| BackgroundInflarmmatory bowel disease(IBD),comprised of Crohn’s disease(CD),ulcerative colitis(UC)and unclassified IBD(IBDU),is a chronic relapsing disease of the gastrointestinal tract,with increasing morbidity.As the lifestyle of developing countries becomes "westernization",the incidence of IBD has been increasing gradually over the last decades.China’s epidemiological studies also show that the incidence of IBD has risen year by year.The pathogenesis of IBD is unclear that not only relate to the genetic gene and abnormal immune system,in which environmental factors also play a very important role.Gut microbiota,as one of the most important environment factors,is getting more and more attention.Gut microbiota,the biggest microbial symbiosis in our community,coexists with its host in variable concentrations throughout the gastrointestinal tract.It contains about 1011-1012 cells per gram of intestinal contents in the colon,being referred to as the "super organ".This"super organs" performs a series of powerful functions for the host,including digesting substrates inaccessible to host enzymes,educating the immune system,and repressing the growth of harmful microorganisms.Throughout the past ten years,there has been much progress in understanding the structure,functions,and interactions with the host of gut microbiota,since it has led to a surge of research.Depends on the culture-independent techniques,including metagenomics and functional"omic" methods,such as meta-transcriptomics,metabolomics,and proteomics,the composition and functions of the gut microbiota in IBD,as well as the interactions gut microbiota and its host,have been gradually uncovered.99.1%of the genes detected by metagenomics are derived from bacteria,most of the remaining from the archaea,and only 0.1%from eukaryotes and viruses.More than 1000 species of bacteria coexist with the involving in the host’s nutrition metabolism,essential nutrients absorption,protection pathogens,the activation of immune responses and development of immune system.It been show that once the balance between the host and gut microbiota was broken,potential pathogens,such as gene mutation,abnormal immune system and intestinal barrier could lead to IBD.Genome-wide association study(GWAS),other genetic analyses and epigenetic researches have revealed links between changes in the gut microbiota in IBD and an aberrant immune response,all these alterations being influenced by environment,including diet.Recent studies show that,both diversity and stability of mucosa-associated and fecal microbiota declined overall in patients with CD and UC vs HCs.Since fecal bacteria are combined with shed mucosal bacteria and a separate nonadherent luminal population that are different from mucosa-associated flora,with the latter being more important.Intestinal flora is different in active stage and symptomatic relief stage,inflammatory site and normal site.Diet is one of the most important env:ironmental factors affecting gut microbiota,which has been shown a decisive role of microbial composition in mammal evolution.Although no specific food or dietary pattern has been found as directly cause,effective prevention or treatment for IBD,it is important to take interactions between diet and microbiota into account when studying the role of the microbiome in disease.So far,there are little direct evidences have been found on how diet affects host gut microbiota,thus large-scale controlled diet study would need to gather more information.Studies have shown that long-term dietary pattern affect the proportion of bacteroides,Prevotella,and Firmicutes,however,short-term dietary pattern may not play a key role in microbiota composition.In addition,Bacteroides,Bifidobacterium,and the Enterobacteriaceae have been reported markedly reduced in a strict vegan or vegetarian diet,whereas the total bacteria load remained unaltered.Given the complexity of dietary effects,it is very valuable to focus on the influence of long-term and short-term dietary patterns in patients with IBD in the future.Objectives(1)Creating an IBD patients information database.(2)To find out if dietary components and nutrients are different between control group and IBD group.In addition focus on association of IBD and diet.(3)Gathering gut microbiota composition information of IBD patients and the control group by high-throughput sequencing,analyzing the distribution and diversity of mucosa-associated microbiota and fecal microbiota,and comparing gut microbiota in different disease type,different disease stages,different parts,inflammatory site and normal site of IBD patients.(4)To understand the effects of dietary factors on gut microbiota in IBD patients by correlation analysis.Materials and MethodsParticipants:A total of 280 IBD patients(173 with Crohn’s disease and 107 with ulcerative colitis)and a control population composed of 42 healthy individuals were recruited between March 15,2014 and May 31,2015 in the IBD Unit of Gastroenterology Department and Physical Examination Center,Southern medical university NanFang hospital.A total of 113 participants attend the sequencing of intestinal flora.Informed consent was obtained from all subjects in accordance with the local research ethics committee guidelines.The diagnosis of IBD was based on clinical,endoscopic,radiographic and pathological criteria.Methods:(1)Design the questionnaire to collect IBD patients’ demographic data and clinical medical records.(2)The 2010 Chinese residents of nutrition and health status monitoring semi-quantitative food questionnaires(CNHS2010-J1)was used for diet investigation and completed in a one-to-one manner.Participants were investigated how often and how much at a time they had consumed food or beverage items on an average during the past year.The mini nutritional assessment(MNA)was used as a screening and assessment tool to identify subjects at risk of malnutrition.(3)Database of IBD patients was established to analysis the clinical characteristics and dietary pattem.(4)Fresh feces were stored at-80 C in less than 30 minutes before DNA extraction to prevent DNA degradation and anaerobic bacteria from being exposed to oxygen.Biopsies were taken from inflammatory and non-inflammatory location of patients as well as from the terminal ileum and rectum of healthy individuals using standard endoscopic forceps and placed into a sterile cryovial on ice in the Endoscopy Suite and stored at-80℃ before DNA extraction.(5)Nucleotides were isolated from these feces and biopsies using the QIAamp DNA Mini Kit(Qiagen).(6)Once the DNA samples were received,a quality test has been done first,and all qualified DNA were used to construct a library.For PCR product,the jagged ends of DNA fragment would be converted into blunt ends by using T4 DNA polymerase,Klenow Fragment and T4 Polynucleotide Kinase.Then add an ’A’ base to each 3’end to make it easier to add adapters.After all that,fragments too short would be removed by Ampure beads.For genomics DNA,we use fusion primer with dual index and adapters for PCR,fragments too short would be removed by Ampure beads too.In both cases,only the qualified library can be used for sequencing.The bioinformatics analysis will be carried on with sequencing data.ResultAge at diagnosis of CD patients was younger than UC patients(t=6.965,P=0.000).percentage of CD patients whose BMI<18 was higher than UC patients.29.9%of UC and 41.0%of CD patients belongs to technological academy and above.29.9%of UC pati and 43.4%of CD patients were rolling stone.Average monthly income of 57%UC pati and 62.4%CD patients was in 3000 yuan or less.More UC patients suffered diarrhea(χ2=23.039,P=0.000),hematochezia(χ2=81.889,P=0.000),mucoid stool(χ2=19.682,P=0.000).,and tenesmus(χ2=32.530,P=0.000).As well as more CD patients suffered from abdominal pain(χ2=10.529,P=0.001),abdominal mass(P=0.026),and fecal incontinence(P=0.001).There was no difference in the overall proportion of patients with systemic manifestations between the two groups,whereas the incidence of fever(χ2=5.620,P=0.018),night sweats(χ2=8.873,P=0.003),anemia(χ2=8.131,P=0.004)was significantly higher in CD patients.The most common symptom of extra-intestinal manifestation was oral ulcer.The incidence of extra-intestinal manifestation(χ2=11.480,P=0.001)and complication(χ2=33.826,P=0.000)were significantly higher in CD patients than UC patients.The incidence of complication(χ2=33.826,P=0.000)were significantly higher in CD patients than UC patients,including perianal disease(χ2=13.257,P=0.000),fistula(P=0.026),intestinal obstruction(χ2=9.115,P=0.003)and intestinal stenosis(P=0.033).Laboratory examination results showed that there were significantly differences of hemoglobin,glutamate pyruvate transaminase,serum glutamic-oxaloacetic transaminase,globulin,total bilirubin,indirect bilirubin,blood urea nitrogen and serum iron between two groups.All indicators except globulin were lower in patients with UC than patients with CD.There were more CD patients suffered from anemia(χ2=-89.629,P=0.000),with 17 patients in UC group(15.9%)and 51 patients in CD group(29.5%).Fe of CD group was obviously lower than reference value(7.52-11.82)(t = 4.853,P=4.853),however,P was obviously higher than reference value.Ca and Mg of both UC and CD group was obviously lower than reference value.There was no difference UC and CD set of No obvious difference was found in blood vitamin B12,folic acid,and vitamin D between UC and CD group.The average amount of vitamin B12 and folic acid were within the reference range except vitamin D.Diet investigation showed that daily intake of cereal,tuber crops and mixed bean were within dietary reference intake.Daily intake of poultry&meat in IBD patients and control group as well as fat intake in control group were beyond the reference intake.Instead,daily intake of vegetables、fruits、milk and dairy products、beans and nuts in all groups,fish and shrimpin IBD patients were lower than the reference intake in different degree.Daily intake of vegetables,fish and shrimp,milk and dairy products were significantly difference in three groups.Nutrient daily intake show that energy,fat,carbohydrate were in the reference intake range whereas dietary fiber was below reference intake.Daily intake of vitamin A,vitamin B1,vitamin B2,vitamin B6,folic acid,vitamin C,vitamin E,and vitamin D were below the reference intake with the exception of nicotinic acid.Daily intake of folic acid(χ2=12.923,P=0.002),niacin(χ2=7.676,P=0.022),vitamin C(χ2=1.203,P=0.004),and vitamin D(χ2=9.053,P=0.011)were significantly different in the three groups.The intake of folic acid and vitamin C was lowest in CD group.The intake of niacin acid and vitamin C was lowest in UC group and vitamin D intake was lowest in the control group.Daily intake of Calcium,potassium,sodium,magnesium and selenium were lower than the reference intake,while the intake of iron,copper,manganese were higher than reference intake.The intake of zinc and iodine were near to reference intake.Significant differences were observed in calcium(χ2=6.661,P=0.036)and selenium intake(χ2=12.273,P-0.002)within three groups,Both the lowest intake of calcium and selenium group was the UC group.42.1%of UC patients and 49.7%of CD patients believed that certain foods were IBD etiology or contributing factors,such foods include:seafood,spicy or fried food,wine,milk,beef,lamb,frozen food,beans and bean products,fruit and vegetable.A source of dietary advice mainly includes:the relatives and friends,medical personnel,network,books,and one’s own experience.The correlation analysis between IBD and dietary factors found vegetables,poultry&meat,fish and shrimp were negatively related to IBD while milk dairy products were positively correlation.Nutrients contains protein,folic acid,vitamin C,calcium,potassium,magnesium,zinc,selenium and manganese were negatively related to IBD while vitamin D was positive correlation.Vegetables were seems like a protection factor for CD activity(P= 0.05).High-throughput sequencing analysis showed that the dominant sequences belonged to two phyla that included Firmicutes and Bacteroidetes.The amount of Proteobacteria,Firmicutes and Bacteroidetes were given equal priority to mucosa-associated microbiota in the control group and UC group.Proteobacteria was the dominant bacteria in CD followed by Bacteroidetes and Firmicutes.Bacteroides was the core bacteria both in mucosa-associated and fecal microbiota on genus level.There was significant difference in adiversity of fecal microbiota among three groups(P<0.05).No significant difference was found in a diversity of microbiota.Comparing subgroups of UC group,we found that adiversity was richer in active stage than in remission stage and richer in non-inflammatory site than in inflammatory site of active stage(P<0.05).No significant difference was found in a diversity among different CD subgroups.After comparing different groups of fecal flora we found that:(1)UC patients in active stage demonstrated a marked decrease in the relative abundance of Bacteroidetes.(2)Significantly differences were observed in the relative abundance of Tenericutes,Verrucomicrobia,Firmicutes,Fusobacteria and Thermi among the control group,the active UC group and the active CD group.(3)There were significant differences in the relative abundance of Thermi,Firmicutes,Planctomycetes and Tenericutes among the control group,the remittent UC group and the remittent CD group.The relative abundance of Tenericutes and Firmicutes were highly enriched in the control group compared with CD group and UC group,while Planctomycetes was highly enriched in UC group.After comparing different groups of mucosa-associated microbiota we found that:(1)CD patients in active stage demonstrated highest abundance of Verrucomicrobia compared with control group and CD patients in remission stage,whereas demonstrated lowest abundance of Firmicutes.(2)Significantly differences were observed in the relative abundance of Synergistetes and Nitrospirae among the control group,the active UC group and the remittent UC group.The abundance of Synergistetes decreased markedly in the remittent UC group and Nitrospirae decreased in the active UC groupAfter comparing different subgroups of mucosa-associated microbiota we found that Synergistetes,Chloroflexi,Tenericutes,Fusobacteria were significantly higher in inflammatory site of CD active group compared with UC active group.Thermi showed lower abundance in the remittent CD group than in the_remittent UC group.Verrucomicrobia was highly enriched in ileac inflammatory site compared with colonic inflammatory site of remittent CD group,whereas demonstrated lowest abundance of Firmicutes.The correlation analysis between fecal microbiota and dietary factors found that the abundance of Actinobacteria was negatively related to vegetables,beans and nuts.Proteobacteria could be related to milk and dairy products(P=0.05)while Cyanobacteria was negatively related to beans and nuts.Relationship between nutrients and fecal microbiota showed that Cyanobacteria was negatively related to sodium,and positively related to manganese.There was negative correlation between selenium and Bacteroidetes and positive correlation between selenium and Proteobacteria.In addition,Fusobacterium was negatively related to vitamin D.Foods correlated to mucosa-associated microbiota included cereal,tuber crops,mixed bean,fruits,poultry&meat,eggs,milk and dairy products.Nutrients correlated to mucosa-associated microbiota included energy,fat,carbohydrates,dietary fiber,cholesterol,thiamine,folic acid,niacin,vitamin C,vitamin E,vitamin D,calcium,magnesium,iron and zinc.Mucosa-associated microbiota correlated to dietary factors included Chloroflexi,Verrucomicrobia,Actinobacteria,Cyanobacteria,Plancto,mycetes,Thermi,Bacteroidetes Chlamydiae,Proteobacteria,Gemmatonadetes,Synergistetes,Firmicutes and FusobacteriaConclusionThrough the investigation of IBD patients’ demographic data,clinical data,dietary components and gut microbiota,we learned about the clinical features of patients with IBD,the abundance of diet and nutrient intake.Then we analyzed the relationship among diet,the gut microbiota and IBD that could provide advice on diet and nutrition treatment for IBD patients.Furthermore,the role of gut microbiota in IBD pathogenesis could be understood better. |
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