Application of general bacteroides and general bacteroides metabolite cholic acid in inhibiting molecular mechanism of obesity induced by high fat diet

Abstract

The invention discloses application of bacteroides vulgaris and bacteroides metabolite cholic acid in inhibiting a molecular mechanism of obesity induced by high fat diet, belongs to the technical field of biological medicines, and aims at solving the problem of how to regulate and control the synthesis molecular mechanism of 5-HT by intestinal flora and metabolite thereof through a series of researches. B, the vulgutus strain and the bacteroid metabolite CA reduce lipid absorption during high-fat diet by inhibiting generation of 5-HT, and play a role in resisting obesity caused by high-fat diet. Intervention of the structure and abundance of intestinal flora may become a means for preventing obesity, and a B.vulgaris strain-metabolite CA-5-HT signal axis may become an important target for revealing the regulation of the obesity generation mechanism by the intestinal flora and researching and developing related treatment strategies.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a molecular mechanism of Bacteroides vulgaris and bile acid, a metabolite of Bacteroides vulgaris, in inhibiting obesity induced by a high-fat diet. Background Art

[0002] With the improvement of human living standards, the daily dietary structure has gradually changed from a plant-based diet to a high-fat, low-carbohydrate diet. The increase in high-calorie food intake caused by changes in dietary habits can induce obesity and a series of related complications (such as chronic non-communicable diseases such as cardiovascular disease, diabetes, and some cancers), among which overweight / obesity is the main risk factor for obesity-related complications. It is reported that since 2000, the overweight rate, obesity rate and the prevalence of related chronic diseases in the Chinese population have risen rapidly, and the prevention and control of overweight / obesity have become a top priority for global public health. Therefore, it is crucial to clarify the underlying mechanism of obesity and find prevention / treatment methods. Among the current prevention / treatment programs for obesity, changing the dietary structure is the preferred method for controlling obesity. It is reported that by adjusting the species richness in the diet, the structure of the intestinal flora of the body can be significantly changed, thereby affecting the occurrence and development of obesity. Therefore, finding a prevention and treatment strategy for obesity guided by intestinal flora is a major key issue that needs to be solved urgently, which provides a theoretical basis for the development of anti-obesity drugs that can be used safely and for a long time, and has important clinical significance.

[0003] The human gastrointestinal flora plays an important role in nutrient metabolism and development. Its composition and function are established from infancy and continue to develop and change throughout life. Due to changes in many physiological processes and lifestyles, especially changes in diet, metabolism, energy balance and immunity, the structure of the intestinal flora also changes. In a normal body, the intestinal flora can be mainly divided into four categories: Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria. Among them, the changes in the proportion of Bacteroidetes and Firmicutes are most closely related to the occurrence and development of obesity. Studies have shown that changing the content of certain substances in the diet (such as dietary fiber) can increase the proportion of Bacteroidetes and Firmicutes in the intestine, thereby avoiding the occurrence and development of obesity. However, this study focuses on the bacterial phylum level and includes many types of bacteria, which makes it difficult to accurately formulate obesity prevention and treatment strategies. Therefore, a more detailed study of the structural changes of the intestinal flora and the exploration and regulation of specific bacterial strains for overweight / obesity can theoretically become a precise target for improving obesity and related metabolic diseases.

[0004] 5-hydroxytryptamine (5-HT) is a neurotransmitter synthesized from the essential amino acid tryptophan by tryptophan hydroxylase (TPH) and aromatic amino acid decarboxylase, also known as serotonin. 5-HT exerts its biological effects by binding to the 5-hydroxytryptamine receptor (5-HTR). The action of the receptor is then terminated by the uptake of 5-HT into the cell through the serotonin transporter (SERT). The hydroxylation of tryptophan is the rate-limiting step in the synthesis of 5-HT. The production of 5-HT is regulated by TPH activity and tryptophan supply. It has been reported that intestinal flora is the main factor regulating the production of intestinal 5-HT; the level of 5-HT in the serum of germ-free mice or mice treated with antibiotics is significantly reduced. At the same time, the metabolites of intestinal flora (such as short-chain fatty acids) can increase the expression of TPH1 to regulate the synthesis of 5-HT. Not only that, 5-HT can also affect the concentration of glycolipid-related metabolites in peripheral blood and liver, and accelerate the metabolism of bile acid; the secondary bile acid deoxycholic acid (DCA) in the bile acid metabolism pathway is positively correlated with the synthesis of 5-HT. At the same time, the role of 5-HT in energy homeostasis has been reported in many papers. In the intestine, intestinal flora and its metabolites can regulate the synthesis of 5-HT, and 5-HT is involved in the regulation of the body's energy homeostasis, which provides a theoretical basis for the participation of intestinal flora in regulating the body's energy homeostasis. However, the mechanism of how intestinal flora and its metabolites regulate the synthesis of 5-HT is unclear. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of common Bacteroides and Bacteroides metabolite bile acid to inhibit the molecular mechanism of obesity induced by a high-fat diet.

[0006] The present invention provides an application of the molecular mechanism of Bacteroides vulgaris and bile acid, a metabolite of Bacteroides vulgaris, in inhibiting obesity induced by a high-fat diet. Bacteroides vulgaris and bile acid, a metabolite of Bacteroides vulgaris, inhibit obesity induced by a high-fat diet by inhibiting the 5-HT content in the intestine and serum.

[0007] Preferably, Bacteroides vulgaris and its metabolite bile acid inhibit the 5-HT content in the intestine and serum by inhibiting the number of EC cells.

[0008] Preferably, the inhibition of 5-HT content inhibits the secretion of jejunal chylomicrons into intestinal lymph, thereby inhibiting obesity induced by a high-fat diet.

[0009] Preferably, the inhibition of 5-HT content promotes the number of lipid droplets and chylomicrons in intestinal epithelial cells, thereby inhibiting obesity induced by a high-fat diet.

[0010] Preferably, the 5-HT content inhibition inhibits the secretion of jejunal chylomicrons into intestinal lymph by inhibiting the calcium ion concentration in intestinal epithelial cells, thereby inhibiting obesity induced by a high-fat diet.

[0011] The present invention also provides the use of Bacteroides vulgaris and a bile acid inhibitor of a Bacteroides vulgaris metabolite in the preparation of a drug for treating obesity induced by a high-fat diet.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present study found that mice with different obesity phenotypes have different intestinal flora structures and compositions. The common Bacteroides strain B. vulgauts and the Bacteroides metabolite CA with high abundance in the intestinal flora of lean mice can inhibit the synthesis of intestinal 5-HT, reduce the calcium ion concentration in intestinal epithelial cells, inhibit the secretion and transport of chylomicrons, and thus inhibit obesity. Therefore, intervening in the structure and abundance of intestinal flora may become a means of preventing obesity. The B. vulgatus strain-metabolite CA-5-HT signaling axis may become an important target for revealing the mechanism of intestinal flora regulating obesity and developing related treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The relevant results of the HIGH and LOW groups of mice fed with HFD for 12 weeks;

[0015] in, Figure 1 A is the statistics of food and water intake (n=4); Figure 1 B is a body shape comparison chart; Figure 1 C body weight change (n=5); Figure 1 D is the statistical diagram of body composition detected by nuclear magnetic resonance (n=5);

[0016] Figure 2 The figure shows the glucose tolerance and insulin tolerance test results of the nutritional obesity mouse model;

[0017] in, Figure 2 A is the glucose tolerance curve of mice in the HIGH and LOW groups (n=10); Figure 2 B is the area under the curve (AUC) statistic for (A); Figure 2 C is the insulin tolerance curve of mice in the HIGH (n=5) and LOW (n=3) groups; Figure 2 D is for Figure 2 Area under the curve (AUC) statistical graph of C;

[0018] Figure 3 This is a diagram of the target organ detection of fat metabolism in the nutritional obesity mouse model;

[0019] in, Figure 3 A is the liver morphology of mice in the HIGH and LOW groups; Figure 3 B is the statistical graph of liver weight of mice in the HIGH and LOW groups (n=5); Figure 3 C is for Figure 3 Frequency statistics of the area of ​​individual fat cells in E; Figure 3 D is the H&E staining result of liver tissue of mice in the HIGH and LOW groups; the scale bar is 100 μm; Figure 3 E is the H&E staining result of epididymal adipose tissue of mice in the HIGH and LOW groups, the scale bar is 100 μm;

[0020] Figure 4 This is the result diagram of the effect of FMT on the body weight and body composition of the nutritional obesity mouse model;

[0021] in, Figure 4 A shows the weight changes of mice in the HIGH FMT and LOW FMT groups (n=7); Figure 4 B is a statistical diagram of body composition (ratio of fat mass, lean mass to body mass) detected by nuclear magnetic resonance of mice in the HIGH FMT (n=5) and LOW FMT (n=4) groups;

[0022] Figure 5 This is the result diagram of the effect of FMT on glucose tolerance in the nutritional obesity mouse model;

[0023] in, Figure 5 A is the glucose tolerance test of mice in the HIGH FMT and LOW FMT groups (n=4); Figure 5 B is for Figure 5 The area under the curve (AUC) statistic of A;

[0024] Figure 6 The effect of FMT on the target organs of fat metabolism in the nutritional obesity mouse model;

[0025] in, Figure 6 A is the liver morphology of mice in the HIGH FMT and LOW FMT groups; Figure 6 B is the statistics of liver weight of mice in the HIGH FMT (n=4) and LOW FMT (n=3) groups; Figure 6 C is for Figure 6 Frequency statistics of the area size of individual fat cells in E; Figure 6 D shows the H&E staining results of liver tissues of mice in the HIGH FMT and LOW FMT groups, with a scale of 100 μm; Figure 6E is the H&E staining result of epididymal adipose tissue of mice in the HIGH FMT and LOW FMT groups, the scale bar is 100 μm;

[0026] Figure 7 Metagenomic analysis of intestinal flora of mice in the HIGH and LOW groups;

[0027] in, Figure 7 A is the principal component analysis (PCA) of the intestinal flora of mice in the HIGH and LOW groups; Figure 7 B is the NMDS analysis of the intestinal flora of mice in the HIGH and LOW groups; Figure 7 C is the difference in the bacterial phylum levels of the intestinal flora between the HIGH and LOW groups of mice; Figure 7 D is the difference in the bacterial genus levels of the intestinal flora between the HIGH and LOW groups of mice; Figure 7 E is the LDAEffect Size (LEFse) analysis of the bacterial species level of the intestinal flora of the HIGH and LOW groups of mice;

[0028] Figure 8 The results of body weight and body composition test of mice in the control group and B. vulgatus strain supplementation group fed with HFD for 12 weeks;

[0029] in, Figure 8 A is a body shape comparison chart; Figure 8 B is the change in body weight (n=9); Figure 8 C is the statistics of body composition detected by NMR (n=6);

[0030] Fig. 9 This is a graph showing the effect of B. vulgatus strains on glucose tolerance and insulin tolerance;

[0031] in, Fig. 9 A is the glucose tolerance curve of mice in the control group and the B. vulgatus strain complementation group (n=5); Fig. 9 B is for Fig. 9 The area under the curve (AUC) statistic of A; Fig. 9 C is the insulin tolerance curve of mice in the control group and the B. vulgatus strain complementation group (n=5); Figure D is the area under the curve (AUC) statistics for (C);

[0032] Fig.10 Effects of B. vulgatus strains on target organs of fat metabolism

[0033] in, Fig.10 A is the liver morphology of mice in the control group and the B. vulgatus strain complementation group; Fig.10 B is the statistics of liver weight of mice in the control group and B. vulgatus strain complementation group; Fig.10 C is for Fig.10 Frequency statistics of the area size of individual fat cells in E; Fig.10 D is the H&E staining result of liver tissue of mice in the control group and B. vulgatus strain complementation group, the scale bar is 100 μm; Fig.10 E is the H&E staining result of epididymal adipose tissue of mice in the control group and B. vulgatus strain complementation group, the scale bar is 100 μm;

[0034] Fig.11 The results of the detection of various components in the serum of mice in the control group and the B. vulgatus strain supplementation group;

[0035] in, Fig.11 A is the TC content of serum (n=7); Fig.11 B is the serum TG content (n=6); Fig.11 C is the serum HDL content (n=7); Fig.11 D is the serum LDL content (n=7);

[0036] Fig.12 The figure shows the body weight and body composition of mice in the control group and the C. beijerinckii strain complementation group;

[0037] in, Fig.12 A is a comparison of the body shape of mice in the control group and the C. beijerinckii strain complementation group fed with HFD for 12 weeks; Fig.12 B is a comparison of the weight changes of mice in the control group and the C. beijerinckii strain complementation group (n = 3); Fig.12 C is the statistical diagram of body composition detected by nuclear magnetic resonance of mice in the control group and the C. beijerinckii strain complementation group (n = 3);

[0038] Fig.13 This is a graph showing the effect of strain C. beijerinckii on glucose tolerance in mice;

[0039] in, Fig.13 A is the glucose tolerance curve of mice in the control group and the C. beijerinckii complementation group (n = 6); Fig.13 B is for Fig.13 Statistical graph of the area under the curve (AUC) of A;

[0040] Fig.14 This is a diagram showing the effect of C. beijerinckii strain on target organs of mouse fat metabolism;

[0041] in, Fig.14 A is the liver morphology of mice in the control group and the C. beijerinckii strain complementation group; Fig.14 B is a statistical graph of liver weights of mice in the control group and the C. beijerinckii strain complementation group (n = 5);

[0042] Fig.15 The results of bile acid targeted metabolomics analysis of fecal samples of mice in the HIGH and LOW groups are shown in the figure;

[0043] in, Fig.15 A is the PCA analysis result of bile acid in fecal samples of mice in the HIGH and LOW groups (n=4); Fig.15 B is the volcano plot analysis of the difference in bile acids in fecal samples of mice in the HIGH and LOW groups (n=4); Fig.15 C is the cluster analysis result of differential bile acid in fecal samples of mice in the HIGH and LOW groups (n=4);

[0044] Fig.16 The figure shows the body weight and body composition test results of mice in the HFD and HCA groups;

[0045] in, Fig.16 A is the body weight change of mice in the HFD group (n=5) and HCA group (n=5); Fig.16 B is the statistical diagram of body composition detected by nuclear magnetic resonance of mice in the HFD group (n=5) and HCA group (n=5);

[0046] Fig.17 This is a graph showing the effect of metabolite CA on glucose tolerance in mice;

[0047] in, Fig.17 A is a graph showing the glucose tolerance test results of mice in the HFD group (n=5) and the HCA group (n=5); Fig.17 B is for Fig.17 Area under the curve (AUC) statistical graph of A;

[0048] Fig.18 This is a diagram showing the effect of metabolite CA on target organs of mouse fat metabolism;

[0049] in, Fig.18 A is the H&E staining results of liver tissues of mice in the HFD group and HCA group, the scale bar is 100 μm; Fig.18 B is the H&E staining result of epididymal adipose tissue of mice in the HFD group and HCA group, the scale bar is 100 μm; Fig.18 C is a frequency statistical diagram of the area size of a single fat cell in 18B;

[0050] Fig.19 The expression results of CgA and 5-HT in the jejunum tissue of mice in the HIGH and LOW groups;

[0051] in, Fig.19A is the immunofluorescence results of CgA and 5-HT in the jejunum tissue of mice in the HIGH (n=4) and LOW (n=5) groups (in the figure: red fluorescence represents CgA, green fluorescence represents 5-HT, and blue-green fluorescence represents cell nuclei); Fig.19 B is a statistical diagram of the number of CgA positives per unit area;

[0052] Fig. 20 The expression diagram of CgA and 5-HT in the jejunum tissue of B. vulgatus strain complemented mice;

[0053] in, Fig. 20 A is the immunofluorescence result of CgA and 5-HT in the jejunum tissue of B. vulgatus complemented mice (n=10) (in the figure: red fluorescence represents CgA, green fluorescence represents 5-HT, and lake blue fluorescence represents cell nuclei); Fig. 20 B is a statistical diagram of the number of CgA positives per unit area; Fig. 20 C is the number of 5-HT positive cells per unit area; Fig. 20 D is the content of 5-HT in the jejunum tissue of B. vulgatus complementation model mice (n=7); Fig. 20 E is a graph showing the 5-HT content in the serum of mice with the B. vulgatus complementation model (n=6);

[0054] Fig.21 The expression results of CgA and 5-HT in the jejunum tissue of mice supplemented with metabolite CA;

[0055] in, Fig.21 A is the immunofluorescence result of CgA and 5-HT in the jejunum tissue of CA-supplemented mice (n=3) (in the figure: red fluorescence represents CgA, green fluorescence represents 5-HT, and lake blue fluorescence represents cell nuclei); Fig.21 B is a statistical diagram of the number of CgA positives per unit area; Fig.21 C is the number of 5-HT positive cells per unit area; Fig.21 D is the content of 5-HT in the jejunum tissue of CA supplementation model mice (n=5) Fig.21 E is the graph of 5-HT content in serum of CA replenishment model mice (n=5);

[0056] Fig. 22 This is a diagram showing the identification results of intestinal epithelial-specific Tph1 knockout mice;

[0057] in, Fig. 22 A: PCR identification of Tph1 fl / fl and Tph1 △IEC Mouse genomic DNA sequence (n=3); Fig. 22 B: RT-qPCR detection of Tph1 fl / fland Tph1 △IEC Relative expression results of TPH1 in mouse intestinal epithelial tissue (n=4); Fig. 22 C: Western blot detection of Tph1 fl / fl and Tph1 △IEC Results of TPH1 expression in mouse intestinal epithelial tissue (n=2);

[0058] Fig.23 The results show the effects of intestinal epithelial-specific Tph1 knockout on body weight and glucose tolerance in mice under HFD conditions.

[0059] in, Fig.23 A is Tph1 fl / fl Group (n=4) and Tph1 △IEC Body weight changes of mice in each group (n=5); Fig.23 B is Tph1 fl / fl Group (n=5) and Tph1 △IEC Glucose tolerance test of mice in group (n=5); Fig.23 C is for Fig.23 Area under the curve (AUC) statistical results of B;

[0060] Fig.24 This is a graph showing the effect of intestinal epithelial-specific Tph1 knockout mice on chylomicron transport;

[0061] in, Fig.24 A: Tph1 observed under a stereo microscope fl / fl Mouse and Tph1 △IEC Results of mouse jejunal chyle uptake (in the figure: the yellow arrows indicate the lacteals filled with chylomicrons); Fig.24 B is Tph1 fl / fl Mice (n=6) and Tph1 △IEC Statistical graph of the number of jejunal lacteals in mice (n=7);

[0062] Fig.25 This is a graph showing the effect of model mice on lipid uptake in intestinal epithelial cells;

[0063] in, Fig.25 A is the transmission electron microscopic image of the jejunal tissues of mice in the HIGH and LOW groups; Fig.25 B is the transmission electron microscopic image of the jejunal tissues of mice in the control group and the B. vulgatus strain complementation group; Fig.25 C is Fig.25 Figure 1 shows the statistical results of the number of CLDs in A (n=4); Fig.25 D is for Fig.25 Figure B shows the statistical results of the number of CLDs (n=4); Fig.25 E is the transmission electron microscopic image of the jejunal tissues of mice in the control group and the CA supplementation group; Fig.25 F is the transmission electron microscopy image of the jejunal tissue of the control group and intestinal epithelial-specific knockout Tph1 mice; Fig.25 G is for Fig.25 Figure E shows the statistical results of the number of CLDs (n=3); Fig.25 H is for Fig.25 Statistical results of the number of CLDs in F (n=3);

[0064] Fig.26 This is a graph showing the effect of B. vulgatus strain complementation on intestinal epithelial cell lipid transport-related molecular markers;

[0065] Fig. 27 The results of transcriptomic analysis of intestinal epithelial tissues in the Control group and the B. vulgatus group of mice;

[0066] in, Fig. 27 A is the volcano plot analysis result of differentially expressed genes in intestinal epithelial tissue (n=3); Fig. 27 B is the result of cluster analysis of differentially expressed genes in intestinal epithelial tissue (n=3); Fig. 27 C is the result of principal component analysis of differentially expressed genes in intestinal epithelial tissue (n=3); Fig. 27 D is the KEGG analysis result of differentially expressed genes in intestinal epithelial tissue (n=3); Fig. 27 E is the result of GO enrichment analysis of differentially expressed genes in intestinal epithelial tissue (n=3);

[0067] Fig.28 This is the dose-effect curve of 5-HT in the jejunal mucosa of wild-type mice;

[0068] Fig.29 The effect of 5-HT on the short-circuit current of the jejunal mucosa of model mice;

[0069] in, Fig.29 A is the real-time recording curve of short-circuit current of jejunal mucosal epithelium in the HIGH and LOW groups of mice; Fig.29 B is the changes of 5-HT-induced short-circuit current in the jejunal mucosal epithelium of mice in the HIGH and LOW groups (n=7); Fig.29 C is the real-time recording curve of short-circuit current of jejunal mucosal epithelium in control group and B. vulgatus strain-replenished mice; Fig.29 D is a graph showing the changes in short-circuit current of the jejunal mucosal epithelium in the control group and B. vulgatus strain complemented mice induced by 5-HT (n=6); Fig.29 E is the real-time recording curve of short-circuit current of jejunal mucosal epithelium in the control group and the metabolite CA supplementation group; Fig.29 F is the change of short-circuit current of jejunal mucosal epithelium in mice of control group and metabolite CA replenishment group induced by 5-HT (n=5);

[0070] Fig.30 The results of transcriptomic intersection analysis of intestinal epithelial tissues in the obesity model HIGH and LOW groups, B. vulgatus strain complementation model mice, metabolite CA complementation model mice, and intestinal epithelial-specific knockout Tph1 mice;

[0071] in, Fig.30 A is the result of Venn diagram analysis of differentially expressed genes in intestinal epithelial tissue; Fig.30 B is the KEGG enrichment analysis result of differentially expressed genes in intestinal epithelial tissue; Fig.30 C is the result of GO enrichment analysis of differentially expressed genes in intestinal epithelial tissue;

[0072] Fig.31 This is a graph showing the effect of 5-HT on intracellular calcium concentration in NCM 460 cells;

[0073] in, Fig.31 A is the curve of the change of the average Fluo-4 fluorescence intensity in NCM 460 cells with time by the solvent control DMSO (n=6); Fig.31 B is a graph showing the change of the average Fluo-4 fluorescence intensity in NCM 460 cells with time after 5-HT (10 μM) (n=6); Fig.31 C is for Fig.31 A and Fig.31 B is a statistical diagram of the maximum Fluo-4 fluorescence intensity change fold difference under the action of drugs;

[0074] Fig.32 Schematic diagram of the regulatory mechanism by which B. vulgatus strain and metabolite CA affect nutritional obesity by regulating 5-HT. DETAILED DESCRIPTION

[0075] The following is a description of the embodiments of the present invention. Figures 1 to 32 , the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0076] 1. Experimental Materials

[0077] 1.1 Experimental animals: Male C57BL / 6J mice, SPF grade, 8 weeks old, weighing 22±1g, purchased from the Experimental Animal Department of Capital Medical University. △IEC CRISPR / Cas9 conditional knockout mice were prepared by Saiye (Suzhou) Biotechnology Co., Ltd. The use of experimental animals followed the Capital Medical University Laboratory Animal Management Rules and was approved by the Capital Medical University Animal Ethics Committee.

[0078] 1.2 Experimental cell line: NCM 460 human normal colon mucosal epithelial cells were purchased from ATCC Cell Collection Center.

[0079] 1.3 Experimental strains: Bacteroides vulgatus JCM 5826 was purchased from ATCC. Clostridium beijerinckii CGMCC 5077 was purchased from China General Microbiological Culture Collection.

[0080] 2. Experimental Methods

[0081] 1. Construction of experimental model mice

[0082] 1.1. Construction of nutritional obesity model mice

[0083] Wild-type 8-week-old C57BL / 6J male SPF mice were fed a high-fat diet (HFD) containing 60% fat calories for 90 days. Animal housing conditions: temperature 22±2℃, humidity 40-70%, 12h light / 12h dark cycle, free access to feed and water. Body weight changes were recorded every 2 weeks. After 12 weeks, the mice were divided into a high weight gain group (HIGH) and a low weight gain group (LOW) according to the following conditions:

[0084] a) Weight gain ratio: The weight gain ratio greater than 0.5 is the HIGH group, and less than 0.5 is the LOW group.

[0085] The weight gain ratio = (weight of the mouse at the 12th week - weight of the mouse at the 1st week) / weight of the mouse at the 12th week.

[0086] b) The weight of the lowest-weight mouse in the HIGH group minus the weight of the highest-weight mouse in the LOW group. The value obtained must be greater than 8 g.

[0087] 1.2. Construction of fecal microbiota transplantation (FMT) mouse model

[0088] Weigh 500 mL of ultrapure water with a measuring cylinder and place it into a volumetric flask. Add a magnetic rotor and sterilize by high pressure. When the water temperature drops to room temperature, add the antibiotics listed in Table 1 below in sequence and mix thoroughly on a magnetic stirrer. Prepare before use and store at room temperature for one week.

[0089] Wild-type 8-week-old C57BL / 6J male SPF mice were fed with mixed antibiotic water for 2 weeks to eliminate the original intestinal flora and randomly divided into 2 groups:

[0090] (a) HIGH FMT group, mice were gavaged with feces mixture from the high weight gain group (HIGH) and fed a high-fat diet;

[0091] (b) The LOW FMT group was gavaged with a mixture of feces from the low weight gain group (LOW) mice and fed a high-fat diet. The fecal gavage solution was prepared by dissolving 100 mg of mixed feces in 5 mL of sterile PBS, vortexing thoroughly and standing at room temperature for 10 minutes, and taking the supernatant for gavage. In order to ensure the effect of microbiota transplantation, 200 μL of gavage was performed every two days (1st, 3rd, 5th, and 7th days) in the first week after antibiotic water feeding. Starting from the second week after antibiotic water feeding, gavage was performed twice a week for 8 weeks.

[0092] Table 1 Broad-spectrum antibiotics in water antibiotic formula

[0093] Element Dosage Neomycin Sulfate 0.25 Metronidazole 0.25 Ampicillin Sodium 0.25 Vancomycin 0.125

[0094] 1.3. Construction of strain complementation mouse model

[0095] 1.3.1. Activation, culture and cryopreservation of B. vulgatus and C. beijerinckii

[0096] Table 2. Culture medium for B. vulgatus strains

[0097] Element Dosage Lab-Lemcopowder(Oxoid) 2.4g Proteosepeptone No.3 (BD-Difco) 10.0g Yeastextract (BD-Difco) 5.0g Na2HPO4 4.0g Glucose 1.5g Solublestarch 0.5g L-Cystine 0.2g L-Cysteine·HCl·H2O 0.5g FBS 50mL Distilledwater 950mL

[0098] Add L-Cystine to 50 mL of 1N hydrochloric acid, vortex to mix, and pour into a volumetric flask. Weigh the remaining reagents in Table 2 and dilute to 950 mL with distilled water, and adjust the pH to 7.6-7.8. Autoclave and cool to room temperature, transfer to a biosafety cabinet, add 50 mL of FBS, mix thoroughly, and dispense into 50 mL centrifuge tubes to obtain B. vulgatus strain culture medium, and store at 4°C for later use.

[0099] Table 3. C. beijerinckii strain culture medium

[0100]

[0101]

[0102] Weigh the above reagents in Table 3 and dilute to 1 L with distilled water, adjust pH to 6.0 ± 0.1, sterilize by high pressure and cool to room temperature to obtain C. beijerinckii strain culture medium, and store at 4°C.

[0103] 1) Strain activation: Take the frozen B. vulgatus strain and C. beijerinckii strain at -80℃ and immediately place them on ice, then transfer them to a biosafety cabinet for operation. Use a pipette tip to pick up a small amount of bacterial freezing solution and add it to 3mL of B. vulgatus strain culture medium and C. beijerinckii strain culture medium respectively, pipetting and mixing.

[0104] (2) Cultivation: The activated strains were cultured in an anaerobic workstation (37°C; 80% N2; 10% CO2; 10% H2) for 24 h and subcultured every other day. The OD value of the strain suspension was detected by a spectrophotometer and the OD 600 of the strain suspension was adjusted to 0.6 ± 0.1. The strain concentration was 6.0 × 10 8 ~7.0×10 8 CFU / mL.

[0105] (3) Cryopreservation: Take the above cultured strain suspension from the anaerobic workstation and transfer it to a biosafety cabinet for operation. Mix the strain suspension with glycerol in a ratio of 1:1 into a cryopreservation tube, seal it, and store it at -80°C.

[0106] (4) Detection of strain suspension concentration: Use the plate colony counting method (CFU). Dilute the bacterial suspension and culture medium in proportion to set up groups:

[0107] (a) Bacterial suspension stock solution; (b) 10-fold bacterial suspension dilution group; (c) 20-fold bacterial suspension dilution group. Take 1 mL of bacterial suspension from each group and pour it onto nutrient agar plates. Incubate for 48 hours and record the number of colonies formed on each plate. Calculate the total number of live bacteria contained in each mL of the original sample according to CFU / mL = average colony count of 3 replicates at the same dilution multiple × dilution multiple × 5.

[0108] 1.3.2. B. vulgatus and C. beijerinckii repopulation and modeling

[0109] Wild-type 8-week-old C57BL / 6J male SPF mice were fed with mixed antibiotic water for 2 weeks to eliminate the original intestinal flora and randomly divided into 2 groups: (a) Control group: B. vulgatus and C. beijerinckii strain culture media were gavaged and fed with a high-fat diet; (b) B. vulgatus group and C. beijerinckii group: 6.0×10 8 ~7.0×10 8CFU / mL of the strain suspension was gavaged and the mice were fed a high-fat diet. To ensure the colonization effect of the strain, 200 μL of the suspension was gavaged 4 times every two days (1st, 3rd, 5th, and 7th days) in the first week after feeding with antibiotic water. Starting from the second week after feeding with antibiotic water, the mice were gavaged twice a week for 8 weeks. The weight changes were recorded every week.

[0110] 1.4. Construction of a bile acid replenishment mouse model

[0111] Wild-type 8-week-old C57BL / 6J male SPF mice were randomly divided into 2 groups:

[0112] (a) HFD group: fed with a high-fat diet; (b) HFD+CA group: fed with a high-fat diet containing 60% calories from fat and supplemented with 0.5% bile acid. Body weight changes were recorded every two weeks.

[0113] 2. Phenotypic analysis of each model mouse

[0114] 2.1. Food and water intake test

[0115] Mice were housed in individual cages at a temperature of 22±2°C, a humidity of 40%-70%, and a 12-h light / 12-h dark cycle. 30 g of high-fat feed containing 60% fat calories and 100 g of distilled water were weighed. After 24 h of feeding, the remaining food and water were weighed. The weights were continuously weighed for 5 days, the average value was taken, and the 24-h consumption was calculated.

[0116] 2.2 Body composition detection

[0117] After each model mouse was established, the mouse was placed in a small animal body composition analysis and imaging system (small animal PET / CT / SPECT three-in-one system) for analysis, and the total body fat and lean body mass (LBM) were detected in vivo.

[0118] 2.3 Intraperitoneal glucose tolerance test (IPGTT)

[0119] Each model mouse was fasted but not watered for 16 h (new cages and bedding to ensure no food residues), and the working concentration of glucose solution was 1.5 g / 10 mL. Glucose solution was injected intraperitoneally at 80 μL / 10 g of mouse body weight (i.e., the glucose dosage was 12 mg / 10 g of mouse body weight), and blood was collected from the tail vein at 0, 15, 30, 60, 90, and 120 min after injection. The blood glucose concentration was detected with a portable blood glucose meter, and the blood glucose curves of each group at each time point were drawn, and the area under the curve (AUC) was calculated.

[0120] 2.4 Intraperitoneal insulin tolerance test (IPITT)

[0121] Each model mouse was fasted but not watered for 6 h, and insulin (1 U / kg) was injected intraperitoneally. Blood was collected from the tail vein at 0, 15, 30, 60, 90, and 120 min after injection. Blood glucose concentration was measured with a portable blood glucose meter. The blood glucose curves of each group at each time point were plotted, and the AUC was calculated.

[0122] 2.5 Sample Collection

[0123] 2.5.1. Collection of stool and serum samples

[0124] When each model mouse was fed with a high-fat diet for 10-11 weeks, fecal samples were collected at 10:00 AM every day, quickly placed in liquid nitrogen, and then transferred to -80°C for 7-10 consecutive days.

[0125] Blood samples were collected from the orbital vein of each model mouse and placed at 4°C for 1 hour, followed by centrifugation at 4°C and 3000 rpm for 15 minutes. The upper serum was taken and the above centrifugation operation was repeated to finally obtain serum samples, which were stored at -80°C.

[0126] 2.5.2. Collection of liver and epididymal adipose tissue samples

[0127] Each model mouse was killed by cervical dislocation, the surgical area was disinfected with 75% alcohol, the abdominal cavity was opened, and the complete liver and part of the epididymal adipose tissue were immediately separated and placed in ice-cold Kreb's solution. The surface moisture was absorbed with filter paper, and the liver was weighed and then placed in paraffin embedding boxes respectively. It was fixed with 10% neutral formalin for 48 hours in preparation for hematoxylin-eosin (H&E) staining.

[0128] 2.5.3. Intestinal sample collection

[0129] Each model mouse was killed by cervical dislocation, the surgical area was disinfected with 75% alcohol, the abdominal cavity was opened, the intestinal tissue was separated from the pylorus to the cecum, the mesentery around the intestine was removed as much as possible, and the intestinal length was measured from the pylorus. The 10-10.5 cm after the pylorus was cut for tissue transmission electron microscopy detection; the 10.5-11.5 cm after the pylorus was cut for high performance liquid chromatography (HPLC) detection; the 11.5-15.5 cm after the pylorus was cut for immunofluorescence detection, and another part of the samples was used for short-circuit current detection; the 15.5-19.5 cm and 19.5-23.5 cm after the pylorus were cut for RT-qPCR and Western blotting to detect the mRNA and protein expression levels of each functional protein. The above-mentioned intestinal tissues were placed in ice-cold Kreb's solution. For intestinal tissues used for cryo-TEM detection, the intestine was cut open longitudinally, rinsed with ice-cold Kreb's solution to remove the residue in the intestinal cavity, and the water stains were absorbed by filter paper and immediately placed in a mixture of 2% paraformaldehyde (PFA) + 2.5% glutaraldehyde (GA) for fixation for 24 hours and stored at 4°C; for intestinal tissues used for immunofluorescence detection, the residue in the intestinal cavity was slowly washed out with ice-cold Kreb's solution, and care was taken not to damage the intestinal structure. After the water stains were absorbed by filter paper, it was immediately placed in 10% neutral formalin for fixation for 48 hours; for the remaining intestinal tissues, the intestine was cut open longitudinally, rinsed with ice-cold Kreb's solution to remove the residue in the intestinal cavity, and the intestinal epithelial tissue was scraped with a broken piece, quickly frozen in liquid nitrogen, and then transferred to -80°C.

[0130] 2.6 Hematoxylin-eosin (H&E) staining

[0131] After killing the mice by cervical dislocation, the abdominal cavity was opened, and the freshly separated liver tissue and epididymal fat tissue were taken out and placed in ice-cold Kreb's solution. The water stains were absorbed by filter paper, and then placed in a paraffin embedding box and fixed with 10% neutral formalin for 48 hours. Then, the following operations were carried out in sequence: dehydration and wax immersion → embedding and sectioning → dewaxing and hydration → H&E staining → dehydration, transparency and sealing → scanning → using ImageJ software to analyze and process the scanned images to obtain the area of ​​fat cells.

[0132] 2.7 Immunofluorescence staining of paraffin sections

[0133] (1) Dewaxing and hydrating: Place the paraffin sections in a 60°C oven for 2 h, and then perform the following operations in sequence: dewaxing and hydrating → antigen repair → blocking → tissue permeabilization → primary antibody incubation → secondary antibody incubation → DAPI staining of cell nuclei → sealing → scanning → image result export (using cellSens Dimension software for export) → Tiff image processing (using ImageJ software to analyze and process Tiff images). Under a high-power microscope (20×), three fields of view were randomly selected for each sample, and the number of positive cells per unit area was counted.

[0134] 3. RT-PCR experiment

[0135] 3.1. Extraction of total RNA from tissues

[0136] Using FastRNA from MP Biomedicals TM The SPIN Kit for Yeast was used to extract RNA from jejunal intestinal epithelial tissue. For specific procedures, please refer to the kit instructions.

[0137] 3.2. RNA concentration detection

[0138] The RNA concentration was detected using an ultra-micro high-precision nucleic acid protein concentration meter.

[0139] 3.3 RNA integrity detection

[0140] Gel electrophoresis was used to detect the integrity of RNA.

[0141] 3.4. mRNA Reverse Transcription-cDNA Synthesis

[0142] The extracted mRNA was reverse transcribed into cDNA using the FastKing RTKit (With gDNase) kit from China Tiangen Biotechnology Co., Ltd. The experimental operation was carried out according to the instructions. The cDNA solution obtained by reverse transcription was stored in a -20°C / -80°C refrigerator for subsequent experiments.

[0143] 3.5 PCR amplification reaction

[0144] RT-PCR and RT-qPCR experimental primer design: Primers were designed online using Blast-Primer on the NCBI website. Primer synthesis was completed by Beijing Aoke Dingsheng Company. The designed primers are shown in Table 4 below. The RT-PCR experimental reaction amplification system is shown in Table 4 below.

[0145] Table 4. Mouse RT-PCR and RT-qPCR primers

[0146]

[0147] Table 5. RT-PCR experimental reaction amplification system

[0148]

[0149]

[0150] Vortex the components in Table 5, centrifuge briefly and then use in the next PCR amplification reaction.

[0151] 3.6. RT-PCR Amplification Conditions

[0152] (a) Pre-denaturation: 98℃, 5min; (b) Denaturation: 98℃, 30s. Annealing: 59℃, 30s; Extension: 72℃, 45s. This step is cycled 40 times. (c) Post-extension: 72℃, 5min; (d) The product can be used for agarose gel electrophoresis or stored in a 4℃ refrigerator.

[0153] 3.8 qPCR amplification reaction

[0154] The mRNA extraction steps and mRNA reverse transcription-cDNA synthesis steps in the experiment are the same as before, see above for details, and the qPCR experimental reaction amplification system is shown in Table 6 below.

[0155] Table 6. qPCR experimental reaction amplification system

[0156]

[0157] After preparing the reaction system according to Table 6, use a vortex oscillator to fully vortex mix, and briefly centrifuge, then dispense 20 μL per tube into 8-row PCR tubes (set 2 replicate wells for each sample, and set a set of negative controls without DNA for each primer), cover the 8-row tube caps with clean PE gloves (avoid direct contact of the skin or rubber gloves with the middle transparent area to avoid affecting the fluorescence signal reading), briefly centrifuge on an 8-row centrifuge, ensure that there are no bubbles in the tube, and then use it for the next experiment.

[0158] 3.9. RT-qPCR Amplification Conditions

[0159] Place the 8-tube strips into the ABI Steponeplus PCR instrument and set the qPCR experimental reaction conditions:

[0160] ① Pre-denaturation: 50℃, 2min, 95℃, 2min. ② Denaturation: 95℃, 15s. Annealing: 60℃, 1min. This step was repeated 40 times.

[0161] 4. Western Blot Experiment

[0162] 4.1. Extraction of tissue proteins

[0163] 4.1.1. Extraction of total protein from intestinal epithelial tissue

[0164] (a) For freshly isolated tissue samples or tissue samples frozen in liquid nitrogen, add 400 μL ice-cold RIPA working solution to about 50-70 mg of jejunal epithelial tissue, use a fast sample preparation instrument (speed 5 m / s, 20 s), place on ice for 5 min, and repeat 3 times. (b) Then place on ice for lysis for 50 min. (c) Centrifuge at 12000 rpm at 4°C for 15 min. (d) Transfer the supernatant to a new EP tube. The protein sample can be tested for concentration using the BCA method or stored in a -80°C refrigerator.

[0165] 4.2. BCA method to detect protein concentration

[0166] 4.2.1. Detection of sample protein concentration

[0167] Thermo Scientific TM Pierce TM BCA protein detection kit, the extraction process was carried out according to the instructions.

[0168] 4.2.2 Protein denaturation

[0169] After mixing the protein sample and 5× loading buffer at a ratio of 4:1, place the sample in a metal bath at 100°C for 10 min to fully denature the protein sample. The denatured sample is stored in a -20°C refrigerator.

[0170] 4.3. Western Blot Electrophoresis and Immunoreaction

[0171] Perform electrophoresis at a constant voltage of 80 V for 40 min. When the protein sample enters the separation gel and the protein marker band is clear, increase the voltage to 120 V and continue constant voltage electrophoresis until the bromophenol blue dye reaches the bottom of the separation gel. Then prepare 6 filter papers and 0.45 μm PVDF membrane for transfer. After the transfer, perform immune reaction.

[0172] 5. Genomic DNA extraction and metagenomic sequencing of fecal samples

[0173] 5.1. Extraction of genomic DNA from stool samples

[0174] MagBeads from MP Biomedicals, USA Kit for Feces was used to extract genomic DNA from stool samples. For specific procedures, see the instruction manual.

[0175] 5.2 DNA concentration detection

[0176] DNA concentration was detected by ultra-micro high-precision nucleic acid protein concentration meter combined with NanoDrop software Nucleic Acid, and DNA concentration was detected using QubitTM 1×dsDNA HS Assay Kits from Thermo Fisher, USA. The process was carried out according to the instructions.

[0177] 5.3 DNA integrity test

[0178] Gel electrophoresis was used to detect the integrity of DNA.

[0179] 5.4 Metagenomic sequencing library construction

[0180] Native barcoding genomic DNA (with EXP-NBD104, EXP-NBD114, and SQK-LSK109) from Oxford Nanopore Technologies Ltd., UK, was used to construct the metagenomic sequencing library.

[0181] 6. Targeted metabolome analysis of bile acids

[0182] The metabolite extraction, on-machine detection and data analysis of bile acid targeted metabolomics analysis were completed by Shanghai Aqu Biotechnology Co., Ltd.

[0183] 7. High Performance Liquid Chromatography

[0184] The target compound was chromatographed using a Waters e2695 / 2475 high performance liquid chromatograph and a Waters SunFire C18 (150*4.6mm, 5μm, Waters) liquid chromatography column. The liquid chromatography phase A was a buffered salt solution, and the phase B was methanol (phase A: phase B = 12.8:87.2). The column oven temperature was 35°C, the sample tray was set at 25°C, and the injection volume was 10μL.

[0185] 8. Cryo-TEM

[0186] Transmission electron microscopy sample preparation was completed by the electron microscopy room of the cell platform of the Biomedical Testing Center of Tsinghua University. Sample photography was completed in the electron microscopy room of the cell platform of the Biomedical Testing Center of Tsinghua University. The results were statistically analyzed using ImageJ software.

[0187] 9. Observation of jejunal mesenteric lacteals under stereoscope

[0188] Mice were fasted for 14 h, given 200 μL of milk by gavage, and fed a high-fat diet containing 60% calories from fat for 6 h. After euthanasia and laparotomy, the mesenteric lacteals around the jejunum were exposed and images were acquired using an optical microscope. The number of lacteals filled with chylomicrons was counted using ImageJ software.

[0189] 10. Serum sample testing

[0190] 10.1. Serum 5-HT content detection

[0191] The 5-HT enzyme-linked immunosorbent assay kit (Huaying, Beijing) was used to detect 5-HT in mouse serum samples, and the detection process was carried out according to the instructions.

[0192] 10.2. Serum biochemical index detection

[0193] Triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL) in mouse serum samples

[0194] and low-density lipoprotein (LDL) were tested according to the kit instructions of Beijing Huaying Biotechnology Research Institute.

[0195] 11. Short circuit current

[0196] Take 1-2cm of freshly separated small intestine and put it in ice-cold Kreb's solution to maintain tissue activity. Cut it open along the mesentery and separate the serosal layer and muscular layer under a microscope, and keep the mucosal layer (with submucosal layer attached). The mucosal specimen is vertically placed in a constant temperature perfusion incubation system with an area of ​​0.3cm2 slide. 5mL of Kreb's solution is injected on both sides, and a mixture of 95% oxygen and 5% carbon dioxide is introduced at the same time. The specimen is placed in a 37℃ water bath for 30min to stabilize the electrical parameters. The voltage clamp fixes the epithelium at zero potential, forming a short circuit on both sides of the tissue. The transepithelial current measured at this time is the short-circuit current. Through automatic voltage pulse (ΔVt) stimulation, observe the change in membrane current (ΔIt), and calculate the membrane resistance (Rt) according to Ohm's law Rt=ΔVt / ΔIt. The change in transmembrane current before and after stimulation is expressed in the form of current passing through the unit area of ​​the mucosa (μA / cm2) or current area (μA×min).

[0197] 12. Mouse tail genotyping

[0198] 12.1. Sample processing steps

[0199] (a) High-pressure equipment: several 1.5 mL EP tubes, one pair of ophthalmic scissors and one pair of small tweezers.

[0200] (b) Restrain the mouse, gently cut the distal 1-2 mm of the mouse tail and place it in a 1.5 mL EP tube, add 200 μL of mouse tail lysis buffer and 10 μL of proteinase K, and shake in a metal bath at 55°C overnight.

[0201] (c) The next day, take out the EP tube from the metal bath, vortex mix and centrifuge, then put it back into the metal bath and shake at 100°C for 12 min.

[0202] (d) Cool to room temperature, prepare the PCR reaction system according to Table 7 below, vortex mix, briefly centrifuge and then use in the next PCR amplification reaction.

[0203] Table 7. RT-PCR experimental reaction amplification system

[0204] Element Dosage 2×TaqPCR premix 12.5μL ForwardPrimer 1μL(10μM) ReversePrimer 1μL(10μM) cDNA 1.5μL RNase-free deionized water Make up to 25 μL

[0205] (e) RT-PCR amplification conditions

[0206] Pre-denaturation: 94℃, 3min; Denaturation: 94℃, 30s. Annealing: 62℃, 35s. Extension: 72℃, 35s. This step was cycled 40 times. Post-extension: 72℃, 5min. The product can be used for agarose gel electrophoresis experiment or stored in a 4℃ refrigerator, and the primer identification results of the product are shown in Table 8 below.

[0207] Table 8. Mouse tail genotyping primers

[0208]

[0209] 13. Transcriptomics experiments

[0210] Transcriptomics library construction and testing were completed by Beijing Novogene Technology Co., Ltd.

[0211] 14. Recovery, passaging and cryopreservation of normal human colon mucosal epithelial cells NCM 460

[0212] 14.1. Recovery of NCM 460 cells

[0213] 14.2 Passaging of NCM 460 cells

[0214] 15. NCM 460 Cell Calcium Imaging

[0215] 15.1 Calcium Imaging Experimental Procedure

[0216] (a) NCM 460 cells were seeded into Confocal dishes, and Fluo-4AM staining was performed when the cells grew to 80% confluence. (b) The original culture medium was discarded, and the cells were gently washed twice with D-PBS. (c) Fluo-4AM working solution was added, and the cells were incubated at 37℃ and 5% CO2 for 30min. (d) The liquid was discarded, the cells were gently washed once with D-PBS, and 2mL of 4.5g / L Glucose DMEM was added, and the cells were incubated at 37℃ and 5% CO2 for 20min. (e) The Confocal dish was placed under a laser scanning confocal microscope (OLYMPUS FLUOVIEW FV3000), and real-time image acquisition (excitation wavelength 488nm) was performed every 3.22s to record the changes in fluorescence signals. Images of 30s-60s were collected before drug addition as a control.

[0217] 2. Experimental Results

[0218] 1. Differences in glucose and lipid metabolism characteristics, intestinal flora and their metabolites in the nutritional obesity mouse model

[0219] 1.1 Screening and construction of nutritional obesity mouse model

[0220] 1.1.1. Body weight and body composition of nutritionally obese mouse models

[0221] The present invention gave 8-week-old male C57BL / 6J mice the same high-fat diet for 12 weeks, and found that under the conditions of no significant difference in food intake and water intake [(HIGH Food intake: 3.275±0.7884, n=4), (LOW Food intake: 3.280±1.085, n=4), (HIGH water intake: 3.545±0.5183, n=4), (LOW water intake: 3.470±0.4999, n=4)], the weight changes of mice were inconsistent ( Figure 1 A). The present invention divides mice with inconsistent weight changes into a high weight growth group (HIGH) and a low weight growth group (LOW) ( Figure 1 B and 1C). The results of body composition test showed that compared with the HIGH group, the fat mass of the mice in the LOW group was significantly reduced, and the lean body mass (LBM) was significantly increased [(HIGH Fat: 46.38±1.608, n=5), (LOW Fat: 29.85±1.330, n=5), (HIGH LBM: 53.62±1.608, n=5), (LOWLBM: 70.15±1.330, n=5)]( Figure 1 D).

[0222] 1.1.2. Glucose tolerance and insulin tolerance tests in the nutritionally obese mouse model

[0223] In order to clarify whether there is a difference in the glucose metabolism phenotype between the HIGH and LOW groups of mice, the present invention conducted an intraperitoneal glucose tolerance test (IPGTT) and an intraperitoneal insulin tolerance test (IPITT) on the HIGH and LOW groups of mice at the 11th week of HFD administration. The results of the IPGTT test showed that compared with the HIGH group, the blood glucose levels of the LOW group of mice at 60 minutes, 90 minutes and 120 minutes after glucose administration were significantly lower ( Figure 2 A). The area under the glucose tolerance curve (AUC) results showed that the glucose tolerance of the HIGH group mice was impaired compared with that of the LOW group mice, and the glucose tolerance of the LOW group mice was significantly improved compared with that of the HIGH group mice [(HIGH: 28377±3394, n=10), (LOW: 20561±2290, n=10)]( Figure 2 B). The results of IPITT test showed that compared with the HIGH group, the blood glucose level of the LOW group mice was lower at 30 minutes after insulin administration ( Figure 2 C), the area under the insulin tolerance curve (AUC) decreased accordingly [(HIGH: 25170±553.6, n=5), (LOW: 21750±1403, n=3)] ( Figure 2 D) The results showed that the insulin sensitivity of mice in the LOW group returned to normal and the obesity phenotype was not obvious.

[0224] 1.1.3 Phenotypic detection of target organs of fat metabolism in the nutritionally obese mouse model

[0225] In order to clarify the difference in lipid metabolism between the HIGH and LOW groups of mice, the weight of the liver was counted. The results showed that compared with the LOW group of mice, the liver weight of the HIGH group of mice was significantly increased, and there was obvious fat accumulation in the liver [(HIGH: 2.425±0.1256, n=5), (LOW: 1.378±0.1246, n=5)]( Figure 3 A, B), and further H&E staining results showed that the liver tissue structure of mice in the LOW group was clear and the morphology was normal, while the liver parenchymal cells of mice in the HIGH group showed fatty degeneration, forming lipid droplet vacuoles, and the fatty liver phenotype was obvious ( Figure 3 D). H&E staining and statistical results of epididymal adipose tissue showed that the number of adipocytes in large areas of mice in the HIGH group increased significantly ( Figure 3C, E). Even if they ate the same diet, the mice responded differently to the high-fat diet. The HIGH group mice gained significant weight, had impaired glucose tolerance, decreased insulin sensitivity, and showed obvious obesity phenotypes in the liver and adipose tissue, while the LOW group mice did not have the above phenomenon, indicating that under the same feeding conditions, compared with the HIGH group mice, the LOW group mice did not show obesity phenotypes in the liver and epididymal adipose tissue.

[0226] 1.2 Fecal microbiota transplantation (FMT) experiment in a mouse model of nutritional obesity

[0227] 1.2.1 Effects of FMT on body weight and body composition in a mouse model of nutritional obesity

[0228] The results of the FMT experiment showed that starting from the 7th week of high-fat diet feeding, there was a difference in body weight between the high-weight gain group mice that underwent fecal microbiota transplantation (HIGH FMT) and the low-weight gain group mice that underwent fecal microbiota transplantation (LOW FMT). Figure 4 A). MRI examination of the body composition of mice revealed that compared with LOW FMT mice, the body fat mass of HIGH FMT mice increased significantly ( Figure 4 B). The intestinal microbiota of mice with different obese phenotypes has a regulatory effect on reconstructing the obese phenotype of wild-type mice, that is, the intestinal microbiota can regulate the obese phenotype.

[0229] 1.2.2 Effect of FMT on glucose tolerance in a mouse model of nutritional obesity

[0230] In the 10th week of HFD, the present invention conducted an IPGTT test on the two groups of mice, and the results showed that the blood glucose level of the mice in the LOW FMT group was significantly lower than that in the HIGH FMT group ( Figure 5 A), The area under the glucose tolerance curve (AUC) results showed that the glucose tolerance of mice in the LOW FMT group was significantly improved compared with that in the HIGHFMT group ( Figure 5 B).

[0231] 1.2.3 Effects of FMT on the phenotype of target organs of fat metabolism in the nutritionally obese mouse model

[0232] The present invention dissected the mice, freed the intact liver and counted the liver weight. The results showed that compared with the LOWFMT group mice, the livers of the HIGH FMT group mice had a slight fat accumulation phenotype and a significantly increased weight [(HIGH FMT: 1.906±0.09970, n=4), (LOW FMT: 1.582±0.03768, n=3)]( Figure 6A, B), and further H&E staining results showed that the liver tissue structure of mice in the LOW FMT group was clear and normal in morphology, while the liver parenchymal cells of mice in the HIGH FMT group showed partial fatty degeneration and formed smaller lipid droplet vacuoles ( Figure 6 D). H&E staining was also performed on epididymal adipose tissue, and statistically it was found that the number of small-area adipocytes in the LOW FMT group was greater than that in the HIGH FMT group ( Figure 6 C, E), indicating that lipid accumulation in epididymal adipose tissue of mice in the LOW FMT group was reduced. The above results suggest that the intestinal flora of mice with different obesity phenotypes has an important regulatory effect on lipid metabolism.

[0233] 2. Screening of Different Intestinal Bacteria between HIGH and LOW Groups

[0234] 2.1 Metagenomic analysis of intestinal flora of mice in the HIGH and LOW groups

[0235] In order to clarify the specific differences in the intestinal flora of mice with different obesity phenotypes, the present invention collected feces from the HIGH and LOW groups of mice and extracted fecal DNA for metagenomic analysis. Principal Component Analysis (PCA) means that the more similar the community structure of the samples is, the closer their distance in the PCoA graph is. Figure 7 As shown in A, the points of the HIGH group and the LOW group are partially separated, indicating that there are differences in the intestinal flora structure between the HIGH and LOW groups, and the similarity is low. The advantage of non-metric multi-dimensional scaling (NMDS) is that it corrects the linear model shortcomings of PCoA and can better reflect the nonlinear structure of ecological data. Figure 7 As can be seen from B, the points of the HIGH and LOW groups are not completely separated, which also suggests that there are some differences in the intestinal flora between the HIGH and LOW groups, that is, the similarity of the intestinal flora composition of the two groups of mice is low.

[0236] The top five bacterial species with the largest abundance at the phylum level in each group were further selected. The results are shown in Table 9 below. A species relative abundance bar graph was generated. The results showed that the differential bacteria were mainly concentrated in the following five phyla, namely Verrucomicrobia, Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Among them, Firmicutes and Bacteroidetes had the highest content in the intestinal flora of the two groups of mice, accounting for more than 50%.

[0237] Table 9 Distribution of bacterial content at the phylum level

[0238] bacteria HIGH (percent) LOW(percent) Verrucomicrobia 0.030 0.056 Actinobacteria 0.117 0.095 Proteobacteria 0.260 0.240 Firmicutes 0.308 0.225 Bacteroidetes 0.285 0.385

[0239] The results of the metagenomic analysis of the present invention indicate that the change in the proportion of Firmicutes and Bacteroidetes is closely related to obesity. At the same time, Firmicutes accounted for the highest proportion in the HIGH group, and Bacteroidetes accounted for the highest proportion in the LOW group ( Figure 7 C). In order to further clarify the detailed bacterial genus under this phylum, the present invention selects the top 10 species with the largest abundance at the genus level in each group, as shown in Table 10 below, and generates a species relative abundance bar graph. The results show that the following three genera in the Firmicutes phylum of differential bacteria have high expression abundance, namely Clostridium, Lachnoclostridium and Lactobacillus; there are mainly changes in the following two genera in the Bacteroidetes phylum, namely Alistipes and Bacteroides ( Figure 7 D).

[0240] Table 10 Distribution of bacterial content at genus level

[0241] bacteria HIGH (percent) LOW(percent) Enterobacter 0.024 0.034 Akkermansia 0.055 0.101 Clostridium 0.060 0.031 Streptomyces 0.044 0.037 Pseudomonas 0.052 0.043 Lactobacillus 0.105 0.041 Bifidobacterium 0.103 0.044 Lachnoclostridium 0.066 0.043 Alistipes 0.079 0.092 Bacteroides 0.412 0.535

[0242] At the same time, the LDA value distribution bar chart of metagenomic sequencing (LEfSe analysis) shows the different bacterial communities (i.e., dominant bacterial communities) between the groups. Among them, one-third of the top 30 dominant bacterial species in the HIGH group were species of the genus Clostridium, while among the top 30 dominant bacterial species in the LOW group, there were several species of the genus Bacteroidetes ( Figure 7E). The above results show that there are indeed significant differences in the intestinal flora of HIGH and LOW mice with different obesity phenotypes at the genus and species level. The HIGH group mice expressed high abundance of Firmicutes species: Lactobacillus johnsonii, Lactobacillus reuteri, Clostridium botulinum, Clostridium chauvoei, Clostridium bolteae, Clostridium beijerinckii, Clostridium butyricum, Clostridium saccharolyticum, Clostridium perfringens, Clostridium baratii, etc. The LOW group mice expressed high abundance of Bacteroides species including Bacteroides dorei, Bacteroides vulgatus, Alistipesshahii, Bacteroides ovatus, Parabacteroidesdistasonis, Bacteroides cellulosilyticus, Bacteroides helcogenes, Porphyromonas levii, and Bacteroides sartorii.

[0243] 2.2. Complementation experiment of high-abundance expression in the LOW group B. vulgatus strains

[0244] 2.2.1 Effects of different B. vulgatus strains on body weight and body composition

[0245] In order to verify whether the high-abundance intestinal differential B. vulgatus strains in the LOW group found in metagenomic analysis have a negative regulatory effect on the obesity phenotype, the present invention conducted an in vivo differential B. vulgatus strain complementation experiment. The present invention found that compared with the control group, the weight of mice was significantly reduced in the 5th week of B. vulgatus complementation, and it lasted until the 12th week of high-fat diet feeding ( Figure 8A, B), which suggests that the B. vulgatus strain supplementation of the present invention can improve obesity induced by a high-fat diet. Body composition detection found that compared with the control group, the fat mass of the mice in the B. vulgatus strain supplementation group was significantly reduced, and the lean mass (the sum of muscle and free water) was significantly increased.

[0246] 2.2.2 Effects of B. vulgatus strains on glucose tolerance and insulin tolerance

[0247] The present invention conducted IPGTT and IPITT tests in the 11th week of HFD administration. The results of the IPGTT test showed that compared with the control group, the blood glucose level of the supplemented B. vulgatus strain was significantly reduced at 30 minutes after glucose administration ( Fig. 9 A). The results of the area under the glucose tolerance curve (AUC) showed that supplementation with B. vulgatus strain significantly improved the glucose tolerance of mice ( Fig. 9 B). The results of the IPITT test showed that after the B. vulgatus strain was supplemented, the blood glucose level showed a downward trend from 60 to 120 minutes after the administration of insulin, and it was most obvious at the 90th minute ( Fig. 9 C). The area under the insulin tolerance curve (AUC) also decreased accordingly ( Fig. 9 D) The above results indicate that the glucose tolerance of mice supplemented with B. vulgatus strain was significantly improved compared with the control group.

[0248] 2.2.3 Effects of B. vulgatus strains on target organs of fat metabolism

[0249] The present invention then dissected the mice, freed the complete liver and counted the liver weight. The results showed that there was fat accumulation in the liver of the control group mice, and the phenotype of liver fat accumulation was significantly improved after the B. vulgatus strain was supplemented ( Fig.10 A), and the liver weight was significantly reduced compared with the control group ( Fig.10 B). H&E staining of the liver tissues of the two groups of mice revealed that the liver cells of the control group mice had fat accumulation, lipid droplet vacuoles, and obvious fatty liver phenotypes, while the liver cells of the mice in the B. vulgatus strain complementation group had a relatively normal structure, complete morphology, fewer lipid droplet vacuoles, and tended to be normal liver cells ( Fig.10 D). This result indicates that supplementation with B. vulgatus strains has a protective effect on liver damage in HFD mice. The results of H&E staining of epididymal adipose tissue showed that the number of small-area adipocytes in the B. vulgatus strain supplementation group mice increased significantly ( Fig.10C, E), indicating that the lipid accumulation in the epididymal adipose tissue of mice was reduced after B. vulgatus strain complementation. The above results suggest that complementation of B. vulgatus strains can control body weight gain and inhibit obesity induced by a high-fat diet.

[0250] 2.2.4 Effects of B. vulgatus strains on blood lipids

[0251] In order to clarify whether the B. vulgatus strain has a regulatory effect on the blood lipids of HFD mice, the present invention detected the serum biochemical indicators of mice at the 12th week of supplementation with B. vulgatus strain and HFD. The results showed that compared with the control group, the serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) of mice supplemented with B. vulgatus strain were significantly reduced ( Fig.11 AD). The results suggest that B. vulgatus strain has the effect of lowering blood lipids, further illustrating its negative regulatory effect on obesity caused by a high-fat diet.

[0252] 2.3. Complementation experiment of the highly abundant differentially expressed strain C. beijerinckii in the HIGH group

[0253] 2.3.1 Effects of different strains of C. beijerinckii on body weight and body composition

[0254] Similarly, the present invention found in the metagenomic screening that the high-abundance strains in the HIGH group were mostly Clostridium bacteria, so the present invention selected Clostridium beijerinckii, a high-abundance Clostridium strain in the HIGH group in the metagenomic screening, for differential strain complementation experiments. The present invention found that compared with the control group, there was no significant difference in the weight gain of mice after C. beijerinckii complementation ( Fig.12 A, B), which suggests that the supplemented strain C.beijerinckii has no significant effect on promoting body weight. Body composition detection found that compared with the control group, the fat mass of mice in the C.beijerinckii supplemented group increased, and the lean mass (the sum of muscle and free water) decreased ( Fig.12 C).

[0255] 2.3.2 Effect of C. beijerinckii strains on glucose tolerance

[0256] The present invention conducted an IPGTT test in the 11th week of HFD administration. The IPGTT test results showed that compared with the control group, the complement strain C.beijerinckii had no significant difference in the change of blood sugar level after glucose administration. Fig.13A). The area under the glucose tolerance curve (AUC) results showed that the glucose tolerance of mice supplemented with the C. beijerinckii strain did not change ( Fig.13 B) The above results suggest that supplementation with C. beijerinckii strain has no significant effect on the glucose metabolism of mice, indicating that the obesity-inducing phenomenon of supplementation with C. beijerinckii strain is not obvious.

[0257] 2.3.3 Effects of C. beijerinckii on target organs of fat metabolism

[0258] Subsequently, the present invention counted the liver weights, and found that there was no significant difference in the liver weights of mice in the control group and the C. beijerinckii strain-supplemented group, which was consistent with the body weight and glucose tolerance results of the C. beijerinckii strain-supplemented group mentioned above. The above results indicate that the phenotypic changes of obesity after the C. beijerinckii strain-supplemented group were not obvious. The present invention considers that the possible reason is that the mice were given a high-fat diet containing 60% fat for 12 weeks, which had caused the mice to show a more obese phenotype. On this basis, it was difficult to obtain a more obese phenotype after the C. beijerinckii strain-supplemented group was given a high-fat diet containing 60% fat.

[0259] 3. Screening of metabolites in fecal samples of mice in the HIGH and LOW groups

[0260] 3.1 Targeted metabolomics analysis of fecal metabolites in mice in the HIGH and LOW groups

[0261] The present invention collected fecal samples from the HIGH and LOW groups of mice for targeted metabolomics analysis of bile acids. Comparing the PCA analysis of the HIGH and LOW groups of mice, it can be seen that the composition of bile acids in the fecal samples of the two groups of mice is different, and all samples are within the 95% confidence interval ( Fig.15 A). The results of differential bile acid were visualized in the form of a volcano plot. Each point in the volcano plot represents a metabolite. The horizontal axis represents the differential expression fold [log2 (Fold change)]. A value greater than 0 indicates that the metabolite is upregulated, and a value less than 0 indicates that the metabolite is downregulated; the vertical axis represents the significance of the difference [-log10 (p-value)]. The larger the vertical axis value, the smaller the P value and the greater the difference. Compared with the control HIGH group, metabolites with reduced expression are located to the left of zero on the X-axis, while metabolites with increased expression are located to the right of zero. Metabolites with statistically significant differential expression are above the horizontal threshold. Therefore, red scatter points represent metabolites that are significantly upregulated in the HIGH group compared with the LOW group, and blue represents metabolites that are significantly downregulated in the HIGH group compared with the LOW group. Among them, 11 metabolites are upregulated and 25 metabolites are downregulated ( Fig.15 B). Cluster analysis of differential bile acids ( Fig.15C), the horizontal axis represents different experimental groups, and the vertical axis represents the differential metabolites compared in the group. Red represents metabolites with a high expression trend, and blue represents metabolites with a low expression trend. The results show that primary bile salt is highly expressed in the LOW group, and the main components are α-Muricholic Acid (α-MCA), β-Muricholic Acid (β-MCA), Cholic Acid (CA), Ursodeoxycholic Acid (UDCA) and Chenodeoxycholic Acid (CDCA). The above results show that compared with the HIGH group, the primary bile salt content of mice in the LOW group is higher, which is also consistent with the results of metagenomic analysis in the previous article. The high abundance of Bacteroides in the LOW group is closely related to the high content of primary bile salt in the LOW group. That is, for mice in the HIGH and LOW groups with different obese phenotypes, the high abundance of Bacteroides in the LOW group will participate in the regulation of the obese phenotype by increasing the content of its metabolite primary bile salt, inhibiting the occurrence and development of obesity.

[0262] 3.2 Replenishment experiment of bile acid, a differential metabolite in the intestine

[0263] In order to verify that the Bacteroides metabolite CA has a regulatory effect on the obesity phenotype, the present invention selected the primary bile salt CA highly expressed in the LOW group to conduct an in vivo differential metabolite replenishment experiment.

[0264] 3.2.1 Effects of bile acid metabolites on body weight and body composition

[0265] The present invention found that compared with the HFD group, the body weight of mice in the bile acid CA supplementation group (HFD+CA, HCA) was significantly reduced at the second week, which lasted until the 12th week of high-fat diet feeding ( Fig.16 A), and body composition detection found that compared with the HFD group, the fat mass of the HCA group mice was significantly reduced [(HFD Fat: 35.23±0.5324, n=5), (HCAFat: 29.55±1.230, n=9), (HFD LBM: 30.94±0.5568, n=5), (HCALBM: 34.71±1.154, n=9)]( Fig.16 B) The above results indicate that supplementation of CA can inhibit the increase of body weight and body fat in mice induced by a high-fat diet.

[0266] 3.2.2 Effect of metabolite CA on glucose tolerance

[0267] Then, the present invention conducted an IPGTT test on the two groups of mice in the 11th week of HFD. The results of the IPGTT test showed that compared with the HFD group, the blood glucose levels of the HCA group mice at 60min, 90min and 120min after glucose administration were significantly lower ( Fig.17 A). The results of the area under the glucose tolerance curve (AUC) showed that the HFD group showed impaired glucose tolerance, and bile acid significantly improved the glucose tolerance of HFD mice [(HFD: 48816±2917, n=5), (HCA: 34056±881.4, n=9)]( Fig.17 B).

[0268] 3.2.3 Effects of metabolite CA on target organs of fat metabolism

[0269] In order to further observe the effect of bile acid on the target organs of fat metabolism, the present invention performed H&E staining on the liver tissues of the two groups of mice. H&E staining of liver tissue sections of mice in the HFD group showed fatty degeneration of hepatic parenchymal cells, the formation of lipid droplet vacuoles, and obvious fatty liver phenotype; the liver tissue structure of mice in the HCA group was clear, the morphology was normal, the vacuoles were significantly reduced, and the liver cells tended to be normal ( Fig.18 A), which indicates that bile acid has a protective effect on liver damage in HFD mice. The results of H&E staining of epididymal adipose tissue showed that the number of small-area adipocytes in HCA group mice increased ( Fig.18 B, C), indicating that lipid accumulation in epididymal adipose tissue of mice was reduced after CA replenishment. The above results suggest that bile acid can inhibit the increase of body weight and body fat induced by high-fat diet.

[0270] 4. Effect of peripheral 5-HT synthesis on obesity

[0271] 4.1. Enterochromaffin cells and 5-HT expression in nutritional obesity model mice

[0272] In order to clarify whether peripheral 5-HT has a regulatory effect on obese model mice, the present invention first detected the expression of 5-HT in the jejunum tissue of nutritional obesity model mice by immunofluorescence experiment. Fig.19 It can be seen that after high-fat feeding, the number of enterochromaffin cell marker chromogranin A (CgA) in the jejunal mucosa of mice in the HIGH group increased compared with that in the LOW group ( Fig.19 A, B), and the 5-HT positive cells also increased significantly ( Fig.19 A, C), that is, obesity increases the number of enterochromaffin cells and 5-HT expression in the jejunal mucosal layer. Then, the present invention detected the content of 5-HT in the jejunal tissue and serum of each model mouse by HPLC experiment and Elisa experiment. The results are as follows: In the nutritional obesity mouse model, compared with the HIGH group, the content of 5-HT in the jejunal tissue and serum of the LOW group was reduced ( Fig.19 D, E), further suggesting that peripheral 5-HT synthesis is closely related to the obesity phenotype.

[0273] 4.2 Effects of B. vulgatus strains on enterochromaffin cells and 5-HT

[0274] In order to clarify whether B. vulgatus strains regulate intestinal 5-HT, the present invention detected the expression of 5-HT in the jejunal tissue of B. vulgatus complemented mice by immunofluorescence experiment. The results showed that after high-fat feeding, the number of enterochromaffin cell marker CgA in the jejunal mucosa layer of B. vulgatus complemented mice was significantly reduced compared with the control group ( Fig. 20 A, B), and the 5-HT positive cells also decreased significantly ( Fig. 20 A, C); The levels of 5-HT in the jejunum tissue and serum of mice in the B. vulgatus strain complementation group were lower than those in the control group, which further demonstrated that the B. vulgatus strain complementation of mice fed high-fat diet inhibited the peripheral 5-HT content and had a regulatory effect on the synthesis of 5-HT.

[0275] 4.3 Effects of metabolite CA on enterochromaffin cells and 5-HT

[0276] In order to clarify whether the metabolite CA in the bile acid metabolic pathway also regulates the synthesis of peripheral 5-HT, the present invention detected the expression of 5-HT in the jejunal tissue of CA-supplemented mice by immunofluorescence experiment. The results showed that after high-fat feeding, the number of enterochromaffin cell marker CgA in the jejunal mucosa of CA-supplemented mice was significantly reduced compared with the control group ( Fig.21 A, B), and the 5-HT positive cells also decreased significantly ( Fig.21 A, C); The levels of 5-HT in the jejunum and serum of mice in the CA supplementation group were lower than those in the control group ( Fig.21 D, E), suggesting that Bacteroides metabolite CA can also inhibit peripheral 5-HT content.

[0277] 5. Effects of intestinal epithelial-specific knockout of Tph1 on HFD-induced obesity

[0278] 5.1. Construction of intestinal epithelial-specific Tph1 knockout mice

[0279] In order to clarify whether the intestinal flora and its metabolites affect obesity by regulating intestinal 5-HT, the present invention uses CRISPR / Cas9 technology (chromosome homologous recombination method) to flox the Tph1 gene (Tph1 transcript is ENSMUST00000049298, flox targets the exon 3 site of Tph1, and 2 pairs of sgRNA sequences are designed before and after) to achieve the purpose of conditional knockout of Tph1 in the intestinal epithelium (Tph1△IEC; TPH1CKO), so that EC cells lack the function of producing 5-HT. In order to verify the knockout effect, the present invention performs RT-PCR detection on the genomic DNA extracted from the mouse tail tissue, and the electrophoresis results are as follows Fig. 22 As shown in A, through Fig. 22 As can be seen from A, the product band of Flox homozygous mice appears at around 265bp, while the wild-type mouse band appears at around 216bp; the product band of Cre-positive mice appears at around 204bp, while Cre-negative and wild-type mice have no band at this position. At the same time, the present invention confirms the expression loss of Tph1 gene at the mRNA level through RT-qPCR detection ( Fig. 22 B), Western blot confirmed that the expression of Tph1 gene at the protein level was significantly reduced ( Fig. 22 C) The above results indicate that the Tph1 gene intestinal epithelial conditional knockout mice were successfully constructed.

[0280] 5.2 Effects of intestinal epithelial-specific Tph1 knockout on body weight and glucose tolerance in mice on HFD

[0281] After being fed a high-fat diet for 12 weeks, the results showed that compared with Tph1fl / fl, The mice showed a significant improvement in obesity induced by a high-fat diet, and their weight gain was not significant ( Fig.23 A). The results of IPGTT test showed that compared with Tph1fl / fl, The blood glucose levels of mice decreased significantly at 60min, 90min and 120min after glucose administration ( Fig.23 B). The results of the area under the glucose tolerance curve (AUC) showed that Tph1 fl / fl Impaired glucose tolerance in mice Mice showed significant improvement ( Fig.23 C). Compared with the control group, the liver weight of Tph1 knockout mice was significantly reduced. H&E staining results showed that the liver morphology of Tph1 knockout mice was intact, with significantly fewer vacuoles, tending to normal hepatocytes. The above results suggest that inhibiting the synthesis of intestinal 5-HT can inhibit obesity induced by a high-fat diet.

[0282] 5.3 Effects of intestinal epithelial-specific knockout of Tph1 on jejunal chylomicron transport in mice

[0283] Most of the nutrients in food cannot be directly absorbed and utilized by animals. They must be processed in the digestive tract and converted into soluble small molecules before they can pass through the digestive tract epithelium into the blood and lymph for use by body tissues. Therefore, the nutrients we take in must first be digested and absorbed in the intestines, which is related to the normal development and health of the body and is of great significance. Material transport is the most important biological function of the small intestinal mucosal epithelium. Relying on this function, intestinal epithelial cells can transport digested nutrients from the intestinal cavity to the blood and lymphatic system and provide them to extraintestinal tissues for use. The lacteal duct is a capillary lymphatic vessel located in the small intestinal mesentery. This capillary lymphatic vessel absorbs digested fat droplets and is milky white. In experiments, the present invention found that when the same high-fat diet is given and the same eating time is ensured, compared with Tph1 fl / fl Mouse, The uptake of jejunal mesenteric chyle in mice was significantly reduced ( Fig.24 A). By counting the number of lacteals (milky white lymphatic vessels) filled with chylomicrons in the jejunum, we found The number of lacteals filled with chylomicrons in the jejunum of mice was also significantly reduced ( Fig.24 B) indicates that knocking out intestinal epithelial Tph1 reduces intestinal 5-HT synthesis, which in turn hinders the transport of chylomicrons from the jejunum to the lacteals.

[0284] The present invention detected the content of 5-HT in the small intestine tissue and serum of mice with different obesity phenotypes, mice supplemented with B. vulgatus strains and mice supplemented with metabolites CA through immunofluorescence experiments, HPLC experiments and Elisa experiments. The results confirmed that intestinal 5-HT is involved in regulating the process of B. vulgatus strains inhibiting obesity through Bacteroides metabolites CA. In order to clarify the regulatory effect of intestinal 5-HT on B. vulgatus strains and Bacteroides metabolites CA on obesity, the present invention constructed intestinal epithelial-specific knockout Tph1 mice, which lost the function of intestinal EC cells to synthesize 5-HT, and observed The mice did have a phenotype of lean body weight, low blood glucose levels, and impaired transport of chylomicrons from the jejunum to the lacteals.

[0285] 6. Mechanism of B. vulgatus and Bacteroides metabolites CA regulating obesity by inhibiting peripheral 5-HT synthesis

[0286] 6.1. Detection of lipid absorption function of intestinal epithelial cells in model mice

[0287] In order to clarify whether the intestinal lipid absorption of model mice changes under different obesity phenotypes, the present invention uses cryo-transmission electron microscopy to observe the lipid transport of intestinal epithelial cells in model mice. By counting the number of lipid droplets in a single intestinal epithelial cell, it is found that compared with the HIGH group mice, the number of lipid droplets (Intracellular lipid droplets, CLD) in a single intestinal epithelial cell in the LOW group mice is larger, and there are a large number of chylomicrons (Chylemicroparticle, CM) ( Fig.25 A, B); also compared with the control group, the number of CLD and CM in a single intestinal epithelial cell of mice in the B. vulgatus group was also higher ( Fig.25 C, D). In the mice supplemented with metabolite CA, compared with the control group, the number of CLD in a single intestinal epithelial cell in the CA group mice increased significantly, and a large number of CMs were also present at the edge of the intestinal epithelial cells and retained in the intestinal epithelial cells ( Fig.25 E, F). The present invention also observed similar phenomena in intestinal epithelial-specific knockout Tph1 mice. Compared with Tph1fl / fl mice, The number of CLD in a single mouse intestinal epithelial cell was high, and there was also retention of CM, which was consistent with the observation The results of the study on the filling status of chylomicrons in the jejunal lacteals of mice indicated that the number of CLD and CM in intestinal epithelial cells of mice with phenotypically thin was increased.

[0288] 6.2 Effects of B. vulgatus complementation in mice on lipid transport-related molecular markers in intestinal epithelial cells

[0289] The present invention uses RT-qPCR experiments to relatively quantitatively analyze the molecular markers CD36, Fatp4, FABP1, and FABP2 related to intestinal epithelial cell lipid uptake; the molecular markers APOB and MTP related to intestinal epithelial cell lipid chylomicron assembly; and the molecular markers HSL and ATGL related to intestinal epithelial lipid decomposition. The results showed that there was no significant change in the above-mentioned molecular markers related to intestinal epithelial lipid absorption in mice supplemented with B. vulgatus strains ( Fig.26 ), which suggests that after the B. vulgatus strain was supplemented, the uptake of lipids by the small intestinal epithelial cells, the synthesis and decomposition of lipids in the intestinal epithelial cells, and the assembly process of chylomicrons did not change significantly.

[0290] In order to further clarify the mechanism by which B. vulgatus regulates obesity by inhibiting the synthesis of peripheral 5-HT, the present invention extracted mRNA from the intestinal epithelial tissue of mice in the control group and the B. vulgatus strain complementation group, and performed transcriptomics (RNA-seq) analysis. The DESeq2 method was used to detect the differentially expressed genes (DEGs) between the two groups. After RNA-seq detection, the obtained data were analyzed. In order to prove the repeatability of the sequencing experiment and the reliability of the evaluation results, the RNA-seq data quality check was performed on the two groups of 6 samples. The results are shown in Table 11 below. The Q30 percentage in each sample is not less than 92%, the error rate of sample base recognition is less than 0.03%, and the GC content of the sample is 49.47%-50.53%. The above test results show that the samples in this group meet the requirements of library construction and detection, the credibility and repeatability of the sequencing data results are high, and the evaluation results are reliable.

[0291] Table 11 Summary of transcriptomic data quality of B. vulgatus group

[0292]

[0293] The present invention uses log2FoldChange(DESeq2)≥2 or log2FoldChange(DESeq2)≤-2 as the threshold for significant differential expression. The results of RNA-seq analysis showed that a total of 22,073 differentially expressed genes were screened out between the B. vulgatus group and the control group, of which 10,375 genes were upregulated and 11,698 genes were downregulated ( Fig. 27 A), the cluster analysis results and principal component analysis results of intestinal epithelial tissues of B. vulgatus group and control group are shown in Fig. 27 B and Fig. 27 C. Subsequently, the 22073 differentially expressed genes were subjected to KEGG enrichment analysis based on the KEGG database. The results are as follows: Fig. 27 D. According to the results of KEGG enrichment analysis, no pathways related to lipid synthesis metabolism were enriched after complementation of B. vulgatus strains. Subsequently, the differentially expressed genes were enriched in biological process (BP), molecular function (MF) and cellular component (CC) based on the GO database. The results are shown in Fig. 27 E. The results of GO enrichment analysis showed that there was no enrichment of pathways related to lipid anabolism after complementation of the B. vulgatus strain, which confirmed the results of RT-qPCR detection at the level of small intestinal epithelial tissue in the previous article.

[0294] At the same time, the present invention found in the results of GO enrichment analysis that the differentially expressed genes were enriched in the plasma membrane invagination pathway in the BP enrichment analysis; and were enriched in the channel activity pathway and the passive transmembrane transporter activity pathway in the MF enrichment analysis ( Fig. 27 E). This suggests that the previous experimental results of the present invention ( Fig.24 ) may be closely related to the transport of substances on the cell membrane.

[0295] 6.3 Effect of 5-HT on short-circuit current in jejunal mucosa of model mice

[0296] 6.3.1. Dose-effect curve of 5-HT

[0297] First, the present invention found that 5-HT caused significant changes in transepithelial ion transport in the jejunal mucosa of wild-type mice in a dose-dependent manner, with an EC50 of 0.59×10 -6 M Fig.28 As shown, through Fig.28 The results showed that the intensity of the short-circuit current produced in the mouse jejunal mucosa was positively correlated with the dose of 5-HT.

[0298] 6.3.2 Effect of 5-HT on short-circuit current in jejunal mucosa of model mice

[0299] The same concentration of 5-HT was administered to the jejunal mucosa of mice to observe the short-circuit current generated. The present invention found that 10 μM 5-HT promoted the increase of short-circuit current of jejunal mucosal epithelium in HIGH group mice to a much greater extent than that in LOW group ( Fig.29 A, B), indicating that the sensitivity of the jejunal epithelium to 5-HT in the HIGH group of mice increased, which enhanced the transport of substances in the jejunal epithelium. The B. vulgatus strain complementation experiment found that after complementation with the B. vulgatus strain, the magnitude of the increase in the short-circuit current of the jejunal epithelium promoted by 5-HT was reduced compared with the control group. The metabolite CA complementation experiment found that after complementation with CA, the magnitude of the increase in the short-circuit current of the jejunal epithelium promoted by 5-HT was also reduced compared with the control group ( Fig.29 E, F). The above results suggest that the replenishment of B. vulgatus strains and Bacteroides metabolite CA will reduce the sensitivity of 5-HT in the jejunal epithelium of mice and reduce the material transport of the jejunal epithelium.

[0300] 6.4. RNA-seq screening experiment of intestinal epithelial tissues of various mouse models

[0301] In order to clarify the reasons why the intestinal 5-HT synthesis is inhibited after the B. vulgatus strain is complemented, resulting in the accumulation of intestinal epithelial cell lipid droplets and chylomicrons in intestinal epithelial cells, causing chylomicron transport disorders, the present invention collects the jejunal epithelial tissues of the obesity model HIGH group and LOW group, B. vulgatus strain complementation model mice, metabolite CA complementation model mice and intestinal epithelial specific knockout Tph1 mice for transcriptomics (RNAseq) analysis. In order to prove the repeatability of the sequencing experiment and the reliability of the evaluation results, the RNA-seq data quality check was performed on 6 groups of 18 samples, and the results are shown in Tables 12-14 below. It can be seen from the results of Tables 12-14 that the Q30 percentage in each sample is not less than 90%, the error rate of sample base recognition is less than 0.03%, and the GC content of the sample is 49.47%-50.53%. The above test results show that the samples in this group meet the requirements of library construction and detection, the credibility and repeatability of the sequencing data results are high, and the evaluation results are reliable.

[0302] Table 12 Summary of transcriptomic data quality of HIGH and LOW groups

[0303]

[0304] Table 13 Summary of transcriptomic data quality of HFD and HCA groups

[0305]

[0306]

[0307] Table 14Tph1 fl / fl Group and Tph1 △IEC Summary table of transcriptomics data quality

[0308] sample Grouping RawReads CleanReads Error(%) Q20(％) Q30(％) GC (%) 467 <![CDATA[Tph1 fl / fl ]]> 45163304 43612706 0.03 96.81 91.71 50.37 468 <![CDATA[Tph1 fl / fl ]]> 43235204 41652118 0.03 96.39 90.84 50.62 474 <![CDATA[Tph1 fl / fl ]]> 50854846 49194912 0.03 96.85 91.80 50.28 462 <![CDATA[Tph1 △IEC ]]> 43457900 42206534 0.03 96.94 91.97 50.14 463 <![CDATA[Tph1 △IEC ]]> 49194912 42131528 0.03 96.83 91.74 50.46 470 <![CDATA[Tph1 △IEC ]]> 47628836 45671856 0.03 96.92 91.96 50.34

[0309] The DESeq2 method was used to detect differentially expressed genes (DEGs) between the two groups. The obtained data were analyzed based on log2FoldChange(DESeq2)≥2 or log2FoldChange(DESeq2)≤-2 as the threshold for significant differential expression. Fig.30 A shows the intersection of differentially expressed genes in the intestinal epithelial tissue transcriptome screening of the four groups of mouse models. Based on the intersection of these differentially expressed genes, the present invention performed KEGG enrichment analysis ( Fig.30 B), the results showed that the intersection differentially expressed genes were significantly enriched in calcium signaling pathway (P < 0.0001) and cAMP signaling pathway (P < 0.0001). Subsequently, the intersection of these differentially expressed genes was subjected to BP, MF and CC enrichment analysis based on the GO database. Fig.30As shown in C, the intersection differential genes in the GO BP enrichment analysis results were significantly enriched in the cytoplasmic calcium ion concentration regulation pathway (P < 0.0001). Studies have shown that intracellular calcium ion concentration is closely related to the secretion and transport of chylomicrons. Combining the KEGG analysis and BP analysis results of the intersection differential genes, it was shown that the inhibition of intestinal 5-HT synthesis after the B. vulgatus strain was complemented, resulting in the transport disorder of chylomicrons in intestinal epithelial cells, which was related to the change of intracellular calcium ion concentration. The present invention speculates whether the inhibition of intestinal 5-HT synthesis after the B. vulgatus strain was complemented will reduce the calcium ion concentration in the intestinal epithelial cells, reduce the secretion and transport of chylomicrons in the intestinal epithelial cells, and then cause a thin phenotype.

[0310] 6.5 Effect of 5-HT on calcium concentration in intestinal epithelial cells

[0311] According to the results of KEGG and BP analysis of the intersection differential gene transcriptomics, the present invention uses normal human colon epithelial cells (NCM 460) to detect the effect of 5-HT on the calcium ion concentration in intestinal epithelial cells at the cellular level. The calcium imaging results show that the administration of 10 μM 5-HT can increase the average fluorescence intensity of the calcium ion indicator Fluo-4 in most NCM 460 cells in the field of view ( Fig.31 A, B) and the maximum fluorescence intensity change of Fluo-4 in a single cell [(Control: 0.0232±0.0195, n=6), (5-HT: 0.1974±0.0311, n=6)] ( Fig.31 C). The results suggest that 5-HT can increase the concentration of calcium in intestinal epithelial cells. The present invention confirms that 5-HT can increase the concentration of calcium ions in intestinal epithelial cells, thereby enhancing the secretion and transport of chylomicrons in intestinal epithelial cells, promoting the absorption of nutrients, and showing an obese phenotype.

[0312] In summary, if Fig.32 As shown, the present study found that mice with different obesity phenotypes have different intestinal flora structures and compositions. The high abundance of Bacteroides vulgauts and Bacteroides metabolite CA in the intestinal flora of lean mice can inhibit the synthesis of intestinal 5-HT, reduce the calcium ion concentration in intestinal epithelial cells, inhibit the secretion and transport of chylomicrons, and thus inhibit obesity.

[0313] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. The application of Bacteroides vulgaris and its metabolite bile acid to inhibit the molecular mechanism of obesity induced by high-fat diet, characterized in that: Bacteroides vulgaris and its metabolite bile acid inhibit high-fat diet-induced obesity by suppressing 5-HT content in the intestine and serum.

2. The use of Bacteroides vulgaris and Bacteroides metabolite bile acid in inhibiting the molecular mechanism of obesity induced by a high-fat diet according to claim 1, characterized in that: Bacteroides vulgaris and its metabolite bile acid inhibit the 5-HT content in the intestine and serum by inhibiting the number of EC cells.

3. The use of Bacteroides vulgaris and the Bacteroides vulgaris metabolite bile acid in inhibiting the molecular mechanism of obesity induced by a high-fat diet according to claim 2, characterized in that: Inhibition of 5-HT content inhibits the secretion of jejunal chylomicrons into the intestinal lymph, thereby inhibiting obesity induced by a high-fat diet.

4. The use of Bacteroides vulgaris and the Bacteroides vulgaris metabolite bile acid in inhibiting the molecular mechanism of obesity induced by a high-fat diet according to claim 2, characterized in that: Inhibition of 5-HT content promotes the number of lipid droplets and chylomicrons in intestinal epithelial cells, thereby inhibiting obesity induced by a high-fat diet.

5. The use of Bacteroides vulgaris and the Bacteroides vulgaris metabolite bile acid in inhibiting the molecular mechanism of obesity induced by a high-fat diet according to claim 3, characterized in that: 5-HT content inhibition inhibits the secretion of jejunal chylomicrons into the intestinal lymph by inhibiting the calcium ion concentration in the intestinal epithelial cells, thereby inhibiting high-fat diet-induced obesity.

6. Use of Bacteroides vulgaris and bile acid inhibitors of Bacteroides vulgaris metabolites in the preparation of drugs for treating obesity induced by a high-fat diet.

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