The Effect Of Qiweibaizhusan On The Intestinal Mucosal Bacteria And Lactase Gene Diversity Of Dysbacterial Diarrhea Mice

Objective:The current research was tried to investigate the effect of Qiweibaizhu powder（QWBZP）on intestinal mucosa bacteria and bacterial lactase gene diversity from dysbacterial diarrhea mice,the best primer of bacterial lactase gene in intestinal mucosa bacteria was designed,as the good therapeutic mechanism of QWBZP on dysbacterial diarrhea from the perspective of the diversity of intestinal mucosa bacteria and bacterial lactase gene.Methods:The modeling experiment was set up in the normal group and the model group.The treatment group was set up in the normal group,the model group and the QWBZP group.Dysbacterial diarrhea mice model was constructed by gastric perfusion with mixture of antibiotic solution(23.33 mL·kg^-1·day^-1)twice a day for 5 days,and the mice in normal group were gavaged with 0.35 mL sterile water.After the success of establishing diarrhea,the model group mice were divided into the natural recovery group and the treatment group.The mice in the treatment group were administered QWBZP decoction at the dose of 0.16 g·kg^-1·day^-1,the normal group and the natural recovery group were given the same amount of sterile water twice a day for 3 days.After the modeling and treatment,intestinal mucosa from jejunum to ileum in each group was collected,the microgenomic DNA of intestinal mucosa bacteria was extracted,the V3+V4 variable region of bacterial 16S rDNA and the bacterial lactase gene were amplified by PCR,the universal primers of bacterial lactase gene were designed,and the diversity of bacteria and bacterial lactase genes in intestinal mucosa were analyzed by Miseq high-throughput sequencing.Results:（i）The analysis of bacterial 16S rDNA sequencing showed that alpha diversity index in intestinal mucosa bacteria was increased after the dysbacterial diarrhea model,but there was no statistical difference.In the beta diversity analysis,the samples in the model group were relatively discrete,and they were far away from the samples in the normal group,indicating the poor similarity.From the perspective of bacterial abundance,intestinal mucosa bacteria were mainly originated from Firmicutes,Proteobacteria,Bacteroidetes,Actinobacteria and Tenerictutes,and Firmicutes was the most abundant.At the phylum level,Proteobacteria and Actinobacteria were increased,Firmicutes and Tenerictutes were decreased significantly,F/B value was increased,and B/E value was decreased significantly.At the genus level,Lactobacillus,Mycoplasma,Bacteroidales_s24-7 and Helicobacter were significantly reduced,while Enterococcus,Stenotrophomonas,Citrobacter,Blautia,Carnobacterium,Glutamicibacter and Clostridium were significantly increased in the model group（P<0.01 or P<0.05）.After treatment with QWBZP,alpha diversity index in the treatment group was close to that in the natural recovery group,and the ACE,Chao and Shannon indices were slightly lower but the Simpson index was slightly higher than the normal group,but there were no statistical differences.In the beta diversity analysis,samples in treatment and normal groups were relatively concentrated,while samples in the natural recovery group were relatively dispersed.From the perspective of bacterial abundance,at the phylum level,the proportion of Firmicutes,Bacteroidetes and Tenerictutes were increased,while the proportion of Proteobacteria and Actinobacteria were decreased.Among them,Firmicutes were higher while Proteobacteria and Bacteroidetes were lower than the normal group,but there was no significant difference.The F/B ratio was decreased and returned to the normal group.At the genus level,Lactobacillus and Bacteroidales_s24-7 were increased to normal level.Stenotrophomonas showed no significant difference between treatment and natural recovery groups,which was lower than the normal group.Enterococcus continued to increase in the treatment group,which was significantly different from the natural recovery group and the normal group.（ii）The analysis of bacterial lactase gene sequencing showed that alpha diversity index in intestinal mucosa bacterial lactase genes was increased significantly（P<0.01 or P<0.05）after the dysbacterial diarrhea model.From the PCA and box plot analysis,the distance between the samples in the normal group was closer,which was far away in the model group,and the distance between the two groups was far.From the abundance of bacterial communities containing lactase gene in intestinal mucosa,bacterial lactase genes were mainly constituted of Proteobacteria,Actinobacteria and some unclassified bacteria.At the phylum level,the abundance of Actinobacteria and unclassified bacteria were increased and the abundance of Proteobacteria was decreased after being induced by antibiotics.At the genus level,except for Stenotrophomonas,the relative abundance of other bacterial lactase genes was increased,and Ewingella,Methyloversatilis,Mycobacterium,Microbacterium,Beutenberqia and Actinomyces were added in the model group.After treatment with QWBZP,alpha diversity index in the treatment group was close to that in the normal group,and there was no statistical difference.From the PCA and box plot analysis,the distribution was relatively concentrated in both normal and treatment groups,the sample distance was close.From the species abundance,the composition structure and abundance of species between the treatment group and the normal group were very similar at the phylum and genus level,while the natural recovery group reduced the abundance of lactase gene originated from Proteobacteria and Stenotrophomonas,and increased many unclassified lactase genes.Conclusion:Antibiotics destroyed the structure of bacteria and bacterial lactase gene in the intestinal mucosa,increased their diversity and abundance.After treatment with QWBZP,the intestinal mucosal bacterial diversity of mice did not change greatly,but the diversity of bacterial lactase genes in intestinal mucosa restore to normal level.The mucosal bacteria and its lactase gene structure were effectively repaired and the mucosal microbiota was recovered to balance.Specifically,QWBZP increased the bacterial abundance of Lactobacillus and Bacteroidales_S24-7,and increased the lactase gene abundance of Lysobacter and Eukaryota.