| Objective As a traditional ancient prescriptions in our country,Zengye decoction(ZYD)is widely used in the treatment of constipation and other chronic diseases.However,most relative studies focus on ZYD’s therapeutic effect from the perspective of pharmacology.It still lacks an integrative and systematical research which can provide fundamental support for further profound research in a specific pathway or potential target.In recent years,the existence of intestinal microecology associated with intestinal physiological processes has been gradually recognized,which suggesting us.The therapeutic target of ZYD may be associated with intestinal flora.Therefore,to access the impact of gut microbiota on metabolism in the condition of aged constipation with fluid-deficiency,an intergrated approach of polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)combined with 16S rDNA sequencing and nuclear magnetic resonance(NMR)based metabonomics was performed in rat model for studying the coeerlation between the gut microbial composition and host metabolites.Methods (1)The model of aged constipation with fluid-deficiency was established by usingthemethodofwaterlimitation,intragastricadministrationof Loperamide Hydrochloride and subcutaneous injection of D-galactose.After the model was successfully established,the rats were treated with ZYD.And then the model and therapeutic effect was evaluated by macro indicators、biochemical indexes、physiological indexes and pathological section.(2)Collected feces and urine samples from rats at different time points during animal experiments.Some of the feces were used for bioinformatics analysis,including PCR-DGGE and 16S rDNA sequencing.Another part of fecal samples and urine samples were profiled with the method of ~1H-NMR after the preparation of urine and faeces samples.The peaks of metabolites were ascribed according to the chemical shift values,peak splitting case,the coupling constant and othe references.The data were analyzed by using SIMCA-P 13.0 with the method of principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).Then the relevant the biomakers were screened and trace back to the metabolic pathways.In the intestinal microecology analysis,we first use of PCR-DGGE research the intestinal flora fingerprint of aged constipation with fluid-deficiency model and ZYD intervention,and discusses the trend of intestinal flora changes over time.The structure and diversity of intestinal flora were analyzed by 16S rDNA sequencing technology.Finally,through LEfSe analysis and PICRUSt analysis,we screened the intestinal flora biomarkers of rats with aged constipation model and predicted its function and used metagenomics sequencing to verify metabolic function.Results (1)After modeling,rats showed an abnormal condition including weight loss and poor appetite.According to the characteristics of constipation,the frequency and/or amount of defecation in model group decreased significantly,which can also explain for the significant decrease in ink propelling rate of the small intestine and thickness of colonic mucosa.The results indicate that the difficult defecation was caused by the slow bowel motility and insufficient water in intestinal tract.And lower level of SOD and higher level of MDA in model suggest that the body was heavily oxidized.Therefore,the aged constipation model with syndrome of fluid-deficiency was successfully established.After the drug intervention,the above indicators were improved to varying degrees.(2)Analysis of urine and faeces by metabonomics:Totally,105 metabolites were detected,74 in urine samples and 56 in faeces samples.After multivariate statistical analysis,we found the difference metabolites in urine samples and fecal samples.There were 27 urine samples and23 feces.In metabolic pathway analysis,In the analysis of metabolic pathways,we found the effect of the model on the host,respectively interfere with energy metabolism;inhibit the synthesis of butyric acid;interfere with the biological activities of choline and methane;disrupt the function of liver and kidney;interfere with the metabolism of amino acids in the body.After ZYD intervention,ZYD led to 20 metabolites changed significantly in the model group(12 in urine,9 in fecal samples,common 1).And the trend of these metabolites is the opposite of the model period,In pathway analysis,ZYD can effect the metabolic pathways for energy metabolism,methane metabolism,amino acid metabolism and bacterial toxins.(3)After the modeling,there were significant differences in the intestinal flora structure of the control group and the model group,and the difference gradually decreased after the intervention of ZYD.In the analysis of biological diversity,theα-diversity of the model group was the lowest and there was significant difference with the control group,and there was no significant difference between the ZYD group and the control group.Similarly,theβ-diversity of the model group was significantly different from that of the control group,while the control group had less difference with the ZYD group.In LEfSe analysis,compared with the model group,the ZYD group and the control group showed lower levels of Helicobacteraceae,Desulfovibrionaceae(including Desulfovibrio),Ruminococcaceae(including Ruminococcus),Lactobacillaceae(including Lactobacillus),Prevotellaceae(including Prevotella)and Dorea.And showed a higher levels of Aeromonadaceae,Oxalobacteraceae(including Oxalobacter),Veillonellaceae,Clostridiaceae(including Clostridium)and Roseburia.By analyzing PICRUSt function projections for biomarkers community,had a great effect on the whole process of building and give medicine metabolism of metabolism of glucose and mannose,methane metabolism,lysine biosynthesis,pyruvate metabolism,pentose phosphate pathway,bacterial toxin,valine,leucine and isoleucine degradation and glutamic acid metabolism.Conclusion Our study successfully established the model of aged constipation with fluid-deficiency in SD rats.The effects of model and drug intervention on rats were studied from the perspective of metabolomics and intestinal microecology,and identify related metabolic pathways and intestinal flora.By discussing the composition distribution of intestinal flora in different states,we can elucidates the relationship between intestinal flora and host metabolism and the regulation effect of ZYD on intestinal flora and metabolism in rats,which are of particular important for understanding the function of gut micobiota in human health and disease. |
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