DSS Induced The Imbalance Of The Environmental Flora In The Intestine Of UC Model Mice And The Regulating Effect Of The Mongolian Medicine Garidi San On The Imbalance Of The Flora

Aim:Ulcerative colitis（UC）is a chronic,spontaneous inflammatory bowel dis-ease affecting the innermost mucosa lining of the colon and rectum.Because of its relapsing nature,involvement of the entire colon,poor prognosis,and a risk factor for colorectal cancer,UC has been listed as a refractory disease by the World Health Organization.Extensive studies over the past decade have demon-strated that environmental factors,especially bacterial microflora,genetic and immunological factors play a substantial role in the pathogenesis of UC.Much more studies found that the dysbiosis of gut microbiota plays crucial roles in the pathogenesis of UC.Mongolian medicine GaRiDi pulvis is a Mongolian classic prescription and is usually used in the treatment of UC.GaRiDi pul vis has good effect,low toxicity,and hardly relapse after cure.Previous studies have also found that GaRiDi pulvis has a good therapeutic effect in the treatment of UC.To further explore the possible mechanism of GaRiDi pulvis in the treatment of UC and the characteristics of intestinal microflora in UC model mice,the study combined the latest research progress with recent results of modern medicine treatment on UC,used the imbalance of intestinal flora as the research target and used high-throughput sequencing technology and bioinformatics analysis to study the imbalance of intestinal flora in the DSS-induced UC model mice.The regulation effect of GaRiDi pulvis on the imbalanced flora was studied in the study.The study also revealed the correlation between different intestinal struc-tures and the pathogenesis of UC.Methods:The mice UC model was replicated by using DSS drinking water circularly and the mice colons were drawed under sterile condition.After drawing materi-als,the total DNA extraction was performed on the outer mucus layer,inner mucus layer and the intestinal contents of the colon in the control group,model group,sulfasalazine group,Bupiyichangwan group and GaRiDi pulvis group.Afterwards,high-throughput sequencing and PCR purification of the V3+V4 re-gion of 16SrRNA gene in the colony were performed and operational taxonomic units clustering,species annotation and diversity analysis were analyzed.The species composition and community structure differences of the grouped samples were analyzed by statistical analysis methods such as MetaStat,LefSe,multivar-iate analysis of variance and spatial statistics.Results:1.For colonic contents of bacteria,this study screened the blank grou p and model group and got UC-related differences in a total of 141 OTU s.Analysis of 141 different OTUs and UC for Bayesian causality analysis found that there were 3 OTUs directly related to UC and the 3 OTUs w ere Bacteroidales family S24-7,Anaerotruncus and Ruminococcaceae.Whe n UC occurs,the abundance of Anaerotruncus may be significantly increas ed,while the reduction of Bacteroidales family S24-7 and Ruminococcacea e may promote the occurrence of UC.2.The contents of sulfasalazine and model groups were screened.The re were 24 OTUs related to UC.Analysis of 24 different OTUs and a tot al of 141 OTUs in blank-model group showed that there were 10 commo n differential bacteria.They were Peptostreptococcaceae,Roseburia,Clostri dium_sensu_stricto₁,Lachnospiraceae,Ruminococcaceae,Thalassospira,La chnospiraceae,Blautia,Lachnospiraceae,Alistipes sp.AP11.Bayesian caus ality analysis showed that sulfasalazine had a direct effect on UC by influ encing the Alistipes sp.AP 11 in the intestinal contents directly.The Alisti pes_sp._AP11 could not only directly affect UC but also had an effect on the inferior Peptostreptococcaceae.And the Peptostreptococcaceae may ha ve an indirect effect on UC by Lachnospiraceae.3.The contents of Bupiyichangwan and model groups were screened.There were 16 OTUs related to UC.Analysis of 16 different OTUs and a total of 141 OTUs in blank-model group showed that there were 8 com mon differential bacteria.They were parts of Lachnospiraceae,Holdemania_filiformis_DSM₁2042,Christensenellaceae,Alistipes sp.AP11.Bayesian causality analysis showed that Bupiyichangwan had no direct effect on UC.It means that Bupiyichangwan could regulate the intestinal content flora i n UC model mice.But it may not affect UC by regulating the intestinal f lora in intestinal content.4.The contents of GaRiDi pulvis and model groups were screened.T here were 10 OTUs related to UC.Analysis of 10 different OTUs and a total of 141 OTUs in blank-model group showed that there were 5 comm on differential bacteria.They were parts of Lachnospiraceae,Blautia,Alisti pes_sp._AP11.Bayesian causality analysis showed that GaRiDi pulvis had a direct effect on UC by influencing the OTU253 in the intestinal content s directly.5.The diversity of the microbiota from luminal content,the outer mu cus layer,and inner mucus layer were significantly different in both contr of and UC model groups.The differences between any two groups weremore substantial than those within any individual group.At the same taxo nomy level,the richness of bacteria differed to some extent.Multiple logi stic regression analyses of OTU richness in each intestinal layer and UC r evealed that the statistically different OTUs,which belonged to Lachnospir aceae and Ruminococcaceae families within the order Clostridiales,were m ainly localized to the outer mucus layer.6.The outer mucus layer of sulfasalazine and model groups were scr eened.There were 26 OTUs related to UC.Analysis of 26 different OTU s and a total of 204 OTUs in blank-model group showed that there were 9 common differential bacteria.They were parts of Ruminococcus,Erysipe lotrichacea,Clostridium sensu stricto 1,Lachnospiraceae,Bacteroides,Enter obacteriaceae,Acetatifactor.Bayesian causality analysis showed that sulfasa lazine had no direct effect on UC.It means that sulfasalazine could regula te the intestinal flora of outer mucus layer in colon in UC model mice.B ut it may not affect UC by regulating the flora in intestinal outer mucus 1 ayer.7.The inner mucus layer of sulfasalazine and model groups were scr eened.There were 26 OTUs related to UC.Analysis of 26 different OTU s and a total of 110 OTUs in blank-model group showed that there were 2 common differential bacteria.They were parts of Clostridiales vadinBB60,Ruminococcaceae.Bayesian causality analysis showed that sulfasalazine had no direct effect on UC.It means that sulfasalazine could regulate the intestinal flora of inner mucus layer in colon in UC model mice.But it may not affect UC by regulating the flora in intestinal inner mucus layer.8.The outer mucus layer of Bupiyichangwan and model groups were screened.There were 7 OTUs related to UC.Analysis of 7 different OT Us and a total of 204 OTUs in blank-model group showed that there wer e 2 common differential bacteria.They were parts of Lachnospiraceae.Ba yesian causality analysis showed that Bupiyichangwan had no direct effect on UC.It means that Bupiyichangwan could regulate the intestinal flora of outer mucus layer in colon in UC model mice.But it may not affect UC by regulating the flora in intestinal outer mucus layer.9.The inner mucus layer of Bupiyichangwan and model groups were screened.There were 35 OTUs related to UC.Analysis of 35 different OTUs and a total of 110 OTUs in blank-model group showed that there were 5 common differential bacteria.They were parts of Lachnospiraceae,Allobaculum.Bayesian causality analysis showed that Bupiyichangwan had no direct effect on UC.It means that Bupiyichangwan could regulate the intestinal flora of inner mucus layer in colon in UC model mice.But it may not affect UC by regulating the flora in intestinal inner mucus layer.10.The outer mucus layer of GaRiDi pulvis and model groups were screened.There were 37 OTUs related to UC.Analysis of 37 different O TUs and a total of 204 OTUs in blank-model group showed that there we re 16 common differential bacteria.They were parts of Thalassospira,Lac hnospiraceae,Eubacterium desmolans,Blautia,Lactobacillus,Turicibacter,Gastranaerophilales,Roseburia,Enterobacteriaceae,Streptococcus.Bayesian causality analysis showed that GaRiDi pulvis had a direct effect on UC b y influencing the Lachnospiraceae in intestinal inner mucus layer directly.11.The inner mucus layer of GaRiDi pulvis and model groups were screened.There were 58 OTUs related to UC.Analysis of 58 different O TUs and a total of 110 OTUs in blank-model group showed that there we re 5 common differential bacteria.They were parts of Lachnospiraceae,Ps eudomonas_putida,Erysipelotrichaceae,Bacteroides.Bayesian causality anal ysis showed that GaRiDi pulvis had no direct effect on UC.It means that GaRiDi pulvis could regulate the intestinal flora of inner mucus layer in colon in UC model mice.But it may not affect UC by regulating the flor a in intestinal inner mucus layer.Conclusion:This study found that the luminal content flora of UC model mice w as disordered and there is a certain correlation among the disordered flora.Among them,the bacteria most closely related to UC were,Ruminococca ceae,Anaerotruncus and Bacteroidales family S24-7.And the bacteria indir ectly related to UC were in total of 141,such as Turicibacter,Erysipelotri chaceae,Bacteroides.The reduction of Bacteroidales family S24-7 and Ru minococcaceae families may promote the occurrence of UC.And the prom otion of Anaerotruncus may promote the occurrence of UC.When differen t drugs were given to the UC model mice,luminal content flora was adju sted to varying degrees.It indicates that the occurrence of UC is related t o intestinal flora imbalance.The mechanism of the commonly used drug o f UC is not clear,but its mechanism may be related to the regulation of intestinal flora imbalance.And the flora regulated by the drugs were differ ent.This study found that the Lachnospiraceae were directly related to UC after the UC model mice were given GaRiDi pulvis.Therefore,it is spec ulated that GaRiDi pulvis can play a therapeutic role in the treatment of UC by regulating the bacteria of Lachnospiraceae（Firmicutes）in the intest inal tract.Further experimental verification is needed in the future.Further study on the relationship between the intestinal flora and the colon show ed that the changes in intestinal contents were not significant when compa red with the mucus layer.Changes in flora structure and species were mai nly concentrated in the outer mucus layer.It is more important to pay att ention to the characteristics of intestinal mucus layer flora especially the c haracteristics of outer mucus layer flora for exploring the common features of IBD and the relationship between disease and intestinal flora.In additi on,the imbalance of intestinal mucus layer flora under different drug inter vention appears to varying degrees of adjustment,indicating that commonl y used UC therapeutic drugs can have a regulatory role in colonic mucosa 1 flora.Different drugs involved in the regulation of different bacteria,but all the bacteria were superior bacteria.GaRiDi pulvis can regulate the ba cteria of Bacteroidetes,Proteobacteria,Actinobacteria,Firmicutes in the int estinal mucous layer and play a therapeutic role in the treatment of UC b y regulating the bacteria of Lachnospiraceae（Firmicutes）.The study of intestinal flora is limited to studying the changes of thecontents flora only which cannot fully reveal the law of intestinal flora.The characteristics of intestinal flora change should be studied in combinat ion with the change rule of intestinal mucous layer flora.GaRiDi pulvis n ot only regulate the intestinal content flora,but also have a certain regulat ing effect on the microflora of the colonic mucous layer.Lachnospiraceae may be an important direct target for the study of the mechanism of UC treatment by GaRiDi pulvis.