Studies On Intervention Effects Of Zuojin Wan On Stomach Heat Syndrome In Rats Based On Metabonomics And Gut Microbiota

To investigate the machanismst of Zuojin Wan(ZJW)in the intervention of Stomach Heat Syndrome(SH)based on nuclear magnetic resonance(~1H-NMR)and Ultra High Performance Liquid Tandem Chromatography Quadrupole Time of Flight Mass Spectrometr(UHPLC-QTOF/MS)of metabonomics approach and 16S rRNA high-throughput sequencing approach,from the perspective of metabolic network and gut microbiota,explore the essence of“syndromes”and provide objective evidence for clinical diagnosis and treatment of SH.Explain the scientific connotation of ZJW from a global perspective and provide scientific basis for the rational application of this prescription.The main contents of the study are as follows:(1)24 rats were randomly divided into control group,model group and ZJW group.The SH models were established by gavage of decoction of 8%pepper and 1.5 ml absolute ethanol in rats,The basic signs,body weight,anal temperature and gastric tissue pathology of all rats were observed and recorded daily.The contents of IL-2?IL-8 and PGE2?EGF in rat gastric tissue and serum were determined by ELISA.The results showed that compared with the conrtol group,the body weight was significantly reduced(P<0.05),the anal temperature and water consumption were significantly increased(P<0.05),scanty yellowish urine,constipation,pathological changes in gastric tissue,the contents of IL-2?IL-8 in serum of rats significantly increased(P<0.05),and PGE2and EGF in gastric tissues significantly decreased(P<0.05)of the rats in the model group.After intervention with ZJW,the stool and anal temperature were close to the control group,the contents of IL-2?IL-8?PGE2 and EGF tended to be normal.It is initially proven that the model of SH was established successfully and ZJW has certain curative effect.(2)A proton nuclear magnetic resonance(~1H-NMR)based metabonomics approach was developed to profile SH-related metabolic perturbations in rat stomach.According to the variable importance projection(VIP)>1 in the Orthogonal partial least-squaresdiscriminant analysis(OPLS-DA)and t-test(P<0.05),the potential biomarkers between the control group and model group were screened.Metaboanalyst and KEGG databases were used to analyze metabolic pathways of potential biomarkers and establish metabolic networks.The results showed that compared with the conrtol group,the contents of3-D-hydroxybutyric acid was significantly increased(P<0.05),glutamic acid,lysine,choline phosphate,glyceryl phosphate choline,taurine,inositol,creatine,serine,lactic acid,inosine,phenylalanine and hypoxanthine were significantly reduced(P<0.05)in stomach of rats with SH.ZJW could significantly reverse 8 of these potential biomarkers,mainly involving regulated amino acid metabolism,energy metabolism,and Phospholipid metabolism.(3)Ultra performance liquid chromatography-quadrupole-time offlight-mass spectrometry(UPLC-QTOF/MS)based metabonomics approach inpositive and negative ion mode was developed to profile SH perturbations in rat serum.Principal component analysis and Orthogonal partial least-squaresdiscriminant analysis were used to study the metabolic differences between control group,model group,and ZJW group,the variable importance projection(VIP)and t-test were used to screen which potential biomarkers e between the control group and model group.Metaboanalyst and KEGG databases were used to analyze metabolic pathways of potential biomarkers and establish metabolic networks.The results showed that 27 potential markers were selected in the positive ion mode and 14 potential markers were selected in the negative ion mode.18 potential biomarkers related to SH were significantly reversed by administration of ZJW,which was probably regulated energy metabolism,Vitamin metabolism,phospholipid metabolism,purine and pyrimidine metabolism,bile acid metabolism.(4)We performed high throughput sequencing analysis in region(V3+V4)of bacterial 16S rRNA through the Illumina Hiseq 2500platform,analyze changes in diversity of gut microbiota,the LEf Se analysis to identified the biomarkers.Due to excessive reproduction of harmful bacteria,the rats of SH had structural disturbances of gut microbiota,ZJW can regulate the gut microbiota by promoting beneficial bacteria and reducing the abundance of beneficial bacteria.