Application of quercetin in preparation of medicine for regulating intestinal microbiological disorder caused by bladder cancer

Abstract

The invention provides application of quercetin in preparation of a medicine for regulating intestinal microbiological disorder caused by bladder cancer, and belongs to the technical field of medical technology. It is found for the first time that quercetin regulates intestinal flora in the occurrence and development process of bladder cancer, effectively enriches tumor-related intestinal beneficial bacteria, can reverse disorder of in-vivo metabolic markers and reduces the level of L-serine, and therefore the effect of inhibiting the progress of bladder tumor is achieved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of quercetin in the preparation of drugs for regulating intestinal microbial disorders caused by bladder cancer. Background Technology

[0002] Currently, interventions for bladder cancer tend towards multidisciplinary comprehensive treatment, including surgical resection, intravesical chemotherapy, neoadjuvant therapy, radiotherapy, and immunotherapy. Although these clinical interventions can partially alleviate tumor recurrence and progression, a large proportion of patients develop high-grade or metastatic disease after receiving cisplatin-based cytotoxic chemotherapy, resulting in a poor prognosis (5-year progression rate between 0.8% and 45%). Due to their limited specificity and unclear clinical efficacy, few drugs are widely used in clinical practice. Therefore, it is crucial to find and develop safe, effective drugs with low side effects to improve bladder cancer.

[0003] Plants have long been used to treat diseases, and some remain standard treatments for several conditions. Numerous plant components and compounds with varying biological and pharmacological activities have been isolated and identified from medicinal plants. Quercetin (molecular weight: 302.236), classified as a flavonol, is one of the six subclasses of flavonoids and is a major polyphenolic flavonoid found in various vegetables and fruits. In recent years, quercetin has shown antitumor activity in various malignant tumors without adverse effects on normal cells. This lays the foundation for its potential as an effective anticancer drug and adjuvant therapy. At the cellular level, quercetin inhibits the proliferation of bladder cancer cells and promotes apoptosis. However, the molecular mechanism by which quercetin improves bladder tumors remains unclear.

[0004] The human gut is home to a vast number of microorganisms, numbering nearly 100 trillion. This number far exceeds the combined total of all other microbial communities associated with the body's surface. Therefore, the gut microbiota can be described as a unique and powerful organ. With the increasing incidence of tumors, the relationship between gut microbiota and specific tumors has gradually become a research hotspot. Firstly, some gut microbiota are themselves carcinogens, meaning that the gut microbiota itself or its metabolites can directly promote cancer. Secondly, gut microbiota can also indirectly promote tumors by inducing inflammation or exerting immunosuppressive effects. Furthermore, changes in the composition of the gut microbiota can also affect tumor development. The gut microbiota may be one of the important factors involved in the development and progression of bladder tumors; therefore, developing new drugs targeting the gut microbiota and its metabolic pathways may be a potential strategy for treating bladder cancer. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the use of quercetin in the preparation of a medicament for regulating intestinal microbial dysbiosis caused by bladder cancer.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] Application of quercetin in the preparation of drugs that regulate intestinal microbial dysbiosis caused by bladder cancer.

[0008] Preferably, the quercetin enriches beneficial gut bacteria associated with tumor inhibition.

[0009] Preferably, the beneficial gut bacteria include Rikenella, Gordonibacter, Candidatus Arthromitus, Eubacterium siraeum, Coridextribacter, Dubosie, Eubacterium fissicatena, Erysipelatocostridium, Clostridium innocuum, Blautia, Parabacteroides, Parasutterella, and Anaerostipes.

[0010] Preferably, the beneficial intestinal bacteria inhibit the activity of bladder cancer cells.

[0011] Preferably, the bladder cancer cells include T24 cells and UMUC3 cells.

[0012] Preferably, the quercetin inhibits the level of the intestinal flora metabolite L-serine.

[0013] Preferably, the quercetin inhibits the expression of the PSPH gene and the PSPH protein.

[0014] Another object of the present invention is to provide the application of quercetin in the preparation of a drug for the prevention and treatment of bladder cancer. Quercetin is administered to a nude mouse xenograft model of human bladder tumor, and feces are collected after administration. Microorganisms are extracted from the feces, and the microorganisms are used to prepare a drug for the treatment of bladder cancer.

[0015] Preferably, the steps for extracting microorganisms from feces include: mixing feces with PBS at a ratio of 1g:5mL, centrifuging, and collecting the supernatant.

[0016] Preferably, the centrifugation speed is 800 rpm and the centrifugation time is 3 minutes.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides the application of quercetin in the preparation of drugs that regulate intestinal microbial dysbiosis caused by bladder cancer. This invention is the first to discover that quercetin regulates the intestinal flora during the occurrence and development of bladder cancer, effectively enriches beneficial intestinal bacteria associated with tumors, and can reverse the disorder of metabolic markers in tumor model mice, reduce the level of L-serine, thereby playing a role in improving the progression of bladder tumors. Attached Figure Description

[0019] Figure 1 Example 1 describes the process of establishing a human bladder tumor xenograft model in nude mice;

[0020] Figure 2 The study aimed to investigate the inhibitory effect of quercetin on the progression of bladder tumors in nude mice. In the data, A represents tumor size after different drug administrations; B represents tumor volume (P < 0.0001); C represents tumor weight (P < 0.001, P < 0.0001); D represents Ki 67 staining; E represents the proportion of Ki 67-positive cells (P < 0.01, P < 0.001); F represents PCNA staining (P < 0.0001); G represents the proportion of PCNA-positive cells; H represents the in vitro drug test results of three bladder cancer organoids; I represents the mean tumor cell viability of the three bladder cancer organoids 5 days after drug administration (P < 0.0001); J represents the proliferation capacity of SW780 cells and the IC50 inhibitory effect of quercetin on cancer cell progression. 50 Value; K represents the proliferation capacity of UMUC3 cells and the IC50 value of quercetin in inhibiting cancer cell proliferation. 50 value;

[0021] Figure 3 To assess the safety of quercetin treatment in nude mice, A represents no significant difference in body weight and food intake between the high-dose quercetin group and the control group; B represents the effect of high-dose quercetin on liver and spleen indices in nude mice; C represents the effect of high-dose quercetin on serum alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine in nude mice, with ns representing no significant difference.

[0022] Figure 4 The effect of quercetin on the gut microbiota of nude mice with bladder tumors is shown in Figure A, where A represents the Shannon-Wiener curve and species accumulation curve; B represents the tumor-associated beneficial gut bacteria that were significantly enriched after quercetin administration, *P<0.05, **P<0.01.

[0023] Figure 5The effects of quercetin on the gut microbiota of nude mice with bladder tumors were investigated. A represents the α-diversity of gut microbiota (***P < 0.001, ****P < 0.0001); B represents the β-diversity of gut microbiota: the left figure shows principal component analysis results; the right figure shows principal coordinate analysis results; C represents the heatmap analysis of gut microbiota genera in each group after quercetin treatment; D represents the relative abundance of gut microbiota phyla in each group; E represents the relative abundance of gut microbiota families in each group; F represents the enrichment of Parabacteroides sp. by quercetin treatment (***P < 0.001). 01; Figure G, from left to right, shows the effect of the intestinal probiotic Parabacteroides_sp. on T24, UMUC3, and SV-HUC-1 cells, **P<0.01; H represents the enrichment of Clostridium_cocleatum by quercetin treatment, ***P<0.001; Figure I, from left to right, shows the effect of the intestinal probiotic Clostridium_cocleatum on T24, UMUC3, and SV-HUC-1 cells, *P<0.05, **P<0.01;

[0024] Figure 6 The study investigated the effects of quercetin on the gut microbiota of nude mice with bladder tumors. A represents orthogonal-partial least squares discriminant analysis; B is the control group; C is the model group; and Q is the quercetin-treated group (dose: 100 mg / kg / d). B shows the S-spot map constructed using OPLS-DA. C represents metabolic markers in feces. D shows the KEGG enrichment pathway map of differentially expressed metabolites among the groups. E shows the KEGG enrichment pathway map of differentially expressed metabolites among the groups. F shows the correlation between intestinal metabolites and tumor size in nude mice. G shows the changes in serine content in each group of nude mice; the top left shows L-serine content in feces, the top right shows L-serine content in blood, the bottom left shows L-serine content in urine, and the bottom right shows L-serine content in tumors. *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0025] Figure 7 The study investigated the effects of quercetin on L-serine, a metabolite of gut microbiota. Specifically, A represents the correlation between gut microbiota metabolites and gut microbiota after quercetin treatment (*P < 0.05); B represents the correlation between L-serine and gut microbiota after quercetin treatment; C represents the effect of quercetin on L-serine levels in vitro; D represents the growth rate of three bladder tumor cell lines in different culture media; E represents the regulation of bladder tumor cell line phenotypic proteins by L-serine using Western blotting; F represents the gross specimens of SW780 bladder cancer cell xenografts in nude mice treated with L-serine; G represents the growth curve inhibition (***P < 0.001); and H represents tumor weight statistics (***P < 0.001).

[0026] Figure 8 To investigate the effect of quercetin on bladder cancer progression, the following diagrams are presented: A represents the serine synthesis process; B is a volcano plot showing differential gene expression in tumor samples from groups M and LQ after quercetin administration; C is a heatmap showing differential gene expression in tumor samples from groups M and LQ after quercetin administration; D represents PSPH expression (**P < 0.01); E represents PSPH protein expression; F represents PSPH expression after transfection of UMUC3 and T24 cells with PSPH siRNA (****P < 0.0001); G represents PSPH protein expression after transfection of UMUC3 and T24 cells with PSPH siRNA; H represents KEGG enrichment analysis of quercetin's target sites; and I represents functional protein changes caused by downregulation of PSPH expression.

[0027] Figure 9 The study investigated the changes in gut microbiota in nude mice treated with quercetin for bladder cancer. A represents the experimental procedure; B represents tumor size in different groups of nude mice; C represents tumor volume; D represents tumor weight; E represents α-diversity of gut microbiota; F represents β-diversity of gut microbiota; G represents the relative abundance of gut microbiota phyla in each group of nude mice, from left to right: phylum, family, genus; H represents the changes in serine content in each group of nude mice, from left to right: L-serine content in feces, blood, urine, and tumors; *P < 0.05, ***P < 0.001, ****P < 0.0001. Detailed Implementation

[0028] This invention provides the application of quercetin in the preparation of drugs for regulating intestinal microbiota dysbiosis caused by bladder cancer. Due to its low solubility, poor first-pass metabolism efficiency, low systemic metabolic efficiency, and low oral bioavailability, quercetin accumulates in large quantities in the large intestine. This invention is the first to discover that quercetin can effectively regulate intestinal microbiota dysbiosis caused by bladder cancer.

[0029] In this invention, the quercetin enriches and inhibits tumor-associated beneficial gut bacteria. These beneficial gut bacteria include Rikenella, Gordonibacter, Candidatus Arthromitus, Eubacterium siraeum, Coridextribacter, Dubosie, Eubacterium fissicatena, Erysipelatocostridium, Clostridium innocuum, Blautia, Parabacteroides, Parasutterella, and Anaerostipes, with Clostridium innocuum and Parabacteroides being preferred.

[0030] In this invention, the beneficial intestinal bacteria can inhibit the activity of bladder cancer cells, including T24 cells and UMUC3 cells. Quercetin-enriched bacteria help inhibit the progression of bladder cancer cells.

[0031] In this invention, quercetin inhibits the level of L-serine, a metabolite of gut microbiota. This study found that quercetin treatment can reverse the dysregulation of metabolic markers in tumor model mice and effectively inhibit the level of L-serine, thereby inhibiting the progression of bladder cancer.

[0032] In this invention, quercetin inhibits the expression of the PSPH gene and the PSPH protein, thereby inhibiting the progression of bladder tumors.

[0033] Another object of the present invention is to provide the use of quercetin in the preparation of a drug for the prevention and treatment of bladder cancer. A nude mouse xenograft model of human bladder tumor is administered quercetin, and feces are collected after administration. Microorganisms are extracted from the feces, and these microorganisms are used to prepare a drug for treating bladder cancer. As one feasible method, feces are collected two weeks after administration, at a dose of 100 mg / kg / day.

[0034] In this invention, the step of extracting microorganisms from feces includes: mixing feces with PBS at a ratio of 1g:5mL, centrifuging and collecting the supernatant, wherein the centrifugation speed is 800rpm and the centrifugation time is 3min.

[0035] In this invention, after treatment with quercetin, the human bladder tumor xenograft model in nude mice accumulated beneficial microorganisms and metabolites associated with the tumor in the intestines. The supernatant collected by this invention can be used to treat bladder cancer and effectively inhibit the progression of bladder cancer.

[0036] The quercetin used in the specific embodiments of this invention was purchased from Sigma-Aldrich.

[0037] In a specific embodiment of this invention, data processing was performed as follows: GraphPad Prism (GraphPad Software, version 9.0) was used for statistical analysis and graphing. Student's t-test was used to analyze data between two groups; one-way ANOVA was used to analyze data from more than two groups. Regression-probit regression analysis was used to regress quercetin concentration against cell viability, and the IC50 value of quercetin was calculated. All results are expressed as mean ± standard deviation (SD), and P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns represent no significant difference.

[0038] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1

[0040] Application of quercetin in regulating the intestinal flora of bladder cancer

[0041] 1. Experimental animals.

[0042] Specific pathogen-free BALB / c nude mice (4 weeks old, female) were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd., with the license number: SCXK (Su) 2023-0009. The body weight was 19-20 g. The animals were housed in an animal room with a 12h light / dark cycle, the environmental temperature was maintained at 20-24°C, and the humidity was maintained at 40-60%. The food and water sterilized by high-pressure were replaced every day. This invention was carried out under the permission and guidance of the Experimental Animal Ethics Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College. All steps were carried out in accordance with the "Organizational Guidelines and Ethical Guidelines for the Experimental Animal Ethics Committee" (Ethical number: 00004309).

[0043] 2. Establishment of a nude mouse xenograft model of human bladder tumors.

[0044] Sixty BALB / c nude mice (female) were housed in a SPF-level animal room and allowed to adapt to the new environment for 1 week before the experiment; 2×10 6 times the number of human bladder cancer cells SW780 were evenly suspended in a suspension of 2 mL PBS and Matrigel (Corning, USA) (volume ratio 1:1), and inoculated into the right back of the nude mice in front of the front leg under sterile conditions, with an injection volume of 100 μL; The tumor formation of the nude mice was observed daily. Once the tumor size reached 100-150 mm 3 , the nude mice were randomly divided into 6 groups, with 10 nude mice in each group. The 6 groups were the model group, low-dose quercetin treatment group, high-dose quercetin treatment group, antibiotic treatment group, antibiotic + high-dose quercetin treatment group, and control group.

[0045] Model group: Gavage with 0.5% CMC-Na every day (denoted as group M);

[0046] Low-dose quercetin treatment group: Gavage with quercetin every day, with a gavage volume of 100 mg / kg / d (denoted as group LQ);

[0047] High-dose quercetin treatment group: Gavage with quercetin every day, with a gavage volume of 200 mg / kg / d (denoted as group HQ);

[0048] Antibiotic treatment group: Cefadroxil monohydrate, erythromycin and oxytetracycline were administered by gavage daily. The three antibiotics were dissolved together in 0.5% CMC-Na physiological saline and then administered by gavage. The gavage dosage was: cefadroxil monohydrate 100mg / kg / day, erythromycin 300mg / kg / day, and oxytetracycline 300mg / kg / day (referred to as group A).

[0049] The antibiotic + high-dose quercetin treatment group: Cefadroxil monohydrate, erythromycin, oxytetracycline and quercetin were administered by gavage daily. The above antibiotics and quercetin were dissolved together in 0.5% CMC-Na physiological saline before gavage. The gavage dosage was: cefadroxil monohydrate 100mg / kg / day, erythromycin 300mg / kg / day, oxytetracycline 300mg / kg / day, and quercetin 200mg / kg / day (denoted as AQ group);

[0050] Control group: The remaining 10 nude mice served as the control group (referred to as group C). The mice in the control group were not inoculated with human bladder cancer cells SW780.

[0051] The test procedure is as follows Figure 1 As shown. Day 0 was the time when human bladder cancer cells SW780 were inoculated. Four weeks after inoculation, nude mice were euthanized under anesthesia, and the volume and mass of the tumors were observed.

[0052] 3. The effects of quercetin on bladder tumors.

[0053] 3.1 Immunohistochemical staining of bladder tumors in nude mice.

[0054] (1) Dewaxing paraffin sections to water: Place the sections in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then wash with distilled water.

[0055] (2) Antigen retrieval: Tissue slides were placed in a retrieval box filled with EDTA antigen retrieval solution (pH 9.0) and microwaved for antigen retrieval. The microwave was heated on medium heat for 8 minutes until boiling, then turned off and kept warm for 8 minutes before turning on medium-low heat for 10 minutes. During this process, excessive evaporation of the buffer solution should be prevented, and the slides should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time.

[0056] (3) Blocking endogenous peroxidase: Place the slide in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Then place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time.

[0057] (4) Serum blocking: Add 3% BSA evenly to the histochemistry zone and block at room temperature for 30 min (use rabbit serum for primary antibody from goat, and BSA for other sources).

[0058] (5) Add primary antibody: Gently shake off the blocking solution, drop the primary antibody prepared in PBS at a certain ratio onto the slide, and incubate the slide flat in a humidified chamber at 4°C overnight (add a small amount of water to the humidified chamber to prevent antibody evaporation).

[0059] (6) Add secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the section, add the secondary antibody (HRP-labeled) of the corresponding species to the primary antibody to cover the tissue and incubate at room temperature for 50 minutes.

[0060] (7) DAB staining: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. The positive result is brownish-yellow. Rinse the slide with tap water to stop the staining.

[0061] (8) Counterstaining cell nuclei: Counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin blue solution, and rinse with running water.

[0062] (9) Dehydration and mounting: The sections are placed in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and xylene I for 5 min in sequence to dehydrate and become transparent. The sections are then removed from the xylene and allowed to dry slightly before being mounted with neutral resin.

[0063] (10) Microscopic examination and image acquisition and analysis.

[0064] Ki-67 staining: primary antibody Ki67, purchased from Servicebio, GB111499; secondary antibody was HRP goat anti-rabbit, purchased from Servicebio, GB23303.

[0065] PCNA staining: Primary antibody PCNA, purchased from Servicebio, GB12010; Secondary antibody HRP goat anti-mouse, purchased from Servicebio, GB23301.

[0066] 3.2. Establishment of organoids from patient-derived bladder tumors.

[0067] Fresh tumor tissue from the patient was washed in DMEM medium containing 10 μMY-27632 and minced to 1 mm with scissors. 3Then, the tumor tissue was washed in PBS supplemented with 100 μg / mL antibiotics, followed by digestion in DMEM medium containing collagenase II and IV (working concentrations of 4 mg / mL for both) at 37°C for 15-30 minutes. The digested tissue was centrifuged at 350g for 5 minutes, resuspended in 10 mL PBS, and centrifuged again at 350g for 5 minutes. The tissue was then resuspended in 5 mL TrypLE Express and incubated at room temperature for 3 minutes. Digestion was terminated by adding excess PBS supplemented with 10 μM ROCK inhibitor Y-27632 and 100 μg / mL penicillin-streptomycin, followed by centrifugation at 350g. After resuspending the pellet, the digested cells (approximately 1 x 10⁻⁶ cells) were filtered through a 100 μm cell filter. 5 The cells were resuspended in 60 μL of matrix gel and seeded into the center of each well in a 24-well plate to form solid droplets. The plates were then incubated at 37°C and 5% CO2 for 30 minutes to allow the droplets to solidify. 0.7–1 mL of organoid culture medium was added to the plates, and the medium was changed every 3–4 days.

[0068] For passage, the organoids were released by digesting the matrix gel using Recovery technology. The organoids were then centrifuged at 350g for 5 min, washed and resuspended in PBS. 5 mL of LTrypLE Express was added, and the organoids were incubated at room temperature for 3 min, followed by mechanical dissociation into small cell clusters via pipette. The organoids were passaged every 2–3 weeks at a 1:2–3 expansion ratio.

[0069] In a well plate containing organoids, 20 μL of quercetin was added to each well to achieve final concentrations of 200 μM, 100 μM, and 50 μM. Each concentration was tested in triplicate. 20 μL of culture medium was added as a negative control, and the negative control was tested in triplicate. The plates were incubated at 37°C and 5% CO2 for 12 h. CellTiter-Glo 3D assay reagent was added, and the procedure was performed according to the instructions. The luminescence value was detected and the data was processed using a microplate reader that can record luminescence signals. The luminescence value was directly proportional to the amount of ATP in the cells, and the amount of ATP was directly proportional to the number of viable cells in the culture. This confirmed the sensitivity of bladder tumor organoids to various antitumor drugs.

[0070] 3.3. CCK-8 assay of quercetin co-cultured with bladder tumor cells and bladder epithelial cells.

[0071] Logarithmic growth phase SW780 and UMUC3 cells were digested with 0.25% trypsin containing EDTA, and the growth was terminated by adding complete culture medium (500ml DMEM + 5ml penicillin-streptomycin + 50ml fetal bovine serum (FBS),) and cell counts were performed. The cells were then diluted with culture medium to form a cell suspension. 5×10 3 SW780 and UMUC3 cells were rapidly added to each well of a 96-well plate, and quercetin at concentrations of 0, 10, 25, 50, 75, 100, 200, and 300 μM was added, respectively, and the cells were co-cultured for 48 hours. Six replicates were set for each concentration, with a drug-free (0) solution serving as a control. Then, 10 μL of CCK-8 reagent (Sigma-Aldrich, USA) was added to each well, and the culture was terminated after 30 minutes. OD values ​​were measured at 450 nm.

[0072] 3.4 Test results.

[0073] like Figure 2 The results showed that quercetin significantly inhibited the progression of bladder cancer. Figure 2 A and Figure 2 In the B group, the tumor inhibition rates (inhibition rate = (1 - average tumor weight in the treatment group / average tumor weight in the control group) × 100%) in the LQ and HQ groups were 48.5% and 51.417%, respectively, but the AQ group had a lower inhibitory effect on bladder cancer, at only 24.18%. Figure 2 (C) In the AQ group, a large number of intestinal bacteria were eliminated after antibiotic treatment, and the efficacy of quercetin treatment was worse than that of the LQ and HQ groups, indicating that the anticancer effect of quercetin depends on the gut microbiota. According to the assessment of Ki-67 and PCNA staining, low and high doses of quercetin treatment significantly inhibited cell proliferation (P<0.01, P<0.001). Figure 2 D- Figure 2 (G in the middle).

[0074] To further verify the changes in bladder cancer after quercetin treatment, three bladder cancer organoids were constructed using fresh tumor tissue from three clinical patients for in vitro drug testing. Figure 2 As shown in Figure H, after 5 days of culture with the drug, the bladder cancer organoids in the control group were clearly visible as spheroids composed of many cells with very close connections between them. The diameter of a single organoid could reach about 100 μm. In addition to some cell death, the surviving organoids in the quercetin-treated group aggregated into solid spheres, with a morphology significantly different from the control group. After 5 days of culture with the drug, ATP assays of tumor cell activity confirmed that the cell activity in the quercetin-treated group was significantly lower than that in the control group, showing a dose-dependent trend (P<0.001). Figure 2 (I in the middle).

[0075] At the cellular level, the inhibitory effect of quercetin on the proliferation of bladder cancer cells was dose- and time-dependent; with increasing drug concentration and prolonged treatment time, the proliferation capacity of SW780 and UMUC3 bladder cancer cells gradually decreased. The IC50 values ​​of the two bladder cancer cell lines after 72 hours were also discussed. 50 The values ​​were 94.22 μM and 82.22 μM, respectively. Figure 2 J- in Figure 2 K in the middle.

[0076] No adverse reactions were observed in mice treated with quercetin. There was no significant difference in body weight and food intake between the high-dose quercetin group and the control group. Figure 3 A) High-dose quercetin had no effect on liver and spleen indices, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) (liver function), blood urea nitrogen, and creatinine (kidney function) in nude mice. Figure 3 China B- Figure 3 (C) This indicates that quercetin is safe.

[0077] 4. Quercetin regulates gut microbiota dysbiosis in nude mice with bladder tumors.

[0078] 4.1 Analysis of 16S rRNA biodiversity in gut microbiota.

[0079] Fecal samples were collected from each group of nude mice to further analyze the composition and structure of the gut microbiota using bacterial 16S rRNA gene amplification and sequencing. Genomic DNA was first extracted from fecal samples according to the instructions of the EZNA.Soil DNA Kit. The V3-V4 region of the bacterial 16S rRNA gene was amplified using universal primers 338F: 5'-ACTCCTACGGGAGGCAGCAG-3' (SEQ ID NO.1) and 806R: 5'-GGACTACH VGGGTWTCTAAT-3' (SEQ ID NO.2). The PCR products were used to construct libraries using the NEB NextUltra II DNA Library Prep Kit. Finally, the libraries were sequenced on a high-throughput sequencing platform using a PE250 / PE300 sequencing strategy.

[0080] 4.2 Cell proliferation detection after co-culturing bladder tumor cells and bladder epithelial cells with the supernatant of intestinal characteristic bacteria culture medium.

[0081] Culture of characteristic gut bacteria:

[0082] Parabacteroides sp. (purchased from the German Culture Collection, DSM 24821) was cultured using custom DSM medium (purchased from the German Culture Collection), which contained 500.0 g of ground beef (fat free), 1000.0 mL of distilled water, and 25.0 mL of NaOH 1N. The medium was cultured in an anaerobic environment at 37°C for 48 h using a gas generation bag system (purchased from Shanghai Yiheng Technology Co., Ltd.).

[0083] Clostridium cocleatum (purchased from the German Culture Collection, DSM 1551) was cultured using custom DSM medium (purchased from the German Culture Collection). The medium contained 30.00 g casein peptone, 5.00 g yeast extract, 5.00 g K2HPO4, 0.50 mL sodium resazurin (0.1% w / v), 0.50 g L-cysteine ​​hydrochloride HCl x H2O, 10.00 mL sugar mix, 1.50 g Na2CO3, and 1000.00 mL meat filtrate. The culture was carried out in an anaerobic environment at 37°C for 48 h using a gas generation bag system (purchased from Shanghai Yiheng Technology Co., Ltd.).

[0084] After culturing Parabacteroides sp. and Clostridium cocleatum, the cultures were collected by centrifugation (4℃, 5000×g, 15 min) and washed twice with sterile 0.85% NaCl (w / v). They were then resuspended in PBS solution, and 200 μL of 10... 8 CFU / mL resuspensions of Parabacteroides sp. and Clostridium cocleatum were inoculated into 500 mL of the above-mentioned custom-made medium and incubated at 37°C for 20 hours without stirring. Once the concentration reached 2.9 × 10⁻⁶, the culture medium was incubated. 9To achieve the CFU / mL growth period, live Parabacteroides sp. and Clostridium cocleatum were isolated from the culture medium by centrifugation at 7000 rpm for 30 minutes at 4°C. The Parabacteroides sp. and Clostridium cocleatum bacterial particles were washed twice with sterile phosphate-p-PBS solution (to remove culture medium residue) and resuspended in 500 mL of sterile RPMI-1640 solution. The mixture was incubated at 37°C for 5 hours without stirring to obtain conditioned medium for Parabacteroides sp. and Clostridium cocleatum. Subsequently, the conditioned medium was centrifuged at 7000 rpm for 30 minutes at 4°C to separate the conditioned medium from the bacterial particles, and then sterilized by filtration through a 0.22 μm bottle filter. The cell-free RPMI-1640 supernatant was replenished to a volume of 200 mL with FBS and L-glutamine to obtain a complete Parabacteroides sp. and Clostridium cocleatum conditioned supernatant for use in in vitro experiments, following the same experimental method as in 3.3.

[0085] 4.3 Fecal metabolomics analysis.

[0086] Accurately weigh 2 mg of sample into a 2 mL centrifuge tube, add 600 μL of methanol containing 2-chloro-L-phenylalanine (4 ppm), vortex for 30 s; add steel beads, place in a tissue homogenizer, and homogenize at 50 Hz for 120 s; sonicate at room temperature for 10 min; centrifuge at 12000 rpm at 4℃ for 10 min, collect the supernatant and filter through a 0.22 μm membrane, add the filtrate to a sample vial for LC-MS detection. Then use a device equipped with ACQUITY... Untargeted metabolomics analysis was performed using a Thermo Vanquish ultra-high performance liquid chromatography system with an HSS T3 (2.1×100mm, 1.8μm) column and a Thermo Orbitrap Exploris 120 mass spectrometer detector. Chromatographic conditions: positive ion mode; mobile phase: 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (A2); gradient elution program: 0–1 min, 8% B2; 1–8 min, 8%–98% B2; 8–10 min, 98% B2; 10–10.1 min, 98%–8% B2; 10.1–12 min, 8% B2. In negative ion mode, the mobile phase consisted of acetonitrile (B3) and 5 mM ammonium formate aqueous solution (A3). The gradient elution program was as follows: 0-1 min, 8% B3; 1-8 min, 8%-98% B3; 8-10 min, 98% B3; 10-10.1 min, 98%-8% B3; 10.1-12 min, 8% B3. ESI source conditions were set as follows: data were acquired in both positive and negative ion modes. The positive and negative ion spray voltages were 3.50 kV and -2.50 kV, respectively, with a sheath gas of 40 arb and an auxiliary gas of 10 arb. The capillary temperature was 325 °C. A first-stage full scan was performed at a resolution of 60,000 m / z, with a first-stage ion scan range of 100-1000 m / z. Second-stage fragmentation was performed using HCD with a collision energy of 30% and a second-stage resolution of 15,000 m / z. The first four ions acquired were fragmented.

[0087] 4.4 Qualitative and quantitative analysis methods for L-serine.

[0088] High-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS 8060) was used to analyze serine from various samples in in vitro and in vivo studies. Chromatographic separation was performed using an ACQUITY BEH Amide Column (2.1×100mm, 1.7μm) at a column temperature of 35℃. The mobile phase consisted of A: 20mM ammonium acetate aqueous solution: acetonitrile = 15:85 (containing 0.5% formic acid); B: 20mM ammonium acetate aqueous solution (containing 0.5% formic acid). The elution gradient from phase A to phase B was as follows: 0.50 min (100% A and 0% B); 2.50 min (96% A and 4% B); 3.50 min (80% A and 20% B); 4.00 min (62% A and 38% B); 6.00 min (55% A and 45% B); 6.50 min (100% A and 0% B); 8.00 min (Stop). The flow rate was 0.6 mL / min. The autosampler temperature was set to 10 °C. Analysis was performed in ESR mode with the following parameters: nebulizer gas flow rate 3.0 L / min, drying gas flow rate 10.0 L / mL, interface voltage -4.5 kV, DL temperature 250 °C, CID pressure 230 kPa, and heating block temperature 400 °C. The mass spectrometer was used to optimize the ion pairs in multiple reaction monitoring (MRM) mode for quantification. The ion pair for serine was 160.05→60.05 (m / z).

[0089] 4.5 In vitro incubation test of quercetin and mouse intestinal flora.

[0090] C57BL / 6J mice were fed for 7 days prior to the experiment with a normal diet. Mice were fasted for 12 hours before the experiment but had free access to water during the experiment. To prepare intestinal microbiota cultures, anaerobic culture medium (Solbio, Cat: LA0740) was prepared according to the instructions (1.9g medium + 200mL purified water). The resulting medium was then autoclaved at 0.1MPa and 121℃ for 20 minutes and cooled before use. C57BL / 6J mice were euthanized by cervical dislocation. Under anaerobic conditions, the abdomen was opened, the colon was removed, and the colonic contents were mixed with the anaerobic culture medium at a ratio of 1:20 [weight (g):volume (mL)]. After thorough mixing, the intestinal microbiota culture was filtered through two layers of sterile gauze, pre-incubated at 37℃ under anaerobic conditions for 30 minutes, and a portion was used for later use. Another portion was sterilized at high temperature for later use.

[0091] The samples were divided into four groups: a negative control group, a quercetin (10 μg / mL) group, a quercetin (50 μg / mL) group, and a heat-sterilized group (Heated gutmicrobiota). 1 mg of glipizide was accurately weighed, dissolved in methanol, and diluted to 100 ng / mL, then stored at 4°C. 10 mg of quercetin was accurately weighed and dissolved in the aforementioned methanol solution containing glipizide, and stock solutions of 1 mg / mL and 5 mg / mL were prepared for the experimental groups, respectively.

[0092] Con group: 10 μL of methanol was added to a centrifuge tube, and 990 μL of the above-mentioned negative control group mouse intestinal flora mixed culture medium was added;

[0093] LQ group: 10 μL of the stock solution with a concentration of 1 mg / mL was added to a centrifuge tube, and 990 μL of the mixed culture medium of the intestinal flora of mice in the above quercetin (10 μg / mL) group was added.

[0094] HQ group: 10 μL of the stock solution with a concentration of 5 mg / mL was added to a centrifuge tube, and 990 μL of the mixed culture medium of the intestinal flora of mice in the above quercetin (50 μg / mL) group was added.

[0095] Heatedgutmicrobiota group: 10 μL of stock solution with a concentration of 5 mg / mL was added to a centrifuge tube, and 990 μL of the mixed culture medium of mouse intestinal flora from the above high-temperature sterilization group was added.

[0096] Seal the centrifuge tubes with sealing film and incubate them at 37℃ for 6h, 12h, and 24h respectively. After incubation, remove the centrifuge tubes and take 100μL of sample. Immediately add 300μL of pre-cooled methanol (containing 100ng / mL glipizide). Vortex for 30s and centrifuge at 14800×g at 4℃ for 10min. Take 100μL of the supernatant and add it to a vial for LC-MS / MS analysis. Inject 1μL to determine the serine content. The concentrations of the mixed working solution standard curve for serine are as follows: 100, 200, 500, 1000, 5000, and 10000ng / mL.

[0097] 4.6 Test Results.

[0098] To determine whether quercetin regulates the gut microbiota during the development and progression of bladder cancer, we performed 16S rRNA sequencing on feces from each group of nude mice. Figure 4 The A curve in the figure shows that both the Shannon-Wiener curve and the species accumulation curve tend to flatten out, indicating that the detection can reflect most of the microbial information in the sample.

[0099] like Figure 5As shown in Figure A, compared with Group M, both LQ and HQ groups showed significant downregulation in α-diversity-related indicators, and exhibited a trend towards recovery towards Group C (P < 0.05), indicating that quercetin administration altered the gut microbiota of tumor model mice and tended to revert to normal. Lower levels of α-diversity were observed in both Group A and AQ, suggesting successful pseudo-sterile modeling and indicating that antibiotics kill gut microbes.

[0100] like Figure 5 As shown in B, principal component analysis (PCA) and principal coordinate analysis (PCoA) revealed that, in terms of β diversity, the gut microbiota of nude mice in each group showed a high degree of aggregation within the group, but a significant degree of separation between the groups.

[0101] Further analysis at the species level was conducted to assess the effects of quercetin on gut microbiota. For example... Figure 5 As shown in D, at the phylum level, the gut microbiota of nude mice is mainly composed of Firmicutes and Bacteroidota, which are the absolutely dominant strains, but significant differences also exist. Compared with group M, group LQ has a lower abundance of Bacteroidota, while Firmicutes and Proteinaceria are higher, showing a trend of recovery towards group C. Figure 5 As shown in E, a similar pullback trend was observed at the science level. This indicates that quercetin fully restores the body's healthy microbiome. For example... Figure 5 As shown in Figure C, at the genus level, the heatmap of the top 50 genera clearly illustrates the changes in the composition of the gut microbiota for each group. Notably, quercetin administration significantly enriched a variety of tumor-associated beneficial gut bacteria, including Rikenella, Gordonibacter, Candidatus Arthromitus, Eubacterium siraeum, Colidextribacter, Dubosie, Eubacterium fissicatena, Erysipelatocostridium, Clostridium innocuum, Blautia, Parasutterella, and Anaerostipes, etc. (P<0.05). Figure 4 (B in the middle).

[0102] The levels of quercetin-enriched gut probiotics Parabacteroides_sp and Closterdium_cocleatum in nude mouse feces were also investigated. Figure 5 (F, H, P < 0.001). For example... Figure 5 G in Figure 5As shown in Figure I, the conditioned medium supernatant of Parabacteroides_sp and Closterdium_cocleatum significantly inhibited the viability of BCa cells (T24, UMUC3) but had no effect on normal bladder epithelial cells (SV-HUC-1). This suggests that quercetin-enriched bacteria help inhibit BCa progression.

[0103] Given the important role of metabolites secreted by the gut microbiota in regulating disease and health, a metabolomics analysis was performed on the feces of nude mice in each group. Orthogonal partial least squares discriminant analysis (OPLS-DA) of the three groups showed significant separation of metabolites among them. Figure 6 (A) OPLS-DA was also used to create an S-spot plot to screen for differences among the three groups ( Figure 6 (B in the middle).

[0104] Next, the VIP value is used to reflect the contribution of each group's metabolite score to the inter-group differences in the OPLS-DA plot. Metabolites with VIP > 1.0 and P < 0.05 are candidate metabolites in feces. Figure 6 As shown in C, sorted by P-value, the metabolites with significant differences between group M and group LQ include: biliverdin, (1S,2R,4S)-(-)-borate acetate, methyl β-D-galactoside, N-formyl-L-methionine, 5'-dehydroadenosine, pyridoxine 5'-phosphate, 2-arachidonic acid glycerol, pyridoxine, galactitol, and L-serine. Metabolites with significant differences between group M and group C include: N-methyl-2-pyrrolidone, isovaleric acid, triethylamine, betaine aldehyde, ethylmethylacetic acid, choline, L-serine, benzaldehyde, m-cresol, and hydroquinone. Finally, we further compared the relative changes between the groups and found that the levels of 127 metabolites among all the differentially expressed metabolites showed a significant trend of reverting to group C after quercetin treatment. This indicates that quercetin treatment can reverse the dysregulation of metabolic markers in tumor model mice.

[0105] like Figure 6 D and Figure 6As shown in E, based on pathway enrichment analysis, the changes in the three main metabolic pathways were concentrated in Aminoacyl-tRNA biosynthesis, D-Amino acid metabolism, Arginine and proline metabolism, Phenylalanine, tyrosine and tryptophan biosynthesis, Alanine, aspartate and glutamate metabolism, Lysine degradation, and Glycine, serine and threonine metabolism. This indicates that amino acid metabolism processes were strongly disrupted during the development and progression of bladder cancer and during quercetin treatment.

[0106] To clarify the association between quercetin-induced intestinal bacterial metabolites in nude mice and the antitumor effect of quercetin, a correlation analysis was performed between intestinal metabolites and tumor size in nude mice. The results are as follows: Figure 6 As shown in F, (1S, 2R, 4S)-(-)-borate acetate, 5'-dehydroadenosine, pyridoxine, L-serine, and chenodeoxycholic acid were significantly positively correlated with tumor weight and volume in xenograft mouse models (P < 0.05), with L-serine showing the strongest correlation (r = -0.935). Changes in serine content in nude mice across groups showed that group M had a higher L-serine content compared to group C (P < 0.05). Furthermore, the L-serine content in feces, blood, urine, and tumors was significantly lower in groups LQ and HQ than in group M (P < 0.05). Figure 6 (G in the middle).

[0107] Based on the above research, it is known that quercetin can improve the progression of bladder tumors by regulating the gut microbiota and affecting the level of its metabolite L-serine. Therefore, a comprehensive analysis was conducted on the altered bacteria and metabolites. Figure 7 A and Figure 7 As shown in B, L-serine levels in nude mice treated with quercetin were significantly correlated with multiple gut microbiota. For example, L-serine was significantly negatively correlated with Blautia, Lachnoclostridium, Erysipelatoclostridium, Escherichia-Shigella, and Enterobacter (P < 0.05), while it was significantly positively correlated with Lachnospiraceae_NK4A136_groupg, Alistipes, and Lactobacillus (P < 0.05).

[0108] like Figure 7 As shown in Figure C, to investigate the effect of quercetin on L-serine levels in vitro, we conducted an in vitro incubation experiment with quercetin and gut microbiota. The L-serine concentration in the Con group gradually decreased with increasing time. Meanwhile, compared to the Con group, the L-serine concentration in the Heated gut microbiota group (without quercetin) remained essentially unchanged at the same incubation time, indicating that L-serine can be synthesized and metabolized by the gut microbiota itself. At the same incubation time, the L-serine concentrations in the LQ and HQ groups were lower, and the decrease was more pronounced with increasing quercetin concentration. When the quercetin concentration reached 50 μg / mL, the L-serine levels at each incubation time point were significantly lower than those in the Con group (10.54%, 27.86%, and 40.26%, P < 0.05). As a non-essential amino acid, L-serine can be taken up from the extracellular environment.

[0109] Cancer cells voraciously consume serine and depend on exogenous serine for optimal growth. This study utilizes a custom-designed culture medium (lacking serine / glycine) to investigate the dependence of tumor cell lines on exogenous serine. Figure 7 As shown in Figure D, in the three bladder tumor cell lines (T24, UMUC3, and EJ), the growth rate of cells in serine / glycine-deficient medium was significantly lower than that in complete medium (P < 0.05). To further clarify the reason for the L-serine-targeted anti-bladder tumor cell line proliferation, the expression of function-related proteins was also examined. Figure 7 As shown in Figure E, in the T24 and UMUC3 cell lines, the expression of proliferation-related protein PCNA and cell cycle-related protein Cyclin D1 was significantly downregulated in the -S / G group compared to the Con group, while the expression of apoptosis-related proteins (cCaspase-3 and cParp) was significantly upregulated. This indicates that a decrease in L-serine levels can inhibit the proliferation of bladder tumor cells.

[0110] Based on the above results, an animal model was used to modify the univariate L-serine to verify its anti-tumor effect.

[0111] Group M: The model group in "2. Establishment of a human bladder tumor xenograft model in nude mice", fed normally;

[0112] -S / G group: The model group in "2. Establishment of a human bladder tumor xenograft model in nude mice", fed a diet lacking serine / glycine.

[0113] like Figure 7 F- Figure 7As shown in Figure H, after 4 weeks, the tumor volume in the serine / glycine-deficient diet group was significantly smaller than that in the normal diet group, with a tumor inhibition rate of 32.58% (P < 0.05). The tumor inhibition effect was worse than that of direct oral quercetin, indicating that quercetin treatment of bladder cancer depends on changes in L-serine metabolism induced by gut microbiota.

[0114] 5. Library construction and sequencing.

[0115] RNA was extracted from tumor tissues of nude mice in groups M and LQ in step 2. After all samples passed the tests, 1.5 μg of RNA from each group was sent to Beijing Aovisen Gene Technology Co., Ltd. for library construction and sequencing.

[0116] Library construction was performed using the accompanying kit: mRNA was enriched using magnetic beads with Oligo(dT) inclusions. Subsequently, fragmentation buffer was added to break the mRNA into short fragments. One-stranded cDNA was synthesized using reverse transcription with mRNA as a template via hexabase random primers. Then, buffer, dNTPs, and DNA polymerase I were added to synthesize two-stranded cDNA. The double-stranded cDNA was then purified using AMPure XP beads. End repair, A-addition, and adapter addition were performed on the purified double-stranded cDNA. Fragment size selection (200-250 bp) was achieved using AMPure XP beads, and PCR amplification was performed to construct the cDNA library. After the library passed quality control, pooling was performed according to the required effective concentration and desired data volume. Finally, sequencing was performed using a Novaseq 6000 with a PE 15 sequencing strategy.

[0117] Several cancer subtypes overactivate glycolytic anabolic metabolism, leading to serine and glycine synthesis side branches and resulting in addiction to these substances. Figure 8 As shown in A, the synthesis of serine consists of two processes: the synthesis of serine from glucose and the reversible interconversion of serine into glycine. De novo serine synthesis is a branch of glycolysis, where the glycolytic intermediate 3-phosphoglycerate (3-PG) is converted into serine through three consecutive enzymatic reactions catalyzed by phosphoglycerate dehydrogenase (PHGDH), phosphoserine transaminase 1 (PSAT1), and phosphoserine phosphatase (PSPH).

[0118] Transcriptome sequencing was performed on tumor tissue from nude mice to analyze the molecular-level regulation of serine synthesis by quercetin. The results showed that 1965 genes were upregulated and 2211 genes were downregulated. Figure 8(B and C in the text). There was no significant difference in the expression of PHGDH and PSAT1 in the tumor tissues of the M and LQ groups, but PSPH expression was significantly downregulated after quercetin administration compared to the M group (P < 0.05). Figure 8 (D in the text). Simultaneously, PSPH protein expression yielded the same result (…). Figure 8 E in the text). Enrichment analysis showed that differentially expressed genes, in addition to focusing on tumor-related molecular pathways, were also associated with multiple amino acid metabolic pathways (E in the text). Figure 8 (H in the text).

[0119] Since elevated PSPH expression is directly associated with bladder tumor progression, PSPH siRNA transfection into UMUC3 and T24 cells showed a significant knockdown efficiency (P < 0.05). Figure 8 In addition to the F in the middle, PSPH protein was also downregulated (P < 0.05). Figure 8 (G in the middle). Figure 8 The I-value in the study showed that downregulation of PSPH expression led to changes in functional proteins, including upregulation of cPARP and Ccasp-3, and downregulation of Cyclin D1 and PCNA, confirming the correlation between PSPH expression and tumor progression.

[0120] Example 2

[0121] Pharmacodynamic study and grouping scheme of fecal microbiota transplantation in nude mice with xenograft tumors.

[0122] Female BALB / c nude mice (4 weeks old, 25 mice) were acclimatized to their environment for 3 days with standard diet and then randomly divided into four groups: donor group (D group), model group (M group), low-dose quercetin administration group (LQ group), fecal microbiota transplantation group (FMT group), and blank control group (C group), with n=5 in each group. Groups D and LQ were treated with oral quercetin at a dose of 100 mg / kg / day, while groups M and C were treated with an equal volume of aqueous solution orally.

[0123] Starting from week 0, a human bladder tumor xenograft model was established in group D using the same method as step 2 in Example 1, while simultaneously starting oral quercetin treatment (100 mg / kg / day). Based on the above results, approximately two weeks after quercetin administration, a difference in tumor size appeared between the model group and the quercetin-treated group. Therefore, starting two weeks of quercetin treatment in group D, fresh feces were collected weekly. Simultaneously, a pseudo-sterile model was established in the FMT group starting from week 2.

[0124] A pseudo-sterile model was established following methods from previous literature (Polymorphisms in drug-metabolizing genes and urinary bladder cancer susceptibility and prognosis: Possible impacts and future management). Cefoxitin, metronidazole, gentamicin, and vancomycin (100 mg / kg) were mixed and dissolved in PBS, and administered to nude mice in the FMT group for a short-term pseudo-sterile model for 6 days to rapidly deplete the intestinal microbiota. Clindamycin hydrochloride (33.3 mg / kg) was administered on day 7.

[0125] Meanwhile, in week 3, human bladder tumor xenograft models were established in nude mice in groups M, LQ and FMT, and corresponding drug treatments were initiated.

[0126] After establishing the pseudo-sterile model, fresh feces were collected from group D and mixed in PBS at a ratio of 1:5 [weight (g): volume (mL)]. The mixture was centrifuged at 800 rpm for 3 min at 4°C, and the particulate matter was discarded. The supernatant was then administered to the FMT group via gavage. The FMT group received treatment at 48-hour intervals, with a treatment cycle of 4 weeks.

[0127] To confirm the direct effect of quercetin-altered gut microbiota and its metabolites on bladder cancer progression, a fecal microbiota transplantation experiment was conducted. Fecal samples from quercetin-treated xenograft nude mice were administered via gavage to sterile xenograft nude mice. After 4 weeks of treatment, the nude mice were examined. Figure 9 (A) Ultimately, it can be clearly seen that the tumors in the FMT group were smaller than those in the M group, but larger than those in the LQ group, with a tumor inhibition rate of 26.16% (P < 0.05). Figure 9 B- Figure 9 (D in the text). Further testing was conducted to examine the consistency of microbial community changes among the groups before and after fecal microbiota transplantation. Similar to the LQ group, the α-diversity in the FMT group was lower than that in the M group (P < 0.05). Figure 9 (E in the text). Furthermore, the β-diversity of the gut microbiota in nude mice treated with FMT also showed high inter-group differentiation and high intra-group aggregation. The LQ group and the FMT group showed significant overlap, indicating a high degree of similarity in their gut microbiota composition. Figure 9The gut microbiota classification and composition of each group of nude mice were further analyzed. The results indicated that at the phylum level, the changes in the FMT group were more consistent with those in the LQ group. Compared to the M group, FMT showed a decrease in Bacteroidota and Verrucomicrobiota, with a more pronounced decrease in Bacteroidota. Simultaneously, the abundance of Fimicutes increased, and the Fimicutes / Bacteroidota ratio was upregulated (F). Figure 9 Although no changes in L-serine levels were detected in urine between the FMT and M groups, we could clearly observe lower levels of serine in the LQ and FMT groups compared to the M group in feces, blood, and tumors (P < 0.05). Figure 9 (H in the text). Therefore, the gut microbiota altered by quercetin can directly exert an anti-tumor effect.

[0128] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of quercetin in the preparation of drugs for regulating intestinal microbial dysbiosis caused by bladder cancer.

2. The application according to claim 1, characterized in that, The quercetin enriches and inhibits tumor-associated beneficial gut bacteria.

3. The application according to claim 2, characterized in that, The beneficial gut bacteria include Rickenella, Gordonibacter, Candidatus Arthromitus, Eubacterium siraeum, Coridextribacter, Dubosie, Eubacterium fissicatena, Erysipelatocostridium, Clostridium innocuum, Blautia, Parabacteroides, Parasutterella, and Anaerostipes.

4. The application according to claim 2 or 3, characterized in that, The beneficial bacteria in the gut inhibit the activity of bladder cancer cells.

5. The application according to claim 4, characterized in that, The bladder cancer cells include T24 cells and UMUC3 cells.

6. The application according to claim 1, characterized in that, Quercetin inhibits the level of L-serine, a metabolite of gut microbiota.

7. The application according to claim 1, characterized in that, Quercetin inhibits the expression of the PSPH gene and the PSPH protein.

8. The application of quercetin in the preparation of drugs for the prevention and treatment of bladder cancer, characterized in that, Quercetin was administered to a nude mouse xenograft model of human bladder tumors. After administration, feces were collected, and microorganisms were extracted from the feces. These microorganisms were used to prepare drugs for the treatment of bladder cancer.

9. The application according to claim 8, characterized in that, The steps for extracting microorganisms from feces include: mixing feces with PBS at a ratio of 1 g: 5 mL, centrifuging, and collecting the supernatant.

10. The application according to claim 9, characterized in that, The centrifugation speed is 800 rpm, and the centrifugation time is 3 minutes.

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