Application of bifidobacterium breve lw01 in preparation of products for prevention or adjuvant therapy of obesity-related tumors

Abstract

The invention relates to the technical field of biological medicine. The invention provides an application of bifidobacterium breve lw01 in preparation of a product for prevention or adjuvant therapy of obesity-related tumors, and also provides an application of the bifidobacterium breve lw01 in a product for regulating intestinal flora and bile acid metabolism level. In the invention, bile acid (such as taurocholic acid TCA) is used as an intervention target, and the bifidobacterium breve lw01 is used for targeted regulation of disordered bile acid metabolism in an obesity state, so that the promotion effect of obesity on tumor growth is remarkably relieved, and finally, the purpose of inhibiting the development of obesity-related tumors is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of Bifidobacterium breve lw01 in preparation of products for preventing or adjuvant treating obesity-related tumors. BACKGROUND

[0002] The prevalence of overweight / obese individuals has sharply increased and is associated with a higher risk of at least 13 cancers, including hepatocellular carcinoma. There is an interaction between gut microbiota and obesity, and obesity-related gut microbiota imbalance can promote cancer progression by affecting systemic immunity or destroying local immune surveillance. More and more evidence confirms that certain symbiotic bacteria can prevent cancer occurrence or improve the efficacy of chemotherapy and immunotherapy. Although probiotics have a significant market position in weight management, their potential role in tumor regulation in obese individuals and their potential mechanisms still need further research.

[0003] Microbiota-derived metabolites are the key hub of the interaction between gut microbiota and tumors that cross the intestinal mucosal barrier. Short-chain fatty acids produced by complex carbohydrate fermentation, and tryptophan derivatives produced by bacterial enzymatic processes, are the most widely studied classes of small molecule metabolites. Although these metabolites are important, their effects on immune cells are different, and some of the effects even contradict each other. Recently, emerging research has focused on bile acids (BAs), a host-microorganism co-metabolite that can act as a messenger of gut microbiota. The effects of bile acids have both beneficial and harmful sides, depending on factors such as the specific type of affected tissue and metabolic state. Although bile acids are recognized to have a bidirectional regulatory role, how specific types of bile acids specifically affect the function, infiltration, and expansion of anti-tumor immune cells (especially CD8 + T cells) in the context of obesity, and whether this effect is a key link in obesity-promoted tumors, is not clear. There is a lack of mature methods that effectively and specifically regulate the levels of harmful bile acids in obese individuals to restore immune surveillance function.

[0004] Although probiotics are widely used in fields such as weight management, and some symbiotic bacteria have been confirmed to have potential anti-tumor or adjuvant therapy effects, there is a serious lack of research on the specific mechanisms by which they regulate tumor occurrence and development in obese individuals. Overweight can change the function of immune cells (such as natural killer (NK) cells or cytotoxic CD8 +The gut microbiota can remodel the tumor microenvironment (TME) by modulating the lipid and amino acid metabolism of T cells. Previous studies have focused on short-chain fatty acids (SCFAs) or tryptophan metabolites, and there is a lack of in-depth exploration and solid evidence on whether and how probiotics can affect obesity-related TME and anti-tumor immunity by regulating key bile acid metabolism. A previous study on a probiotic, Bifidobacterium breve lw01, confirmed its anti-tumor effect on head and neck squamous cell carcinoma (HNSCC), which affects tryptophan metabolism, improves the pre-cancerous inflammatory intestinal environment by guiding the differentiation of immature colonic macrophages, and inhibits tumorigenesis. Therefore, it is valuable to further study the relationship between Bifidobacterium breve lw01 and obesity-related tumors in overweight / obese individuals. SUMMARY

[0005] The present application aims to provide a use of Bifidobacterium breve lw01 in the preparation of a product for preventing or adjuvant therapy of obesity-related tumors. Bifidobacterium breve lw01 can target and regulate the disturbed bile acid metabolism in obesity, thereby significantly reducing the promotion of tumor growth by obesity, and ultimately achieving the purpose of inhibiting the development of obesity-related tumors.

[0006] To achieve the above application purposes, the present application provides the following technical solutions:

[0007] The present application provides a use of Bifidobacterium breve lw01 in the preparation of a product for preventing or adjuvant therapy of obesity-related tumors.

[0008] As a preferred, the classification name of the Bifidobacterium breve lw01 is Bifidobacterium breve, which has been preserved in the China General Microbiological Culture Collection Center on May 8, 2019, the address of the preservation center is No. 1, Beichen West Road, Haidian District, Beijing, China, the Institute of Microbiology of the Chinese Academy of Sciences, and the preservation number is CGMCC NO. 17727.

[0009] The present application provides a use of Bifidobacterium breve lw01 in the preparation of a product for targeting and regulating the intestinal flora in obesity.

[0010] The present application provides a use of Bifidobacterium breve lw01 in the preparation of a product for regulating the bile acid metabolism level in obesity.

[0011] The present application provides a use of bile acid as a target in the preparation of a product for preventing or adjuvant therapy of obesity-related tumors.

[0012] The present application provides an obesity-related tumor immunosuppression product, which comprises the Bifidobacterium breve lw01.

[0013] Preferably, the concentration of Bifidobacterium breve lw01 is (0.8-1.2) x 10 9 CFU / 100 μL.

[0014] By adopting the technical scheme, the present application has the following beneficial effects:

[0015] 1. The technical scheme of the present application discloses a specific molecular mechanism of how abnormal increase of a specific bile acid, Taurocholic Acid (TCA) level, exacerbates tumor growth by directly inhibiting the accumulation, local expansion and function of key anti-tumor immune cells (especially cytotoxic CD8 + T cells) in the tumor microenvironment. It is clear that TCA is a key effector molecule and potential intervention target of obesity-related tumor immunosuppression. TCA down-regulates the expression of chemokine receptor CXCR6 on the surface of CD8 + T cells in the tumor microenvironment by inhibiting the MAPK / ERK pathway, interferes with its recruitment and infiltration to the tumor site, and weakens its cytotoxic function, thereby inhibiting the anti-tumor immune response.

[0016] 2. The technical scheme of the present application studies that Bifidobacterium breve lw01 can effectively reduce the serum and local TCA level of obese individuals, and confirms that the probiotic can target and regulate the disturbed bile acid metabolism in obesity, degrade bile salts by using bile salt hydrolase, especially reduce TCA which has immunosuppressive effect, and then improve the inhibitory tumor immune microenvironment to play a tumor inhibition effect.

[0017] 3. The technical scheme of the present application proves that taking Bifidobacterium breve lw01 can reduce the TCA level, can effectively reverse the inhibition of CD8 + T cell function induced by obesity, restore its infiltration, expansion and killing activity at the tumor site, thereby significantly reducing the promotion of tumor growth by obesity, and ultimately achieving the purpose of inhibiting the development of obesity-related tumors.

[0018] Biological preservation instructions

[0019] The present application relates to Bifidobacterium breve lw01, which is classified as Bifidobacterium breve, and has been preserved in the China General Microbiological Culture Collection Center on May 8, 2019, at the address of No. 1, Beichen West Road, Chaoyang District, Beijing, China, at the Institute of Microbiology, Chinese Academy of Sciences, with the preservation number of CGMCC NO. 17727. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Timeline of HFD and CD groups of mice (n = 8 in each group);

[0021] Figure 2 Figure 1. Body weight changes in HFD and CD mice during the experimental period. Figure 2 Figure 1. Body weight changes in HFD and CD mice during the experimental period.

[0022] Figure 3 Figure 2. Fasting blood glucose levels in HFD and CD mice after 8 hours of fasting.

[0023] Figure 4 Figure 3. Oral glucose tolerance test results in HFD and CD mice. Figure 4 Figure 3. Oral glucose tolerance test results in HFD and CD mice.

[0024] Figure 5 Figure 4. Serum total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein levels in HFD and CD mice.

[0025] Figure 6 Figure 5. Wet weights of epididymal white adipose tissue and liver in HFD and CD mice.

[0026] Figure 7 Figure 6. H&E staining of liver cross-sections and AOI statistics in HFD and CD mice. Figure 7 Figure 6. H&E staining of liver cross-sections and AOI statistics in HFD and CD mice.

[0027] Figure 8 Figure 7. Appearance and changes in mass and volume of Hepa1-6 tumors in HFD and CD mice. Figure 8 Figure 7. Appearance and changes in mass and volume of Hepa1-6 tumors in HFD and CD mice.

[0028] Figure 9 Figure 8. Expression levels of proteins and phosphorylated factors in Hepa1-6 tumor tissues in HFD and CD mice. Figure 9 Figure 8. Expression levels of proteins and phosphorylated factors in Hepa1-6 tumor tissues in HFD and CD mice.

[0029] Figure 10 Figure 9. Diversity of fecal microbiota in HFD and CD mice. Figure 10 Figure 9. Diversity of fecal microbiota in HFD and CD mice.

[0030] Figure 11 Figure 10. Experimental timeline for HFD+PBS and HFD+B. breve groups (n=8 per group).

[0031] Figure 12 Figure 11. Body weight changes in HFD+PBS and HFD+B. breve mice during the experimental period.Figure 12 A represents the comparison of body weight at the 16th week, and B represents the body weight change curve.

[0032] Figure 13 The fasting blood glucose level of HFD+PBS group and HFD+B.breve group mice after fasting for 8 hours;

[0033] Figure 14 The results of oral glucose tolerance test of HFD+PBS group and HFD+B.breve group mice Figure 14 A represents the blood glucose curve, and B represents the AUC statistics of different groups.

[0034] Figure 15 The wet weight of epididymal white adipose tissue and liver of HFD+PBS group and HFD+B.breve group mice

[0035] Figure 16 H&E staining of HFD+PBS group and HFD+B.breve group mice liver cross-section and AOI statistics Figure 16 A represents H&E staining, scale bar=100 μm; B represents AOI statistics.

[0036] Figure 17 The appearance and mass, volume change of Hepa1-6 tumor of HFD+PBS group and HFD+B.breve group mice Figure 17 A represents the appearance of tumor, B represents the volume change curve, and C represents the mass of tumor tissue at the end of the experiment.

[0037] Figure 18 The expression level of protein and phosphorylation related factors in Hepa1-6 tumor tissue of HFD+PBS group and HFD+B.breve group mice Figure 18 A represents the expression band, and B represents the band intensity ratio statistics.

[0038] Figure 19 The expression of NK cells, NKT cells, T cells, Tc cells and Th cells in total single live cells in Hepa1-6 tumor Figure 19 A represents CD group and HFD group, and B represents HFD+PBS group and HFD+B.breve group.

[0039] Figure 20 The proportion of Tc cells and Th cells in T cells Figure 20 A represents CD group and HFD group, and B represents HFD+PBS group and HFD+B.breve group.

[0040] Figure 21The expression of DCs, TAMs and their subsets, and bone marrow-derived MDSCs in the total single living cells of Hepa1-6 tumors ( Figure 21 A represents the CD group and the HFD group, and B represents the HFD+PBS group and the HFD+B.breve group);

[0041] Figure 22 The ratio of pro-inflammatory (M1) TAMs and alternatively activated (M2) TAMs in TAMs ( Figure 22 A represents the CD group and the HFD group, and B represents the HFD+PBS group and the HFD+B.breve group);

[0042] Figure 23 Results and statistical graphs of H&E staining and immunohistochemical staining of tumor tissues ( Figure 23 A in the figure shows the staining images of the CD group and the HFD group, B shows the statistical graph of marker expression in the CD group and the HFD group, C shows the staining images of the HFD+PBS group and the HFD+B.breve group, and D shows the statistical graph of marker expression in the HFD+PBS group and the HFD+B.breve group; scale bar = 50 μm);

[0043] Figure 24 Serum metabolomics analysis of mice in CD group, HFD group and HFD+B.breve group ( Figure 24 A represents the principal component analysis (PCA) diagram, B represents the Venn diagram, C represents the normalized heat map of the relative abundance of differential serum metabolites, D represents the absolute quantification of taurocholic acid (TCA) and taurodeoxycholic acid (TDCA) in serum, and E represents the correlation analysis between Bifidobacterium and TCA in diet-induced obese (DIO) mice);

[0044] Figure 25 The decomposition of TCA by Bifidobacterium breve in the MRS group and the MRS+B.breve group (n=4 in each group);

[0045] Figure 26 Schematic diagram of the experimental timeline of mice in the PBS and TCA groups (n=5 per group in the PBS group, n=6 per group in the TCA group);

[0046] Figure 27 The changes in appearance, mass and volume of Hepa1-6 tumors in mice in the PBS group and TCA group ( Figure 27 A represents the appearance of the tumor, B represents the volume change curve, and C represents the tumor tissue quality at the end point of the experiment);

[0047] Figure 28The expression of NK cells, NKT cells, T cells, Tc cells, and Th cells in the total single living cells of Hepa1-6 tumors in mice in the PBS group and TCA group;

[0048] Figure 29 The ratio of Tc cells and Th cells in T cells of mice in PBS group and TCA group;

[0049] Figure 30 The expression of DCs, TAMs and their subsets, and bone marrow-derived MDSCs in total single living cells in Hepa1-6 tumors of mice in the PBS and TCA groups;

[0050] Figure 31 The ratios of pro-inflammatory (M1) TAMs and alternatively activated (M2) TAMs in TAMs in mice of PBS group and TCA group;

[0051] Figure 32 The results and statistical graphs of H&E staining and immunohistochemical staining of tumor tissues of mice in the PBS group and TCA group are shown ( Figure 32 A in the figure shows the staining image, and B shows the statistical graph of marker expression; scale bar = 50 μm);

[0052] Figure 33 The BSH activity of Bifidobacterium breve lw01 in the MRS+B.breve group and the MRS+B.breve+AAA-10 group;

[0053] Figure 34 Schematic diagram of the experimental timeline of mice in the MRS+B.breve group and the MRS+B.breve+AAA-10 group (n=6 in each group);

[0054] Figure 35 The changes in appearance, mass and volume of Hepa1-6 tumors in mice in the MRS+B.breve group and the MRS+B.breve+AAA-10 group ( Figure 35 A represents the appearance of the tumor, B represents the volume change curve, and C represents the tumor tissue quality at the end point of the experiment);

[0055] Figure 36 The body weight changes of mice in the MRS+B.breve group and the MRS+B.breve+AAA-10 group during the experimental period ( Figure 36 A represents the weight comparison at week 16, and B represents the weight change curve);

[0056] Figure 37 The wet weights of epididymal white adipose tissue and liver of mice in the MRS+B.breve group and the MRS+B.breve+AAA-10 group;

[0057] Figure 38 H&E staining images and AOI statistics of liver cross sections of mice in MRS+B.breve group and MRS+B.breve+AAA-10 group ( Figure 38 A in the figure represents H&E staining, scale bar = 100 μm; B represents AOI statistical graph);

[0058] Figure 39 The expression of NK cells, NKT cells, T cells, Tc cells, and Th cells in the total single living cells of Hepa1-6 tumors in mice in the MRS+B.breve group and the MRS+B.breve+AAA-10 group;

[0059] Figure 40 The ratios of Tc cells and Th cells in T cells of mice in the MRS+B.breve group and the MRS+B.breve+AAA-10 group;

[0060] Figure 41 The expression of DCs, TAMs and their subsets, and bone marrow-derived MDSCs in total single living cells of Hepa1-6 tumors in mice in the MRS+B.breve group and the MRS+B.breve+AAA-10 group;

[0061] Figure 42 The ratios of pro-inflammatory (M1) TAMs and alternatively activated (M2) TAMs in TAMs in mice of MRS+B.breve group and MRS+B.breve+AAA-10 group;

[0062] Figure 43 The results and statistical graphs of H&E staining and immunohistochemical staining of tumor tissues of mice in the MRS+B.breve group and MRS+B.breve+AAA-10 group are shown. Figure 43 A in the figure shows the staining image, and B shows the statistical graph of marker expression; scale bar = 50 μm);

[0063] Figure 44 For the genome analysis of Bifidobacterium breve lw01 ( Figure 44 A represents genome sequence, and B represents KEGG analysis);

[0064] Figure 45 Bsh gene sequence and phylogenetic tree ( Figure 45 A in the figure represents the Bsh gene sequence, and B represents the Bsh gene phylogenetic tree);

[0065] Figure 46 For BSH amino acid sequence alignment and protein structure prediction ( Figure 46 A represents the 3D structure prediction model, and B represents the amino acid sequence alignment result);

[0066] Figure 47 CD8 T cells from mice + Timeline of T cell exhaustion experiment (n=6 per group);

[0067] Figure 48 Appearance and weight / volume changes of Hepa1-6 tumors in different groups of mice Figure 48 A: appearance of tumors, B: volume change curve, C: weight of tumor tissues at the end of the experiment;

[0068] Figure 49 Flow cytometry analysis of tumor infiltrating lymphocytes

[0069] Figure 50 Apoptosis of CD8 T cells treated with TCA +

[0070] Figure 51 Flow cytometry evaluation of cell proliferation, cytotoxic cytokine expression, and chemokine receptor expression

[0071] Figure 52 Gene expression of cytotoxic cytokines and chemokine receptors in CD8 T cells in vitro +

[0072] Figure 53 mRNA analysis of CD8 T cells isolated from Hepa1-6 tumors in TCA and PBS groups of mice +

[0073] Figure 54 Relative expression heatmap of characteristic genes in CD8 T cells from TCA and PBS groups of mice +

[0074] Figure 55 KEGG pathway enrichment analysis of down-regulated genes in CD8 T cells from different groups of mice +

[0075] Figure 56 GO-BP analysis of down-regulated genes in CD8 T cells from different groups of mice +

[0076] Figure 57 Western blot analysis of ERK, P38, JNK, and P65 phosphorylation

[0077] Figure 58 ERK phosphorylation in CD8 T cells from different groups of mice treated with TCA +

[0078] ​​​​​​​Figure 59 Figure 1 shows the results of flow cytometry and qPCR detection of TCA on CD8 + Figure 1 shows the results of flow cytometry and qPCR detection of TCA on CD8 Figure 59 Figure 1 shows the results of flow cytometry and qPCR detection of TCA on CD8

[0079] Figure 60 Figure 1 shows the results of flow cytometry and qPCR detection of TCA on CD8 DETAILED DESCRIPTION

[0080] The application provides application of Bifidobacterium breve lw01 in preparation of a product for preventing or adjuvant treating obesity-related tumors.

[0081] In the application, the Bifidobacterium breve lw01 is named Bifidobacterium breve, and has been preserved in the China General Microbiological Culture Collection Center on May 8, 2019, and the address of the preservation center is No. 1, Beichen West Road, Haidian District, Beijing, and the Institute of Microbiology of the Chinese Academy of Sciences, and the preservation number is CGMCC NO. 17727.

[0082] The application provides application of Bifidobacterium breve lw01 in preparation of a product for preventing or adjuvant treating obesity-related tumors.

[0083] The application provides application of Bifidobacterium breve lw01 in preparation of a product for preventing or adjuvant treating obesity-related tumors.

[0084] In the application, the Bifidobacterium breve lw01 is named Bifidobacterium breve, and has been preserved in the China General Microbiological Culture Collection Center on May 8, 2019, and the address of the preservation center is No. 1, Beichen West Road, Haidian District, Beijing, and the Institute of Microbiology of the Chinese Academy of Sciences, and the preservation number is CGMCC NO. 17727.

[0085] The application provides application of Bifidobacterium breve lw01 in preparation of a product for preventing or adjuvant treating obesity-related tumors.

[0086] The application provides an obesity-related tumor immunosuppression product, which comprises the Bifidobacterium breve lw01.

[0087] In the application, the concentration of the Bifidobacterium breve lw01 is preferably (0.8-1.2) x 10 9 CFU / 100 muL, further preferably (0.9-1.1) x 10 9 CFU / 100 muL, and more preferably 1 x 10 9 CFU / 100 muL.

[0088] The technical solutions provided by the present application will be described in detail below in combination with embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0089] Example 1

[0090] (I) Tumor cell treatment

[0091] 1. Cell culture

[0092] The Hepa1-6 cell line (ATCC CRL-1830 TM ) was selected, and the cells were cultured in a 37℃ constant temperature incubator containing 5% CO2 using DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin, and the cells adhered and grew, appeared epithelial cell-like under a microscope, and were polygonal at low density; at high density, they appeared uniform and pavement-like, with good periphery refraction, homogeneous and transparent cytoplasm, and visible mitotic figures.

[0093] 2. Drug treatment

[0094] Logarithmic growth phase cells were inoculated in corresponding culture dishes or plates, and drug treatment was performed when the cells grew to 70-80%.

[0095] TCA was dissolved in PBS to prepare a 10mM solution, and when used, it was diluted with DMEM medium to 1μM, 10μM, 100μM and 500μM.

[0096] (II) Bacterial treatment

[0097] 1. Bacterial culture

[0098] Bifidobacterium breve lw01 was cultured in an anaerobic 37℃ constant temperature incubator using MRS liquid medium containing 20g / L raffinose and 2.5g / L L-cysteine.

[0099] Logarithmic growth phase bacteria were mixed with 400μL of 50% glycerol in a cryogenic tube, and then stored in a -80℃ refrigerator.

[0100] The cryogenic glycerol bacteria were taken out from the -80℃ refrigerator, thawed on ice, and 10μL was streaked on RCM agar plates. The culture dishes were inverted and cultured in an anaerobic 37℃ constant temperature incubator for 48h, and single colonies were picked and inoculated in liquid medium for further culture.

[0101] 2. Bacterial solution preparation

[0102] (1) Logarithmic growth phase bacteria were vortexed and 200μL of bacterial solution was taken in a 96-well plate, and 200μL of MRS medium was taken as a blank control;

[0103] (2) Using EnSpire multimodal microplate detector, the absorbance of bacteria solution and blank medium at 600 nm wavelength was detected. When the absorbance difference between the two was 0.8, the concentration of the bacteria solution was about 1 x 10 9 CFU / mL;

[0104] (3) Centrifugation at room temperature for 2 min at 5000 x g, discard the supernatant, resuspend the bacteria in PBS, and obtain the bacterial precipitate after rinsing twice;

[0105] (4) Resuspend the bacterial precipitate in PBS, and make the final concentration of the bacteria solution 1 x 10 9 CFU / 100 μL.

[0106] 3. Bacterial culture treatment

[0107] (1) Bifidobacterium breve lw01 TCA degradation grouping and treatment:

[0108] MRS group: 5 mL of 20 g / L raffinose and 2.5 g / L L-cysteine containing MRS medium containing 10 μM TCA;

[0109] MRS+B.breve group: 5 mL of 20 g / L raffinose and 2.5 g / L L-cysteine containing MRS medium containing 10 μM TCA + 50 μL of B.breve bacteria solution in the logarithmic growth phase (B.breve bacteria solution);

[0110] After anaerobic culture in a 37°C constant temperature incubator for 36 h, centrifugation at room temperature for 2 min at 5000 x g, the supernatant was collected and filtered with a 0.22 μm filter membrane, and stored in a -80°C refrigerator.

[0111] (2) Bifidobacterium breve lw01 inhibits bile salt hydrolase (Bile Salt Hydrolase, BSH) experimental grouping and treatment:

[0112] MRS+B.breve group: 3 mL of 20 g / L raffinose and 2.5 g / L L-cysteine containing MRS medium containing 100 μM TCA + 30 μL of B.breve bacteria solution in the logarithmic growth phase + 100 μM DMSO;

[0113] MRS+B.breve+AAA-10 group: 3 mL of 20 g / L raffinose and 2.5 g / L L-cysteine containing MRS medium containing 100 μM TCA + 30 μL of B.breve bacteria solution in the logarithmic growth phase + 100 μM AAA-10 (purchased from MCE company, item number: HY-145147);

[0114] After 24h anaerobic incubation at 37℃, centrifuge at 5000xg for 2min at room temperature, collect the supernatant and filter through 0.22μm filter membrane, store at -80℃.

[0115] (II) Mouse grouping

[0116] (1) For high-fat diet-induced obesity model, after 1 week of adaptive feeding, mice were randomly divided into groups of 8-10, and the groups were as follows:

[0117] CD group: normal laboratory mice were given maintenance feed (Medience MD17121);

[0118] HFD group: 60kcal% high-fat diet (HFD, Research Diets D12492, 60kcal% fat) was given.

[0119] After maintaining the respective diet for 16 weeks, a subcutaneous tumor xenograft model was established, and the respective diet was maintained until the end of the experiment.

[0120] The construction process of the tumor-bearing mouse model: the right flank skin of each group of mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was depilated the day before inoculation of tumor cells. The above-mentioned log phase Hepa1-6 cells were digested and counted, resuspended in PBS at 1.2x10 6 After intraperitoneal injection of 1% sodium pentobarbital to anesthetize the mice, 100μL of cell suspension was injected subcutaneously into the right flank of the depilated mice using a 1mL insulin syringe.

[0121] (2) For oral B. breve liquid tumor inhibition model, after 1 week of adaptive feeding, mice were randomly divided into groups of 8-10, and the groups were as follows:

[0122] HFD+PBS group: PBS, 100μL per mouse, was given daily by gavage during the high-fat period;

[0123] HFD+B. breve group: B. breve liquid, 1x10 9 CFU / 100μL per mouse, was given daily by gavage during the high-fat period.

[0124] After 16 weeks of high-fat diet, a subcutaneous tumor xenograft model was established (the model establishment steps were the same as above), and the high-fat diet was continued until the end of the experiment.

[0125] (3) For TCA tumor-promoting model, after 1 week of adaptive feeding, mice were randomly divided into groups of 6-8, and the groups were as follows:

[0126] PBS group: intraperitoneal injection of PBS, 100 μL per mouse per day during normal diet;

[0127] TCA group: intraperitoneal injection of TCA, 0.25 mg / kg per mouse per day during normal diet.

[0128] After 8 weeks of continuous administration / control solvent, a subcutaneous tumor transplantation model was established (the model establishment procedure was the same as described above), and the administration / control solvent was continued until the end of the experiment.

[0129] (4) For the BSH inhibition model of Bifidobacterium breve lw01, the mice were randomly divided into groups after 1 week of adaptive feeding, with 8-10 mice in each group, and the groups were as follows:

[0130] HFD+B.breve group: B.breve bacterial solution, 1 × 10 9 CFU / 100 μL per mouse, was given by gavage daily during high-fat diet, and control solvent (20% SBE-β-CD saline) was given by gavage daily at 200 μL per mouse for 3 days before tumor inoculation;

[0131] HFD+B.breve+AAA-10 group: B.breve bacterial solution, 1 × 10 9 CFU / 100 μL per mouse, was given by gavage daily during high-fat diet, and AAA-10, 0.7 mg / 200 μL per mouse, was given by gavage daily for 3 days before tumor inoculation.

[0132] After 16 weeks of maintaining high-fat diet, a subcutaneous tumor transplantation model was established (the model establishment procedure was the same as described above), and AAA-10 / control solvent was given until the end of the experiment.

[0133] Five days after the tumor cells were inoculated into the mice, the tumorigenicity was observed, and mice with good tumorigenicity were selected for subsequent experimental analysis.

[0134] Example 2 Influence of high-fat diet-induced obesity on tumor development and body homeostasis (I)

[0136] During the entire experimental period of the above-constructed HFD group and CD group mice (e.g. Figure 1 ), the body weight of the mice was measured once a week, and the body weight of the mice was measured after fasting for 12 h the day before, and the results are shown in Figure 2 .

[0137] (II) Changes in obesity-related metabolic indicators were evaluated by measuring fasting blood glucose, oral glucose tolerance, and blood biochemistry / serum lipidomics. 1.

[0139] The above-constructed HFD group and CD group mice were fasted for 8 hours, the mouse tail tip was cut off 1-2 mm, the first drop of blood was discarded, and the second drop was used to detect the fasting blood glucose level, and the results are as followsFigure 3 shown.

[0140] The HFD group and CD group mice were given 2g / kg glucose solution at a dose of 2g / kg and subjected to an oral glucose tolerance test (OGTT). Blood glucose was monitored before the test and at 15, 30, 60, and 120 minutes after the test. Blood glucose curves were drawn and the area under the OGTT curve (AUC) was calculated. Figure 4 shown. 2.

[0142] The experiment was terminated two weeks after tumor loading, and mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital. Blood was collected from the eyeballs and collected in 1.5 mL Eppendorf tubes. After standing at room temperature for 1 hour to separate layers, the samples were centrifuged at 3000 rpm for 10 minutes at 4°C. The clear, pale yellow serum layer was collected and stored in aliquots at -80°C for determination of serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) levels.

[0143] After the serum was diluted 1:1, it was tested using the automatic biochemical analyzer with the kits for Glu, TC, TG, HDL-C, and LDL-C. Figure 5 shown. (three)

[0145] The mice in each group were sacrificed by cervical dislocation and then dissected in the supine position. The perigonal white adipose tissue, distal ileum, liver and spleen were separated and slices were prepared.

[0146] The wet weight of epididymal white adipose tissue (eWAT) and liver of each group of mice was measured. Figure 6 The liver cross sections were stained with H&E, and Image-J (Version 1.53c) was used to statistically analyze the liver fat vacuolar area ratio and white adipose tissue adipocyte diameter, and the lipid area percentage (%-AOI) was calculated. The results are shown in Figure 7 shown. (Four)

[0148] Tumor tissues from the sacrificed mice in the above groups were collected and cut into three parts for flow cytometry analysis (RPMI1640 culture medium on ice), protein extraction (quick freezing in liquid nitrogen) and paraffin section preparation (formalin fixation).

[0149] After the tumors were formed in each group of mice, they were observed every other day, and the long diameter, short diameter and mass of the tumors were measured to calculate the tumor volume. Figure 8 shown.

[0150] The formula for calculating tumor volume is: tumor volume (mm 3) = (long diameter x short diameter2) / 2

[0151] The relative expression levels of proteins Bax, Bcl-2, phosphorylated signal transduction and transcription activator 3 (p-STAT3), total signal transduction and transcription activator 3 (t-STAT3), phosphorylated protein kinase B (p-AKT), protein kinase B (AKT), Caspase-3 and cleaved Caspase-3 in Hepa1-6 tumor tissues were analyzed by Western blotting.

[0152] Extraction of tumor tissue proteins: (1) Place the tumor tissue block stored in liquid nitrogen into a 2 mL thick-walled centrifuge tube, add 200 μL of RIPA lysis buffer containing 1x protease inhibitor and 1x phosphatase inhibitor, and place a clean 5 mm diameter steel bead;

[0153] (2) Place the thick-walled centrifuge tube into a 4°C pre-cooled module, and after balancing, place the module on a high-throughput tissue crusher, with a frequency of 30 Hz and a shock time of 100 sec;

[0154] (3) Centrifuge at 12000 x g for 30 min at 4°C, and transfer the supernatant to a new 1.5 mL centrifuge tube. Complete the Western blotting procedure.

[0155] Statistical analysis of the bands using ImageJ. The results are shown in Figure 9 . (Five)

[0157] The alpha diversity (Chao1 richness index) and beta diversity (weighted UniFrac distance matrix) of the microbial community in the feces of HFD group and CD group mice were determined and compared.

[0158] Collect the mouse feces in a cryogenic tube, quickly freeze in liquid nitrogen, and store in a -80°C refrigerator. Then send the collected feces to Hangzhou Lianchuan Biotechnology Co., Ltd. for 16S rRNA gene sequencing analysis.

[0159] Extraction and quantification of total fecal microbiome DNA: (1) Select the CTAB method to extract the total DNA of the mouse fecal sample, and detect the V3-V4 region of the total DNA by agarose gel electrophoresis, and use a UV spectrophotometer to quantify the DNA;

[0160] (2) Take 200 μL of PCR tube, add 50 ng of DNA, 12.5 μL of 2x Phusion Hot Start Flex MasterMix, 5 μL of primer (upper and lower primer premix, final concentration 10 μM), and fill up to 25 μL with Milli-Q water, mix gently, and centrifuge; the primer sequences are shown in Table 1;

[0161] (3) Put into PCR instrument for amplification: 98℃, 30s; 98℃, 10s, 54℃, 30s, 72℃, 45s (cycle 35 times); 72℃, 10min; 4℃, 5min;

[0162] (4) Collect the PCR product, detect by 2% agarose gel electrophoresis, recover by AMPure XT beads kit, and quantify by Qubit.

[0163] Table 1 primer sequence

[0164]

[0165] The purified PCR product was evaluated using Agilent 2100 bioanalyzer and Illumina library quantification kit. The qualified sequencing library was gradient diluted, mixed according to the required sequencing amount, denatured to single strand by NaOH, and sequenced. NovaSeq 6000 sequencer was used for 2x250bp double-end sequencing.

[0166] Data processing and analysis of fecal microbiome total DNA: (1) According to the barcode information, the data of each sample was split, and the adapter and barcode sequences were removed;

[0167] (2) Using cutadapt_v1.9 to remove primer sequences and balanced base sequences of RawData;

[0168] (3) Using FLASH_v1.2.8 to splice each pair of paired-end reads into a longer tag according to the overlap region;

[0169] (4) Using fqtrim to scan the sequencing reads by window method, the scanning window is 100bp by default, when the average quality value in the window is less than 20, the part from the start of the window to the 3' end of the reads is cut off;

[0170] (5) Using Vsearch_v2.3.4 to remove sequences with length less than 100bp after cutting, sequences with N (uncertain ambiguous bases) content more than 5% after cutting, and chimeric sequences;

[0171] (6) Through qiime dada2 denoise-paired, DADA2 was called to filter length and denoise, to obtain amplicon sequence variant (ASV) feature sequence and ASV abundance table, and to remove singletons ASVs;

[0172] (7) Based on the obtained ASV feature sequence and ASV abundance table, alpha diversity analysis and beta diversity analysis are performed; according to the ASV feature sequence, species annotation is performed by using the SILVA database and the NT-16S database, and the abundance of each species in each sample is counted according to the ASV abundance table; based on the obtained species abundance statistical information, difference analysis between groups is performed.

[0173] The results are shown in FIG. 2, wherein the linear discriminant analysis effect size (LEfSe) results show that there are significant differences in the abundance of bacteria between the normal diet and high-fat diet mice. Figure 10

[0174] In summary, high-fat diet-induced obesity induces metabolic disorders and reduces the beneficial bacteria Bifidobacterium in the intestinal tract, which significantly promotes the progression of liver cancer in mice.

[0175] Example 3

[0176] During the entire experimental period of the HFD+PBS group and the HFD+B. breve group mice (as shown in FIG. 1), the body weight of the mice was measured once a week. The results are shown in FIG. 2. Figure 11 Figure 12

[0177] As in Example 2, the fasting blood glucose level of the mice in each group was measured after fasting for 8 hours, and the results are shown in FIG. 3. Figure 13

[0178] As in Example 2, oral glucose tolerance test (OGTT) was performed, and the blood glucose level before and after the test and the area under the OGTT curve (AUC) were measured, and the results are shown in FIG. 4. Figure 14

[0179] As in Example 2, the HFD+PBS group and the HFD+B. breve group mice were sacrificed, and the wet weight of the epididymal white adipose tissue and the liver of the mice in each group was measured, and the results are shown in FIG. 5. H&E staining was performed on the liver cross-section, and the lipid area percentage (%-AOI) was calculated, and the results are shown in FIG. 6. Figure 15 Figure 16

[0180] As in Example 2, the tumor tissue of the HFD+PBS group and the HFD+B. breve group mice was collected, and the tumor length, width, and weight were measured, and the tumor volume was calculated, and the results are shown in FIG. 7. Figure 17

[0181] ​​​​​​​​As in Example 2, the relative expression levels of proteins Bax, Bcl-2, phosphorylated signal transduction and transcription activator 3 (p-STAT3), total signal transduction and transcription activator 3 (t-STAT3), phosphorylated protein kinase B (p-AKT), protein kinase B (AKT), Caspase-3 and cleaved Caspase-3 in Hepa1-6 tumor tissues were analyzed by Western blotting, and the results are shown in Table 2. Figure 18

[0182] In summary, taking B. breve lw01 bacterial liquid can inhibit tumor growth promoted by high-fat diet and improve glucose and lipid metabolism.

[0183] Example 4

[0184] Based on Example 2 and Example 3, tumor tissues of CD group, HFD group, HFD+PBS group and HFD+B. breve group mice on day 14 after inoculation of Hepa1-6 tumor were collected, tumor infiltrating immune cells were isolated, and flow cytometry was performed for evaluation.

[0185] The tumor tissue was cut into rice grain size in a 6-well plate, 2 mL of serum-free RPMI1640 was added to each well, the suspension was collected into a 15 mL centrifuge tube, the scissors and the plate were washed with serum-free RPMI1640, the liquid was collected into a 15 mL centrifuge tube, and the volume was made up to 4 mL with serum-free RPMI1640, and the divided collagenase I was added to make the enzyme concentration 1.5 mg / mL; (2) the centrifuge tube was placed in a metal bath at 37°C and 500 rpm, and after digestion for one hour, 2 times the volume of serum-containing RPMI1640 was added to terminate the digestion; (3) the digested liquid was blown and the unfiltered tissue was ground with a 5 mL syringe (a small amount of serum-free RPMI1640 was used for washing), the liquid was collected into a 50 mL centrifuge tube, and 650 x g centrifugation was performed at room temperature for 7 min; (4) the supernatant was discarded, the cells were mixed, 2 mL of red cell lysing solution was added, 20 s later 10 times the volume (20 mL) of serum-free PBS was added to terminate the reaction, and 650 x g centrifugation was performed at room temperature for 7 min; (5) the supernatant was discarded, the remaining liquid was transferred to a 1.5 mL EP tube, 1 mL of serum-free PBS was added for washing once, and 500 x g centrifugation was performed at 4°C for 5 min; (6) the supernatant was aspirated, 100 μL of cell death and viability staining working solution was added to each sample, and incubation was performed at room temperature for 30 min in the dark; (7) washing was performed twice with 3% serum-containing PBS, and 500 x g centrifugation was performed at 4°C for 5 min; (8) the supernatant was discarded, the cells were resuspended with the remaining liquid, and for myeloid cell staining: 1 μL of Fc receptor blocker was added to each experimental tube, the mixture was incubated at 4°C for 10 min, 50 μL of B V staining buffer was then added, and the mixture was mixed; for lymphoid cell staining, no pretreatment was performed; (9) according to the following two sets of staining indexes, lymphoid cells (CD45-APC / Fire​TM 750, CD3-PE, CD4-FITC, CD8-PerCP, NK1.1-BV421) and myeloid (CD45-BV510, CD11b-FITC, F4 / 80-APC / Cy7, I-A / I-E-APC, CD11c-PE / Cy7, Ly-6C-BV421, Ly-6G-BV650, CD86-BV605, CD206-PE), blank control and single staining sample tubes were set up, the staining system was 100 μL, the blank control did not add any antibody, 1 μL of single antibody was added to each single staining sample tube, and 1 μL of each antibody was added to the experimental tube, and after mixing, it was incubated at room temperature for 30 min in the dark.(10) Wash twice with PBS containing 3% serum at 500xg for 5 min at 4°C; (11) Discard the supernatant, resuspend with an appropriate amount of PBS containing 3% serum, and filter onto the machine.(12) Set the voltage of each channel according to the blank control, adjust the compensation between each channel according to the single staining sample tube, and after determining two sets of staining template parameters, collect and record the detection samples respectively. The tumor-infiltrating CD8 + T cells into a 1.5 mL EP tube containing 1 mL of TRIzol, and placed on ice for standby. The proportion of each cell subpopulation was analyzed using FlowJo_v10.8.1.

[0186] The expression of natural killer (NK) cells, natural killer T (NKT) cells, T cells, cytotoxic T (Tc) cells, and helper T (Th) cells in total single live cells in Hepa1-6 tumors is shown in Figure 19 The proportion of Tc cells and Th cells in T cells is shown in Figure 20 The expression of dendritic cells (DCs), tumor-associated macrophages (TAMs) and their subsets, and myeloid-derived suppressor cells (MDSCs) in total single live cells in Hepa1-6 tumors is shown in Figure 21 The proportion of pro-inflammatory (M1) TAMs and alternatively activated (M2) TAMs in TAMs is shown in Figure 22

[0187] H&E staining was performed on tumor tissues of each group, and immunohistochemical staining was performed on tumor sections. The main tumor-infiltrating immune cells were identified by detecting the expression of corresponding surface markers such as CD3, CD4, CD8, F4 / 80, CD86 and CD206, and quantitative analysis was performed using ImageJ software. The results are shown in Figure 23

[0188] In summary, taking Bifidobacterium breve lw01 bacterial liquid can improve the tumor immune suppression microenvironment under obesity.

[0189] Example 5 ​​(I)

[0191] The changes of serum metabolites in mice were detected by liquid chromatography-tandem mass spectrometry, and the differential metabolites were found by correlation analysis.

[0192] The mouse serum was sent to Wuhan Maiteer Biological Technology Co., Ltd. for targeted metabolomics analysis. The serum metabolite compositions of the normal diet (CD) group, high-fat diet (HFD) group and HFD+B. breve group mice were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the results are shown in Figure 24 .

[0193] It can be seen that TCA has a tumor-promoting effect, and taking B. breve lw01 bacterial liquid can inhibit tumor growth by debonding TCA. (II)

[0195] Based on the above prepared MRS group and MRS+B. breve group under in vitro culture conditions, a certain concentration of TCA (specifically as in Example 1) was added to the liquid culture medium of B. breve in vitro, and then the culture supernatant was detected for high-throughput targeted bile acid metabolites to determine the decomposition of TCA by B. breve lw01. The results are shown in Figure 25 . (III)

[0197] During the above-mentioned whole experimental period of PBS group and TCA group mice (such as Figure 26 ), from tumor formation, the tumor growth was measured and observed every other day, and the tumor weight was measured, and the results are shown in Figure 27 .

[0198] As in Example 4, the expression of natural killer (NK) cells, natural killer T (NKT) cells, T cells, cytotoxic T (Tc) cells, and helper T (Th) cells in total single live cells in Hepa1-6 tumors was measured, as shown in Figure 28 ; the proportion of Tc cells and Th cells in T cells, as shown in Figure 29 ; the expression of dendritic cells (DCs), tumor-associated macrophages (TAMs) and their subsets, and myeloid-derived suppressor cells (MDSCs) in total single live cells in Hepa1-6 tumors, as shown in Figure 30 ; the proportion of pro-inflammatory (M1) TAMs and alternative-activated (M2) TAMs in TAMs, as shown in Figure 31 .

[0199] As in Example 4, H&E staining of tumor tissues and immunohistochemical staining of tumor sections were performed, the corresponding surface markers were detected, and quantitative analysis was performed using ImageJ software. The results are as followsFigure 32 Results showed that TCA exacerbated tumor development and limited CD8 + T cell infiltration.

[0200] In summary, tauroursodeoxycholic acid (TCA) is a key differential metabolite, and taking Bifidobacterium breve lw01 regulates intestinal flora and reduces the level of serum tauroursodeoxycholic acid in obese mice.

[0201] Example 6 (I)

[0203] Based on the above-prepared MRS+B.breve group and MRS+B.breve+AAA-10 group under in vitro culture conditions, high-throughput targeted metabolome detection was performed on the BSH activity of Bifidobacterium breve lw01. The results are shown in Figure 33 . (II)

[0205] During the entire experimental period of the above-constructed MRS+B.breve group and MRS+B.breve+AAA-10 group mice (such as Figure 34 ), from tumor formation, tumor growth was measured and observed every other day, and tumor weight was measured. The results are shown in Figure 35 .

[0206] As in Example 2, the body weight of mice in each group was measured, and the results are shown in Figure 36 .

[0207] As in Example 2, the MRS+B.breve group and the MRS+B.breve+AAA-10 group mice were sacrificed, and the wet weight of the epididymal white adipose tissue and liver of the mice in each group was measured. The measurement results are shown in Figure 37 . H&E staining was performed on the liver cross-section, and the lipid area percentage (%-AOI) was calculated. The results are shown in Figure 38 .

[0208] As in Example 4, the expression of natural killer (NK) cells, natural killer T (NKT) cells, T cells, cytotoxic T (Tc) cells, and helper T (Th) cells in total single live cells in Hepa1-6 tumors was measured, as shown in Figure 39 ; the proportion of Tc cells and Th cells in T cells, as shown in Figure 40 ; the expression of dendritic cells (DCs), tumor-associated macrophages (TAMs) and their subsets, and myeloid-derived suppressor cells (MDSCs) in total single live cells in Hepa1-6 tumors, as shown in Figure 41 ; and the proportion of pro-inflammatory (M1) TAMs and alternative-activated (M2) TAMs in TAMs, as shown in Figure 42 .

[0209] As in Example 4, H&E staining was performed on each group of tumor tissues, and immunohistochemical staining was performed on tumor sections to detect the corresponding surface markers, and quantitative analysis was performed using ImageJ software. The results are shown in Figure 43 .

[0210] In summary, inhibiting the BSH activity of B. breve lw01 can weaken its anti-tumor effect; B. breve lw01 degrades tauroursodeoxycholic acid through bile salt hydrolase to suppress its tumor-promoting effect.

[0211] Example 7

[0212] Genome analysis of B. breve lw01 and annotation of its bile salt hydrolase Bsh gene were performed to analyze the dissociation of tauroursodeoxycholic acid by the strain.

[0213] (I) Genome analysis of B. breve lw01

[0214] The whole genome sequence of B. breve lw01 (available on GenBank, accession number CP034192) was annotated and visualized using Bakta (https: / / bakta.computational.bio / ). The corresponding amino acid sequences generated were annotated by KEGG and GO using eggNOG-Mapper (http: / / eggnog-mapper.embl.de / ). The results are shown in Figure 44 .

[0215] (II) Collection of Bsh genes and construction of phylogenetic tree

[0216] The Bsh gene sequences of the Bifidobacterium species used in the study were retrieved from NCBI Nucleotide, and the Bsh gene sequence of B. breve lw01 was obtained according to the annotated position of the genome analysis and verified by NCBI Blastx. The Bsh gene sequences were aligned and a phylogenetic tree was constructed using the Neighbor-Joining method (Kimura 2-Parameter model) in MEGA software. iTOL (https: / / itol.embL.de / ) was used to beautify the phylogenetic tree, and the labels and fill colors were adjusted as needed. The results are shown in Figure 45 .

[0217] (III) BSH amino acid sequence alignment and protein structure prediction

[0218] 3D structure prediction models of unknown structures were generated from amino acid sequences by alignment with known protein structures using Phyre2 (https: / / www.sbg.bio.ic.ac.uk / phyre2 / html / page.cgi?id=index). 3D structure comparison and root mean square distance (RMSD) spatial variation between BSH of B. breve lw01 and type 4 BSH were calculated using Chimera. The results are shown in FIG. 6. Figure 46

[0219] In summary, B. breve lw01 encodes a type 4 bile salt hydrolase.

[0220] Example 8 (I)

[0222] Mouse CD8 + T cell depletion experiment (as shown in FIG. 5), mice were randomly divided into 4 groups after 1 week of adaptive feeding, 6-8 mice in each group, the specific grouping as follows: Figure 47

[0223] (1) PBS+isotype group: intraperitoneal injection of PBS, 100 μL per mouse, during normal diet, intraperitoneal injection of isotype control antibody IgG2b, 250 μg per mouse, every 2 days, 3 days before tumor-bearing;

[0224] (2) TCA+isotype group: intraperitoneal injection of TCA, 0.25 mg / kg per mouse, during normal diet, intraperitoneal injection of isotype control antibody IgG2b, 250 μg per mouse, every 2 days, 3 days before tumor-bearing;

[0225] (3) PBS+αCD8 group: intraperitoneal injection of PBS, 100 μL per mouse, during normal diet, intraperitoneal injection of CD8α monoclonal antibody, 250 μg per mouse, every 2 days, 3 days before tumor-bearing;

[0226] (4) TCA+αCD8 group: intraperitoneal injection of TCA, 0.25 mg / kg per mouse, during normal diet, intraperitoneal injection of CD8α monoclonal antibody, 250 μg per mouse, every 2 days, 3 days before tumor-bearing.

[0227] After 8 weeks of continuous administration / control solvent, a subcutaneous tumor transplantation model was established (the model establishment procedure was the same as in Example 1), and the administration / control solvent was continued until the end of the experiment.

[0228] Five days after the mice were inoculated with tumor cells, the tumorigenicity was observed, and mice with good tumorigenicity were selected for subsequent experimental analysis.

[0229] As in Example 5, the macroscopic performance of Hepa1-6 tumors was observed, and the tumor growth curve and tumor weight were determined, and the results are shown in FIG. 6.​​Figure 48 Figure 6 shows the results of flow cytometry analysis of tumor infiltrating lymphocytes.

[0230] Figure 7 shows the results of flow cytometry analysis of tumor infiltrating lymphocytes. Figure 49 (II)

[0232] Figure 8 shows the results of flow cytometry analysis of tumor infiltrating lymphocytes. + T cells were activated and expanded with IL-2 and a-CD3 / CD28 antibodies.

[0233] Figure 9 shows the results of flow cytometry analysis of tumor infiltrating lymphocytes. + T cells were activated and expanded with IL-2 and a-CD3 / CD28 antibodies. 8 T cells were activated and expanded with IL-2 and a-CD3 / CD28 antibodies. 7 T cells were activated and expanded with IL-2 and a-CD3 / CD28 antibodies. 7 T cells were activated and expanded with IL-2 and a-CD3 / CD28 antibodies. + T cells were activated and expanded with IL-2 and a-CD3 / CD28 antibodies.

[0234] Figure 10 shows the results of flow cytometry analysis of tumor infiltrating lymphocytes. + ​Activation of T cells: Cells were resuspended to 3 x 10 6 6-well plates at 37 °C with 5% CO2 for 48 h.

[0235] Mouse primary CD8 + Treatment of T cells: The obtained activated mouse primary CD8+T cells were pretreated with 5 mM and 10 mM JTE-013 (S1PR2 inhibitor) RPMI 1640 complete medium for 1 h or 5 mM C16-PAF (ERK agonist) RPMI 1640 complete medium for 2 h, and then treated with 0 mM, 500 mM or 1000 mM TCA RPMI 1640 complete medium or 2.5 mM and 5 mM CYM-5520 (S1PR2 agonist) RPMI 1640 complete medium at 37 °C with 5% CO2.

[0236] 1. CD8 + Detection of T cell apoptosis

[0237] After 24 h of TCA stimulation, the cell suspension was collected by blowing and centrifuged at 500 x g for 5 min at 4 °C. The supernatant was discarded, and the cells were washed twice with PBS at 500 x g for 5 min at 4 °C. According to the staining index (Live / Death-Zombie Violet, CD8-APC), blank control and single staining sample tubes were set up. The blank tube was added with 100 mL PBS, the Live-Death and experimental tubes were added with 100 mL cell death and live staining working solution, and the CD8 single positive tube and experimental tube were added with 1 mL of the corresponding antibody. After mixing, the mixture was incubated at room temperature for 30 min in the dark. The cells were washed twice with 3% serum-containing PBS at 500 x g for 5 min at 4 °C. The supernatant was discarded, and the cells were resuspended with an appropriate amount of 3% serum-containing PBS and filtered onto the machine. According to the blank control, the voltage of each channel was set, and according to the single staining sample tube, the compensation between channels was adjusted. After determining two sets of staining template parameters, the detection samples were collected and recorded, respectively. The proportion of apoptotic cells was analyzed using FlowJo_v10.8.1. The results are shown in Figure 50

[0238] 2. CD8 + Detection of T cell proliferation, cytokines and chemokines

[0239] ​After 36h of TCA stimulation, a cell activation mixture containing Brefeldin A was added for 6h to stimulate cytokine production and inhibit their secretion outside the cell. The cell suspension was collected by pipetting, centrifuged at 500 x g for 5 min at 4°C. The supernatant was discarded, and the cells were washed twice with PBS, centrifuged at 500 x g for 5 min at 4°C. The blank control and single staining sample tubes were set according to the surface staining index (CD8-APC, CXCR6-FITC), and the staining system was 100 μL. The blank control did not add any antibody, and each single staining sample tube was added with 1 μL of single antibody. Each antibody was added to the experimental tube at 1 μL, and the mixture was incubated at room temperature in the dark for 30 min. The cells were washed twice with PBS, centrifuged at 500 x g for 5 min at 4°C. The supernatant was discarded, and 500 μL of fixing solution was added to each tube, which was incubated at 4°C in the dark for 30 min. The cells were centrifuged at 500 x g for 5 min at 4°C. The supernatant was discarded, and 500 μL of membrane-breaking solution was added to each tube, which was incubated at room temperature in the dark for 30 min. The cells were centrifuged at 500 x g for 5 min at 4°C. The blank control and single staining sample tubes were set according to the intracellular and nuclear staining index (Ki67-BV650, TNF-α-PE / Cy7, IFN-γ-BV421, Gramzyme B-PE, Perforin-APC), and the staining system was 100 μL. The blank control did not add any antibody, and each single staining sample tube was added with 1 μL of single antibody. Each antibody was added to the experimental tube at 1 μL, and the mixture was incubated at room temperature in the dark for 30 min. The cells were washed twice with PBS, centrifuged at 500 x g for 5 min at 4°C. The supernatant was discarded, and the cell pellet was resuspended with an appropriate amount of PBS, and then filtered onto the machine. The voltage of each channel was set according to the blank control, the compensation between each channel was adjusted according to the single staining sample tube, and after the parameters were determined, the detection samples were collected and recorded. The expression levels of intracellular factors, cytokines, and chemokines were analyzed using FlowJo_v10.8.1. The results are shown in Figure 51

[0240] 3. The expression levels of the cytotoxic cytokine and chemokine receptors of CD8 + T cells were determined by referring to β-actin.

[0241] The total RNA of CD8 + T cells was extracted and quantified: (1) After 8h of TCA stimulation, the cell suspension was collected by pipetting, centrifuged at 500 x g for 5 min at 4°C; (2) The supernatant was discarded, and the cells were washed twice with PBS, centrifuged at 500 x g for 5 min at 4°C; (3) The supernatant was discarded, and 1 mL of TRIzol was added, and the mixture was uniformly mixed and incubated at room temperature for 5 min before RNA extraction. The RNA concentration was determined using a Nanodrop, and the experiment was continued or the sample was stored in a -80°C refrigerator.

[0242] ​Reverse transcription PCR: (1) Take 200 μL PCR tube, add 2 μg RNA, 8 μL 5x PrimeScript RT Master Mix, and use DEPC water to make up to 40 μL, mix gently, centrifuge; (2) put into PCR instrument for reverse transcription: 37℃, 15 min; 85℃, 5 s; 4℃, 5 min; (3) collect cDNA product, continue the experiment or store in -80℃ refrigerator.

[0243] Real-time quantitative PCR: (1) Take 1.5 mL centrifuge tube, according to the number of samples, the number of detection indexes and the number of sub-wells, 1 μL cDNA template per well, 8 μL Milli-Q water, 10 μL SYBR Green premix, on ice; (2) prepare 100 μL eight-row PCR tube, add 19 μL mixture to the bottom of the tube, and add 1 μL primer (upstream and downstream premix, final concentration 10 μM) to the wall of the tube. The primer sequence is shown in Table 2; (3) cover the eight-row PCR tube tightly, and centrifuge for 1 min; (4) put the eight-row PCR tube into the real-time fluorescence quantitative PCR instrument: pre-denaturation, 95℃, 10 min; denaturation, 95℃, 15 s; annealing / extension, 60℃, 1 min; denaturation and annealing / extension stage for 40 times; (5) use Actb as the internal reference gene, calculate the average Ct value of the internal reference gene in each group according to the Ct value of each well, calculate ΔCt = target gene Ct value - average Ct value of internal reference gene in each group, ΔΔCt = experimental group ΔCt value - control group average ΔCt value, 2 -ΔΔCt The relative expression level of each gene.

[0244] Table 2 Primer sequence used in real-time quantitative PCR experiment

[0245]

[0246] The determination results are shown in Table 3. Figure 52 Table 3 Determination results

[0247] In summary, tauroursodeoxycholic acid inhibits the MAPK / ERK pathway, limits the local CD8 + T cell infiltration in tumor, and weakens its cytotoxic function, promoting tumor growth.

[0248] Example 9

[0249] The collected tumor infiltrating CD8 + T cells were sent to Shenzhen Huada Gene Technology Co., Ltd. for mRNA sequencing analysis.

[0250] The collected tumor infiltrating CD8 +Data processing and analysis of T cell mRNA: The raw data obtained by sequencing was filtered using SOAPnuke to filter out reads containing adapters, reads with unknown base N content greater than 5%, and low-quality reads (reads with a proportion of bases with quality values less than 15 accounting for more than 20% of the total number of bases in the reads). Clean data was obtained. Subsequently, data analysis, plotting, and mining were performed using Dr. Tom's multi-omics data mining system. (2) Clean data was aligned to the reference genome using HISTA2 software. Then, Ericscript was used for gene fusion detection, and rMATS was used for variable splicing and differential variable splicing detection. (3) Clean data was aligned to the reference gene set using Bowtie2. Gene expression quantification was performed using RSEM (v1.3.1) [6] software, and pheatmap was used to plot the expression quantity clustering heat map of genes in different samples. DESeq2 was used for differential gene detection with a condition of Q value≤0.05 or FDR≤0.001. (4) Further exploration of the functions of genes related to phenotype changes, based on hypergeometric test, Phyper was used for GO and KEGG enrichment analysis of differential genes, with Qvalue≤0.05 as the threshold, and genes meeting this condition were defined as significantly enriched in candidate genes.

[0251] CD8 + T cells isolated from Hepa1-6 tumors of TCA group and PBS group mice were analyzed by RNA sequencing. The results of RNA sequencing analysis are shown in Figure 53 . The relative expression of characteristic genes in CD8 + T cells from TCA-treated and PBS group mice is shown in Figure 54 . The results of KEGG pathway enrichment analysis are shown in Figure 55 . The results of biological process (GO-BP) analysis of down-regulated genes in TCA-treated CD8 + T cells are shown in Figure 56 . The top 20 pathways identified are shown.

[0252] CD8 + T cells isolated from WT mice were treated with different doses of TCA, and the phosphorylation of ERK, P38, JNK, and P65 was analyzed by immunoblotting (the same as in Example 2). The relative expression of ERK, P38, JNK, and P65 was determined by normalization to RPS-18, and the results are shown in Figure 57 .

[0253] The effect of TCA on CD8 +The effects of ERK phosphorylation, proliferation and cytotoxic cytokine and chemokine receptors expression at the transcriptional and protein levels in T cells were manipulated as in Example 8, and the results are shown in Figure 58 and Figure 59 .

[0254] In summary, TCA inactivates CD8 + T cells through the MAPK / ERK pathway.

[0255] In summary, oral B. breve lw01 degrades the elevated circulating taurocholic acid in obese mice using bile salt hydrolase, rescues the impaired chemotaxis and cytotoxic effect of CD8 + T cells by restoring their ERK phosphorylation levels, improves the tumor immunosuppressive microenvironment, and inhibits tumor development in obese mice (as shown in Figure 60 .

[0256] The above only is the preferred embodiment of the present application, it should be pointed out that, for those skilled in the technical field, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Application of Bifidobacterium breve lw01 in the preparation of products for preventing or assisting in the treatment of obesity-related tumors.

2. The use according to claim 1, characterized in that The Bifidobacterium breve lw01 is classified as Bifidobacterium breve and was deposited in the General Microbiology Center of China Culture Collection Committee on May 8, 2019. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCCNO.17727.

3. The application of Bifidobacterium breve lw01 in the preparation of a product for targeted regulation of intestinal flora, characterized in that: Targeted regulation of intestinal flora in obese states.

4. Use of Bifidobacterium breve lw01 in the preparation of a product for regulating bile acid metabolism, characterized in that: Regulate bile acid metabolism levels in obese states.

5. The use of bile acids as targets in the preparation of products for the prevention or adjuvant treatment of obesity-related tumors.

6. An obesity-related tumor immunosuppressive product, characterized in that: The invention comprises the Bifidobacterium breve lw01 according to claim 1.

7. The obesity-related tumor immunosuppressive product according to claim 6, characterized in that: The concentration of the Bifidobacterium breve lw01 is (0.8-1.2)×10 9 CFU / 100μL.

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