| Objective Diabetes mellitus(DM)is a chronic metabolic disease characterized by hyperglycemia.At present,in addition to the treatment of diabetes mellitus with insulin and other drugs,patients mostly also use dietary regulation or natural functional ingredients for intervention.Cuscuta chinensis Lam,as a natural plant with homology of medicine and food,has been shown to have a certain therapeutic effect on diabetes,but its mechanism is still unclear.Therefore,our study was conducted to investigate the intervention effect and mechanism of Cuscuta chinensis Lam extract on diabetes mellitus in rats by using metabolomics and 16 S r DNA gene sequencing of gut microbiota,in order to provide an experimental basis for the application of Cuscuta chinensis Lam in diabetes mellitus.Methods 1.Methods for the establishment of diabetic animal model: A total of 48 male Wistar rats aged 4 to 5 weeks were randomly divided into 6 groups(8 rats/group)after one week of adaptive feeding.They were model control group,model low-dose group(Cuscuta chinensis Lam extract 2g/kg),model high-dose group(Cuscuta chinensis Lam extract 4g/kg),metformin group(100mg/kg),blank control group,and blank high-dose group(Cuscuta chinensis Lam extract 4g/kg).The blank group was fed normal diet,and the model group was given high fat diet for 4 weeks,and the body weight of 35mg/kg was converted into a single intrabitoneal injection of 1% streptozotocin(STZ)solution to establish a rat diabetic animal model.One week after STZ injection,the establishment of the animal model was determined by fasting blood glucose≥11.1mmol/L.The modeling group was treated with gavage of Cuscuta chinensis Lam extract for a total of 40 days,during which blood glucose and body weight were measured every 10 days.2.Establishment of UPLC-Q-TOF/MS method and metabolomic analysis: UPLC-Q-TOF/MS was used to establish metabolic profile analysis of rat plasma,urine and feces,principal component analysis(PCA),orthogonal partial least squares discriminant analysis(OPLS-DA)were used to investigate the differences in metabolite profiles among different groups and using the online website metaboanalyst(https://www.metaboanalyst.ca/)for pathway analysis to investigate the effects of Cuscuta chinensis Lam on metabolic pathways in diabetic rats in vivo.3.16 S r DNA gene sequencing method for gut microbiota analysis: Total gut microbiota DNA was extracted using TIA Namp Stool DNA.After genomic DNA was extracted by 1% agarose gel electrophoresis,the gut microbiota DNA V3-V4 region was amplified,and then Illumina library was constructed and sequenced.PE reads were obtained by Illumina sequencing.Firstly,overlap is used for splicing,quality control and filtering for sequence quality,and OTU cluster analysis and species taxonomic analysis are carried out after samples are distinguished.Based on OTU,a variety of diversity index analysis and OTU cluster analysis results can be carried out.Based on taxonomic information,statistical analysis of community structure can be performed at various taxonomic levels.On the basis of the above analysis,a series of in-depth statistical and visual analysis can be conducted on the community composition and phylogenetic information of multiple species and the difference significance test,so as to analyze the mechanism of Cuscuta chinensis Lam’s intervention in diabetes through gut microbiota.4.Gut microbiota and endogenous metabolites of spearman correlation analysis conducted by Omic Studio tool at https://www.omicstudio.cn/tool.Results 1.Effect of Cuscuta chinensis Lam on blood glucose and body weight of diabetic rats: The statistical difference analysis of blood glucose and body weight of rats after 40 days of successful modeling showed that the difference between blood glucose and body weight of rats treated with Cuscuta chinensis Lam and rats in the non-intervention diabetic model group was statistically significant.2.The results of the metabolomic studies of plasma,urine and feces showed significant changes in the plasma,urine and fecal metabolites in both the low and high dose groups of the model.The model group was far away from the blank group,while the model low-dose and high-dose groups were closer to the blank group.Thirty-four differential metabolites were screened in the rat plasma metabolome for L-Tryptophan,20- hydroxy-eicosatetraenoic acid,leukotriene A4,5(S)-hydroperoxyeicosatetraenoic acid,PGH3,16(R)-hydroxyeicosatetraenoic acid,15(S)-hydroxyeicosatetraenoic acid,oleic acid,palmitic acid,8,11,14-eicosatrienoic acid,arachidonic acid,alpha-linolenic acid,eicosapentaenoic acid,Androsterone,5-acetylamino-6-formylamino-3-methyluracil,theobromine,6-mercaptopurine ribonucleoside triphosphate,D-galactose,linoleic acid,PC(18:2(9Z,12Z)/P-18:1(11Z)),L-tyrosine,9-cis-retinoic acid,4-hydroxyretinoic acid,docosahexaenoic acid,Androstenedione,11B-hydroxyprogesterone,12-oxo-20-hydroxyleukotriene B4,arachidonate,lyso-PC(18:1(9Z)),sphingosine,phenylpyruvic acid,retinyl ester,21-deoxycortisol,19-hydroxytestosterone.Twenty biomarkers associated with kidney disease were screened in urine for NPC,D-glucuronide,2-phenylethanol glucosinolate,5-hydroxy-6-methoxyindole glucosinolate,tyramine glucosinolate,D-ribose,2-methylmarmaluronic acid,L-tyrosine,phenylacetylglycine,all-trans-13,14-dihydroretinol,theobromine,D-galactose,4-phosphopantenoyl cysteine,4-phosphopanthenylmercaptoethylamine,pantothenic acid,5-L-glutamyl taurine,5a-cholesterol-8-ethylenediamine-3b-ol,7a-hydroxydehydroepiandrosterone,estrone sulfate,3-O-sulfogalactosylceramide(d18:1/24:1(15Z)).Twenty-five differential metabolites were screened in feces for 15(S)-HETE,palmitic acid,stearic acid,oleic acid,linoleic acid,8,11,14-eicosanotrienoic acid,eicosapentaenoic acid,PE(24:1(15Z)/ 20:2(11Z,14Z)),13-L-hydroperoxy linoleic acid,PGH3,15(S)-HPETE,3α,7α,26-trihydroxy-5β-cholestane,3β,7α-dihydroxy-5-cholesteneester,cholic acid,3-o-sulfogala-ctoseceramide(d18:1/24:1(15Z)),SM(d18:0/12:0),cholesterol sulfate,4acarboxyl-4B-methyl-5a-cholester-8,24-diene-3b-alcohol,tetrahydrocorticosterone,SM(d18:0/20:2(11Z,14Z),4-(2-aminophenyl)-2,4-dioxy-butyric acid,prostaglandin G2,24-methylene cholesterol,dehydroepiandrosterone sulfate,Geranylgeranyl pyrophosphate were associated with the biosynthesis of unsaturated fatty acids,arachidonic acid metabolism,phenylalanine metabolism,pantot-henic acid and coenzyme A,respectively.3.The results of gut microbiota composition analysis showed that Cuscuta chinensis Lam extract could restore gut microbiota homeostasis by regulating microbial composition and abundance.Improved Romboutsia,unclassified_f_Lachnospiracea,Lachnospiraceae_NK4A136_group abundance,lowered the Lactobacillus and Akkermansia abundance.The results showed that Cuscuta chinensis Lam extract is beneficial to restore gut microbiota and maintain the balance of gut microbiota in rats,and it is possible to alleviate diabetes in rats by regulating the composition and structure of key microorganisms.4.Spearman correlation analysis predicted that some of the affected bacteria had statistically significant correlations with metabolites in various metabolic pathways.Conclusions In this paper,the effects and mechanisms of Cuscuta chinensis Lam extract on blood glucose regulation in diabetic rats were studied by metabomics and gut microbiota analysis.The results showed that Cuscuta chinensis Lam extract can significantly regulate the changes of blood glucose and body weight in diabetic rats.The correlation analysis between in vivo metabolic mass profile and gut microbiota indicated that Cuscuta chinensis Lam may interfere with blood glucose and endogenous metabolic pathways in diabetic rats through gut microbiota may be a potential target for to interfere with blood glucose in diabetic rats. |
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