| Objective: Through animal experiments,this project discussed the influence of Buqitongqiao prescription on the diversity and richness of stool flora in rats with allergic rhinitis,further explored the effect mechanism of Buqitongqiao prescription on the prevention and treatment of allergic rhinitis,and provided scientific basis for the promotion of Buqitongqiao prescription.Methods: Thirty-five clean healthy Wistar rats were divided into three groups by random number table method: blank group,AR model group and Buqitongqiaofang group.There were 10,10,15 rats in each group.Except blank group,AR model group and Buqitongqiaofang group were all induced to establish allergic rhinitis(AR)rat models.AR rats in Buqi Tongqiao formula group were given gavage with Buqi Tongqiao formula at 57.55g/kg/d every day,and blank group and AR model group were given gavage with purified water of 4m L/d for 7 consecutive days.The behavioral scores of the rats were recorded every day to identify the success of modeling.HE staining was used to observe the pathological injury of nasal mucosa in each group.Intestinal contents samples were collected for 16 Sr RNA amplification and sequencing.The sequencing results were analyzed and processed,and the intestinal flora characteristics of rats in each group were analyzed and compared,including the species percentage accumulation histogram,cluster heat map,phylogenetic evolutionary tree,and LDA Effect Size for species composition analysis.Alpha diversity index,significance analysis of differences between groups and sparse curve were used to analyze Alpha diversity.Beta diversity was compared based on PCo A analysis,Anosim analysis and PLS-DA analysis.We use the network analysis to analyses the network diagram of species interaction.Prediction of microbial community function based on PICRUSt2 software.Finally,the difference of intestinal species composition in each group was statistically analyzed.Results:(1)HE staining of the nasal mucosa: In the blank group,the nasal mucosa was pseudomultilayered ciliated columnar epithelium with intact cilia,no edema,congestion and other changes were observed in the lamina propria,and no glandular hyperplasia.In the model group,cilia of nasal mucosa were disordered,exfoliated and collapsed,more lymphocytes and eosinophils were infiltrated,the number of submucosal glands was significantly increased,and small blood vessels were hyperplastic and dilated,which was the manifestation of membrane formation compared with the blank group.The lamina propria in the Buqitongqiao formula group was relatively intact,with no obvious inflammatory cell infiltration and mild glandular hyperplasia.Compared with the model group,the number was significantly reduced,and the pathological damage was significantly improved.(2)Differences in species composition among the sample groups:(1)The percentage accumulation column of species at class,family,and genus level:The intestinal microorganisms of the three groups were different at class,family,and genus level.The species distribution ratio of the model group was significantly reduced compared with the normal group when Bacilli,Lactobacillaceae and Lactobacillus,and showed a slightly increasing trend after the treatment with Buqitongqiao formula.The bacteria genera of Clostridia,Unspecified＿Clostridiales,Lachnospiraceae and Ruminococci in the model group increased significantly compared with the normal group.The abundance of Buqi Tongqiao prescription decreased obviously.(2)Heat map of species number distribution between groups: The top 50 absolute abundations of intestinal microorganisms in the three groups were significantly different at the phylum and genus levels.(3)Further LDA Effect Size showed that lactobacillus was characteristic of normal group from order to genus level.Clostridium and Prevotella were the characteristic bacteria of the model group.Bacteroides from phylum to genus level and spirochaeta from phylum to family level were the characteristic bacteria of Buqitongqiao recipe group.(3)Analysis of OTU clustering results of intestinal microorganisms: The total number of OTU samples in the 3 groups was 9295,and the number of OTU samples in the normal group,the model group and the Buqitongqiaofang group were 4921,3220 and 3626,respectively.The number of OTU samples in the normal group was the largest,and the number of OTU samples in the model group was the least,indicating that the number of OTU samples in the intestinal flora of AR rats was significantly reduced compared with the normal group.Gavage buqi Tongqiao formula can affect the OTU number of intestinal flora in AR rats to a certain extent.(4)Alpha diversity analysis: the Observed species index and Chao1 index of the normal group were larger than those of the model group and Buqitongqiaofang group,but there was no significant difference between the two Alpha diversity indexes among the three samples(p > 0.05).Shannon index and Simpson index of normal group were significantly lower than those of model group and Buqi Tongqiao prescription group(p < 0.05),that is,species richness of normal group was significantly lower than that of model group and Buqi Tongqiao prescription group,and there was no significant difference in Alpha diversity index between model group and Buqi Tongqiao prescription group(p > 0.05).The dilution curve of Alpha diversity index reflected that the sequencing data volume was reasonable and sufficient to reflect the diversity of intestinal flora in the samples.(5)Beta diversity analysis: The results of PCo A analysis based on Bray Curtis distance and Unweighted Unifrac distance showed that there was a significant separation trend among the three groups,indicating significant differences in intestinal microbial composition and structure among all groups(q < 0.05,R > 0).weighted Unifrac distance PCo A analysis showed that there was no significant aggregation trend among the three groups,suggesting that there were differences in intestinal microbiome composition among the three groups.There was no significant difference in species diversity between Buqitongqiaofang group and model group,q =0.055;Species diversity of normal group was significantly different from that of model group and Buqitongqiaofang group(q < 0.05).PLS-DA analysis showed the separation of intestinal flora in the three sample groups,suggesting the presence of a unique intestinal flora in the three groups.(6)network analysis showed that there was a certain relationship between the bacteria enriched in the sample group.(7)The results of functional prediction analysis of microbial community showed that the microbial community samples were closely related to metabolic pathways,especially carbohydrate metabolism,amino acid metabolism,cofactor and vitamin metabolism.Conclusion(s):(1)Buqitongqiao can repair the injured nasal mucosa of rats with allergic rhinitis,inhibit glandular hyperplasia and inflammatory cell infiltration,thus alleviating the nasal histopathological injury of rats with allergic rhinitis,and achieve the effect of treating allergic rhinitis.(2)The intestinal flora composition of AR rats and healthy rats was different,and Bacilli,Lactobacillaceae and Lactobacillus decreased.With the increase of Clostridia,Unspecified＿Clostridiales,Lachnospiraceae,Ruminococcaceae＿Ru minococcus,the microecology of AR rat intestinal flora changed obviously.Microflora is closely related to metabolic pathways,among which carbohydrate metabolism,amino acid metabolism,cofactor and vitamin metabolism are prominent.(3)There were significant differences in βdiversity among normal group,model group and Buqitongqiaofang group.(4)This study speculated that Buqitongqiao decoction can relieve nasal mucosal inflammation and control the progression of AR disease by regulating the structure and composition of intestinal flora in AR rats. |
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