Studies On Intervention Effects Of Huoxiang Zhengqi Oral Liquid On Dampness Obstructing Spleen-stomach Syndrome In Rats Based On Metabonomics And Gut Microbiota

To elucidate the intervention effects of Huoxiang Zhengqi Oral Liquid(HZOL)in rats with Dampness Obstructing Spleen-Stomach Syndrome(DOSS)and the underlying mechanism based on metabonomics and gut microbiota.The main contents of the study are as follows:Firstily,we have established the model of Dampness Obstructing Spleen-Stomach Syndrome.Male SD rats were randomly divided into normal group,model group and HZOL group.Model group and the HZOL group rats were put in the manufacturing model box,under the temperature of 18~25℃,humidity of(90±5)%.The rats of model group and HZOL group were put in the 4 cm deep-water everyday.First day fasting with 4℃water 2 mL once a day by gavage and next day supply sufficient and given each rats with cooked lard 4 mL,hold this for 20 days.On the 11th day,the,HZOL group began to dosewith the 10 mL·kg~-11 Huoxiang Zhengqi Oral Liquid,and normal group and model group were filled with same volume physiological saline.The results show that the DOSS could significantly suppress the locomotor activity of model rats,weight,body length,tail length,abdominal index decreased.And the villi of the colon were shed,the intestinal tract dilated,the intestinal glands decreased,and the mucosa layer and the natural developed severe edema.decreased level of spleen and thymus and IgG,increased level of IL-6demonstrate the success of the model.It was obvious that the inttervention effects of Huoxiang Zhengqi Oral Liquid(HZOL)in rats with Dampness Obstructing Spleen-Stomach Syndrome(DOSS)is available.Secondly,a proton nuclear magnetic resonance(1H-NMR)based metabonomics approach was developed to profile DOSS-related metabolic perturbations in rat urine,serum and fecal.Orthogonal partial least-squares discriminant analysis(OPLS-DA)was carried out on the metabolites,the variable importance projection(VIP)and t-test were used to screen which significant difference between the normal group and model group.Results:(1)HZOL could effectively reverse the disorder of 19 biomarkers related to DOSS in serum(LDL/VLDL、isoleucine,3-hydroxybutyric,alanine,n-acetylglycoprotein,glutamine,creatineandphosphocreatine,trimethylamine oxide),in urine(alpha ketopamyl diacid,dimethylamine,creatinine,trimethylamine oxide,fumaric acid),in fecal(leucine,valine,isoleucine,propionic acid,lactic acid,acetic acid,N-acetyl glucosamine,beta glucose,alpha glucose,uracil,tyrosine,phenylalanine).(2)The metabolic pathways involved in DOSS were mainly related to the tricarboxylic acid cycle,lipid metabolism,amino acid metabolism and intestinal flora metabolism.Thirdly,ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry(UPLC-QTOF-MS)based metabonomics approach in positive and negative ion mode was developed to profile DOSS-related metabolic perturbations in rat urine,serum and fecal.Orthogonal partial least-squares discriminant analysis(OPLS-DA)was carried out on the metabolites,the variable importance projection(VIP)and t-test were used to screenwhich significant difference between the normal group and model group.Results:(1)9 markers were selected in the positive ion mode(histamine,glutathione,goose deoxycholate,LysoPE(16:0/0:0),LysoPC(14:0),LysoPE(20:3),serine,N-Acetyglutamine,sphingosine),and 6 markers were selected in the negative ion mode(Cysteine-S-sulfate,deoxycytidine,S-Methyl-5~’-thioadenosine,PS(18:0),Choline phosphate,erucic acid).(2)The HZOL can produce a correction of 10 potential biomarkers except L-serine,n-acetyglutamine,sphingosine,phosphocholine and erucic acid.The metabolic pathways involved in the DOSS were mainly related to amino acid metabolism,bile acid metabolism and phospholipid metabolism.Fourthly,the abundance and alpha diversity and the following cluster analysis of fecal microbiota were analyzed by pyrosequencing of 16S r RNAgenesontheIlluminahigh-throughputsequencing technology(Illumina Hiseq).Throughthemethodof double-end sequencing(pin-end),the small fragment library was constructed for sequencing.Through the filtering of Reads,the Operational Taxonomic Units cluster,and the species annotation and abundance analysis are carried out to reveal the species composition of the samples.Through the analysis of PcoA and ANOVA,t test found the potential microbial biomarkers.The results show that:(1)intestinal microflora structure and composition werechanged and the intestinal microbiota diversity was reduced in DOSS rats.(2)4 potential microbial biomarkersrelated to DOSS on phylum level were selected(Cyanobacteria,Tenericutes,Verrucomicrobia,Bacteroidetes),and 4 potential microbial biomarkers relatedtoDOSSongenuslevelwereselected(Arthrobacter,Corynebacterium,Butrycricimonas,Kurthia).Gutmicrobiota perturbations were reverted in DOSS rats by HZOL,and bring it back to normal gradually.