Pharmacokinetics Of Shenling Baizhu San Syndrome And Its Mechanism In The Treatment Of Spleen Deficiency With Dampness Retention-ulcerative Colitis

Objective:This topic is based on the difference of the efficacy of Shenling Baizhu San in the treatment of spleen deficiency with dampness retention-ulcerative colitis（SDDR-UC）and pure-ulcerative colitis（P-UC）rats.Firstly,the differences of pharmacokinetic parameters of Shenling Baizhu San in normal group,SDDR-UC group and P-UC group were discussed from the perspective of pharmacokinetics.Then,the therapeutic difference of Shenling Baizhu San on SDDR-UC and P-UC rats and its mechanism were clarified from the perspective of metabolomics,intestinal flora and transcriptomics,in order to provide reference for the clinical treatment of ulcerative colitis of spleen deficiency and dampness stagnation type.Method:1.Pharmacokinetics of effective components of SLBZS in normal and ulcerative colitis ratsThe rats were randomly divided into three groups:normal group,SDDR-UCand P-UC group.Each group was divided into low,medium and high dose groups(0.945,1.89,3.78 g·kg^-1·d^-1).The drug concentrations were 0.625,1.25 and 0.25 g·m L^-1respectively.Subsequently,approximately 0.2 m L blood was collected from retinal vein plexus of rats into heparinized tubes at predetermined time points（0,0.083,0.25,0.5,1,1.5,2,4,6,8,12,24 h）after drug administration.Then,more than 100μL plasma was obtained by centrifugation at 13000 r·min^-1for 10 min and stored at-20^℃ until analysis.The analysis was performed using the UHPLC-MS/MS system.Winnonlin 4.1 pharmacokinetic software was used to calculate the pharmacokinetic parameters of plasma concentration time in each group according to non atrioventricular model.The Student’s t-test was used to compare the pharmacokinetic data,and the statistically significant difference was set at a value of P<0.05（Graph Pad Prism software package,Version 8.0）.2.Metabolomics Study of SLBZS in the treatment of spleen deficiency with dampness retention-ulcerative colitisRats were randomly divided into normal group,SDDR-UC group,P-UC group,low,medium and high dose group of SLBZS(0.945,1.89,3.78 g·kg^-1·d^-1)and treatment group of P-UC(1.89 g·kg^-1·d^-1).The.csv format file containing compound retention time and m/z information is obtained by peak extraction,peak alignment,peak matching and peak intensity correction based on Progenesis QI software.The processed data matrix was imported into SIMCA 14.1 software.PLS-DA was performed on the results of normal group,model group and administration group.Differential metabolites were screened according to the Variable Importance Projection（VIP）value>1 and t-test（P<0.05）with Graph Pad Prism 8.0.Through the identification of Human Metabolome Database（HMDB）,the identified potential biomarkers were introduced into Metabo Analyst 5.0 database for metabolic pathway analysis.3.Study on intestinal flora of SLBZS in the treatment of spleen deficiency with dampness retention-ulcerative colitis.The cecal contents of rats in normal group,SDDR-UC group,P-UC group,low,medium and high dose groups of SLBZS and treatment group of P-UC were collected.Sequencing of sample microbial diversity was completed by Shanghai Meiji Biomedical Technology Co.,Ltd.After the genomic DNA is extracted,primers are designed according to the designated sequencing area,and sequencing adapters are added to the ends of the primers,PCR amplification is performed,and the products are purified,quantified and homogenized to form a sequencing library.The built library is first subjected to library quality Illumina Mi Seq is used to sequence the libraries that have passed the quality inspection.4.Transcriptomic study of SLBZS in the treatment of spleen deficiency with dampness retention-ulcerative colitis.The colon tissues of 3 rats in each group were collected from the normal group,SDDR-UC group,P-UC group,low,medium and high dose groups of SLBZS and the treatment group of P-UC.Eukaryotic m RNA sequencing is based on the Hi Seq platform,which sequences all the m RNAs transcribed from specific eukaryotic tissues or cells in a certain period.The sequencing experiment uses the Illumina Truseq TM RNA sample prep Kit method for library construction.Results:1.Panaxadiol（PAN）,ginsenoside Rg1（Rg1）,atractylenolide I（ATA-I）,atractylenolideⅢ（ATA-III）,pachymicacid（PA）,neferine（NEF）,nuciferine（NUC）,diosgenin（DG）,platycodin D（PD）and isoliquiritigenin（ISL）have good linearity in 0.44-397.50,0.63-388.50,0.44-400.50,0.54-490.00,0.31-279.00,0.41-367.50,0.38-355.50,0.50-447.00,0.42-382.50,0.39-356.10ng·m L^-1.The validation parameters,including intra-/inter-day precisions,accuracy,recovery,matrix effect,and stability were within acceptable ranges,and all meet the requirements of biological sample analysis.2.The results of metabolomics PLS-DA showed that under the two modes,each group was well separated.The results of plasma metabolomics showed that 14 potential biomarkers were identified in positive ion mode and 9 potential biomarkers were identified in negative ion mode.The above 23 potential biomarkers were introduced into Metabo Analyst 5.0 for pathway enrichment analysis.Six metabolic pathways were found:purine metabolism,pentose phosphate pathway,pyrimidine metabolism,retinol metabolism,steroid hormone synthesis and primary bile acid biosynthesis.The results of urine metabolomics showed that 30 potential biomarkers were identified by positive ion mode and 18 potential biomarkers were identified by negative ion mode,The above 48 potential biomarkers were introduced into Metabo Analyst 5.0for pathway enrichment analysis.Nine metabolic pathways were found,including glutathione metabolism,niacin and nicotinamide metabolism,pyrimidine metabolism,mannose O-glycan biosynthesis,tryptophan metabolism,arginine and proline metabolism.The results of fecal metabolomics showed that 26 potential biomarkers were identified by positive ion mode and 10potential biomarkers were identified by negative ion mode,The above 36 potential biomarkers were introduced into Metabo Analyst 5.0 for pathway enrichment analysis.Six metabolic pathways were found,including folate biosynthesis,arachidonic acid metabolism,steroid hormone biosynthesis,tryptophan metabolism.3.Bacterial sequencing results showed that the intestinal flora of the two model groups was disordered compared with the normal group of rats,and the unique flora of SDDR＿UC rats was found:Morganell,Bacillaceae.The unique flora of P＿UC rats:Proteobacteria.After adjustment by SLBZS,andit promoted the recovery of intestinal flora in rats.Compared with the two model groups,at the phylum level,the administration group mainly showed a decrease in the proportion of Proteobacteria and Bacteroides,and an increase in Firmicutes;at the genus level,the main manifestations were the decrease of Desulfovibrio and Ruminiclostridium＿9,the increase of Ruminococcaceae＿UCG-005.4.Transcription sequencing results show,compared with the normal group,there are 420significantly differentially expressed genes in the SDDR-UC group（P<0.05）,of which 209genes are significantly up-regulated and 211 genes are significantly down-regulated.Compared with the SDDR-UC,there were 1060 significantly differentially expressed genes in the administration group（P<0.05）,of which 554 genes were significantly up-regulated and 506genes were significantly down-regulated.Compared with the normal group,the P-UC group had a total of 366 significantly differentially expressed genes,of which 162 genes were significantly up-regulated,and 204 genes were significantly down-regulated.Compared with the P-UC group,theadministration group had a total of 529 significantly differentially expressed genes（P<0.05）,of which 407 genes were significantly up-regulated and 122 genes were significantly down-regulated.KEGG enrichment analysis was performed on 189differentially expressed genes in the normal group,SDDR-UC group and administration group.The results showed that they were mainly involved in tryptophan metabolism,NF-κB signaling pathway,NOD-like receptor signaling pathway,etc.KEGG enrichment analysis was carried out on 113 differential genes in the normal group,P-UC group and administration group,and the results showed that they were mainly involved in tryptophan metabolism,pyrimidine metabolism,and steroid hormone biosynthesis.5.Combined 16S r DNA sequencing,fecalmetabonomics,and transcriptome sequencing,we further discovered that SDDR-UC was primarily linked to 6 related metabolites（i.e.,Sepiapterin,6-Hydroxymelatonin,5-Hydroxy-L-tryptophan,Tryptophan,Arginine,20-OH-Leukotriene B4）,5 gut microbiotas（i.e.,Bacteroidetes,Lachnospiraceae＿NK4A136＿group,Ruminococcaceae＿UCG-005,Lactobacillus,Ruminiclostridium＿9）and 5 genes（Aco1,Inmt,Cyp2j4,Pla2g12b,Akr1b7）mediated various metabolic disorders（folate biosynthesis,tryptophan metabolism,arginine biosynthesis,arachidonic acid metabolism）.P-UC could negotiate 6 related metabolites（i.e.,5-Hydroxy-L-tryptophan,tryptophan,arginine,tryptamine,dihydrofolic acid and aldosterone）and 5 kind of microbes（i.e.,Desulfovibrio,Lachnospiraceae＿NK4A136＿group,Ruminococcaceae＿UCG-005,Lactobacillus,Ruminiclostridium＿9）and 3 genes（Cyp1b1,Inmt,Cyp17a1）.Mainly involved tryptophan metabolism,arginine biosynthesis,folate biosynthesis,steroid hormone biosynthesis.Conclution:1.A selective,sensitive and reliable uhplc-ms/MS method was established and successfully applied to the pharmacokinetics of bioactive components of Shenling Baizhu powder in plasma.The results of pharmacokinetics showed that the state of animals with different diseases would affect the absorption of drugs2.Metabonomic results showed that the therapeutic effect of SLBZS on SDDR-UC rats may be related to the activation of FXR receptor,oxidative stress,cytochrome P₄₅₀expression,activate SIRT1 receptor,inhibition of NF-κB signaling pathway,reduction of inflammatory factors,anti-lipid peroxidation and oxidative stress damage,repair damaged barriers.And through the relative content changes of bile acid,phosphoribosyl pyrophosphate in plasma;arginine,tryptophan,gamma-glutamylcysteine in urine and 5-hydroxy-L-tryptophan,6-hydroxymelatonin in fecal showed that:after the intervention of Shenling Baizhu San,these differential metabolites showed a more obvious trend of returning to normal state in sddr-uc rats.3.The results of intestinal flora showed that SLBZS mainly improves SDDR-UC and P-UC by reducing Desulfovibrio and Ruminiclostridium＿9,and increasing Ruminococcaceae＿UCG-005.The regulating effect of intestinal flora in SDDR-UC rats was better than that P-UC rats.4.Transcriptome results showed that the two different model groups of SDDR-UC group and P-UC group have different differential genes and are enriched in different pathways.The intervention of SLBZS mainly affected the metabolism of tryptophan metabolic pathway,NF-κB signaling pathway,etc.Most of the differential genes regulated by Shenling Baizhu San only appear in SDDR-UC model,such as Cyp2j4、Aoc1、Akr1b7、Pla2g12b,indicating that most of the differential genes play a role in SDDR-UC rats after Shenling Baizhu San intervention.5.The combined analysis of metabolomics,intestinal flora and transcriptomics indicates that SLBZS may improve the metabolic disorder of SDDR-UC by acting on intestinal microorganisms and regulating genes.And tryptophan metabolic pathways have been found in intestinal function prediction,metabolic pathways and gene function enrichment analysis.