The Study Of Tannins And Its Metabolite Gallic Acid In The Progression Of Inflammatory Bowel Disease

BackgroundInflammatory bowel disease(IBD)is a chronic inflammatory intestinal disorder,including Crohn’s disease and ulcerative colitis.It is generally agreed that this condition is a multifactorial disease initiated by the accumulation of genetic,environmental,and immunological factors.Currently the drugs for alleviating IBD symptoms and preventing recurrence mainly include glucocorticoids,immunosuppressants and antibiotics.However,these drugs have serious side effects and poor tolerance,so it is an urgent task to find new potential anti-IBD drugs that can replace traditional drugs.Tannin(TA),a phenolic compound derived from plants,has many biological activities beneficial to human health,such as antioxidant activity,antibacterial activity,anticancer activity and immunomodulatory activity.In recent years,it has attracted much attention due to its potential to regulate major inflammatory pathways.ObjectiveDietary Tannin possesses diverse biological functions,yet,their role in inflammatory bowel disease is still not clear.Our study aimed to evaluate the effect of TA in the progression of IBD.Materials and methods1.DSS-induced colonic colitis and treatmentColonic colitis was induced in 6-week-old male C57BL/6 mice with 2.5%DSS in drinking water.Mice were orally treated with different doses of TA,GA or PBS daily.Stools of mice were collected for further analysis.This experiment protocol was approved by the institutional animal care and use committee(IACUC)of Army Medical University.All procedures adhered to the guidelines approved by the IACUC of the Army Medical University.2.Fecal microbiota transplantation(FMT)experimentTo analyze the effect of gut microbiota in feces from mice treated with TA,6-week-old germ free(GF)mice were gavaged with feces(in sterile PBS)collected from TA-treated mice twice(in a week).After that,the recipient GF mice were kept in GF isolator package for another week.Feces from GF mice were collected to examine bacterial colonization.The GF mice with bacterial colonization were then administrated with 2.5%DSS.This study was approved by the IACUC of Army Medical University.3.Clinical score and histological analysisBody weight was recorded and the changes of body weight were indicated as loss of baseline body weight as a percentage.Colon length was measured when mice were sacrificed.Colon tissues was fixed with 4%paraformaldehyde(PFA)and embedded in paraffin.Sections of colon tissue were stained with H&E according to standard protocol.Histological scoring was assessed in a blind way by a pathologist.The scoring standard was referred as previously described.4.Transmission electron microscope analysisColon tissues(about 1mm~3)of TA-treated mice were collected and fixed in 2.0%glutaraldehyde in 0.1mol/L sodium cacodylate(Electron Microscopy Sciences,Hatfield,PA).Reichert Ultracut E ultramicrotome was used to prepare the ultrathin section which was then examined using a Philips CM100 transmission electron microscope.5.Measurement of tannase activityTannase activity in feces collected from TA-,GA-treated or control mice were tested using the tannase activity assay kit(BC4075,Solarbio),according to manufacturer’s instruction.6.Statistical analysisAll statistical tests were performed using Graph Pad Software(Version 9.1).Data are presented as mean±SD.Comparison between two groups was performed using Student’s t-test.Kaplan-Meier analysis was applied to analyze the survival of GF mice.P values<0.05 were considered statistically significant.Results1.In vivo and in vitro studies showed that TA has dual effects on DSS-induced IBD.2.Metabolomic and metagenomic analysis revealed that TA-induced enrichment of microbial metabolite gallic acid(GA),instead of microbial alteration,was responsible for TA to exert its function.Further investigation demonstrated that TA-induced activation of microbial tannase leading to the enrichment of GA in feces and colonic tissues.3.In vitro and in vivo studies showed that GA is the main down-stream effector molecule for TA to exert its function in the progression of IBD,and it reproduced the dual effects of TA.4.Mechanistically,protective dose of GA promoted secretion of mucus and inhibited penetration of intestinal bacteria into colonic epithelium,thereby reduced the expressions of inflammatory cytokines.Additionally,protective dose of GA ameliorated DSS-induced epithelial injury through inhibiting p53 signaling.In contrast,toxic dose of GA resulted in epithelial injury through promoting cell cycle arrest.5.Therapeutic experiment demonstrated that protective dose of GA promoted recovery of DSS-induced colonic inflammation.Conclusion1.TA has a dose-dependent dual effects in the pathogenesis of IBD,with a protective dose of 50 mg/kg and a pathogenic dose of 250 mg/kg in DSS-induced IBD model.2.GA,the main metabolite of TA in the feces,is the key effector molecule for TA to exert its function,fully reproduced the dual effects of TA in the pathogenesis of IBD;The protective dose of GA in the DSS-induced mouse IBD model was 50 mg/kg,and the toxic dose was 250 mg/kg.3.A protective dose of GA can promote the recovery of IBD induced by DSS.The main mechanism of its protective effect is to promote the secretion of colonic mucin,reduce the expressions of inflammatory factors,and inhibit the apoptosis of colonic epithelial cells.4.The present study demonstrated that the pathogenic or probiotic effects of tannase-containing bacteria were determined by the amount of GA transformed from TA,highlighting the interaction between microbial enzyme and diet in affecting IBD.