Effects Of Faeces Bombycis On Intestinal Microflora And Metabonomics In Rats With Syndrome Of Damp Retention In Middle-jiao

Objective: To investigate the effects of Faeces Bombycis(FB)on intestinal microflora and metabolomics in rats with syndrome of damp retention in middle-jiao.The mechanism of FB for “harmonize the stomach” is described,which expand the scope of clinical application of FB and promote the inheritance and development of traditional Chinese medicine of feces.Methods: 1.Effects of FB on intestinal microflora The rat model with syndrome of damp retention in middle-jiao was established by multi-factor combination of external wet invasion,swimming exhaustion and over-feeding method.After the treatment of FB,the feces of rats were collected,and the total DNA of the samples was extracted and amplified;meanwhile,the total DNA of the microorganisms in the FB was extracted and amplified;Polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE),16 S r DNA high-throughput sequencing,real-time fluorescence quantitative PCR(q PCR)were used to investigate intestinal microflora of rats with syndrome of damp retention in middle-jiao.2.Effects of FB on metabonomics UPLC-Q-TOF-MS technology was used to measure the metabolites in feces and urine.The metabolic profiles of control rats and rats with syndrome of damp retention in middle-jiao were compared to find out the differential metabolites;and study the effect of FB on rats with syndrome of damp retention in middle-jiao;The Metabo Analyst soft was used to analyze metabolic pathways,and clarify the regulation of FB on metabolism.3.Correlation analysis between intestinal microflora and metabolites SPSS software was used to carry out Pearson correlation analysis on intestinal differential microflora and differential metabolites,From the perspective of “intestinal microflora-host metabolism”,the effect of FB on syndrome of damp retention in middle-jiao is discussed,and the mechanism of action of FB “harmonize the stomach” is explained.Results: 1.Effects of FB on intestinal microflora PCR-DGGE results showed that compared with the control group,the Shannon-Wiener diversity index,genotypic richness and Pielou evenness index of the rats in the model group were significantly decreased(P<0.05);Compared with the model group,the Shannon-Wiener diversity index and Pielou evenness index of the middle and high dose groups of FB were significantly increased(P<0.05),and the genotypic richness of the high dose group was also significantly increased(P<0.05).Cluster analysis showed that the bacterial communities in the high dose group and the control group clustered closely to one another,and the bacterial communities in the low and medium dose groups were clustered together,while the bacterial communities in the model group was clustered into a category.The microflora showed that the similarity coefficient between the model group and the control group was significantly different(P<0.05),and compared with the model group,the similarity coefficient of the low,medium and high dose groups were significantly increased(P<0.05);while the similarity coefficient of the microflora in FB were no apparent differences compared with the low,medium and high dose groups(P>0.05).High-throughput sequencing results showed that compared with the control group,the relative abundance of Proteobacteria,Acinetobacter, Anaerobacter,Pseudomonas and Parabacteroides were significantly increased(P<0.05),while the Marvinbryantia was significantly decreased(P<0.05).Compared with the model group,the Proteobacteria was significantly decreased in the low and high dose groups(P<0.05),and the Acinetobacter,Anaerobacter,Pseudomonas,Parabacteroides were significantly decreased in the low,medium and high dose groups(P<0.05),while the Lachnoanaerobaculum,Intestinimonas and Marvinbryantia were increased significantly in the high dose group(P<0.05).Sequencing analysis of FB showed that compared with the microflora in rat faeces,the relative abundance of Staphylococcus,Leclercia,Pantoea,Brachybacterium,Hartmannibacter,Klebsiella,Aurantimonas,Paenibacillus and Bacillus were high in the FB(P<0.05),but they were basically not present or little in the rat feces.q PCR results showed that compared with the control group,the expression of Bifidobacteria and Lactobacillus in the model group was significantly reduced(P<0.05),while the expressions of Escherichia coli and Enterococcus faecalis were significantly increased(P<0.05);Compared with the model group,The expressions of Bifidobacteria and Lactobacillus in the middle and high dose groups significantly increased(P<0.05),while the expressions of Escherichia coli in the low,medium,and high dose groups significantly decreased(P<0.05).The expression of Enterococcus faecalis in high dose group was significantly reduced(P<0.05).2.Effects of FB on metabonomics Metabolomics results showed that 17 and 11 differential metabolites were identified in feces and urine.Compared with the control group,the relative content of 6Z,9Z-octadecadienoic acid,Oleamide,Petroselinic acid,14,15-Di HETr E,MG(P-18:0e/0:0/0:0),Tetracosapentaenoic acid(24:5n-6),7-Ketodeoxycholic acid,Alpha-Tocotrienol,Lyso PE(0:0/15:0),Lyso PE(0:0/18:1(11Z)),Lyso PC(18:3(6Z,9Z,12Z)),Cysteic acid,Indoxyl sulfate,Ricinoleic acid,Cyclic GMP in the model group were significantly increased(P<0.05),Whereas the relative content of 1H-Indole-3-carboxaldehyde,4,6-Dihydroxyquinoline,L-Phenylalanine, Kynurenic acid,Alpha-Linolenic acid,Glycocholic acid,2-indolecarboxylic acid,Acetylphosphate,Homovanillic acid,Xanthurenic acid,Palmitic acid,Linoleic acid,Oleic acid in the model group were significantly reduced(P<0.05).And FB can effectively interfere with fecal and urinary metabolic disturbances in rats with syndrome of damp retention in middle-jiao,and “call back” 11 kinds of biomarkers in feces and 6 kinds of biomarkers in urine;pathway enrichment results showed that FB play a role in “harmonize the stomach” through the regulation of biosynthesis of phenylalanine,tyrosine,and tryptophan,phenylalanine metabolism,α-linolenic acid metabolism,glycerophospholipid metabolism,primary bile acid biosynthesis and other pathways to treat syndrome of damp retention in middle-jiao.3.Correlation analysis between intestinal microflora and metabolites Correlation analysis of intestinal microflora and metabolites showed that the Proteobacteria,Acinetobacter,Anaerobacter,Pseudomonas and Parabacteroides were related to metabolites involved in neurotransmitter metabolism,bile acid metabolism,arachidonic acid metabolism,and glycerophospholipid metabolism.Conclusion: The project is to study the effects of FB on syndrome of damp retention in middle-jiao from the perspective of intestinal microflora and metabolomics.The results showed that FB play a role in effect of “harmonic stomach” by balancing the intestinal microflora,regulating the microflora and host metabolism,and treating the syndrome of damp retention in middle-jiao.The research results of this project expand the scope of clinical application of FB and promote the inheritance and development of traditional Chinese medicine of feces.