Patchouli Alcohol Ameliorates Mice Colitis By Suppressing Tryptophan Metabolism And Regulating Gut Microbiota

ObjectiveInflammatory bowel disease?IBD?is a chronic relapsing intestinal disease with clinical symptoms such as diarrhea,abnormal pain and rectal bleeding,which mainly includes the subtypes of ulcerative colitis?UC?and Crohn's disease?CD?.The pathogenesis of IBD is still unclear.Recent studies showed that Tryptophan?Try?accelerates the rate of catabolism into Kynurenine?Kyn?,and the increased expression of rate-limiting enzyme indoleamine 2,3-dioxygenase?IDO-1?and high level of Kyn can inhibit the immune response to alloantigens,which is closely related to the occurrence of IBD.Try is an endogenous free radical scavenger and antioxidant which has anti-oxidative and anti-inflammatory activity.While Kyn-related metabolites have immune-suppressive effects,which can induce apoptosis in immune cells and weaken its secretory function.In addition,studies revealed that IBD was triggered by disrupted intestinal barrier with participation of gut microbiota,which leads to autoimmune inflammation eventually.Therefore,it is of great significance to further understand the pathogenesis of IBD and search for effective drugs for the treatment of IBD based on tryptophan metabolism and gut microbiota regulation.Pogostemon cablin?Blanco?Benth has been applied in treatment of gastrointestinal disorders via preventing vomiting and relieving summer-heat with its aromatic capability for thousand years in China.Previous studies showed that Pogostemon cablin and its active components including patchouli oil and pogostone attenuated the symptoms of IBD.Patchouli alcohol?PA?is identified and isolated as the major active component and index component of Pogostemon cablin and patchouli oil.PA processed multiple bioactivities including anti-inflammation,anti-gastriculcer and immune-modulation.This makes PA a promising alternative agent for treatment of IBD.Therefore,in this thesis,the anti-IBD effect of PA was investigated and verified by tryptophan metabolism and gut microbiota analysis.It will help us further elucidate the pathogenesis of IBD.MethodsExperimental groups were given 3%DSS?drink freely?to establish experimental IBD model,and mice in Control group were supplied with distilled water.Mice in Control and DSS group received equal volume of vehicle from day 1 to 7,while mice in PA and SASP groups were orally administered with PA?10,20 and 40 mg/kg,respectively?or SASP?200mg/kg?,respectively.The body weight,stool consistency and rectal bleeding of all mice were observed and recorded daily.Then comprehensive score of disease activity index?DAI?was calculated.On the 8^th day of experiment,the colons were taken from the mice in all groups.The colon length was assessed,which can determine the therapeutic effect of PA on IBD along with DAI score.Also,plasma was collected for deproteinization,lyophilization and volumetric determination,followed by global metabolic analysis which was performed by UPLC-MS.OPLS-DA was used to identify patterns among all groups.Differences between DSS group and PA-treated groups were based on the results of UPLC-MS analysis and the search on Human Metabolome Database.Then we examined the expression of relavant protein in tryptophan metabolism?IDO-1 and TPH-1?,and we conducted molecular docking experiment to verify the combination between PA and the active site of IDO-1,which all help us validate metabolic biomarkers related to DSS-induced IBD model to evaluate PA's regulation on IBD.Then the feces were collected for gut microbiota analysis.The total DNA was extracted and the 454 pyrosequencing analysis platform was used for sequencing.And species classification,abundance analysis,OTU Wenn diagram,species evolution analysis and cluster analysis were used to observe the differences among all groups,which help us further study PA'effect on microbiota diversity and population structure.ResultsIn 3%DSS-induced IBD mice colitis,PA-treated groups significantly reduced DAI score and restored the colon length compared to DSS group.In plasma global metabolomics analysis,OPLS-DA analysis was used to perform data pre-processing and model building.The results showed that the samples of each group were clustered and distributed in different quadrants,suggesting that there was a significant difference between DSS group and Control group as well as between DSS group and PA-or SASP-treated groups.Combined with UPLC-MS and Human Metabolome Databases,19potential endogenous differential metabolites were screened out,which proved that the occurrence of IBD is closely related with amino acid metabolism,fatty acid metabolism,tryptophan metabolism,tricarboxylic acid cycle and bile acid metabolism.At the same time,we identified seven metabolites that used to distinguish normal animals with IBD animals.Among those,the prominent change is tryptophan metabolism.Then we further conducted targerted metabolism analysis on the upstream and downstream metabolites including Try,Kyn,5-HT and 5-HTP.The results showed that compared to DSS group,Try in PA-treated groups were significantly increased,but Kyn and 5-HT were significantly decreased.The results of western blot showed that PA-treated group reduced the expression of related rate-limiting enzymes?IDO-1 and TPH-1?in tryptophan metabolism.The results of molecular docking showed that PA binded to the active site of IDO-1 and inhibited the over-expression of IDO-1.Gut microbiota analysis of PA on DSS-induced mice colitis showed that PA significantly inhibited the growth of pathogenic bacteria like Proteobacteria,Desulfovibrio and Helicobacter.However,the relative abundance of dominant bacteria was significantly improved including Bacteroides and Lactobacillus.ConclusionPA has excellent anti-IBD activity and is a potential lead compound for the treatment of IBD.Its possible mechanism may inhibit relavant rate-limiting enzymes and slow down the rate of tryptophan metabolism to alleviate IBD.Meanwhile,PA regulated the composition and structure of gut microbiota to protect the gut microflora and its stability.