Sennoside A Upsets The Balance Of Intestinal Microbial Composition And Damages Colonic Mucosal Barrier

Research background and purposeRhubarb,Chinese formula which contain rhubarb and rhubarb health products have been widely used worldwide,but their safety has long been controversial.Anthraquinones as the main medicinal substance in rhubarb,academic circles have paid much attention to their tumorgenic potential.Few studies have shown that anthraquinones cause damage to the intestinal epithelial tissues.Prospective data also demonstrated the long-term use of anthraquinone laxative is associated with colon cancer development.However,the existing fragmentation information cannot positively answer whether anthraquinones in rhubarb promote cancer.This study confronts this scientific confusion,with Sennoside A,the main component of rhubarb which cause diarrhea,as the main research object.By evaluating the direct effects of Sennoside A,we want to explore whether it is directly pro-inflammatory and carcinogenic?With the help of classical chemical-induced two-stage colon cancer model(AOM/DSS),we further assessed whether Sennoside A could promote the development of colon cancer.With the development of intestinal microbiological research methods,the correlation between gut microbiota and the development of colitis or colon cancer has been confirmed.Among them,the destruction of the integrity of the intestinal mucosal barrier is the key to gut microbiota affecting the development of colitis and colon cancer.Gut microbiota plays an important role in the purgative effect of Sennoside A,it needs to be hydrolyzed by beta-glucosidase secreted by gut microbiota,suggesting Sennoside A inevitably interacts with the microbiota in the intestine.So whether Sennoside A correlate with microbiota?This study takes the integrity of the intestinal mucosal barrier as an entry point and focuses on the role of gut microbiota.It aims to clarify whether Sennoside A destroys the integrity of the colonic epithelial barrier and promotes the development of colitis and colon cancer.This study could provide a basis for determining the potential toxic substances that may have the pro-inflammatory risk in rhubarb.Research contentsThis study evaluated the effect of Sennoside A on the two physical intestinal barrier,mechanical barrier and mucus barrier for the first time.The third part focused on the detection and analysis of the influence of Sennoside A on the structure of gut microbiota and found the biomarker of Sennoside A long-term administration.The forth part focused on the direct inhibiting effects of Sennoside A on some strain of bacteria and subsequently caused the change of colon pathology.Based on the above,the damage process of intestinal mucosal barrier caused by Sennoside A was further confirmed by the fecal microbiota transplantation experiment,and the disorder in microbiota played an important role.In section sixth and seventh,we further evaluated the effects of Sennoside A on low-grade inflammation and metabolic disorders in the colon and promoted colon cancer in the AOM/DSS model.ResultsWe first examined the integrity of colon mechanical barrier.We found that long-term(84 days)administration of Sennoside A caused changes in structure and morphology of colonic crypts in a dose-dependent manner,like reduced numbers of crypt cells and reduced mucus secreted by goblet cells,causing the colonic crypt to shrink and the invasion of lymphocytes in crypt cavity.At the same time,it reduced the expression and distribution of tight junction protein(ZO-1,Claudin-1)and adhesion protein(E-cadherin)in colonic epithelial tissue.In vitro experiments confirmed that Sennoside A did not directly directly affect the colon.Alcian blue staining showed that Sennoside A dose-dependently inhibited the mucus secreted by goblet cells and destroyed the coverage of the intestinal epithelial layer by the inner mucus layer.Immunofluorescence observed the reduction of most important mucin,MUC2,which constitutes the inner mucous layer of the colon.In view of the damage to the colonic physical barrier by Sennoside A,the barrier function of the colonic mucosa was further evaluated.Sennoside A increased the permeability of the colon barrier and promoted bacteria translocation from lumen to mucosa.In the third part we utilized 16s rRNA gene sequencing analysis,and found Sennoside A has no significant change in the Shannon index of gut microbiota.This indicates that Sennoside A ddi not affect the overall component of gut microbiota.However,it significantly affect the beta-diversity of gut microbiota.Sennoside A caused the expansion of Verrucomicrobia,and decreased abundance of Firmicutes.Sennoside A caused the expansion of A.muciniphila and it become the biomarker of Sennoside A treatment.We further used absolute quantitative PCR methods and confirmed the increased A.muciniphila level in Sennoside A group compared to control group.However,in the early time of Sennoside A treatment we did not observe the significant change in A.muciniphila until 56 days later.In vitro experiments showed that Sennoside A,Sennoside A and bacteria mixture and its metabolite rhein did not affect the growth of A.muciniphila,indicating that Sennoside did not directly contribute to the growth advantage of A.muciniphila.In the fourth part we further found that Sennoside A inhibited the abundance of SCFA producing bacteria(Coprcoccus,Prevotella,Oscillospira,Ruminococcus).In the mean time,we observed the decrease of Clostridiales.Sennoside A could directly inhibited the growth of Clostridium tyrobutyrate and Clostridium butyricum by its metabolite rhein.We then assessed the level of short chain fatty acids by GC-MS.Acetate and propionate remain unchanged while the level of butyrate significantly decreased.Luminex experiments showed that Sennoside A can induce elevated levels of various pro-inflammatory factors such as IL-17,IFN-y,and MIP-2.It also promotes the increase in the number of Thl and Th17 cells,and lead to an increased proliferation rate of colonic crypt epithelial cells.The above changes could be reversed by butyric acid supplement.In fifth part we used fecal microbiota transplantation to confirm the key role of gut microbiota in the intestinal mucosal barrier integrity of Sennoside A.Based on the above confirmation that Sennoside A can cause damage to the intestinal mucosal barrier integrity,we further evaluated whether the damage caused by the mucosal barrier produces a pro-inflammatory and cancer-promoting effects.In sixth part we found that 100mg/kg Sennoside A long-term treatment could increase LCN2 level.LCN2 is a marker of low grade inflammation.At the same time,Sennoside A increased the level of pro-inflammatory genes such as Tnf,Il1b and 116,and decreased the level of anti-inflammatory genes such as 1110,suggesting the activation of pro-inflammatory signaling pathways.Metabolomic studies showed that Sennoside A can cause abnormalities in the body's metabolism,including purine metabolism,pyrimidine metabolism,and various amino acid metabolism.Current research suggests that long-term low grade inflammation could promote the development of colon cancer.In seventh part we found Sennoside A could promote the development of colon cancer,which showed earlier adenoma appearance than the AOM/DSS group,accompanied by stronger activation of the STAT3 signaling pathway and the P-catenin signaling pathway.Para-carcinoma tissue showed increased proliferation rate and suggesting the possible malignant transformation.Conclusion and significanceThis study confirmed that Sennoside A,the representative substance of anthraquinones in rhubarb,could destroy the integrity of intestinal barrier and cause long-term low grade inflammation thus promote the development of colon carcinoma.Sennoside A promoted the predominant growth of A.muciniphila and inhibited the growth of SCFA-producing bacteria,demonstrated that the damage of Sennoside A is strongly correlated with the imbalance between mucus degrading bacteria and fiber degrading bacteria.It can be confirmed that Sennoside A is the substance basis of anthraquinones in rhubarb,and this study provides a solid experimental basis to avoid the potential tumorgenic risk of anthraquinones.