Metagenomics Study On The Structure And Function Of Intestinal Microflora Regulated By Modified Renshen Wumei Decoction In Diarrhea Rats

OBJECTIVE:To study the effects of modified Renshen wumei decoction on intestinal microflora structure and function of diarrhea rats based on metagenomic technology,and to further reveal the mechanism of effect of modified Renshen wumei decoction on supplementing qi,promoting jin and preventing diarrhea.METHODS:Forty-eight 4-week-old SD rats were selected for adaptive feeding for two days,and 12 rats were randomly selected as blank group and given normal saline intragastric administration.The other 36 subjects were modeled with the compound factors of diarrhea from bitter cold,fatigue and exhaustion,and hunger and satiety disorders as references.30%senna leaves were given intragastric administration,and placed in water tank for 1 hour to swim with weight until exhaustion,then removed and dried,once a day,for 14 days.After successful modeling,they were randomly divided into model group,Western medicine group and Traditional Chinese Medicine(TCM)group,with 12 mice in each group.Model group was given normal saline intragastric administration,western medicine group was given Bacillus subtilis bivalent viable particles intragastric administration,TCM group was given modified Renshen wumei decoction intragastric administration,once a day,during which the general situation of rats in each group was observed.The rats were anesthetized and killed 7 days later,and samples were taken for examination.?The colon tissue was fixed in 4%paraformaldehyde,stained with H&E,and the pathological morphology of the colon was observed under light microscope.?Serum concentrations of electrolytes(Na⁺,K⁺,CL^-,Ca²⁺),D-Lactate and DAO were detected by ELISA.?Western blot was used to detect the expression levels of Occludin,Claudin-1 and ZO-1in colon.?Metagenomic sequencing of colon contents:sample DNA extraction,database sequencing,data quality control,species annotation and differential species analysis,gene set construction and differential gene analysis,functional annotation analysis and other processes.?Serum untargeted metabolomics analysis:according to the process of metabolite extraction and identification,the differential metabolites and differential metabolic pathway information were analyzed by principal component analysis and partial least square method.?Differential metabolite validation:Serum levels of LTB4,Calcitriol,Taurine and Cortisone were detected by enzyme-linked immunosorbent assay.RESULTS:?Effect index:(1)Diarrhea:after 2 weeks of modeling,the diarrhea symptom was obvious,the diarrhea rate was(90.02±3.59)%,and the diarrhea index was(2.03±0.21).After 1 week of drug intervention,the diarrhea rate and diarrhea index in the TCM and Western medicine groups were significantly lower than those which modeling 2 weeks and the model group(P<0.01).(2)Pathological morphology of colon:Mucosa lamina propria edema,sparse cell arrangement,enhanced light transmittance,intestinal gland atrophy and interstitial inflammatory cells increasein model group and western medicine group;In the TCM group,only slight mucosal edema and slight increase of inflammatory cells were observed.(3)Serum electrolyte:The levels of Na⁺,K⁺,CL^-and Ga²⁺in the model group were significantly decreased compared with that in the blank group(P<0.05),the levels of Na⁺,K⁺and Ga²⁺in the TCM group were significantly increased compared with that in the model group(P<0.05),but no significant improvement was observed in the western medicine group(P>0.05),and the improvement of Ga²⁺in the TCM group was better than that in the western medicine group(P<0.05).(4)Colon injury indexes:The levels of D-lactate and DAO in the model group were significantly higher than those in the blank group(P<0.01).Both TCM and Western medicine could significantly reduce DAO levels(P<0.01),and TCM was better than western medicine in regulating the levels of D-lactate.(5)Colonic mucosal tight junction protein:Compared with the blank group,the expression levels of Occludin,Claudin-1 and ZO-1 in the model group were significantly down-regulated(P<0.01),and the expression levels of Claudin-1 and ZO-1 were significantly up-regulated by both TCM and Western medicine(P<0.01),while the expression levels of Occludin and ZO-1 up-regulated by Chinese medicine were better than those of western medicine(P<0.05).?Metagenomics:(1)Relative abundance of OTU in the flora:OTU number in model group was significantly decreased(P<0.05);the number of OTU in the TCM and Western medicine groups was significantly higher than that in model group(P<0.01).(2)Relative abundance and difference of species:at phylum level,firmicutes increased in the model group,while bacteroidetes and warts micro bacteria decreased significantly;bacteroidetes significantly increased and firmicutes decreased significantly in the TCM and Western medicine groups,and the number of warts micro bacteria in the TCM group was higher than that before.At class level,clostridium and baciliaceae increased,while bacteroidetes decreased in model group.In the TCM and western medicine groups,the bacteriaceae decreased significantly,while the bacteroidetes increased significantly.At the genus level,bacteroidetes and prevotella decreased in the model group,while the relative abundance of lactobacillus increased significantly;the relative abundance of bacteroidetes in TCM and Western medicine groups was significantly increased,while the relative abundance of lactobacillus was significantly decreased.(3)Species cluster analysis:at the level of genus and species,bacteroidetes,bacteroidetes,firmicutes,lactobacillus and bifidobacteria in the TCM group were significantly higher than those in the model group and the Western medicine group.(4)The functional abundance and differences of the flora:KO cluster analysis:the carbohydrate,amino acid,energy metabolism,biosynthesis and metabolism of polysaccharide,and digestive system function annotation in the TCM and Western medicine groups were significantly higher than those in the model group,the clustering analysis degree of energy,carbohydrate,amino acid metabolism and membrane transport in TCM group was higher than that in Western medicine group.Egg NOG function annotation:the biosynthesis,transport and catabolism of secondary metabolites,energy production and conversion,transcription,transport and metabolism of amino acids,signal transduction mechanism and nucleotide transport and metabolism of secondary metabolites in TCM group were significantly higher than those in Western medicine group.CAZy functional annotation:CBMs,GTs,GHs and CEs in the TCM and western medicine groups were significantly higher than those in the model group,while CBMs and GTs in the TCM group were significantly higher than those in the western medicine group.?Untargeted metabolomics:(1)Differential metabolites:compared with model group,phosphoric acid,Taurine,LTB4 and Calcitriol were the main metabolites of difference between TCM and Western medicine groups,and Cortisone was the main metabolite of difference between Western medicine group and model group.(2)Differential metabolic pathway information:compared with the model group,the degradation of valine,leucine and isoleucine,and the metabolism of propionic acid,fructose,mannose and amino acids were the main differential metabolic pathways of the the TCM and Western medicine groups,in which PPARs was the differential metabolic pathway of the Western medicine group,and c AMP/PKA was the differential metabolic pathway of the TCM group.(3)Validation of differential metabolites:the serum levels of Calcitriol,Taurine and Cortisone in model group were significantly lower than those in blank group(P<0.01),both TCM and western drugs could increase the serum levels of LTB4,Calcitrio and Taurinel(P<0.01),TCM significantly improved the metabolism levels of LTB4 and Calcitrio better than western medicine(P<0.05),while western medicine could effectively improve the metabolism level of Cortisone(P<0.01).CONCLUSIONS:1.Modified Renshen wumei decoction has a significant effect of replenishing qi,generating fluid and stopping diarrhea in diarrhea model rats.2.Modified Renshen wumei decoction can effectively regulate the relative abundance of macteroidetes,firmicutes,verrucomicrophyla,Bacilaria,Bacteroidetes,Lactobacillus and Bacteroidetes in the intestinal flora of diarrhea model rats,and functional groups of Bacteroidetes,Bacteroidetes,Firmicutes,Lactobacillus and bifidobacteria at genus and species level.Moreover it regulates the metabolism and transport of host nutrients such as energy,carbohydrates and amino acids through functional flora to play the effect of reinforcing qi shengjin and stopping diarrhea.3.Modified Renshen wumei decoction has a benign regulation effect on serum differential metabolites phosphoric acid,methylmalonic acid,D-mannose 6-phosphate,LTB4,Calcitriol,Taurine and eicosapentaenoic acid in diarrhea model rats.The metabolism of propionic acid,fructose,mannose and amino acid were the main differential metabolic pathways,and PPARs and c AMP/PKA were the key differential metabolic pathways,the degradation of valine,leucine and isoleucine,and the metabolism of propionic acid,fructose,mannose and amino acid are the main differential metabolic pathways,PPARs and c AMP/PKA are key differential metabolic pathways.4.SCFAs may be the main product of covariant effect of Modified Renshen wumei decoction on intestinal flora and host metabolism,and its function may be the main link of multi-target,multi-level and multi-channel effect of Modified Renshen wumei decoction on supplementing qi,promoting jin and preventing diarrhea.