The Research Of Intestinal Micro-ecology Interaction And Isoliquiritigenin Anti-colitis Associated Colorectal Cancer

Background It is well known that the imbalance of intestinal bacteria is closely related to colitis-associated colorectal cancer.It has been reported that traditional Chinese medicines have the ability to adjust gut microbiota.Isoliquiritigenin(ISL),a flavonoid extracted from liquorice,has shown efficacy in the inhibition of cancer growth.However,the effect of ISL on colitis associated colorectal cancer(CAC)and gut microbiota is unknown.Objective(1)To choose one method which can extract high quality DNA from stool and the DNA should tend to reflect the real microbial diversity of stool by compareing four DNA extracted methods.(2)To make sure the scientific and objective of stool samples instead of intestinal contends to analysis the microbial community structure which can help to guide the following research of intestinal microbe associated intestinal disease.(3)CAC mice model were constructed by AOM/DSS.Correspondence analysis based on Terminal restriction fragment length polymorphism(T-RFLP),fluorogenic quantitative PCR,Bacterial 16 S rRNA gene(V3-V5 region)high-throughput sequencing were used to demonstrate the dynamic of microbial change by comparing control treatment and model treatment;we also use different does of ISL to intervene AOM/DSS model,evaluate the CAC inhibition function of ISL and the dynamic of microbial change in the cause of ISL inhibiting CAC development.Methods(1)In the first part,the total microbial DNA were extracted by GITC method,CTAB method,Tiangen and Omega fecal DNA extraction kits;then the yield and purity of the extracted DNA were determined by spectrophotometer of Nano Drop 2000;the microbial community structure diversity index reflected by the DNA were analyzed by PCR-DGGE.(2)In the second part,terminal restriction fragment length polymorphism(T-RFLP)and Fluorescent quantitative polymerase chain reaction(qPCR)were used to compare the difference of bacterial community structure and richness between fecal and the contend in each part of intestinal.(3)In the third part1)six treatments on SPF BALB/c male mice including healthy control mice,CK;isoliquiritigenin control mice,ISL+CK;AOM/DSS induced colitis associated colorectal cancer model,CACM;30,75,150mg/kg isoliquiritigenin intervened CAC treatment(CIL,CIM and CIH)were established to evaluate the anti-cancer effect of isoliquiritigenin and to analyze the dynamic changes of gut microbiota under AOM/DSS and isoliquiritigenin stress.2)Fluorescent quantitative polymerase chain reaction(qPCR)was used to analyze the change of cytokines of intestinal epithelial cell in each treatment,such as IL-6,IL-10,TNF-?,IL-1?,IKK? and COX-2 a kind of proinflammatory factor;HE stain was used to estimate the development of inflammation and colorectal cancer in intestinal of mice in each treatment.3)Terminal restriction fragment length polymorphism(T-RFLP)and high-throught pyrosequencing were use to analyze the microbe change of each mice.4)Combine the results of histopathological analysis and the microbe change to evaluate to function of isoliquiritigenin in the process of initial and development of inflammation associated colorectal cancer.Results(1)The DNA extracted by commercial kits has the highest purity but the lowest concentration when compared with others;on the contrary,the DNA extracted by CTAB method has the highest concentration but the lowest purity;while the DNA extracted by GITC method has the higher purity than CTAB method and higher concentration than commercial kits.(2)The analysis of PCR-DGGE based on bacterial 16 S rRNA suggest that the diversity and richness reflected by DNA extracted by GITC method significant higher than other three methods. (3)The advantage T-RFLP fragments(T-RFs)in feces,rectum and colon were 244 bp,255 bp and 449 bp,however,60 bp,73 bp,261 bp,268 bp and 272 bp T-RFs were enriched in small intestine,including duodenum,jejunum and ileum.The bacterial community composition in various parts of the small intestine was different from each other obviously.(4)To be 6.9 log(copies)/g and 8.3 log(copies)/g,bacterial abundance in the contents of duodenum and jejunum were fewer than others,while the bacterial abundance in the feces,as high as 11.8 log(copies)/g,was about 2 times the duodenum bacteria abundance,which was significantly higher than the bacterial abundance in the duodenum and jejunum(P< 0.05).Bacteria abundance of feces parallel with the large intestine and ileum contents was simila,significant difference has not been found(P>0.05).(5)Histopathological analysis suggested that ISL suppressed inflammatory cytokines and reduced tumor incidence in AOM/DSS model mice.(6)Correspondence analysis based on Terminal restriction fragment length polymorphism(T-RFLP)indicated that gut microbiota were significantly disrupted by AOM/DSS-induced CAC,and this imbalance was alleviated by ISL treatment.(7)Bacterial 16 S rRNA gene(V3-V5 region)high-throughput sequencing revealed that the structure of the gut microbial community shifted significantly in CACM and high dosage ISL treatments.Compared to the control group,Bacteroidetes were decreased and Firmicutes were increased in the process of CAC development.However,ISL reversed the imbalance on phylum level and altered the familial constituents of the gut microbiota.It was found that Helicobacteraceae were increased and Lachnospiraceae and Rikenellaceae were decreased in the high dosage ISL treatment group.At the genus level,ISL reduced the abundance of opportunistic pathogens(Escherichia,Enterococcus),and increased probiotics,especially butyrate-producing bacteria(Butyricicoccus,Clostridium and Ruminococcus).Conclusion(1)The DNA extracted by commercial kits has the highest purity,but has the lowest concentration and high cost,so commercial kits were suited for high through-put sequencing;the DNA extracted by GITC method can more fully response the intestinal microbial diversity and community structure,and the cost is low,so GITC method is suitable for laboratory large-scale research on analysis of the test sample.(2)The inter-mouse variation of bacterial community composition in large intestinal contents and feces were much smaller than those in small intestinal.The bacterial structure and abundances of large intestinal contents were similar with feces.Studies on the relationship between large intestinal especially colorectal microbiota and diseases could be suitable via the fecal sampling.(3)In conclusion,our study confirmed the anti-CAC effects of ISL.Gut microbiota changed when ISL relieved the AOM/DSS induced colitis associated colorectal cancer development.The altered microbiota induced by AOM/DSS were recovered by ISL to a certain extent.The increased probiotics(Prevotella,Butyricicoccus,Clostridium and Ruminococcus)and decreased opportunistic pathogens(Escherichia and Enterococcus)may cooperate with ISL to inhibit colitis associated colorectal cancer development.This study provides new evidence that traditional Chinese medicine prevents cancer development and regulates gut microbiota.Further research should focus on the molecular mechanism of the anti-cancer effects of ISL and effects on microbiota.