| Objective:To analyze the metabolites extracted from serum and the changes of intestinal microflora by metabonomics technology and intestinal microflora detection,and to preliminarily study and summarize the metabolites of Heyi.Pharmacological experiments,metabonomics methods,intestinal microflora detection,metabonomics intestinal microflora association analysis methods and bioinformatics technology to explain the mechanism of action and target of Mongolian medicine ferula.Methods:? Modeling experiment:Mongolian medicine "four Shi(diet,daily life,medicine,therapy)" intervention method,the establishment of Mongolian medicine Heyi syndrome model,and observe the temperature,general state,symptoms,syndrome period change time,death,urine(observation quantity,color,sediment,flocculent,floating,taste,steam).Mobile monitoring of serum gas to determine the establishment standard of Mongolian medicine model.? SD rats were randomly divided into model group and blank control group,with 10 rats in each group.The blank control group was given 1 ml/100(g·d)by gavage at 6 a.m.every day,and the model group was given four kinds of intervention to establish the Mongolian medicine model of Heyi disease.The general behavior,behavior recorder test,urine analysis,serum ast,alt,urea,crep2,trig1 determination,ELISA detection of gas,CK,TSH,T4,T3,ACTH,CRF,CRH,cor,CRE,E2 were observed.?Pharmacological experiment:SD rats were randomly divided into model group,blank control group and ferula medium dose group,with 10 rats in each group.Ferula middle dose group(1.3g/kg)by gavage.Blank control group,model group 1 ml/100(g·d),intragastric administration of distilled water.Except for the blank control group,the other rats were given four interventions(behavioral intervention:playing cat audio at 70 dB volume).Dietary intervention:black tea(5g tea+100ml distilled water,air cooling instead of drinking water for rats),buckwheat flour(1/3 of normal feeding amount).Mongolian medicine intervention:gaburi,1 ml/100(g·d)).Blood letting therapy was performed in the evening(0.1ml).At the end of the experiment,serum ast,alt,urea,crep2,trigl were detected.Gas,CK,TSH,T4,T3,ACTH,CRF,CRH,cor,CRE,E2 were detected by ELISA.?Methods:orthogonal principal component analysis(hplc-upls)and principal component analysis(pca-ms)were used to determine the minimal metabolic profile of ferulic disease.?Intestinal microflora analysis:Based on Illumina Nova sequencing method,the v3-v4 hypervariable region of 16S rRNA was sequenced.According to species annotation,the alpha diversity and beta diversity were further calculated,and the differences between groups were compared to reveal the effect of Hay's disease on intestinal microflora of rats and the regulatory effect of Ferula on intestinal microflora of rats.?The joint analysis of microbiome and metabonomics data verified the correlation between metabonomics data and intestinal flora data of model experiment and ferula pharmacological experiment.Results:1.In the establishment of Mongolian medicine Heyi rat model,the symptoms,etiology and pathogenesis of the model group were consistent with the symptoms,etiology and pathogenesis of Heyi disease.The establishment of the rat model of Mongolian medicine Heyi disease with four intervention methods has the characteristics of simple method,strong stability and high repeatability.According to the basic theory of Mongolian medicine,four interventions were given to rats with Heyi disease,which appeared in the classic literature.2.1 compared with the blank control group,the index of brain organs increased,the contents of pro and pH in urine decreased,the contents of triiodothyronine and gas in serum decreased,and the levels of CK and T3 increased.These changes may be related to the symptoms of Hay's disease.2.2 in the study of metabonomics analysis of Heyi rat model,the metabolites of normal group and model group were significantly separated after positive and negative ions analysis of PCA and pca-da.The above results showed that after 31 days of intervention with Heyi,the metabolism of rats was abnormal.After the analysis of metabolites,it was found that the four intervention methods of Heyi disease were purine metabolism,pyrimidine metabolism,nicotinate and nicotinamide metabolism,tropane,piperidine and pyridine alkaloid biosynthesis,glucosinolate biosynthesis,biosynthesis of plant secondary metabolites,biosynthesis of alkaloids derived from ornithine,Lysine and nicotinic acid metabolism pathway plays a biological role.2.3 the establishment of rat model of hay disease by four intervention methods can cause the disorder of intestinal flora in rats.At the level of phylum,verrucomicrobia,Proteobacteria,Firmicutes and Actinobacteria in the intestinal flora of the model group increased significantly.Spirochaetes and cyanobacteria decreased significantly.The number of selenium,clostridiales and betaproteobacteriales increased significantly at the gene level.Spirochaetales,enterobacteria and bacteria decreased significantly.2.4 the joint analysis of microbiome and metabonomics data showed that guanidinobutanoate was replaced by Lactobacillus,Lactobacillus and Treponema at positive ion level2,lachnospiraceae,turiibacter consumption.Methionine was identified by Lactobacillus,turiibacter,Prevotella9,lachnospiraceae,luminococcaceae,lachnospiraceae,Lactobacillus consumption.L-tryptophan was identified by Roseburia,lachnospiraceae,unculturedbacteriumGBlautia promotes.Na was promoted by phascolarctobacterium.At the negative ion level,betaine was promoted by Lactobacillus and consumed by Lactobacillus.Glycochenodeoxycyclate was promoted by lachnospiraceae,phascolarctobacterium and luminococcaceae,and consumed by Lactobacillus and tricibacter.L-tyrosin was promoted by muribaculaceae.3.1 Mongolian medicine ferula has a therapeutic effect on the rat model established by the four intervention methods of Heyi.After treatment with Mongolian medicine ferula,the levels of serum gas and trigl increased and CK decreased in the model group.The mechanism of ferulic acid in the treatment of Heyi rat model is to improve the function of heart and stomach and regulate fat metabolism.3.2 the results of metabonomics analysis of ferulic acid in the treatment of Heyi rat model were as follows:after PCA and pca-da positive ion and negative ion analysis,the metabolites of normal group and model group,model group and ferulic acid group were significantly separated,and some metabolites of normal group and ferulic acid group were aggregated in the negative ion analysis.The above results showed that the metabolism of rats was abnormal after 31 days of administration of Heyi,The Mongolian medicine ferula can regulate the abnormal metabolism in rats.After analyzing the different metabolites,we found that ferula played its biological role in the treatment of Hay's disease through the metabolic pathways of pyridine metabolism,primary bile acid biosynthesis,arginine and proline metabolism,tryptophan metabolism.3.3 Mongolian medicine ferula can regulate the disturbance of intestinal flora caused by the four interventions of Heyi disease.The following changes can occur.At the level of phylum,ferulic acid caused a significant increase in proteobacteria,Firmicutes and Bacteroidetes.Verrucomicrobia and elusmicrobia decreased significantly.At the level of genus,ferulic acid caused a significant increase in turiciacter,Streptococcus and romboutsia.Klebsiella and akkermansia decreased significantly.3.4 combined analysis of microbiome and metabonomics data showed that guanidinobutanoate was replaced by lachnospiraceae at positive ion levelNK4A136We should promote it.Betaine was promoted by Staphylococcus.At the negative ion level,creatin was promoted by Halomonas.Cytidine is promoted by Lactobacillus,Lactobacillus,family??AD3011It is not consumed.Ecoine was replaced by lachnospiraceaeNK4A136Group,akkermansia,muribaculaceae.Glycochenodeoxycyclate was promoted by Rothia.L-tyrosin was isolated by Desulfovibrio,Lactobacillus,desulfovibrionaceae,unculturedbacteriumFMuribaculaceae,Prevotella9,Lactobacillus,lachnospiraceaeNK4A136Group,akkermansia,Lactobacillus,Lactobacillus consumption.Thiamine by lachnospiraceaeNK4A136We should promote it.Conclusion:The results showed that the brain organ index increased,the contents of pro and pH in urine decreased,the contents of trigl and gas in serum decreased,the levels of CK and T3 increased,and the metabolism and intestinal flora were in disorder.Mongolian medicine ferula has a therapeutic effect on the rat model established by the four intervention methods of Heyi.The mechanism of ferulic acid in the treatment of Heyi rat model is to improve the function of heart and stomach and regulate fat metabolism.It can also regulate the metabolic disorder and the structure of intestinal flora. |
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