Application of intestinal microbiota in stabilizing intestinal glutamine and in preparing drugs for treating cancer

Abstract

The present invention relates to the technical field of anti-cancer drugs, and specifically relates to the application of intestinal microbiota in stabilizing intestinal glutamine and in the preparation of drugs for treating cancer. The large production of glutamine caused by environmental pollutant-induced intestinal microbiota dysbiosis is absorbed through the intestine and enters the body, promoting the development and adverse outcomes of breast cancer in the body. Based on a mouse model, this solution studied the patterns of environmental pollutant-induced intestinal flora dysbiosis, excessive glutamine production, and breast cancer development. Combining metagenomic and database analysis, beneficial microorganisms - Blautia ovata and Clostridium butyricum that can inhibit the progression of breast cancer were screened. The above probiotics can achieve anti-cancer activity by regulating glutamine production and can be used for the treatment of breast cancer. By orally supplementing Clostridium butyricum or Blautia ovata, the intestinal microecology induced by cadmium exposure can be improved and the glutamine content in the intestine can be regulated, thereby effectively antagonizing the occurrence and development of breast cancer, and having ideal application prospects and value.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-cancer drugs, and specifically relates to the application of intestinal microorganisms in stabilizing intestinal glutamine and in the preparation of drugs for treating cancer. Background Art

[0002] Glutamine (Gln) is the most abundant non-essential amino acid in the human body and is also one of the energy sources of cancer cells. Rapidly proliferating cancer cells require a unique metabolic process to support their increasing biomass synthesis for continuous progression. Glutamine can exert biological functions in the following ways, thus affecting tumor progression: Glutamine can serve as a nitrogen donor for purines and pyrimidines and play an important role in the synthesis of nucleotide precursors. Glutamine can play a role as a rate-limiting factor in the proliferation of tumor cells; Glutamine can serve as a nitrogen donor for non-essential amino acids for the synthesis of aspartic acid, alanine, phosphoserine, etc., and thus affect cell proliferation; Glutamine can serve as a carbon donor and directly provide carbon for the synthesis of citric acid and fatty acids, and de novo lipid synthesis plays an important role in tumor growth; Glutamine can affect the electron transport chain and reactive oxygen species, affecting the oxygen consumption rate and ATP production of cells to promote tumor formation; Glutamine can participate in cell signal transduction, and Glutamine can coordinate intracellular signal transduction to promote tumor growth, etc.

[0003] Among different types of cancers, breast cancer's dependence on glutamine metabolism is particularly prominent. HER-2 positive breast cancer is particularly "addicted" to glutamine because HER-2 positive breast cancer cells have higher glutamine consumption and glutamine metabolism levels than ductal subtype cells. The level of glutamine synthesized by the human body itself is insufficient for the growth of malignant tumors, and cancerous cells will retrieve external glutamine to support their rapid proliferation. If the level of glutamine in the body can be stabilized, it will further promote the treatment of cancers that are dependent on glutamine metabolism. Currently, researchers are trying to target glutamine metabolism for cancer treatment. Some studies directly target the synthesis process of glutamine to regulate the synthesis amount of glutamine, thereby affecting tumor cell metabolism; some studies indirectly target some key molecules downstream of the action of glutamine to affect the development of tumor cells. However, there is still a lack of relevant reports on preparations or drugs that can effectively regulate the level of glutamine in the body (such as in the intestine). How to prevent and treat cancer by effectively controlling and stabilizing the level of glutamine in the intestine is an urgent problem in the prior art. Summary of the Invention

[0004] The present invention aims to provide an application of intestinal microorganisms in the preparation of a preparation for stabilizing the intestinal glutamine level, so as to solve the technical problem in the prior art of the lack of reagents or drugs for preventing and treating cancer by stabilizing the glutamine level in the intestine.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] Application of intestinal microorganisms in the preparation of a preparation for stabilizing the intestinal glutamine level, wherein the intestinal microorganisms include Blautia obeum.

[0007] Furthermore, the intestinal microorganisms further include Clostridium butyricum.

[0008] Furthermore, the preservation number of Blautia obeum is DSM25238; the preservation number of Clostridium butyricum is BNCC337239.

[0009] The technical principle and beneficial effects of adopting the above technical solutions are as follows:

[0010] According to prior art reports, the intestinal flora can affect the process of tumor development by changing metabolism, estrogen recirculation, immune response, etc. It has been found that the intestinal flora is closely related to the development of colorectal cancer and the response to treatment. In addition, it has been reported that intestinal flora dysregulation can trigger the development of prostate cancer by increasing the content of lipopolysaccharide (LPS) in tumors. However, regarding the regulatory effect of specific intestinal microorganisms on the glutamine level, there has been no report in the prior art. The inventors have found that oral (gavage) administration of Blautia obeum (B. obeum) and Clostridium butyricum (C. butyricum) can be used to stabilize the intestinal glutamine level, and these two microorganisms are used as preparations for stabilizing the glutamine level (mainly down-regulating the glutamine level).

[0011] During the inventors' research, MMTV-Erbb2 transgenic mice were used. MMTV-Erbb2 transgenic mice overexpress the wild-type Erbb2 gene in their mammary glands. Compared with the subcutaneous tumor implantation model in nude mice, the formation of mammary tumors in MMTV-Erbb2 mice is similar to that in humans, making it an ideal system for studying the risk of mammary tumors. This model has been widely used to study the risk of breast cancer regulated by environmental pollutants or dietary phytoestrogenic compounds (such as BPA and soy isoflavones). The inventors promoted the occurrence and development of mammary tumors in MMTV-Erbb2 transgenic mice by forming low-dose chronic cadmium exposure by adding cadmium ions to the drinking water (Example 1). Experimental studies found that cadmium exposure in drinking water could induce an increase in the level of glutamine in the intestines of MMTV-Erbb2 transgenic mice, thereby exacerbating breast cancer (Example 3). The inventors further conducted a metabolic profile study on MMTV-Erbb2 transgenic mice exposed to cadmium in drinking water and found that glutamine metabolism might be the key pathway involved in cadmium-promoted breast cancer development and deterioration in MMTV-Erbb2 mice (Example 2). Moreover, inhibiting glutamine metabolism reversed the process of cadmium-induced mammary tumors in mice. In addition, based on the above research, the inventors also studied that cadmium exposure changed the metabolic profile of the gut microbiota in MMTV-Erbb2 mice, and the synthesis of glutamine was significantly increased (Example 4). Combining metagenomic and database analysis, it was found that environmental-dose cadmium exposure in drinking water led to gut microbiota dysbiosis in MMTV-Erbb2 mice, disrupted the gut microecological balance by inhibiting bacteria that consume glutamine, induced excessive synthesis of glutamine, and thus promoted the progression of mammary tumors in MMTV-Erbb2 mice (Examples 5 and 6). To screen out the bacterial species that were inhibited by cadmium and decomposed and consumed glutamine, the inventors used the MetOrign online database to search for all bacterial species that were enriched in the alanine, aspartate, and glutamate metabolic pathways and utilized glutamine. The bacterial species in the database were intersected with the significantly reduced differential bacterial species found by metagenomic analysis, and then a correlation analysis was performed with the glutamine content in their corresponding cecal contents. Then, a linear discriminant analysis value analysis was carried out, and it was found that the two bacterial species with the highest LDA value and significantly correlated with the glutamine content were Clostridium butyricum and Blautia ovata (Example 7, Figure 7 D shows the heatmap of Clostridium butyricum and Blautia ovata, and the LDA values of the two bacteria are in the top two).

[0012] To further verify the effects of Clostridium butyricum and Blautia ovata, the inventors further conducted experimental studies on supplementing Clostridium butyricum or Blautia ovata in cadmium-exposed MMTV-Erbb2 transgenic mice. The experimental results are shown in Figure 8 and Figure 9。The experimental results showed that supplementing Clostridium butyricum or Blautia ovata significantly inhibited the increase in glutamine content in intestinal contents induced by cadmium exposure, effectively regulated the disorder of intestinal microecology induced by cadmium exposure, and the effects of the two bacteria were comparable; cadmium exposure led to a significant increase in tumor weight and an increase in the expression level of the malignant tumor marker Ki67 in mice, but the tumor weight of mice supplemented with Clostridium butyricum or Blautia ovata was significantly smaller than that of the cadmium-treated group, and in the immunofluorescence detection of Ki67 in tumors, Clostridium butyricum or Blautia ovata could reduce the percentage of cells labeled with Ki67, and in terms of trend, the effect of Blautia ovata was more excellent. Therefore, supplementing Clostridium butyricum or Blautia ovata can effectively regulate the increase in glutamine content caused by intestinal microbial disorders, which is closely related to the inhibition of breast tumor progression, further verifying the above metagenomic and database analysis results. Clostridium butyricum or Blautia ovata can be used as a preparation to stabilize the intestinal glutamine level and applied to the medical practice of the treatment and prevention of cancers related to glutamine metabolism.

[0013] The present technical solution also provides the use of intestinal microorganisms in the preparation of drugs for preventing or treating cancers, and the intestinal microorganisms include Clostridium butyricum and / or Blautia ovata.

[0014] Furthermore, the cancer is breast cancer.

[0015] Furthermore, the breast cancer is caused by chronic cadmium exposure from drinking water; the cadmium concentration in the drinking water is 3.6 mg / L.

[0016] Furthermore, in mice, the gavage dose of Clostridium butyricum and / or Blautia ovata each time is 2×10 8 colony-forming units, and gavage is performed twice a week.

[0017] The technical principle and beneficial effects of adopting the above technical solution are as follows:

[0018] Animal and population studies have shown that under the influence of exposure to environmental pollutants, changes in the intestinal flora will disrupt the metabolic balance and physiological processes of the host, leading to the occurrence of various diseases, especially cancers. In the present technical solution, the inventor accelerated the development process of breast cancer in a mouse model by cadmium exposure in drinking water, and then treated the mice by gavage with Clostridium butyricum and / or Blautia ovata, greatly reducing the level of glutamine in the intestines of mice exposed to cadmium in drinking water and slowing down the process of tumorigenesis and development. The inventor verified through the animal model of this solution (taking cadmium-exposed MMTV-Erbb2 transgenic mice as an example for research) the significant role of Clostridium butyricum and Blautia ovata in resisting cancers related to glutamine metabolism. Therefore, Clostridium butyricum and Blautia ovata can be used as drugs for preparing to prevent or treat cancers and then applied to the treatment of cancers related to glutamine metabolism including breast cancer.

[0019] The present technical solution also provides a drug for preventing or treating chronic cadmium poisoning, and the drug comprises at least one of 6-diazo-5-oxo-L-norleucine, Clostridium butyricum, and Blautia ovata.

[0020] Furthermore, the chronic cadmium poisoning is caused by daily drinking of water containing low-dose cadmium ions; the chronic cadmium poisoning triggers breast cancer.

[0021] The technical principle and beneficial effects of adopting the above technical solution are as follows:

[0022] Cadmium (Cd) is a metal pollutant toxic to humans and is widely present in drinking water and farmland. The main routes of cadmium exposure are diet and smoking. The International Agency for Research on Cancer classifies cadmium as a Group 1 carcinogen due to its carcinogenicity and ability to increase the risk of lung cancer and prostate cancer. Recent epidemiological studies have shown that cadmium exposure may increase the risk of breast cancer (BC) related to estrogen receptor (ER) and progesterone receptor (PR) status and lead to adverse outcomes. Cadmium increases the proliferation and metastasis ability of breast cancer cells through epigenetic regulation. Therefore, studying the mechanism of cadmium promoting breast tumorigenesis and finding key intervention targets for the occurrence and development of breast cancer are crucial for developing new drugs for treating chronic cadmium poisoning and related cancers (such as breast cancer).

[0023] In the present technical solution, the inventor fed MMTV-Erbb2 transgenic mice with drinking water containing 3.6 mg / L of cadmium, thereby causing chronic cadmium poisoning in the mice. One specific manifestation is that the occurrence and development process of breast cancer in the mice is accelerated and the glutamine level in the intestines of the mice increases. The inventor used 6-diazo-5-oxo-L-norleucine (glutamine inhibitor) in Experimental Example 3 and used Clostridium butyricum or Blautia ovata to treat cadmium-exposed MMTV-Erbb2 transgenic mice in Example 8, and both can relieve the symptoms caused by chronic cadmium poisoning (such as breast cancer, glutamine content, etc.). Therefore, 6-diazo-5-oxo-L-norleucine, Clostridium butyricum, and Blautia ovata can all be used as drugs for preventing or treating chronic cadmium poisoning and have an ideal prospect of popularization and application.

[0024] The present technical solution also provides the application of an intestinal glutamine level inhibitor in the preparation of a drug for preventing or treating cancer or in the preparation of a drug for preventing or treating chronic cadmium poisoning, and the intestinal glutamine level inhibitor comprises at least one of 6-diazo-5-oxo-L-norleucine, Clostridium butyricum, and Blautia ovata.

[0025] The technical principle and beneficial effects of adopting the above technical solution are as follows:

[0026] The inventors' research found that inhibiting glutamine levels can effectively prevent or treat glutamine-dependent cancers, and since this study revealed the relationship between chronic cadmium poisoning and intestinal glutamine levels, inhibiting glutamine levels can also relieve the symptoms caused by chronic cadmium poisoning. Therefore, intestinal glutamine level inhibitors can be used as drugs for preventing or treating cancer or preventing or treating chronic cadmium poisoning, and thus can be applied in related medical practices. Among them, the inventors verified through a large number of experiments the effects of 6-diazo-5-oxo-L-norleucine, Clostridium butyricum, and Blautia ovata in inhibiting glutamine levels, alleviating the occurrence and development process of breast cancer, and relieving the symptoms of chronic cadmium poisoning. Therefore, 6-diazo-5-oxo-L-norleucine, Clostridium butyricum, and Blautia ovata can be used as intestinal glutamine level inhibitors, drugs for preventing or treating cancer, and drugs for preventing or treating chronic cadmium poisoning (cadmium poisoning caused by long-term low-dose cadmium exposure).

[0027] In summary, environmental pollutant cadmium exposure can induce a large amount of glutamine production caused by intestinal microbial dysbiosis, which is absorbed through the intestine and enters the body to promote the development and adverse outcomes of breast cancer in the body. In response, the inventors screened and verified two strains of microorganisms with anti-cancer activity (Clostridium butyricum and Blautia ovata, which can be supplemented orally), which can be used to stabilize the glutamine concentration level in the body and for the treatment of cancers related to glutamine metabolism such as breast cancer. Brief Description of the Drawings

[0028] Figure 1 It is the experimental result of the promotion of the development of mammary tumors in MMTV-Erbb2 mice by cadmium drinking water exposure at environmental concentration in Example 1.

[0029] Figure 2 It is the research result of the effect of cadmium exposure on the metabolite profile in tumors of MMTV-Erbb2 mice in Example 2.

[0030] Figure 3 It is the research result of the effect of inhibiting glutamine metabolism on the process of cadmium-induced mammary tumors in mice in Example 3.

[0031] Figure 4 It is the research result of the effect of cadmium exposure on the metabolic pattern of intestinal microorganisms and the synthesis amount of glutamine in mice in Example 4.

[0032] Figure 5 It is the research result of the effect of drinking water cadmium exposure on intestinal microorganisms in MMTV-Erbb2 mice in Example 5.

[0033] Figure 6 It is the research result of the intestinal microbiota transplantation experiment changed by cadmium exposure in Example 6.

[0034] Figure 7 Bioinformatics research results on the disruption of intestinal microecological balance by cadmium exposure in Example 7.

[0035] Figure 8 Research results on the effect of supplementing Clostridium butyricum or Blautia ovata on the promotion of breast cancer progression in MMTV-Erbb2 mice by drinking water cadmium exposure in Example 8.

[0036] Figure 9 Research results on the effect of supplementing Clostridium butyricum or Blautia ovata on the increase in intestinal glutamine content in MMTV-Erbb2 mice promoted by drinking water cadmium exposure in Example 8. Detailed implementation manners

[0037] The present invention will be further described in detail below in conjunction with examples, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can all be obtained from commercial channels.

[0038] Example 1: Drinking water exposure to environmental pollutants (heavy metal cadmium Cd) can promote the development of breast cancer

[0039] A: Animal experiment design and grouping

[0040] Six-week-old female MMTV-Erbb2 mice were selected, purchased from Jackson Laboratory in the United States, and centrally housed in the IVC animal breeding room of the Experimental Animal Center of XX University. Approximately 3 - 5 mice were housed in each IVC cage. Under a constant temperature environment, a constant light with a simulated day-night ratio of 12h:12h was given. To study the relationship between chronic cadmium exposure and breast cancer, the effect of environmental dose cadmium exposure on breast cancer progression was explored by exposing mice to 3.6 mg / L CdCl2 in drinking water. This concentration is 1 / 120 of the median lethal dose of cadmium and is equivalent to the average daily cadmium intake of women with a high incidence of breast cancer. First, 20 female MMTV-Erbb2 mice were randomly divided into a cadmium exposure group and its corresponding control group with 10 mice in each group. Subsequently, according to the experimental design, after exposing the mice to cadmium in drinking water for 23 weeks, the experiment was terminated when the average diameter of the tumors in the cadmium exposure group reached 1.5 cm. The experimental reagent cadmium chloride (CdCl2, Sigma-Aldrich, 439800) was dissolved in deionized water, filtered and sterilized, and stored at -20 °C.

[0041] B: Experimental methods

[0042] (1) Observation of mammary tumor growth in MMTV-Erbb2 mice

[0043] After a palpable tumor appeared in the mouse mammary gland, the size of the mouse mammary gland tumor was measured and recorded every 1 week using a vernier caliper. The tumor volume was calculated according to the following formula: volume = (length × width) 2 / 2. After the tumor diameter approached 1.5 cm, the mice were sacrificed, and the tumor bodies were dissected and fixed in paraformaldehyde fixative solution.

[0044] (2) Hematoxylin and eosin staining

[0045] (2.1) Dewaxing of paraffin sections: First, place the sections in environment-friendly dewaxing solution I for 20 min, then in environment-friendly dewaxing solution II for 20 min, and then in absolute ethanol for 5 min. Repeat this twice, then treat with 75% alcohol for 5 min and rinse with running water.

[0046] (2.2) Rewarming and fixation of frozen sections: Take the frozen sections out of the -20°C refrigerator and let them return to room temperature. Fix them with tissue fixative for 15 min and then rinse with running water.

[0047] (2.3) Hematoxylin staining: Stain the sections with hematoxylin stain for 3 min. After rinsing with running water, use the differentiation solution to differentiate the sections, rinse with running water, use the blueing solution to blue the sections, and rinse with running water.

[0048] (2.4) Eosin staining: Dehydrate in 85% and 95% gradient concentration alcohols for 5 min each. Put the sections into eosin stain and stain for 5 min, then take them out.

[0049] (2.5) Sealing the sections: Dehydrate the sections in absolute ethanol for 5 min, repeat three times, changing the absolute ethanol each time. Finally, make them transparent in xylene for 5 min, repeat twice, and then seal the sections with neutral gum.

[0050] (2.6) Microscopic examination of the sections and image acquisition and analysis.

[0051] (3) Immunofluorescence of paraffin sections

[0052] (3.1) Dewaxing of paraffin sections: First, place the sections in environment-friendly dewaxing solution I for 20 min, then in environment-friendly dewaxing solution II for 20 min, and then in absolute ethanol for 5 min. Repeat this twice, then treat with 75% alcohol for 5 min and rinse with running water.

[0053] (3.2) Antigen retrieval: Treat the tissue sections with EDTA antigen retrieval solution. Place them in a microwave oven, heat at medium power for 8 min until boiling, then switch to low power and heat for 7 min. After natural cooling, wash the slides three times with decolorizing solution, 5 min each time.

[0054] (3.3) Draw a circle around the tissue and block with serum: To prevent the antibody from flowing away, after the section is dried, use a tissue brush to draw a circle around the tissue, drain the PBS by shaking, add BSA, and block the section for 30 min.

[0055] (3.4) Add the primary antibody: Remove the blocking solution, add the prepared primary antibody dilution to the section, and incubate the section flat in a wet box at 4 °C overnight.

[0056] (3.5) Add the secondary antibody: Wash the slide 3 times with PBS at pH 7.4 for 5 min each time. Incubate with the secondary antibody for 50 min in the dark at room temperature throughout the process. See Table 1 for the technical parameters of the primary and secondary antibodies.

[0057] Table 1: Technical parameters of the primary and secondary antibodies

[0058]

[0059] (3.6) DAPI counterstain the cell nucleus: Wash the slide 3 times with PBS at pH 7.4 for 5 min each time. After draining the section, add DAPI to completely cover the tissue and incubate in the dark at room temperature for 10 min.

[0060] (3.7) Quench the autofluorescence of the tissue: Wash the slide 3 times with PBS at pH 7.4 for 5 min each time. After treating with the fluorescence quencher for 5 min, rinse with running water.

[0061] (3.8) Mount the section: After draining the section, use an anti-fluorescence quenching mounting medium to mount the tissue section.

[0062] (3.9) Microscopic examination and photography: Examine the section under a microscope and perform image acquisition and analysis.

[0063] (3.10) Result interpretation: DAPI shows blue light and Ki67 shows red light.

[0064] (4) Statistical analysis

[0065] Data statistical analysis was performed using GraphPad Prism 9.0. The experimental data are presented as mean ± standard deviation SD. For multiple group comparisons, one-way analysis of variance (ANOVA) was performed, followed by Dunnett's multiple comparison test. For two-group comparisons, an unpaired two-tailed t-test was performed. Differences with a P value less than 0.05 were considered significant.

[0066] C: Experimental results

[0067] The experimental results of this example are shown in Figure 1 . Among them, Figure 1 A is a schematic diagram of the Cd-exposed MMTV-Erbb2 mouse breast cancer development model; Figure 1B is the Kaplan-Meier plot of tumor-free survival for the control and cadmium groups (n = 10); Figure 1 C is the calculation of the average tumor volume starting from palpable tumors (n = 10 control group and n = 10 cadmium group); Figure 1 D is the representative image of tumors for the control and cadmium groups; Figure 1 E is the tumor weight at 29 weeks (n = 10 control group and n = 10 cadmium group, two tumors per mouse); Figure 1 F is the representative image and statistical results of Ki67 immunofluorescence detection of tumors in control and cadmium group mice (scale bar: 50 μm); Figure 1 G is the representative image of HE staining of tumors (scale bar: 1000 μm (original image), 50 μm (magnified image)). Figure 1 In the figure, ** indicates P < 0.01 compared with the control group.

[0068] To evaluate the effect of environmental-dose cadmium on the occurrence and development of breast cancer in mice, MMTV-Erbb2 mice were exposed to cadmium in drinking water at one-twentieth of the median lethal concentration for 23 weeks, and the number of mice with breast cancer and tumor size were recorded weekly ( Figure 1 A). It was found that the onset time of breast cancer in cadmium-exposed mice was advanced, and the difference was statistically significant ( Figure 1 B). The diameter of breast tumors was measured weekly starting from 19 weeks, and the growth of breast tumor volume was recorded. When the tumor diameter reached 1.5 cm, the tumor was surgically removed. It was found that the tumor volume of mice in the cadmium-exposed group was significantly larger than that of the control group after the 24th week, and the difference was statistically significant ( Figure 1 C-D). Subsequently, the isolated mice were weighed for tumors (two tumors were taken from each mouse), and it was found that the tumor mass of mice in the cadmium-exposed group was significantly larger than that of the control group, and the difference was statistically significant ( Figure 1 E). After the isolated tumor bodies were fixed and pathological sections were made, and after HE staining, they were all breast tumors with a high degree of malignancy. The tumors isolated from the cadmium-exposed group showed more focal tumor necrosis and neovascularization, suggesting that the tumors in the cadmium-treated group grew too fast and had a higher degree of malignancy ( Figure 1 G). Subsequently, Ki67 immunofluorescence staining was further performed on the pathological sections, and the results were consistent with the above results. The Ki67-positive cells were significantly higher than those in the control group, and the difference was statistically significant ( Figure 1 F), further indicating that environmental-dose cadmium exposure in drinking water promoted the proliferation of mouse breast tumors and caused the rapid deterioration of breast cancer.

[0069] Example 2: Study on the change of the metabolic profile of spontaneous breast tumors in MMTV-Erbb2 mice by cadmium exposure in drinking water

[0070] A. Experimental method - Untargeted metabolomics analysis

[0071] (1) Sample processing

[0072] Immediately after dissection, the animal tissues were rapidly frozen in liquid nitrogen. Then the tissues were minced on dry ice (about 100 mg), 1 mL of pre-cooled methanol:acetonitrile:water (2:2:1, v / v) was added, and homogenization was carried out using an MP homogenizer (24×2, 6.0 M / S, 20 s, 3 times), followed by low-temperature ultrasonic treatment for 30 min each time, 2 times, standing at -20 °C for 60 min, centrifuging at 13000 g at 4 °C for 15 min, taking the supernatant (aliquoted into 900 μL / tube), vacuum drying, and storing the freeze-dried powder at -80 °C for later use; when performing mass spectrometry analysis, 100 μL of acetonitrile aqueous solution (acetonitrile:water = 1:1, v / v) was added for reconstitution, vortexing, centrifuging at 14000 g at 4 °C for 15 min, and taking the supernatant for injection analysis.

[0073] (2) UHPLC-Q-TOF MS

[0074] Analysis was performed using UHPLC (1290 Infinity LC, Agilent Technologies) and quadrupole time-of-flight (ABSciex TripleTOF 6600) by Shanghai Applied Protein Technology Co., Ltd.

[0075] For HILIC separation, the samples were analyzed using a 2.1 mm×100 mm acquy UPLC BEH Amide 1.7 μm chromatographic column (waters, Ireland). In both positive and negative ESI modes, the mobile phases were A = 25 mM ammonium acetate and 25 mM ammonium hydroxide aqueous solution, and B = acetonitrile. The gradient was 95% B at 0.5 min, linearly decreased to 65% at 6.5 min, then decreased to 40% and held for 1 min at 1 min, then increased to 95% at 0.1 min, and the re-equilibration period was 3 min.

[0076] The ESI source conditions were set as follows: ion source Gas1 (Gas1) was 60, ion source Gas2 (Gas2) was 60, curtain gas (CUR) was 30, source temperature was 600℃, and ion spray floating voltage (ISVF) was ±5500V. In pure mass spectrometry acquisition, the instrument was set to collect 60 1000Da in the m / z range, and the accumulation time of TOF mass spectrometry scanning was set to 0.20s / spectrum. In automatic MS / MS acquisition, the instrument was set to collect 25 1000Da in the m / z range, and the accumulation time of product ion scanning was set to 0.05s / spectrum. Information dependent acquisition (IDA) was used for product ion scanning, and high sensitivity mode was selected. The parameters set were as follows: the collision energy (CE) was fixed at 35V±15eV; the cluster potential DP, 60V(+) and -60V(-); isotopes within 4Da were excluded, and candidate ions were monitored every 10 cycles.

[0077] (3)UHPLC-Q-Exactive Orbitrap MS

[0078] The analysis was performed using UHPLC (Vanquish UHPLC, Thermo) coupled to Orbitrap from Shanghai Zhongke Xinsheng Technology Co., Ltd. For HILIC separation, samples were analyzed using a 2.1 mm × 100 mm acquy UPLC BEH Amide 1.7 μm column (waters, Ireland). In both ESI positive and negative modes, the mobile phases were A = 25 mM ammonium acetate and 25 mM ammonium hydroxide aqueous solution, and B = acetonitrile. The gradient was 98% B at 1.5 min, linearly decreased to 2% at 10.5 min, then maintained for 2 min, and then increased to 98% at 0.1 min, with a re-equilibrium period of 3 min. The ESI source conditions were set as follows: ion source Gas1 (Gas1) was 60, ion source Gas2 (Gas2) was 60, curtain gas (CUR) was 30, source temperature was 600 °C, and ion spray floating voltage (ISVF) was ±5500 V. In pure mass spectrometry acquisition, the instrument was set to acquire within the m / z range of 80-1200Da, the resolution was set to 60,000, and the accumulation time was set to 100ms. In automatic MS / MS acquisition, the instrument was set to acquire within the m / z range of 70-1200Da, the resolution was set to 30,000, the accumulation time was set to 50ms, and the exclusion time was set to within 4s.

[0079] (4) Data processing

[0080] Before importing the XCMS software, the raw MS data was converted to MzXML files using ProteoWizard MSConvert. For peak picking, the following parameters were used: centWave m / z = 10 ppm, peak width = c(10, 60), prefilter = c(10, 100). Peak grouping was performed with bw = 5, mzwid = 0.025, minfrac = 0.5. CAMERA (Collection of Algorithms of MEtabolite pRofile Annotation) was used for isotope and adduct annotation. Among the extracted ion features, only variables with non-zero measurements greater than 50% in at least one group were retained. Metabolites were identified by comparing the accurate m / z values (<10 ppm) and MS / MS spectra using an in-house database established with existing standards.

[0081] (5) Statistical analysis

[0082] After the processed data was normalized, multivariate data analysis was performed using the R package (ropls), including Pareto-scaled principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Seven-fold cross-validation and response permutation tests were used to evaluate the robustness of the model. The variable importance in the projection (VIP) value of each variable in the OPLS-DA model was calculated to represent its contribution to classification. The t-test was used to determine the significance of differences between two independent samples. Differentially expressed metabolites with significant changes were screened with VIP > 1 and p-value < 0.05.

[0083] B. Experimental results

[0084] The experimental results of this example are shown in Figure 2 . Among them, Figure 2 A is the PLS-DA plot of tumor metabolomics analysis of control group mice and cadmium-exposed group mice; Figure 2 B is the volcano plot of differentially expressed metabolites between control group mice and cadmium-exposed group; Figure 2 C is the KEGG pathway analysis of differentially enriched metabolites between control group mice and cadmium-exposed group; Figure 2 D is the heatmap of differentially expressed metabolites in the alanine, asparagine, and glutamate metabolic pathways (n = 8).

[0085] Mammary tumors (n = 8) from control group and cadmium-exposed group mice were taken for untargeted metabolomics detection to detect changes in differentially expressed metabolites. The results of PLS-DA analysis showed that samples within the group were clustered and the distance between samples between groups was far Figure 2 (A), indicating that cadmium exposure in drinking water induced significant changes in metabolites of mammary tumors. The volcano plot separated the differentially expressed metabolites according to their fold change and P value Figure 2B). The results of KEGG analysis of differential metabolites showed that alanine metabolism, aspartate metabolism and glutamate metabolism were the pathways with the highest enrichment ranking. Figure 2 C). Among them, glutamine, L-aspartic acid and N-acetyl-L-aspartic acid were the differential metabolites in this pathway. Figure 2 D). The above results suggested that glutamine metabolism might be the key pathway involved in the promotion of breast cancer development and deterioration in MMTV-Erbb2 mice by cadmium.

[0086] Experimental Example 3: Study on the effect of glutamine metabolism on the progression of cadmium-induced mammary tumors in MMTV-Erbb2 mice

[0087] A. Experimental method

[0088] (1) Experimental design and grouping

[0089] Six-week-old female MMTV-Erbb2 mice were purchased from Jackson Laboratory in the United States and were centrally housed in the IVC animal breeding room of the Experimental Animal Center of XX University. Approximately 3-5 mice were housed in each IVC cage. Under a constant temperature environment, a constant light with a simulated day-night ratio of 12h:12h was provided. The glutamine inhibitor 6-diazo-5-oxo-L-norleucine (DON, MedChemExpress, HY-108357) was used to study whether glutamine plays a key role in the progression of cadmium-induced mammary tumors in MMTV-Erbb2 mice. Mice exposed to 3.6 mg / L CdCl2 in drinking water were used as a mouse model for promoting breast cancer development. The grouping was as follows: 18 female MMTV-Erbb2 mice were randomly divided into a control group, a cadmium exposure group, and a DON+Cd group, with 6 mice in each group. The control group drank pure water and was intraperitoneally injected with PBS; the cadmium exposure group drank pure water containing 3.6 mg / L CdCl2 and was intraperitoneally injected with PBS; the DON+Cd group drank pure water containing 3.6 mg / L CdCl2 and was intraperitoneally injected with DON (injected twice a week, 1 mg / kg each time). The cadmium exposure time and the DON administration time started at the 6th week. Subsequently, according to the experimental design, after the mice were exposed to cadmium in drinking water for 23 weeks, the experiment was terminated when the average diameter of the tumors in the cadmium exposure group of mice reached 1.5 cm. Cadmium chloride (CdCl2, Sigma-Aldrich, 439800) was dissolved in deionized water, filtered and sterilized, and stored at -20°C. 6-diazo-5-oxo-L-norleucine (DON, MedChemExpress, HY-108357) was dissolved in PBS, filtered and sterilized, and stored at -20°C.

[0090] (2) The experimental methods and statistical methods were the same as those in Example 1.

[0091] B. Experimental results

[0092] For the specific experimental results, please refer to Figure 3 , which shows that inhibiting glutamine metabolism reversed the progression of cadmium-induced mammary tumors in mice. Among them, Figure 3 A is a schematic diagram of the experimental design for DON treatment to inhibit the progression of cadmium-promoted breast cancer; Figure 3 B is the Kaplan-Meier plot of tumor-free survival for the control group, Cd group, and DON+Cd group (n = 6); Figure 3 C is the calculation of the average tumor volume starting from palpable tumors (in the control group, Cd group, and DON+Cd group, n = 6); Figure 3 D is a representative picture of tumors from the control group, Cd group, and DON+Cd group; Figure 3 E is the tumor weight harvested at 29 weeks (in the control group, Cd group, and DON+Cd group, n = 6; two tumors per mouse); Figure 3 F is the representative image and statistical results of Ki67 fluorescence detection of tumors in mice in the control group, Cd group, and DON+Cd group (scale bar: 50 μm); Figure 3 G is the representative image of HE staining of tumors (scale bar: 1000 μm (original image), 50 μm (magnified image)). Figure 3 In it, ** indicates P < 0.01 compared with the control group; ## indicates P < 0.01 compared with the Cd group.

[0093] The metabolism of glutamine in mice was inhibited with the glutamine antagonist DON to confirm the key role of glutamine metabolism in the development of mammary tumors in MMTV-Erbb2 mice exposed to cadmium in drinking water. As Figure 3 shown in A, MMTV-Erbb2 mice were exposed to 3.6 mg / L CdCl2 in drinking water for 23 weeks (starting from the 6th week and ending at the 29th week). The number of mice with breast cancer and the tumor size in the control group, cadmium-exposed group, and DON plus cadmium-exposed group were observed and recorded weekly ( Figure 3 A). It was found that the onset time of breast cancer in cadmium-exposed mice was delayed after DON treatment, and the difference was statistically significant ( Figure 3 B). The diameter of mammary tumors was measured weekly starting from the 19th week, and the growth of mammary tumor volume was recorded. When the tumor diameter grew to 1.5 cm, the tumors were surgically removed. It was found that the tumor volume of mice in the DON+Cd group was significantly smaller than that in the cadmium-treated group, and the difference was statistically significant ( Figure 3 C-D). Subsequently, the isolated mice were weighed for tumors (two tumors were taken from each mouse), and it was found that the tumor weight of mice in the cadmium-exposed group was significantly greater than that in the control group, and the DON+Cd group was significantly smaller than the cadmium-treated group.Figure 3 E). After fixing the separated tumor masses and making pathological sections, and after HE staining, they were all breast tumors with a relatively high degree of malignancy. The appearance of a large number of necrotic areas in the tumor center separated from the cadmium exposure group indicated that the tumor grew too fast and had a higher degree of malignancy. There was a slight remission in the DON+Cd group ( Figure 3 G). Subsequently, Ki67 fluorescence staining was further performed on the pathological sections, and the results were consistent with the above results. The Ki67-positive cells were significantly higher than those in the control group. The Ki67-positive cells in the DON+Cd group were significantly lower than those in the cadmium treatment group, and the difference was statistically significant, further indicating that cadmium exposure promoted the proliferation of breast tumors and accelerated the deterioration of breast cancer ( Figure 3 E-F). These results showed that inhibiting glutamine metabolism could significantly inhibit the progression of Cd-exposure-enhanced breast tumors in mice, demonstrating the key role of glutamine in the progression of breast cancer induced by environmental pollutants.

[0094] Example 4: Study on the effects of drinking water cadmium exposure on the metabolic pattern of intestinal microbiota and the amount of glutamine synthesis in MMTV-Erbb2 mice

[0095] A. Experimental method

[0096] (1) Sample treatment

[0097] Using a sterile scalpel, the entire intestine of the mouse was taken out under sterile conditions, and the contents of the cecum segment were cut. The contents were dug out with a sterile scalpel, immediately placed on ice for aliquoting and labeling; after aliquoting, it was immediately frozen in liquid nitrogen. About 100 mg of intestinal content samples were taken, and 1 mL of pre-cooled methanol:acetonitrile:water (2:2:1, v / v) was added. Homogenization and fragmentation were carried out with an MP homogenizer (24×2, 6.0M / S, 20 s, 3 times), low-temperature ultrasound was performed for 30 min each time, 2 times, left standing at -20 °C for 60 min, centrifuged at 13000g at 4 °C for 15 min, and the supernatant was taken (aliquoted as 900 μL / tube), vacuum dried, and the freeze-dried powder was stored at -80 °C for later use; when performing mass spectrometry analysis, 100 μL of acetonitrile aqueous solution (acetonitrile:water = 1:1, v / v) was added for reconstitution, vortexed, centrifuged at 14000g at 4 °C for 15 min, and the supernatant sample was taken for non-target metabolomics analysis.

[0098] (2) The detection and analysis method was the same as that in Example 2.

[0099] B. Experimental results

[0100] The experimental results are shown in Figure 4 , showing that cadmium exposure changed the metabolic profile of intestinal microbiota in MMTV-Erbb2 mice, and the synthesis of glutamine increased significantly. Among them, Figure 4 A is the PLS-DA diagram of the intestinal microbiota metabolomics analysis of control mice and Cd-exposed mice; Figure 4B is the volcano plot of differentially expressed metabolites between the control group and the Cd group; Figure 4 C is the result of KEGG enrichment analysis of differentially expressed metabolites; Figure 4 D is the schematic diagram of glutamine synthesis in nitrogen metabolism; Figure 4 E is the relative content of glutamine in the intestinal contents of the control group and the Cd group. Figure 4 In it, ** indicates P < 0.01 compared with the control group (n = 5).

[0101] Subsequently, non-target metabolomics detection was performed on the cecal contents of mice from the control group and the cadmium-exposed group (n = 5) to detect changes in metabolites therein. The results of PLS-DA analysis showed that the samples within the group were aggregated and the distance between samples between groups was far apart, indicating that cadmium exposure significantly changed the metabolites of the intestinal microbiota in mice ( Figure 4 A), and the volcano plot separated the differentially expressed metabolites according to their fold change and P value ( Figure 4 B). The results of KEGG analysis of differentially expressed metabolites showed that the nitrogen metabolism pathway for synthesizing glutamine was one of the significantly enriched pathways ( Figure 4 C). It is indicated that the intestinal microbiota can synthesize free ammonia into glutamine through the nitrogen cycle pathway ( Figure 4 D), and the metabolomics results showed that the content of glutamine in the cecal contents of the cadmium-treated group was significantly increased ( Figure 4 E).

[0102] Example 5: Study on the effect of drinking water cadmium exposure on the intestinal microbiota of MMTV-Erbb2 mice

[0103] A. Metagenomic sequencing analysis

[0104] (1) DNA extraction and quality control

[0105] DNA was extracted from the cecal contents using Magnetic Soil And Stool DNA Kit (TIANGEN).

[0106] Quality control of DNA samples included two methods: monitoring the degree of DNA degradation and potential contamination on 1% agarose gel; using 2.0 Flurometer (Life Technologies, CA, USA) with dsDNA Assay Kit to measure the DNA concentration. Only DNA with qualified quality control could be used for library construction.

[0107] (2) Library construction

[0108] A total of 1 μg DNA of each sample was used as the input material for DNA sample preparation. Using Ultra TMDNA Library Prep Kit for Illumina (NEB) was used to generate sequencing libraries according to the manufacturer's recommendations, and the index was added to the attribute sequence of each sample. In brief, the DNA samples were fragmented to a size of 350 bp by sonication, and then the DNA fragments were blunt-ended, A-tailed, and ligated with full-length adapters for Illumina sequencing and further PCR amplification. Finally, the PCR products were purified (AMPure XP system) and the size distribution of the library was analyzed by the Agilent2100 Bioanalyzer, and then the library concentration was quantified using the Bio-Rad CFX96 (or CFX384) instrument.

[0109] (3) Sequencing

[0110] Clustering of the Index samples was performed on the cBot cluster generation system according to the manufacturer's instructions. After cluster generation, library preparations were sequenced using the NovaSeq 6000 (or MGISEQ-T7) sequencing platform with PE150 reads.

[0111] (4) Bioinformatics analysis

[0112] Data generated from the Illumina (or BGI) platform were used for bioinformatics analysis. All analyses were performed by Shanghai Zhongke Xinsheng Biotechnology Co., Ltd. The main software and parameters are as follows:

[0113] (4.1) Sequencing result preprocessing

[0114] 1) Use fastp (https: / / github.com / OpenGene / fastp) software default parameters to preprocess the raw data (Raw Data) obtained from the Illumina (or BGI) sequencing platform to obtain valid data (CleanData) for subsequent analysis. The specific processing steps are as follows: a) remove reads containing adapters; b) remove low-quality reads (including reads with N ratio greater than 10%; c) remove reads with a quality value Q≤15 that accounts for more than 50% of the entire read.

[0115] 2) If the sample is contaminated by the host, it needs to be compared with the host database to filter out reads that may come from the host. The default software is BWA.

[0116] (4.2) Metagenome assembly

[0117] Use sequence splicing software MEGAHIT (or IDBA_UD) to splice and assemble the clean data. According to the overlap relationship between kmers, construct a De-Brujin graph, and finally obtain Contigs. Screen Contigs longer than 800bp for data statistics and use them for subsequent analysis.

[0118] (4.3) Gene prediction and abundance analysis

[0119] 1) Use Prodigal software to predict ORFs for the spliced Contigs sequences and translate them into amino acid sequences.

[0120] 2) For the ORF prediction results, use CD-HIT software to remove redundancy to obtain a non-redundant gene catalogue (here, the nucleic acid sequences encoding non-redundant continuous genes are called genes). By default, cluster with identity 95% and coverage 90%, and select the longest sequence as the representative sequence.

[0121] 3) Use bowtie2 software to align the clean reads of each sample with the non-redundant gene set (identity 95%) respectively, and count the abundance information of genes in the corresponding samples.

[0122] 4) Starting from the number of aligned reads and gene length, calculate the abundance information (TPM) of each gene in each sample.

[0123] 5) Based on the abundance information of each gene in each sample, conduct basic information statistics, core-pan gene analysis, sample correlation analysis, and gene number Venn diagram analysis.

[0124] (5) Species annotation

[0125] 1) Use DIAMOND software (V0.9.9.110) to align the non-redundant gene set with the sequences of bacteria, fungi, archaea, and viruses extracted from the NCBI NR database (Version: 2021.11).

[0126] 2) For the alignment results of each sequence, select the results with evalue ≤ 1e-5. Since each sequence may have multiple alignment results, adopt the LCA algorithm (applied to the system classification of MEGAN software) to determine the species annotation information of the sequence.

[0127] 3) Starting from the LCA annotation results and the gene abundance table, obtain the abundance information and gene entry tables of each sample at each taxonomic level (kingdom, phylum, class, order, family, genus, species) on www.aptbiotech.com. For the abundance of a certain species in a certain sample, it is equal to the sum of the gene abundances annotated as that species; for the number of genes of a certain species in a certain sample, it is equal to the number of genes with non-zero abundance among the genes annotated as that species.

[0128] 4) Starting from the abundance tables at each taxonomic level, perform Krona analysis to display the relative abundance profile, and use Gephi software to construct a species association network diagram with a threshold of species correlation coefficient > 0.7 and P < 0.01.

[0129] (6) Functional annotation

[0130] 1) Use DIAMOND software (v0.9.9.110) to align the non-redundant gene set with the functional databases KEGG, eggNOG, and CAZy databases. For the alignment results of each sequence, select the Best Blast Hit result for subsequent analysis.

[0131] 2) Starting from the alignment results, calculate the relative abundances at different functional levels (the relative abundance of each functional level is equal to the sum of the relative abundances of the genes annotated as that functional level).

[0132] 3) Starting from the functional annotation results and the gene abundance table, obtain the gene number tables of each sample at each taxonomic level. For the number of genes of a certain function in a certain sample, it is equal to the number of genes with non-zero abundance among the genes annotated as that function.

[0133] 4) Starting from the abundance tables at each taxonomic level, perform statistical analysis of the annotated gene numbers, display the relative abundance profile, and display the abundance clustering heatmap.

[0134] (7) Resistance gene annotation

[0135] 1) Use ARGs-OAP v2.0 software to align the non-redundant gene set with the SARG2 database (default evalue ≤ 1e-30). Based on the alignment results and combined with the gene abundance information, calculate the relative abundances of each ARG.

[0136] 2) Draw a heatmap based on the resistance gene annotation and abundance information of all samples in the database to display the ARGs abundance profile, and perform species and functional contribution analysis.

[0137] (8) Statistical analysis

[0138] PCA (ade4 package, Version 2.15.3) and NMDS (vegan package, Version 2.15.3) dimensionality reduction analyses were used to compare the species and functional compositions among different groups; ANOSIM and Adonis analyses were used to test the differences between groups; then Wilcoxon (two groups) / Kruskal-Wallis (multiple groups) rank sum tests (or STAMP analysis) and LEfSe analysis were used to find differentially abundant species or functions at each hierarchical level between groups.

[0139] B. Experimental results

[0140] The experimental results are shown in Figure 5 , demonstrating the situation of cadmium exposure-induced intestinal microbiota dysbiosis in mice. Among them, Figure 5 A is a stacked bar chart of the relative abundances of phylum-class-genus-species of the bacterial communities in the control group and the Cd group; Figure 5 B is the Shannon index of the bacteria in the control group and the Cd group; Figure 5 C is an unsupervised PCoA plot of the Unifrac distances between the control group and the Cd group; Figure 5 D is a classification tree of metagenomic sequencing; Figure 5 E is the result of KEGG enrichment analysis of the differentially abundant intestinal microbiota between the control group and the Cd group. Figure 5 In, ** indicates P < 0.01 compared with the control group (n = 5).

[0141] The cecal contents of the mice in the control group and the cadmium-exposed group were subjected to metagenomics detection and analysis (n = 5). The results showed that the intestinal flora composition of the mice in the environmental-dose cadmium treatment group changed significantly, and the α diversity was significantly downregulated ( Figure 5 A-C) The cladogram shows the differentially abundant flora in the control group and the cadmium-exposed group ( Figure 5 D). KEGG analysis showed that the differentially abundant flora was closely related to the breast cancer pathway and the cancer pathway ( Figure 5 E).

[0142] Example 6: Study on the promotion of mammary tumor progression in MMTV-Erbb2 mice by the transplantation of intestinal microbiota altered by cadmium exposure

[0143] A. Experimental grouping and microbiota transplantation method

[0144] To confirm the role of gut microbiota in the development of cadmium-exposure-induced breast tumors, fecal microbiota transplantation experiments were conducted in MMTV-Erbb2 mice. Four-week-old female MMTV-Erbb2 mice were randomly divided into two groups (6 mice per group): the FMT_Control group and the FMT_Cd group. Before oral transplantation, according to previously reported literature (Jian X, Zhu Y, Ouyang J, Wang Y, Lei Q, Xia J, Guan Y, Zhang J, Guo J, He Y, Wang J, Li J, Lin J, Su M, Li G, Wu M, Qiu L, Xiang J, Xie L, Jia W, Zhou W. Alterations of gut microbiome accelerate multiple myeloma progression by increasing the relative abundances of nitrogen-recycling bacteria. Microbiome. 2020 May 28;8(1):74. doi:10.1186 / s40168-020-00854-5.), all MMTV-Erbb2 mice were treated with an antibiotic mixture AbX (0.1 g / L vancomycin, 0.2 g / L ampicillin, 0.2 g / L neomycin, and 0.2 g / L metronidazole) in their drinking water for 2 weeks to ensure the clearance of the original gut microbiota in the mice. Next, fresh feces from control mice not exposed to Cd and mice exposed to Cd for 21 weeks were collected separately, suspended in sterile sodium chloride solution (0.9%), at a ratio of 250 mg / ml. The feces were filtered through a 200-mesh sterile mesh sieve to remove large particles, and then the filtrate was successively passed through 400-mesh and 800-mesh sterile mesh sieves to remove undigested food and smaller particulate matter. The resulting filtrate was collected in a sterile centrifuge tube, and a resuspension was obtained by vortexing for 2 minutes. The resuspension was centrifuged at 500×g for 2 minutes to remove insoluble matter, obtaining a suspension. For gavage transplantation, 200 μL of the fecal suspension was taken. FMT_Control mice and FMT_Cd mice were given gavage transplantation twice a week for a total of two weeks.

[0145] B. Other experimental methods and statistical methods are the same as those in Example 1.

[0146] C. Experimental results

[0147] The experimental results are shown in Figure 6 , demonstrating that the fecal microbiota transplantation experiment shows that the gut microbiota altered by cadmium exposure is a key factor in cadmium exposure promoting the development of breast tumors in MMTV-Erbb2 mice. Among them, Figure 6 A shows an overview of the mouse FMT experiment;Figure 6 Panel B shows the Kaplan-Meier plot for tumor-free survival (n = 6); Figure 6 Panel C shows the mean tumor volume calculated after the appearance of palpable tumors (n = 6 control group, n = 6 Cd group); Figure 6 Panel D shows representative images of mammary tumors in the control group and the Cd group; Figure 6 Panel E shows the tumor weights harvested at 29 weeks (n = 6 control mice, n = 6 mice receiving fecal extracts of Cd-exposed mice; two tumors were extracted from each mouse); Figure 6 Panel F shows representative images and statistical results of immunofluorescence detection of Ki67 in tumors of FMT_Control and FMT_Cd mice (scale bar: 50 μm); Panel G shows representative images of HE staining of tumors (scale bar: 1000 μm (original image), 50 μm (magnified image)). Figure 6 *, ** indicate P < 0.05, P < 0.01 compared with the control group, respectively.

[0148] To verify the key role of Cd-induced gut microbiota dysbiosis in mammary tumor development, the inventors conducted a fecal microbiota transplantation experiment (FMT) using gavage transplantation technology in MMTV-Erbb2 mice. Four-week-old MMTV-Erbb2 mice received antibiotic combination treatment to eliminate the original gut microbiota in the mice. Subsequently, the inventors divided all the mice into two groups, namely the FMT_Control group and the FMT_Cd group, which received transplantation of fecal extracts from control group (FMT_Control) or cadmium-exposed group (FMT_Cd) mice. The fecal donors of the control group and the cadmium-exposed group mice, namely the gut microbiota, were extracted as described in the method for transplantation experiments ( Figure 6 A). Next, the time of tumor appearance, tumor growth kinetics, and tumor weight were evaluated. The results showed that the time of tumor appearance was significantly accelerated in FMT_Cd mice, and the tumor weight was significantly greater than that in FMT_Control mice ( Figure 6 B-E). Immunofluorescence results showed that the proportion of cells positive for Ki67 in mammary tumors of FMT_Cd mice was also significantly increased, indicating that the proliferative activity of mammary tumor cells in FMT_Cd mice was higher ( Figure 6 F), which was consistent with the increased number of neovascularization results ( Figure 6 G). The above results indicate that the microbiota altered by drinking water cadmium exposure can directly promote the proliferation and tumor development process of breast cancer cells in MMTV-Erbb2 mice.

[0149] Example 7: Bioinformatics study on cadmium exposure disrupting the gut microecological balance

[0150] It should be noted that in the original text, there is an error in the description of the significance level in line . It is corrected to the correct format in the translation. Also, the content in the brackets at the end of line in the original text seems incomplete.The gut microbiota is a relatively independent "community" within the organism, and bacteria are widely involved in the synthesis and decomposition of glutamine. Previous research results have shown that environmental-dose cadmium exposure in drinking water induces gut microbiota dysbiosis in mice. Excessive glutamine is synthesized and absorbed into the body through the intestine, providing for the development of mammary tumors in the body. Analysis found that bacteria utilizing glutamine were significantly inhibited by cadmium, presumably an important cause leading to gut microbiota dysbiosis and the production of excessive glutamine. Based on this finding, it is speculated that screening representative glutamine-decomposing bacterial strains and supplementing them may reverse the phenomenon of breast cancer deterioration induced by cadmium exposure in drinking water in MMTV-Erbb2 mice. To screen out bacterial strains that are inhibited by cadmium and decompose and consume glutamine, the inventors used the MetOrign online database (https: / / metorigin.met-bioinformatics.cn / home / ) to search for all bacterial genera that are enriched in the alanine metabolism, aspartate metabolism, and glutamate metabolism pathways and utilize glutamine( Figure 7 A). The bacterial strains in the database were intersected with the differentially abundant bacterial strains with significantly reduced numbers found by the inventors using metagenomic analysis, and 83 strains were screened out( Figure 7 B). The correlation between these 83 strains and the glutamine content in their corresponding cecal contents was analyzed, and 65 strains showed a significant negative correlation( Figure 7 C). The linear discriminant analysis value (LDA score) can reflect the influence of significantly different microorganisms. Sorting by LDA value from high to low, the top 10 microorganisms were used to create a heatmap( Figure 7 D). Among them, the two bacterial strains with the highest LDA value and a significant correlation with the glutamine content were Clostridium butyricum (C. butyricum) and Blautia obeum (B. obeum)( Figure 7 D).

[0151] For the specific experimental results, see Figure 7 , which shows the identification results of glutamine-decomposing bacteria in the gut of MMTV-Erbb2 mice under Cd exposure. Figure 7 A shows a Sankey diagram of microorganisms enriched in the Ko00250 pathway involved in glutamine decomposition in the MetOrign database, including phylum, class, genus, and species levels; Figure 7 B shows a Venn diagram of the intersection of the bacterial strains enriched in the Ko00250 pathway and the microorganisms with decreased abundance among the differentially abundant gut microorganisms; Figure 7 C shows a network diagram of 65 glutamine-decomposing bacteria negatively correlated with the glutamine level in feces; Figure 7Panel D shows a heatmap of the abundances of glutamine-decomposing bacteria in the intestinal microbiota of the control and cadmium-treated groups sorted by LAD.

[0152] Example 8: Study on the effect of supplementing Clostridium butyricum or Blautia obeum on the progression of breast cancer in MMTV-Erbb2 mice exposed to cadmium in drinking water.

[0153] A: Animal experiment design and grouping

[0154] Six-week-old female MMTV-Erbb2 mice were purchased from Jackson Laboratory in the United States and were centrally housed in the IVC animal breeding room of the Experimental Animal Center of XX University. Approximately 3-5 mice were housed in each IVC cage. Under a constant temperature environment, a constant light with a simulated day-night ratio of 12h:12h was provided. To study the antagonistic effect of supplementing Clostridium butyricum (C. butyricum) or Blautia obeum (B. obeum) on the development of breast cancer, the inventors used a mouse model of promoting breast cancer development by exposing mice to 3.6 mg / L CdCl2 in drinking water. The grouping was as follows: Twenty-four female MMTV-Erbb2 mice were randomly divided into a control group, a cadmium-exposed group, a group supplemented with Clostridium butyricum (C. butyricum), and a group supplemented with Blautia obeum (B. obeum), with 6 mice in each group. The control group drank pure water and was gavaged with sterile sodium chloride solution (0.9%); the cadmium-exposed group drank pure water containing 3.6 mg / L CdCl2 and was gavaged with sterile sodium chloride solution (0.9%); the Clostridium butyricum group (C. butyricum) drank pure water containing 3.6 mg / L CdCl2 and was gavaged with Clostridium butyricum; the Blautia obeum group (B. obeum) drank pure water containing 3.6 mg / L CdCl2 and was gavaged with Blautia obeum. The cadmium exposure time and the gavage time started at the 6th week. Subsequently, according to the experimental design, after the mice were exposed to cadmium in drinking water for 23 weeks, the experiment was terminated when the average diameter of the tumors in the cadmium-exposed group of mice reached 1.5 cm. Cadmium chloride (CdCl2, Sigma-Aldrich, 439800) was dissolved in deionized water, filtered and sterilized, and stored at -20°C.

[0155] B: Target bacteria transplantation experiment

[0156] The experimental mice were gavaged orally with 2×10 8Clostridium butyricum (BNCC337239, Beijing NaChuangLian Biotechnology Co., Ltd.) or Blautia ovata (DSM25238, German Collection of Microorganisms and Cell Cultures DSMZ, https: / / bacdive.dsmz.de / strain / 17684) at a concentration of 2×10 8 CFU / 200 μL. The direct source of the strains was purchased from Chongqing Biooni Biotechnology Co., Ltd. Clostridium butyricum or Blautia ovata was anaerobically cultured at 37 °C for about 18 hours under static conditions in thioglycollate fluid medium (Product ID: HB5191, Qingdao Haibo Biotechnology Co., Ltd.). The bacteria were centrifuged at 7000 g for 8 minutes at 4 °C from the bacterial culture solution to obtain bacterial pellets, and the pellets were resuspended with sterile sodium chloride (0.9%) solution and adjusted to a concentration of 2×10 8 CFU / 200 μL for subsequent gavage. Each mouse was gavaged with 200 μL of the bacterial suspension (about 2×10

[0157] C: Experiment for measuring the glutamine content in intestinal contents

[0158] The entire intestine of the mouse was removed under sterile conditions using a sterile scalpel, and the contents of the cecal segment were excised. The contents were scooped out with a sterile scalpel and immediately placed on ice for aliquoting and labeling; immediately after aliquoting, they were snap-frozen in liquid nitrogen. Approximately 500 mg of intestinal contents were suspended in sterile water at a ratio of 500 mg / ml. The intestinal content suspension was filtered successively through sterile mesh sieves of 200 mesh, 400 mesh, and 800 mesh to remove large particles, undigested food, and smaller particles in the feces. The resulting filtrate was collected in a sterile centrifuge tube, vortexed for 2 minutes to obtain a resuspended solution, and the resuspended solution was centrifuged at 500×g for 2 minutes to remove insoluble matter, obtaining a suspension. The glutamine content was detected using a glutamine detection kit (abcam, ab197011) with the suspension.

[0159] D: Other experimental methods and statistical methods were the same as in Example 1.

[0160] E: Experimental results

[0161] For the specific experimental results, please refer to Figure 8 and Figure 9 . Figure 8 It shows that supplementing with Clostridium butyricum or Blautia ovata can effectively antagonize the process of cadmium exposure promoting the development of mammary tumors in MMTV-Erbb2 mice. Among them, Figure 8 A shows the experimental design of supplementing with Clostridium butyricum and Blautia ovata; Figure 8 B is the Kaplan-Meier plot of tumor-free survival (n = 6); Figure 8C shows the average tumor volume calculated after the appearance of palpable tumors (n = 6 in the control group, n = 6 in the Cd group); Figure 8 D shows representative images of mammary tumors in the control group and the Cd group; Figure 8 E) shows the tumor weights harvested at 29 weeks (n = 6 in each group; two tumors were extracted from each mouse); Figure 8 F shows representative images and statistical results of Ki67 detected by immunofluorescence in tumors of FMT_Control and FMT_Cd mice (scale bar: 50 μm); Figure 8 G shows representative images of HE staining of tumors (scale bar: 1000 μm (original picture), 50 μm (magnified picture)). Figure 8 In it, ** indicates P < 0.01 compared with the control group; # indicates P < 0.05 compared with the Cd group; ## indicates P < 0.01 compared with the Cd group. Figure 9 It shows that supplementing Clostridium butyricum or Blautia ovata can effectively reverse the significant increase in intestinal glutamine content induced by cadmium exposure in MMTV-Erbb2 mice. Among them, Figure 9 A shows the comparison results of the relative content of glutamine in the intestinal contents of mice in each treatment group (n = 6); Figure 9 B shows the results of correlation analysis, indicating that the relative content of glutamine is significantly positively correlated with the average tumor weight of mice. Figure 9 In it, ** indicates P < 0.01 compared with the control group; ## indicates P < 0.01 compared with the Cd group.

[0162] To evaluate whether the selected strains can antagonize the breast cancer progression of MMTV-Erbb2 mice induced by chronic low-dose cadmium, Clostridium butyricum or Blautia ovata was used for bacterial transplantation in mice by gavage. Bacteria were supplemented according to the method described. At the same time, MMTV-Erbb2 mice were exposed to 3.6 mg / L cadmium in drinking water for 23 weeks, and the number of mice with breast cancer and tumor size were recorded weekly ( Figure 8 A). The results showed that the onset time of breast cancer in mice with chronic cadmium exposure was delayed after the transplantation of the target bacteria, and there was a statistical difference ( Figure 8 B). The diameter of mammary tumors was measured weekly from the 19th week to record the growth of mammary tumor volume. The results showed that the tumor volume of mice supplemented with Clostridium butyricum or Blautia ovata and drinking cadmium water was significantly smaller than that of mice in the cadmium treatment group ( Figure 8 C-D). Subsequently, the inventor weighed the isolated mouse tumors (two tumors were taken from each mouse), and the results showed that the tumor weight of mice in the cadmium exposure group was significantly greater than that of the control group, but the tumor weight of mice supplemented with Clostridium butyricum or Blautia ovata was significantly smaller than that of the cadmium treatment group ( Figure 8 E). After the isolated tumor tissues were fixed and made into pathological sections and stained with HE, they were all mammary tumors with a high degree of malignancy ( Figure 8G). Ki67 staining revealed that the positive cell rate in the cadmium treatment group was significantly higher than that in the control group. The Ki67 positive cells in the group supplemented with C. butyricum or B. obeum were significantly lower than those in the cadmium treatment group, and the difference was statistically significant, further indicating that cadmium exposure promoted the proliferation of breast tumors, while supplementation with C. butyricum or B. obeum could reduce the development of cadmium-induced breast tumors. The above results suggest that both C. butyricum and B. obeum can effectively inhibit the promotion of breast cancer development by environmental-dose cadmium exposure and can significantly control the development of breast cancer.

[0163] The glutamine content in intestinal contents was detected as described in the experimental method. The results showed that compared with the control group, the glutamine content in the cadmium exposure group was significantly increased, while in the groups supplemented with C. butyricum or B. obeum, the increase in glutamine content in intestinal contents induced by cadmium exposure was significantly inhibited, effectively regulating the disorder of the intestinal microecology induced by cadmium exposure ( Figure 9 A). Further correlation analysis with the average tumor weight of mice found that the glutamine content was positively correlated with the tumor weight ( Figure 9 B). The above results indicate that supplementation with C. butyricum or B. obeum can effectively regulate the increase in glutamine content caused by intestinal microbial disorders, which is closely related to the inhibition of breast tumor progression.

[0164] The above are only examples of the present invention. Specific technical solutions and / or common knowledge such as characteristics known in the art are not described in detail here. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. Use of intestinal microorganisms in the preparation of a drug for preventing or treating breast cancer exacerbated by chronic cadmium exposure in drinking water, characterized in that: The intestinal microorganism is Clostridium butyricum and / or Blautia ovata; the Clostridium butyricum is the strain with the preservation number of BNCC337239 from Beijing Nacron Biotechnology Co., Ltd., and the Blautia ovata is the strain with the preservation number of DSM25238 from the German Collection of Microorganisms and Cell Cultures.

2. The use of intestinal microorganisms in the preparation of a drug for preventing or treating breast cancer exacerbated by chronic cadmium exposure in drinking water according to claim 1, characterized in that: The cadmium concentration in the drinking water is 3.6 mg / L.

3. The use of intestinal microorganisms in the preparation of a drug for preventing or treating breast cancer exacerbated by chronic cadmium exposure in drinking water according to claim 2, characterized in that: In mice, the gavage dose of Clostridium butyricum and / or Blautia ovata each time was 2×10 8 colony forming units, and gavage was performed twice a week.

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