Effects Of Mono Probiotics,Double Probiotics And Triple Probiotics On Intestinal Microbiota Recovery And Glucose And Lipid Metabolism In Mice With Intestinal Microbiota Imbalance

Objective:The mice model of intestinal microbiota imbalance was established by ceftriaxone sodium,and then different combinations of probiotics were given to mice to explore the effects of different combinations of probiotics on the recovery of intestinal microbiota and metabolism in C57BL/6J mice with intestinal microbiota imbalance.Materials and Methods:1.The group of the experimental animalFifty-six 8-week-old SPF C57BL/6J male mice were randomly divided into normal control group（NC,n=16）,antibiotic-treated intestinal bacterial imbalance model group（AB,n=8）,natural recovery group（NR,n=8）,mono probiotics treatment group（MT,n=8）,double probiotics treatment group（DT,n=8）and triple probiotics treatment group（TT,n=8）.The experiment period was 7 weeks.Eight mice in the NC and the NR were sacrificed after 1 week of ceftriaxone sodium treatment.2.Establishment of the mouse model of intestinal microbiota imbalanceIn NC group,each mouse was given 0.3ml normal saline every day for 1 week;in AB group,0.3ml of ceftriaxone sodium solution（at a dose of 8g/kg per animal,the powder was dissolved in normal saline）was gavaged daily for 1 week.Mice feces were collected to detect intretinal microbiota shannon index and richness to determine whether the modeling was successful.3.Treatment methods of each group when probiotics were gavaged to miceIn NC group and NR group,each mouse were given 0.2 ml normal saline d for 6 weeks.In MT group,each mouse was given 0.2 ml DM9811 daily for 6 weeks.In DT group,each mouse was given 0.2ml DM9112 and DM9125 mixture daily for 6 weeks.In TT group,each mouse was given 0.2ml DM9811,DM9112 and DM9125 mixture daily for 6 weeks.4.Analysis of intestinal microbiota compositionStool of mice in each group was collected once a week.E.Z.N.A.^?Stool DNA kit was used to extract intestinal flora DNA from stool for PCR-DGGE analysis and 16S rRNA sequencing.The sequencing results were analyzed on the Meiji Shengxin cloud platform.5.Glucose and lipid metabolism related detection indexesDuring the experiment,water intake and food intake was recorded every day.Body weight and fasting blood glucose were measured every week.At the end of the experiment,mouse serum was obtained by extigation of the eyeball.Then serum was collected to detect cholesterol（TC）,triglyceride（TG）,high density lipoprotein cholesterol（HDL-C）,low density lipoprotein cholesterol（LDL-C）and insulin,and HOMA-IR value was calculated.Cholesterol（TC）and triglyceride（TG）contents of liver were detected.The contents of acetic acid,propionic acid and butyric acid were detected in the stool after 1 week of treatment.Results:1.Changes of intestinal flora in mice with intestinal flora imbalanceAfter intestinal flora imbalance,the cecum of mice was enlarged,fecal soft particles were larger,and shannon index and richness were significantly lower than NC group（P<0.05）.2.Changes of different combinations of probiotics treatment on intestinal flora in mice with intestinal flora imbalancePCR-DGGE analysis showed that after 1 to 2 weeks of intervention,shannon index and richness of mice in NR,MT,DT and TT groups were significantly decreased compared with NC group（P<0.05）.After 1 week of intervention,shannon index and richness of mice in MT,DT and TT groups were significantly decreased compared with those in NR group（P<0.05）.After 3 weeks of intervention,shannon index and richness of mice in MT group were significantly lower than those in other groups（P<0.05）.After 4 to 6 weeks of intervention,there were no differences in shannon index and richness among all groups（P>0.05）.After PCR-DGGE analysis,the mice in each group with significant changes were analyzed by 16S rRNA gene sequencing.The results showed as follows:at the phylum level,after 1 week of intervention,the relative abundance of Verrucobacteria and Actinobacteria in NR group was significantly higher than that of NC group（P<0.05）;Compared with NC group,the relative abundance of Proteobacteria in MT group was significantly increased（P<0.05）.After 6 weeks of intervention,compared with NC group,the relative abundance of Firmicutes,Desulphurates and Actinomyces in MT,DT and TT groups was significantly decreased（P<0.05）,the relative abundance of Bacteroidetes was significantly increased（P<0.05）,and the ratio of Firmicutes to Bacteroidetes（F/B）was also significantly decreased（P<0.05）.At genus level,after 1 week treatment,compared with NC group,the relative abundance of Akkermansia and Blautia in NR group increased significantly（P<0.05）;compared with NC group,the relative abundance of Enterococcus,Robinsoniella and Clostridioides in MT,DT and TT group increased significantly（P<0.05）;compared with NC group,the relative abundance of Lactobacillus and norank_fMuribaculaceae in MT,DT and TT group decreased significantly（P<0.05）.After six week treatment,compared with NC group,the relative abundance of Lactobacillus,Desulfovibrio,Faecalibaculum and Parabacteroides in MT,DT and TT group decreased（P<0.05）;compared with NC group,the relative abundance of Dubosiella in NR group increased（P<0.05）;compared with NC group,the relative abundance of Parabacteroides and g_Rikenellaceae_RC9_gut_group in MT group increased（P<0.05）;compared with NC group,the relative abundance of Bacteroides,Ruminococcus and Lachnospiraceae_NK4A136_group in DT group increased（P<0.05）;compared with NC group,the relative abundance of Ileibacterium and Prevotellaceae UCG-001 in TT group increased（P<0.05）.3.Changes of body weight,food intake and water intake in miceThere were no differences in body weight,food intake and water intake among all groups after intestinal flora imbalance（P>0.05）.After 6 weeks treatment,the body weight of MT,DT and TT groups was lower than that of NC group（P<0.05）.From week 1,ompared with NC group,the water intake of MT,DT and TT groups decreased（P=0.003,P=0.016）.From week 1 to 6 weeks,there was no difference in food intake among all groups after treatment（P>0.05）.4.Changes of glucose metabolism related indicatorsAfter 1 week of intestinal flora imbalance,fasting blood glucose and HOMA-IR were significantly decreased compared with NC group（P=0.037,P=0.016）,and serum insulin was not different（P>0.05）.After 1 week of intervention,compared with NC group,fasting blood glucose in NR group and MT group was significantly decreased（P=0.001,P<0.001）,but there was no significant difference in fasting blood glucose between DT group and TT group（P>0.05）.After 2 to 6 weeks of intervention,there was no difference in fasting blood glucose among all groups（P>0.05）.After 6 weeks of intervention,there were no differences in serum insulin and HOMA-IR among all groups（P>0.05）.5.Changes of lipid metabolism related indicatorsAfter 1 week of intestinal flora imbalance,serum TG and liver were significantly decreased compared with NC group（P=0.045,P=0.031）,but there were no significant differences between serum TC and liver TC,serum HDL-C,serum LDL-C and liver index（P>0.05）.After 6 weeks of intervention,there were no significant differences in serum TG,TC,HDL-C and LDL-C among all groups compared with NC group（P>0.05）.Liver TG in DT group was significantly decreased（P=0.011）,and liver TC in DT and TT groups was significantly decreased（P=0.001,P=0.004）.6.Changes of faecal short-chain fatty acids（SCFAs）after 1 week treatmentThe fecal acetic acid content of NR group was significantly higher than that of NC group and MT group（P<0.05）,and the fecal acetic acid content of DT and TT groups was significantly lower than that of NC group（P<0.05）.Compared with NC group,fecal propionic acid content in DT group and TT group had no significant difference（P>0.05）,while fecal propionic acid content in NR group and MT group was significantly decreased（P<0.05）.Compared with NC group,fecal butyric acid content in NR,MT,DT and TT groups was significantly decreased（P<0.05）.Conclusion:1.Intestinal microbiota imbalance mice intestinal microbiota shannon index and richness decreased,and fasting blood glucose,serum TG and liver decreased.2.Mono probiotics treatment,double probiotics treatment and triple probiotics treatment could restore the balance of intestinal flora of unbalanced mice,and mono probiotics treatment was the slowest.After 6 weeks of treatment,the ratio of Firmicutes to Bacteroidetes（F/B）decreased significantly,and the relative abundance of SCFAs producing bacteria increased.3.Double probiotics treatment and triple probiotics treatment could restore the blood glucose level of mice within 1 week and reduce the liver TC after 6 weeks,but double probiotics treatment could not restore the liver TG of mice.4.After 1 week treatment,fecal acetic acid content in DT and TT group was significantly decreased;fecal propionic acid in MT group was significantly decreased;fecal butyric acid in MT,DT and TT group was significantly decreased.