| Objective:Since the reform and opening up,with the continuous improvement of people’s living standards,Chinese people have gradually adopted a high-fat diet as the main daily dietary pattern.The incidence and mortality of obesity,type 2 diabetes,cardiovascular and cerebrovascular diseases,inflammatory bowel disease,colorectal cancer and other dietary related diseases increased year by year.More and more studies have found that the changes of gut microbiota structure and function play an important role in the occurrence and development of these diseases,and characteristic changes of core microbiota have been observed in some diseases.Based on the demographic,dietary and health characteristics of Chinese populations,our study aims to explore the characteristic changes of gut microbiota in healthy people with high fat diet before serious diseases occurred in the community,and try to improve the intestinal flora structure by adding probiotics and dietary intervention to achieve the purpose of primary prevention.Methods:(1)A questionnaire including dietary pattern,demography and health state from every volunteer was completed and then volunteers were grouped.A total of 214 healthy adults were recruited in two community of Zhouzhuang Town,Jiangyin City,Jiangsu Province.The type,quantity and frequency of food consumed per person per day for the past year were recorded in a self-administered food frequency questionnaire(FFQ)to assess the dietary habits,and the amount of fat,carbohydrate,proteins and dietary fiber in various food products consumed daily were analyzed using the criteria of China Food Composition(Book 1.2nd Edition)and the energy yield of the first three nutrients was derived.Based on the dietary fat content determined using the FFQ,the volunteers were divided into high-fat diet(≥40%of dietary calories came from fat)(HFD)group or low-fat diet(<40%)(LFD)group.Other nine aspects of questionnaire including demographic and health state were investigated and compared.(2)Stool and colonic mucosa were collected.Fresh solid feces from volunteers for the first time were collected in the morning at the same day when the questionnaire was conducted,and stored immediately in-80 ℃ low temperature refrigerator.After intestinal preparation colonic mucosa samples at the ascending colon distant to ileocecal flap by 5 cm were collected using biopsy forceps under colonoscopy.The samples were also preserved at low temperature until the microbiota was detected.(3)Intestinal microbiota structural changes were detected.Genomic DNA from stool sample and colonic mucosa sample were extracted utilizing MicroElute Genomic DNA specimens kit and were examed by 1%agarose gel electrophoresis,then the genomic DNA content for each samples were identified.Extracted genomic DNA was amplified by PCR.16S rDNA V3+V4 variable region were pyrosequenced using Illumina sequencer for PCR amplification products.After that,the sequences were aligned using RDP,Greengenes and NCBI databases,respectively.Various analysis of Alpha diversity index,PCoA,LEfSe was adopted in order to compare the diversity and taxonomy of the intestinal microbiota,and the correlation analysis with the questionnaire data was also carried out.One purpose of this study was to find the difference of fecal and colonic mucosa-associated microflora structure between the two diet patterns and its related factors in high-fat diet population.(4)The effect of probiotics and dietary intervention(DI)was explored on intestinal microbiota structure of high-fat diet population.Volunteers with similar dietary patterns and intestinal flora diversity were recruited from the above HFD group to participate in the intervention study.These subjects were randomly assigned to four groups:HFD group(n=9)-Normal high-fat diet as a control group;DI group(n=9)-HFD replaced by low-fat diet(LFD)in which dietary fat accounted for below 40%of total energy;HFD+Probiotic group(n=9)-high-fat diet and oral administration with daily 2 gram of triple live probiotics including Bifidobacterium longum,Lactobacillus acidophilus and Enterococcus faecalis,the total dose of which is greater than 2.4x109CFU until the end of the intervention;DI+Probiotic group(n=9)-Combination of low-fat diet similar to DI group and oral administration with triple live probiotics the same to HFD+Probiotic group.The intervention lasted for 4 months.After the intervention,the collection of 36 feces and 36 colonic mucosa tissues,microflora detection and bioinformatics analysis were performed the same as before.This section was to compare the differences of fecal and colonic mucosa-associated microbiota among the four groups,and to explore the feasibility of probiotics and dietary intervention to improve intestinal flora in high-fat diet population.Results:(1)The epidemiological data showed that fat energy supply was the main source of energy in HFD group and carbohydrate energy supply was dominant in LFD group.There were significant differences between the two groups(P<0.05)in the daily consumption of rice,buckwheat,leafy vegetables,root vegetables,stem vegetables,pods,nuts,oil,salt,salted meat,etc.The LFD group consumed more vegetables and the proportion of dietary fiber was higher,while daily consumption of vegetable oil was significantly higher in the HFD group and it was positively correlated with the daily dietary fat energy supply proportion through correlation analysis(p=0.511,P<0.001).There was no significant difference in most demographic and health survey data between the two groups.In the HFD group,the main type of work was one with low intensity.The low density lipoprotein level in HFD group was higher than that in LFD group,but the serum creatinine level was lower than that in LFD group.The proportion of patients with high total cholesterol,high triglyceride,and abnormal findings by enteroscopy in HFD group was significantly higher than that in LFD group.(2)The results of 16s rDNA high-throughput sequencing showed that the diversity of fecal microbiota was lower than that of colonic mucosa-associated microbiota.Stratified clustering and LEfSe discriminant analysis also showed that the fecal microbiota structure was different from that of colonic mucosa-associated flora.The highest abundance of fecal bacteria was Bacteroidetes while the highest phylum of colonic mucosa-associated flora was Firmicutes regardless of dietary pattern.The fecal and colonic mucosa-associated microbiota of the two groups were compared,respectively.There was no significant difference between the two groups in the structure of the microflora at the phylum level.The two groups mainly contained the following phyla:Firmicutes,Bacteroidetes,Proteobacteria,Actinobacteria and Fusobacteria.A few of orders and families with very low abundance were different between the two groups,for example,Xanthomonadales(o),Burkholderiaceae(f)and Aerococcaceae(f).Significant difference in microbial community structure was mainly manifested at the genus level.The relative abundance of Prevotella(19.05%vs.4.97%,P=0.035)and Abiotrophia(0.0013%vs.0.0000%,P=0.038)in fecal samples,and S24-7_g_unclassified(0.1%vs.0.0%,P=0.025)in colonic mucosa samples in HFD group was higher than that in LFD group,while The relative abundance of S24-7_g_unclassified(0.19%vs.0.29%,P=0.046),Gemmiger(0.11%vs.0.52%,P=0.018),Akkermansia(0.03%vs.0.19%,P=0.035),Rothia(0.000648%vs.0.000163%,P=0.038)in feces,and Bacteroides(12.96%vs.29.03%,P=O.022),Coprobacter(0%vs.0.05%,P=0.049),Abiotrophia(0.000248%vs.0.020000%,P=0.036),Asteroleplasma(0.00%vs.0.72%,P=0.049)in colonic mucosa samples in HFD group was significantly lower than that in LFD group.The correlation analysis between bacterial abundance and questionnaire data showed that there were a positive correlation between harmful bacteria Abioteophia in feces and fat energy supply proportion(p=0.405,P=0.040),a negative correlation between Abioteophia in feces and HDL level(p=-0.445,P=0.023),beneficial bacteria Gemmiger in feces and serum total cholesterol level(p=-0.542,P=0.004)and triglyceride level(p=-0.580,P=0.002),which may have clinical significance.(3)The intervention study found that the addition of probiotics and dietary intervention could effectively change the diversity,structure and abundance of fecal and colonic mucosa-associated bacteria in high-fat diet population,which showed that intestinal microflora diversity was raised after intervention,the abundance of some potential pathogenic bacteria,including Bacteroides,Fusobacterium,Pseudomonas,was reduced,and the abundance of beneficial bacteria was increased such as Prevotella,Faecalibacterium prausnitzii,Coprococcus,Gemmiger,etc.Conclusion:The diversity of fecal microbiota was lower than that of colonic mucosa-associated microbiota,and there was significant difference in microbiota structure between fecal sample and colonic mucosa tissue.High fat diet dose not have any noticeable effect on the diversity of fecal microbiota and colonic mucosa-associated flora in healthy population,but results in a different microbiota structure from low-fat diet at the genus level,which was related to dietary factor and other healthy state.Probiotic supplementation and dietary intervention can effectively improve the diversity,structure and abundance of fecal and colonic mucosa-associated bacterial communities in high-fat diet population,which have the potential of anti-inflammatory and anti-cancerous. |
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