/day,TT group was given Lactobacillus DM9811,Enterococcus faecalis DM9112 and Lactobacillus plantarum DM9125 bacteria liquid 0.2 ml/only/day.The mice in each group were administered once a day for six weeks.The mice in each group were sacrificed at the 7th week,and the mice in the NC group were not treated specially.Mice in each group had normal diet and drinking water.2.Sample collection and productionFecal specimens of each group were collected at a fixed time every week,and16S r RNA sequencing analysis was performed in the last week.The spleen of mice was collected,and the spleen cell suspension was prepared by grinding and filtering.The spleen cell suspension was divided into tubes.The number of cells in each tube was5×105～1×106 cells/ml,and the cell volume was 0.5～1ml.A blank control tube and a single positive tube were set up,and fluorescent antibodies FITC-CD3,Per CP-Cy5.5-CD4,APC-CD8,and PE-CD19 were added.Fully mix and incubate in the dark for 15-20 min.After centrifugation and PBS resuspension,the T lymphocytes and B lymphocytes in the spleen of mice were detected by flow cytometry,and the CD4/CD8 ratio was calculated to analyze the immune function of mice.The serum levels of interferon-γ(IFN-γ),interleukin-4(IL-4),immunoglobulin(Ig G)and C-reactive protein(CRP)were detected by ELISA.Result:1.Effects of single,double and triple probiotics on basic indexes of mice with intestinal flora imbalanceAfter successful modeling,compared with the NC group,the food and water intake of NR group,MT group,DT group and TT group decreased,but there was no significant difference(P>0.05),except that the weight of NR group decreased significantly(P>0.05).After the intervention of MT,DT and TT probiotics,the water intake of mice in each group gradually recovered compared with that of NC group.The water intake of DT group was always higher than that of MT group and TT group,and gradually approached that of NC group after 4 weeks of intervention.The body weight of mice in NR group,MT group,DT group and TT group gradually recovered.Compared with NC group,the body weight of mice in DT group was different at the first week(P<0.05),and the body weight of mice in TT group was different from the 0th week to the 5th week(P<0.05).2.Effects of single,double and triple probiotics on intestinal flora in mice with intestinal flora imbalance.After successful modeling,compared with the NC group,the PCR-DGGE results of the NR group,MT group,DT group and TT group showed that the intestinal flora richness and aroma concentration index of the mice in each group decreased(P<0.001).The bands and brightness in the DGGE picture were also significantly different from those in the NC group.Compared with NC group,there were significant differences in richness and aroma concentration index between MT,DT and TT probiotics in the first week(P<0.001).At the second week of intervention,the richness and aroma index of DT group were significantly different(P<0.05),the richness and aroma index of NR group were significantly different(P<0.01),and there was no difference between MT group and TT group.At the third week of intervention,there was no significant difference in the richness and fragrance index of MT group,DT group and TT group,indicating that the flora structure of MT group and TT group gradually recovered from the second week,close to the NC group,and the flora structure of DT group gradually recovered from the third week,close to the NC group.The results of 16S r RNA sequencing of fecal samples in the last week showed that after six weeks of probiotics administration,compared with the NR group,the diversity and richness of intestinal flora in the MT group,DT group and TT group were basically restored,and the abundance of Lachnospiraceae＿NK4A136＿group,Parabacteroides,Bacteroides,Bifidobacterium and other genera increased,Enterococcus,Enterothabdus and other genera decreased.3.Effects of single,double and triple probiotics on lymphocytes in mice with intestinal flora imbalanceCompared with NR group,after 6 weeks of intervention,the proportion of CD3~+T lymphocytes in MT group and DT group increased(P<0.05),the proportion of CD3~+CD4~+T lymphocytes in MT group increased(P<0.001),the proportion of CD3~+CD8~+T lymphocytes in MT group decreased(P<0.001),and the CD4/CD8 ratio in MT group increased(P<0.001).MT,DT and TT probiotics had no significant effect on CD3-CD19~+.4.The effects of single,double and triple probiotics on Ig G,CRP,IFN-γand IL-4 in serum of mice with intestinal flora imbalance.After probiotics intervention,compared with NR group,there was no difference in the concentration of Ig G and IL-4 in serum of mice in each experimental group,and the concentration of CRP in serum of mice in MT group and TT group decreased(P<0.05).Compared with NC group,IFN-γin MT group,DT group and TT group decreased(P<0.05).Compared with NR group,the ratio of IFN-γ/IL-4 in each group decreased(P<0.05),and the ratio of IFN-γ/IL-4 in MT group,DT group and TT group was greater than 1.Conclusion:1.The intervention of single,double and triple probiotics can accelerate the recovery of intestinal flora imbalance in mice,which is characterized by the increase of Bacteroidota and the decrease of Desulfobacterota.2.Single,double and triple probiotics can improve the immune function of mice,especially single probiotics(DM9811).3.Single and triple probiotics can affect the inflammatory response in mice.