Differences In The Intestinal Flora Of High-fat-induced Obesity-prone And Obesity-resistant Mice

Background and Objective:Obesity is a chronic metabolic disease caused by multiple factors that lead to excessive accumulation and/or abnormal distribution of body fat.Obese patients are often associated with a variety of complications,such as hypertension,type 2 diabetes,and stroke,which place a huge strain on clinical management and care.The gut flora plays a key role in regulating energy homeostasis.Our preliminary study found that the susceptibility to obesity varied considerably between individuals.It is unclear whether this difference is related to gut flora.In this paper,we aim to construct high-fat-induced obeseprone(OP)and obese-resistant(OR)mouse models,analyze the structural and functional changes in their intestinal flora,identify the key signature microorganisms that distinguish between these two phenotypes,and validate the relationship between intestinal flora and susceptibility to obesity by fecal microbial transplantation(FMT).The identification and differentiation of key signature microorganisms for the OP and OR phenotypes can be used as an indicator for evaluating the effectiveness of health education in clinically obese patients,as well as providing targeted solutions for obesity care and promoting precision in care decisions.Methods:1.Construction of high-fat-induced obese prone and obese resistant mice modelEighty C57BL/6J mice of the same genetic background were randomly divided into two groups: 16 mice in the control group(ND)were given a normal diet and 64 mice in the model group were given a high-fat diet.After 8 weeks of modeling,mice in the upper quartile of weight gain in the high-fat-fed mice were designated as the OP group(n=16),while mice in the lower quartile were designated as the OR group(n=16).A total of 48 C57BL/6J mice were eventually included in the study.The final specimens of blood,liver,fat,ileum,and cecum contents were collected from the mice;the weight of the mice,liver,and adipose tissue were weighed;lipid quadruplicates,blood glucose levels,and histopathological changes in the liver,fat and ileum were measured in each group of mice.2.Analysis of intestinal flora in obese-prone and obese-resistant miceAfter 8 weeks of feeding,feces from ND(n=6),OP(n=6),and OR(n=6)mice were randomly collected and DNA extracted for high throughput sequencing.The characteristics of the intestinal flora of the three groups of mice were analyzed to find species that differed significantly between groups and to compare the relationship between the different species and body weight using correlation analysis.3.Exploring the relationship between intestinal flora and obese prone and obese resistant based on fecal transplantation testThirty-two C57BL/6J mice were randomly divided into 4 groups,including 8 control mice(FMT-PBS),8 transplanted normal mice fecal flora group(FMT-ND),8 transplanted obesity-prone mice fecal flora group(FMT-OP)and 8 transplanted obesity-resistant mice fecal flora group(FMT-OR).A pseudo-sterile mouse model was constructed by giving each group of mice a mixture of antibiotics in drinking water to deplete the intestinal flora.Mice in the FMT-ND group will be given fresh fecal flora from the ND group in the donor mice;mice in the FMT-OR group will be given fresh fecal flora from the OR group in the donor mice;mice in the FMT-OP group will be given fresh fecal flora from the OP group in the donor mice;mice in the control group will be given equal amounts of sterile PBS.After the experiment,blood,liver,fat,and cecum contents were collected from the mice;the weight of the mice,liver,and adipose tissue were weighed;the four lipid items,blood glucose levels,and histopathological changes of the liver were measured in each group.The relative abundance of Ruminococcaceae＿UCG-013 in mice fed with high fat for 8weeks,16 weeks,and 8 weeks of FMT was measured by real-time fluorescence quantitative PCR.Results:1.Construction of high-fat-induced obese-prone and obese-resistant mice modelIn body weight,white fat weight,liver weight,blood glucose level,serum TC,TG,and LDL-C,the OP group was significantly higher than the OR group(p<0.05).H&E staining showed larger brown adipose(BAT)cells and lipid droplets,irregular ependymal fat cells,and mast cell infiltration in the OP group compared to the OR group;the liver had more lipid deposits,inflammatory cell infiltration,and the OP group had lymphocytic infiltration at the myenteric layer of the ileal mucosa and a disorganized arrangement of villi,indicating possible inflammatory symptoms.2.Analysis of intestinal flora in obese-prone and obese-resistant miceThe results of diversity analysis showed that there were significant differences in the intestinal flora structure of the three groups of mice;compared to the ND group,the OP and OR groups showed a decrease in the abundance of the phylum Bacteroides and an increase in the abundance of the phylum Firmicutes and the phylum Proteobacteria.The intestines of the mice in the OP and OR groups showed some differences in the phylum Firmicutes,Bacteroides,Proteobacteria,and Actinobacteria,while the differences were statistically significant in the phylum Deferribacteres and the phylum Verrucomicrobia(p<0.05).Linear discriminant effect size(LEf Se)analysis of the intestinal flora of the OP and OR groups revealed that the key species in the OP group were the Coriobacteriaceae＿UCG＿002,Ruminococcus＿torques＿group,Mucispirillum,and Leuconostocaceae.The key species in the OR group were Akkermansia,Eubacterium＿coprostanoligenes＿group,and Ruminococcaceae＿UCG-013.Random forest analysis revealed Ruminococcaceae＿UCG-013 as the key signature microorganism that distinguished OP and OR mice.Analysis of the correlations between the intestinal bacteria and body weight showed that the Coreiobacteriaceae＿UCG＿002,Ruminococcus＿torques＿group,and GCA＿900066225 were positively correlated with body weight;Akkermansia Ruminococcaceae＿UCG＿013 and Eubacterium＿coprostanoligenes＿group were negatively correlated with body weight.3.Exploring the relationship between intestinal flora and obese-prone and obese-resistant based on fecal transplantation testThe body weight,white fat weight,liver weight,blood glucose level,serum TC and TG of mice in the FMT-OP group were significantly higher than those in the FMT-OR group.H&E staining of the liver showed more lipid deposition,inflammatory cell infiltration,and hepatocyte degeneration in the FMT-OP group.RT-QPCR analysis of the relative abundance of Ruminococcaceae＿UCG-013 in the intestinal flora of mice at different stages showed that the relative abundance of Ruminococcaceae＿UCG-013 was significantly higher in both OR and FMT-OR mice than in the OP and FMTOP groups.Conclusions:1.OR mice have a healthier metabolic phenotype compared to OP mice.2.The intestinal flora of OP and OR mice differ significantly in structure and function and possess their dominant flora,with Ruminococcaceae＿UCG-013 levels being the key signature microorganism that distinguishes OP and OR mice.3.Intestinal flora plays an important role in obese-prone and obeseresistant groups.As a key biomarker in the obesity resistance group,Ruminococcaceae＿UCG-013 plays an important role in delaying weight gain,and it is expected to be used as an indicator to evaluate the effectiveness of health education for clinically obese patients,providing new ideas for clinical precision care.