Application of Prevotella copri and / or its metabolite Cholylcitrulline in the preparation of drugs for treating colorectal cancer

Abstract

This invention belongs to the field of pharmaceutical technology, and particularly relates to the application of Prevotella copri and / or its metabolite Cholylcitrulline in the preparation of a drug for treating colorectal cancer. The drug comprises Prevotella copri and / or its metabolite Cholylcitrulline. The Prevotella copri and Cholylcitrulline of this invention exert a comprehensive anti-cancer effect by remodeling the host intestinal barrier, regulating the expression of tumor-related gene mRNA and proteins, and inhibiting the PI3K / AKT / mTOR signaling pathway, thereby blocking the occurrence and development of colorectal cancer, and without significant toxic side effects on organs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the use of Prevotella copri and / or its metabolite Cholylcitrulline in the preparation of drugs for treating colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors in clinical practice, ranking among the top in incidence and mortality among digestive system tumors. Currently, the conventional treatments for CRC mainly consist of surgical resection, traditional chemotherapy, targeted therapy, and emerging immune checkpoint inhibitors. Regarding drug therapy, existing drugs for treating CRC all have insurmountable shortcomings in clinical application. Traditional chemotherapy drugs such as oxaliplatin, 5-fluorouracil, and irinotecan kill tumor cells by interfering with DNA replication or disrupting cell division. However, chemotherapy drugs lack selectivity and have significant systemic toxicity. Clinically, they often cause severe bone marrow suppression, severe gastrointestinal reactions, peripheral neurotoxicity, and liver and kidney damage. While molecularly targeted drugs such as bevacizumab and cetuximab improve targeting to some extent, they are not only expensive but also easily lead to drug resistance. More seriously, targeted drugs exhibit strong cumulative characteristics when used in the body for a long time or at high doses. This cumulative effect can easily lead to hypertension, proteinuria, arterial thromboembolism, gastrointestinal perforation, and severe skin toxicity, severely limiting its clinical dosage and treatment duration. PD-1 / PD-L1 antibody immune checkpoint inhibitors fight tumors by activating the host's own immune system, but are often only effective in specific mutation populations with mismatch repair defects or high microsatellite instability. Furthermore, they easily induce immune-related adverse reactions, such as immune-related colitis and pneumonia, and may even lead to fatal immune storms. Due to the serious shortcomings of existing colorectal cancer treatments, such as "high toxicity," "strong accumulation in the body," and "high susceptibility to drug resistance," there is an urgent need to find a novel drug that can effectively inhibit colorectal cancer proliferation or enhance the efficacy of conventional therapies, while also being safe, non-toxic, and without harmful accumulation in the body.

[0003] Recent studies in gut microbiota have demonstrated that the gut symbiotic microbiota plays a crucial role in host immune regulation, metabolic homeostasis, and tumor microenvironment remodeling. Utilizing natural human symbiotic bacteria as in vivo biopharmaceuticals has become a cutting-edge direction in modern oncology research. Addressing the significant drawbacks of existing technologies, such as the high toxicity and accumulation of chemically synthesized and targeted drugs, this application aims to screen endogenous probiotic strains with antitumor activity from the perspective of the human natural symbiotic microbiota. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing traditional chemotherapy drugs, molecularly targeted drugs and other anti-colorectal cancer drugs, such as large systemic toxic side effects, strong accumulation in the body, easy development of drug resistance and poor clinical tolerability, and to provide an application of Prevotella copri and its metabolite Cholylcitrulline in the preparation of drugs for the treatment or adjuvant treatment of colorectal cancer.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The application of *Prevotella copri* and / or its metabolite Cholylcitrulline in the preparation of drugs for treating colorectal cancer; *Prevotella copri*, as one of the highly enriched natural core symbiotic strains in the human gut, exhibits extremely high evolutionary adaptability and biocompatibility with the host. Experiments have confirmed that the live bacteria and their metabolites prepared in this application exert anti-tumor effects, improve the intestinal microenvironment, and alleviate chemotherapy-induced colitis, while causing no toxic damage to the host's organs. The strain of this application circulates in vivo through natural intestinal colonization, metabolism, and excretion. Unlike small chemical molecules or monoclonal antibodies, it does not produce pathological drug accumulation in the liver, kidneys, or peripheral tissues, thus completely avoiding delayed organ failure or toxic reactions caused by drug accumulation, exhibiting extremely high safety and providing a novel green strategy for long-term safe treatment and clinical adjuvant enhancement of colorectal cancer. Cholylcitrulline, as a well-defined monomeric metabolite molecule, can be directly developed into an innovative small molecule drug, bypassing the complexity of live bacteria drug development. This application not only discovers the potential of Prevotellacopri for the preparation of anti-colorectal cancer drugs, but also precisely identifies a microecological bile acid derivative (Cholylcitrulline) with great drug potential, providing a new approach for non-genotoxic targeted therapy and immunoadjuvant therapy for colorectal cancer.

[0006] Furthermore, the metabolite Cholylcitrulline is a bile acid derivative, cholylcitrulline. Traditionally, it was believed that primary bile acids synthesized in the liver could only react with glycine or taurine. However, recent studies have shown that gut microbiota (especially specific species such as *Prevotella*) have been shown to possess the ability to catalytically couple bile acids with non-classical amino acids such as citrulline and phenylalanine, thereby generating structurally diverse novel MCBAs, with Cholylcitrulline being a representative molecule. Mechanistically, this molecule can regulate the homeostasis of the gut and tumor microenvironment through paracrine pathways, thus providing a potential biological basis for its application in adjuvant therapy for colorectal cancer.

[0007] Furthermore, the drug comprises *Prevotella copri* and / or its metabolite *Cholylcitrulline*; the main active ingredient of the drug of this invention is *Prevotella copri*, a strain of endogenous commensal bacteria in the human gut. The strain is derived from a universal standard strain preserved in a depository, or isolated and purified from fecal samples of healthy individuals. This invention preferentially uses *Prevotella copri* strains with stable biological characteristics, strong polysaccharide degradation ability, and high anti-inflammatory activity.

[0008] Furthermore, the effective concentration of live Prevotella bacteria in the drug is 1×10⁻⁶. 6 CFU / ml ~ 1×10 14 CFU / ml; the effective dosage of cholylcitrulline is 0.1 mg / d to 5000 mg / d, with a preferred range of 10 mg / d to 1000 mg / d; since Prevotella copri is a strictly obligate anaerobic bacterium, its entire preparation process must be carried out in an anaerobic or hypoxic environment (oxygen concentration <0.1%), or by adding a reducing agent to maintain a low redox potential. The preparation of Prevotella copri first requires the recovery and anaerobic propagation of the seed culture, followed by high-density large-scale anaerobic fermentation, and finally anaerobic harvesting and concentration of the bacterial cells.

[0009] The advantages of this invention are: 1. The Prevotella strain of this application, as a live biological drug, participates in the natural colonization, metabolism and excretion cycle of the host microecology in the intestine; the bacteria and its metabolites do not produce pathological harmful accumulation in the liver, kidneys or peripheral solid organs, thereby fundamentally avoiding delayed toxic reactions caused by drug accumulation, and are suitable for long-term maintenance treatment of colorectal cancer patients. 2. The active ingredients of this invention are derived from natural symbiotic bacteria in the human gut and their derivatives produced in normal physiological metabolic pathways. Compared with traditional cytotoxic chemotherapy drugs, Prevotella copri and Cholylcitrulline exert a strong anti-cancer effect without causing serious non-specific damage to normal cells. Their extremely high biocompatibility and safety make them suitable not only for the acute phase treatment of colorectal cancer, but also for long-term maintenance therapy and recurrence prevention management in the prognostic stage, significantly improving the quality of life of patients. 3. The Prevotella copri and Cholylcitrulline of the present invention exert a comprehensive anti-cancer effect by remodeling the host intestinal barrier, regulating the expression of tumor-related gene mRNA and protein, and inhibiting the PI3K / AKT / mTOR signaling pathway, thereby blocking the occurrence and development of colorectal cancer, and have no obvious toxic side effects on organs. Attached Figure Description

[0010] Figure 1 This is a graph showing the effect of Prevotella copri on the body weight of a CRC xenograft mouse model (n=10).

[0011] Figure 2 This is a graph showing the effect of Prevotella copri on tumor weight in a mouse model of CRC xenograft tumors (n=10).

[0012] Figure 3 This is a graph showing the effect of Prevotella copri on tumor volume in a mouse model of CRC xenograft tumors (n=4, 10).

[0013] Figure 4 This is a graph showing the effect of Prevotella copri on organ indices in a mouse model of CRC xenograft tumors (n=10).

[0014] Figure 5 This is a graph showing the effect of Prevotella copri on gene mRNA expression in tumor-bearing tissues of CRC xenograft mice (n=4).

[0015] Figure 6 This is a graph showing the effect of Prevotella copri on the expression of ZO-1 and p53 proteins in CRC xenograft mice (n=3).

[0016] Figure 7 This is a graph showing the effect of Prevotella copri on the expression of PI3K, AKT, and mTOR proteins in CRC xenograft mice (n=3).

[0017] Figure 8 This is a graph showing the effect of Prevotella copris on the proliferation of HCT116 cells.

[0018] Figure 9 This is a graph showing the effect of Prevotella copris on the migration rate of HCT116 cells (10×, n=4).

[0019] Figure 10 This is a graph showing the effect of Prevotella copris on the clonogenic ability of HCT116 cells (n=4).

[0020] Figure 11 This is a graph showing the effect of Prevotella copris on protein expression in HCT116 cells (150×, scale bar length = 200 μm).

[0021] Figure 12It is a result graph of metabolites of Prevotella copri detected by untargeted metabolomics.

[0022] Figure 13 It is a graph showing the effect of Cholylcitrulline on intestinal tracts of AOM / DSS colorectal cancer mice.

[0023] Figure 14 It is a graph showing the effect of Cholylcitrulline on gene mRNA expression in colorectal tissues of AOM / DSS colorectal cancer mice (n=4). Detailed Description of the Embodiments

[0024] This study found that Prevotella copri and Cholylcitrulline can inhibit tumor growth in colorectal cancer tumor-bearing mice and the malignant phenotype of HCT116 cells. In terms of mechanism, the bacterium exerts an anti-tumor effect by regulating the mRNA and protein expression of tumor-related genes, improving the intestinal mucosal barrier, and inhibiting the PI3K / AKT / mTOR signaling pathway, and has no obvious toxic and side effects on organs.

[0025] Efficacy Experiment The metabolite Cholylcitrulline of Prevotella copri has been verified through multiple models to inhibit the malignant phenotype of colorectal tumors. Prevotella copri is preserved in Beina Chuanglian Biotechnology Co., Ltd., with the preservation number BNCC337399.

[0026] 1. Experimental Materials 1.1 Experimental Animals Forty SPF-grade BALB / c-nu male mice, 5 weeks old, weighing 18-20 g, were divided into 4 groups with 10 mice in each group. The mice were purchased from Beijing Speifu Biotechnology Co., Ltd. (license number: SCXK (Jing) 2024-0001), and the experimental unit use license number is SYXK (Yu) 2021-0015. This experimental protocol was reviewed and approved by the Animal Experimental Ethics Committee of Henan University of Chinese Medicine, and was permitted to be implemented (approval number of experimental animal ethical review: IACUC-S202403135).

[0027] Forty-eight SPF-grade C57BL / 6J male mice, 8 weeks old, weighing 19-22 g, were divided into 4 groups with 12 mice in each group. The mice were purchased from Shandong Jinan Pengyue Experimental Animal Breeding Co., Ltd. (license number: SCXK (Lu) 2022006), and the experimental unit use license number is SYXK (Yu) 2021-0015. This experimental protocol was reviewed and approved by the Animal Experimental Ethics Committee of Henan University of Chinese Medicine, and was permitted to be implemented (approval number of experimental animal ethical review: IACUC-202405027).

[0028] 1.2 Experimental Cells HCT116 colorectal tumor cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0029] 2. Experimental Methods 2.1 Construction of a mouse model of colorectal cancer xenograft and drug treatment After 1 week of acclimatization, HCT116 cells in the logarithmic growth phase were harvested, washed with PBS and digested with trypsin, then resuspended in serum-free RPMI 1640 medium, and the cell density was adjusted to 3 × 10⁻⁶ cells / year. 6 –5×10 6 100 μL / bacterium. Subcutaneous inoculation was performed under the right axilla of mice. BALB / c nude mice were randomly divided into 4 groups: control group, bacterial group (Support of P. copri, 2 × 10⁻⁶), and bacterial group (Support of P. copri, 2 × 10⁻⁶). 6 CFU, PI3K inhibitor group (Dactolisib, 1 mg / kg) and combination therapy group (Combine, 2×10⁻⁶) 6 CFU + 1 mg / kg). All groups received the drug every other day. Except for the control group, all other groups received the corresponding drug via intratumoral injection, while the control group received sterile culture medium via intratumoral injection. During the administration period, the long diameter (L) and short diameter (W) of the tumor were measured periodically, calculated using the formula V = (L × W). 2 ) / 2 Calculate tumor volume and plot growth curve. After the last administration, mice were fasted for 12 h, sacrificed, and subcutaneous tumors were dissected, photographed, weighed, and then stored at -80℃.

[0030] 2.2 Group sampling and determination of tumor volume and weight After cervical vertebrae were removed from mice, subcutaneous tumors were dissected. Four samples were selected from each group, photographed on a blue background, and weighed. The samples were then placed in cryovials and stored at -80°C for subsequent indicator detection.

[0031] 2.3 qRT-PCR detection of the effects of Prevotella copri on gene mRNA in HCT116 cell xenograft mouse model and HCT116 colorectal cancer cells Total RNA extraction and reverse transcription: 10–20 mg of colorectal tissue or cell samples were homogenized at low temperature in RL buffer. Total RNA was extracted strictly according to the kit instructions, and its concentration and purity (A260 / A280 ratio) were determined using a micro spectrophotometer. After passing the determination, the RNA was stored at -80℃. Subsequently, the RNA was reverse transcribed into cDNA according to the instructions. Sequences of genes such as GAPDH, p53, and ZO-1 were retrieved using NCBI, and primers were synthesized by a professional institution. Primer information is detailed in Table 1. The qPCR reaction program was set as follows: 95℃ for 15 s, 60℃ for 30 s, for a total of 40 cycles. GAPDH was used as an internal control gene, and the CT values ​​of each group were recorded. -∆∆CT The relative expression level of the target gene mRNA can be calculated.

[0032] Table 1 Primer Sequences Gene name Primer sequence Human-GAPDH-A 5'-TGATGACCCTTTTGGCTCCC-3' Human-GAPDH-S 5'-GGAAGCTTGTCATCAATGGAAATC-3' Human-p53-A 5'-CTTCAGGTGGCTGGAGTGAG-3' Human-p53-S 5'-CCTCTCCCCAGCCAAAGAAG-3' Human-ZO-1-F 5'-CTGGGCTCTTGGCTTGCTATTCG-3' Human-ZO-1-R 5'-TCTCCAGAAGTCAGCACGGTCTC-3' Human-PI3K-F 5'-GATGCCCTTCTGAACTGGCT-3' Human-PI3K1-R 5'-GCTACACAGTAGCCAGCACA-3' Human-AKT-F 5'-TACGAGATGATGTGCGGTCG-3' Human-AKT-R 5'-GGAAGCGGATCTCCTCCATG-3' Human-mTOR-F 5'-CGATGGCCAGGGATCTCTTC-3' mTOR-R 5'-GGTCTGTGTGACTTCAGCGA-3' 2.4 Effect of IF assay on protein expression in HCT116 cell xenograft mouse model and HCT116 cells Immunofluorescence of tissue: Paraffin sections of 5 μm colorectal tissue were dewaxed and rehydrated, then placed in sodium citrate buffer for heat-induced antigen retrieval. 3% H2O2 was added to block endogenous peroxidase, followed by blocking with 5% BSA at room temperature in the dark for 1 h. Primary antibody (incubated overnight at 4°C) and fluorescently labeled secondary antibody (incubated at room temperature in the dark for 1 h) were added sequentially, with washing three times with PBS between each step. Finally, cell nuclei were counterstained with DAPI for 10 min, and the sections were mounted with anti-fluorescence quenching mounting medium. Images were acquired using a fluorescence microscope to observe the spatial distribution and expression levels of proteins in the tissue.

[0033] Cell immunofluorescence: HCT116 cells (5 × 10⁻⁶) were subjected to immunofluorescence. 4 The target protein (proteins / mL) was seeded into pre-prepared 6-well plates. After 24 h of drug intervention, the plates were fixed with 4% paraformaldehyde (15 min) and permeabilized with 1% Triton X-100 (20 min). After blocking with 5% BSA for 30 min, antibody incubation and DAPI counterstaining were performed according to the above procedure. After washing, the plates were inverted and mounted on glass slides. The subcellular localization and fluorescence intensity differences of the target protein were observed using laser confocal microscopy.

[0034] 2.5 CCK-8 assay for cell proliferation Set up control wells, drug delivery wells, and blank wells for the CCK-8 assay. After the bacterial culture supernatant stock solution was used up, perform serial dilutions at concentration gradients of 0 × 10⁻⁶. 3 CFU, 50×10 3 CFU, 100×103 CFU, 200×10 3 CFU, 500×10 3 CFU, 1000×10 3 CFU, 2000×10 3 CFU, 5000×10 3 CFU and control groups were prepared with complete culture containing 1% DMSO. After 24 h of culture, the old culture medium was discarded, and each well was washed with 100 μL of PBS buffer. 100 μL of the drug was then added to each well at the concentration described above, and the wells were incubated for another 24 h. Following the CCK-8 kit instructions, the solution was brought to room temperature and mixed with the complete culture at a 10% volume ratio to prepare the working solution. The drug-containing culture medium in the culture plate was discarded, and the plate was washed with PBS to remove any residue. 100 μL of 10% CCK-8 working solution was gently added to each well, and the plate was incubated at 37 °C. The OD values ​​at 450 nm were read at 1 h and 2 h of incubation using a microplate reader, and the cell viability of each well was calculated.

[0035] Cell viability = [(OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells)] × 100%.

[0036] 2.6 Effects of Prevotella copri on CRC cell migration ability Logarithmic growth phase CRC cells HCT116 were digested with trypsin, centrifuged, resuspended in complete culture, and the cell density was adjusted to 1×10⁶. 6 At a rate of 0.5 mL / well, cells were seeded into 24-well plates. A control group and a Prevotella copri group (5 × 10⁶ cells / well) were also established. 4 CFU), positive control group (Dactolisib, 3 μM), Prevotella copri + positive control group (5×10⁻⁶) 4 (CFU + 3 μM), with 4 replicates per group. After plating, the plates were placed in a CO2 incubator. Using a sterile steel ruler, a cross was made at the bottom of each well using a 10 μL pipette tip. Exfoliated cells were washed with PBS, and the medium was replaced with 2% FBS containing the drug. Images of the same field of view were taken under an inverted microscope at 0 h, 12 h, and 24 h after drug administration, ensuring that the shooting position and magnification were strictly consistent.

[0037] Image data was statistically analyzed using ImageJ software, and migration rate was calculated using the formula: Cell migration rate (%) = (W 0 h -W t ) / W 0 h ×100% (W) t The area of ​​the scratch at time t (t = 12 / 24 h) represents the area of ​​the scratch at time t.

[0038] 2.7 Effect of Prevotella copri on CRC cell clonogenic ability Logarithmic growth phase CRC cells HCT116 were digested with trypsin, centrifuged, resuspended in complete culture, and the cell density was adjusted to 1×10⁶. 3 Cells were seeded at a rate of 1 mL / well in 6-well plates. Control, Prevotella copri, Dactolisib (3 μM), and Prevotella copri + positive control groups were set up, with 4 replicates per group. After seeding, the plates were incubated in a CO2 incubator. Once cells adhered, the corresponding drug-containing medium was added to each group, and the intervention was carried out in the incubator for 14 days, with the drug-containing medium being changed every 3 days. After culture, the drug-containing medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde solution for 20 min, the fixative was discarded, and the cells were washed three times with PBS. The cells were then stained with crystal violet solution at a volume of 500 μL / well for 30 min. After staining, the staining solution was discarded, the cells were washed three times with PBS, and the plates were drained. The plates were photographed on a white background, ensuring consistent field of view for each well. Images from all groups were compiled, and ImageJ software was used to analyze and count the number of clones.

[0039] Clonal formation rate (%) = Number of clones formed / Number of inoculated cells × 100%.

[0040] 2.8 Non-targeted metabolomics detection of major metabolites in Prevotella copri Take the sterile culture supernatant and the Prevotella copri bacterial culture supernatant, add 500 μL of a methanol:acetonitrile:water mixture (2:2:1), mix thoroughly, sonicate in ice water for 20 min, then freeze at -20℃ for 60 min. Centrifuge at 17000 g for 15 min at 4℃, collect the supernatant, and vacuum centrifuge at 30℃ to dry. Add 200 μL of 50% methanol to the sample and vortex for 60 s to dissolve completely. Centrifuge at 17000 g for 15 min at 4℃, and collect 60 μL of the supernatant into an inner tube. Mix 10 μL of each sample as the quality control sample for instrumental testing.

[0041] The LC-MS / MS mass spectrometer parameters were set as follows: mobile phase conditions: column temperature 40℃, sample loading volume 5 μL; positive ion mode: A: 0.1% formic acid; B: 0.1% formic acid acetonitrile; negative ion mode: A: water (containing 2 mM ammonium acetate); B: acetonitrile. An ABX500R Triple TOF mass spectrometer was used in this study. The primary mass spectrometry acquisition range was set to 50-1200 Da, and the bombardment energy was stabilized at 30 eV. Ten secondary spectra were recorded every 50 ms. ESI ion source parameters were: nebulizer pressure set to 60 Psi, auxiliary gas pressure set to 60 Psi, curtain gas pressure maintained at 35 Psi, and ion source temperature set to 650 ℃. The spray voltage was 5000 V in positive ion mode and -4000 V in negative ion mode.

[0042] Data was converted to .abf format using Analysis Base File Converter. Peak values ​​were detected and aligned using MSDIAL 4.24 software, and ion peaks with a missing value >50% within a group were removed. Simultaneously, based on primary and secondary spectral searches, the databases of Metalin, Mass Bank, MoNA, and HMDB were independently integrated to obtain identification results. Normalization was performed using TIC ion summation, and differential and enrichment analyses were conducted using Metabo Analyst software.

[0043] 2.9 Construction of AOM / DSS-induced colorectal cancer mouse model and drug intervention After 1 week of acclimatization, C57BL / 6J mice were randomly divided into four groups according to body weight: a blank control group, an AOM / DSS model group, a choylcitrulline intervention group (20 mg / kg), and a PI3K inhibitor group (dactolisib, 1 mg / kg). Except for the blank control group, which received an intraperitoneal injection of an equal volume of saline, all other groups received a single intraperitoneal injection of AOM (12.5 mg / kg). One week later, the DSS cycle model was initiated: free access to 2.5% DSS aqueous solution for 1 week, followed by alternating feeding with sterile water for 2 weeks, constituting one cycle, repeated three times. From the first administration of DSS, each treatment group received the corresponding drug via gavage, while the blank control and model groups received an equal volume of saline via gavage, once every other day, until the experimental endpoint. After the last administration, the mice were fasted but allowed free access to water for 12 hours and euthanized by cervical dislocation. Dissection was performed to observe the occurrence of colorectal tumors, and lesion tissue samples were collected.

[0044] 3. Experimental Results 3.1 Effect of Prevotella copri on body weight in a mouse model of CRC xenograft Changes in body weight of mice in each group during the experiment are shown in the figure. Figure 1Compared with the model group, the body weight of mice in each treatment group showed a significant increasing trend (p<0.01). This indicates that Prevotella copri can significantly improve the weight loss induced by transplanted tumors in mice. The experimental results suggest that Prevotella copri may effectively reduce tumor-induced weight loss by optimizing intestinal microecological homeostasis and repairing the intestinal mucosal barrier. This experiment aims to explore the systemic protective role of specific intestinal symbiotic bacteria in alleviating tumor cachexia, thereby demonstrating that Prevotella copri is an effective strategy to reverse body consumption and improve the survival status and quality of life of tumor-bearing hosts.

[0045] 3.2 Effect of Prevotella copri on tumor weight in a mouse model of CRC xenograft The tumor weight of each group of mice was determined by Figure 2 The results showed that, compared with the model group, the tumor weight of mice in each treatment group was significantly reduced (p < 0.01). This suggests that Prevotella copri has an inhibitory effect on tumor growth. Prevotella copri inhibits tumor growth by suppressing tumor cell proliferation and thus reducing tumor weight. This experiment establishes the direct antitumor efficacy of Prevotella copri at the macroscopic phenotypic level, providing crucial in vivo pharmacodynamic evidence for the development of antitumor microecological preparations based on Prevotella copri.

[0046] 3.3 Effect of Prevotella copri on tumor volume in a mouse model of CRC xenograft Tumor volume in each group of mice increased from Figure 3 The results showed that, compared with the model group, the tumor volume in the Prevotella copri group was significantly reduced (p < 0.01). This suggests that Prevotella copri has an inhibitory effect on tumor growth. Prevotella copri may effectively inhibit tumor growth in vivo by targeting and interfering with the tumor microenvironment and the proliferation and energy metabolism network within cancer cells through specific metabolites. This experiment aimed to visually evaluate the anti-tumor potential of Prevotella copri at the whole animal level, providing solid in vivo evidence for its development into an anti-tumor microecological agent.

[0047] 3.4 Effects of Prevotella copri on organ indices in a mouse model of CRC xenograft The organ indices of each group were from Figure 4As can be seen, there were no significant differences in organ indices among the drug-treated groups compared to the model group (p>0.05). Therefore, it can be concluded that CRC xenografts have no effect on the organ status of mice. Prevotella copri effectively exerted its tumor-suppressing effect without causing toxic damage or abnormal proliferation to vital organs in mice, demonstrating good biocompatibility. This experiment aims to systematically evaluate the overall toxicological risk of this intervention, providing necessary safety evidence for its clinical or pharmaceutical translation.

[0048] 3.5 qRT-PCR detection of the effect of Prevotella copri on gene mRNA in tumor-bearing tissues of a mouse model of CRC xenograft The expression of tumor-related genes p53, intestinal barrier gene ZO-1, and mRNAs related to the PI3K / AKT / mTOR signaling pathway in CRC xenograft mouse tumor tissues was detected using qRT-PCR. The results are as follows: Figure 5 As shown, compared with the model group, the expression levels of p53 and ZO-1 gene mRNA in tumor tissues of CRC xenograft mice were significantly increased after Prevotella copri intervention, while the expression levels of PI3K, AKT, and mTOR gene mRNA were significantly inhibited (p<0.01). Prevotella copri exerts its anti-tumor effect by activating the tumor suppressor gene p53, restoring the expression of intestinal mucosal barrier-related genes, and blocking the overactivated PI3K / AKT / mTOR signaling pathway. This experiment aims to deeply analyze the intrinsic molecular mechanism by which this strain inhibits tumor growth at the transcriptional level, providing crucial genomic evidence for elucidating its target as an anti-tumor microecological agent.

[0049] 3.6 IF detection of the effect of Prevotella copri on tumor-related proteins in a mouse model of CRC xenograft The p53 and ZO-1 proteins in CRC xenograft mouse tumor tissues were detected using IF assay, and the results are as follows: Figure 6 As shown, compared with the model group, the expression levels of p53 and ZO-1 proteins in the tumor tissue of CRC xenograft mice were significantly increased after Prevotella copri intervention.

[0050] To further explore the molecular mechanism of Prevotella copri intervention in colorectal cancer, IF was used to detect PI3K, AKT, and mTOR proteins in tumor tissues of CRC xenograft mice. The results are as follows: Figure 7As shown, compared with the model group, the expression levels of PI3K, AKT, and mTOR proteins in CRC xenograft mice were significantly reduced after Prevotella copri intervention. This study simultaneously conducted protein level detection to verify the transcriptional results at the translational level, confirming the anti-tumor mechanism of Prevotella copri and providing a solid basis for the development of this strain into a live biopharmaceutical.

[0051] 3.7 Effects of Prevotella copris on the proliferation of CRC cells To verify the effect of Prevotella copris on CRC cell proliferation, the proliferation of HCT116 cells was detected using CCK-8 assay. Figure 8 As shown, the IC50 of Prevotella copris against HCT116 cells was calculated. 50 It is 69.37×10 3 CFU was used in subsequent studies, therefore Prevotella copris 5×10⁻⁶ was employed. 4 Cellular intervention using CFU. The supernatant of Prevotella copris was able to inhibit the proliferation of HCT116 cells. This experiment directly evaluated the antitumor efficacy of this strain at the cellular level, thus demonstrating that Prevotella copris not only has antitumor potential in vivo, but also possesses the core pharmacological activity of inhibiting tumor cell activity in vitro, providing a basis for its application as a live biopharmaceutical.

[0052] 3.8 Effects of Prevotella copris on CRC cell migration ability Scratch assays were used to investigate the effect of Prevotella copris on the migration ability of HCT116 cells in CRC cells. Results are as follows: Figure 9 As can be seen, the scratch healing rate in the control group was faster, and the scratch area boundary was incomplete after 24 hours. Compared with the control group, the drug-treated group showed significant inhibition of HCT116 cell migration, and the inhibition rate was time-dependent. This experiment evaluated the potential efficacy of Prevotella copris in controlling tumor metastasis, clarifying that Prevotella copri not only inhibits local proliferation but also possesses key biological activities to inhibit tumor invasion and spread, providing cellular kinematic evidence for the study of its anti-tumor mechanism.

[0053] 3.9 Effect of Prevotella copris on CRC cell clonogenic ability A clonogenic assay was conducted to investigate the effects of Prevotella copris on the proliferation and invasion of CRC cells HCT116. Results Figure 10It was observed that the colony formation rate of HCT116 cells in the control group was higher than that in the drug-treated group, indicating that Prevotella copris significantly inhibited HCT116 cell colony formation. Prevotella copris weakened the malignant proliferative phenotype of HCT116 cells by intervening in their sustained proliferation. This experiment evaluated the inhibitory efficacy of this strain on the long-term survival and population proliferation of tumor cells from the perspective of single-cell clonal growth, demonstrating that Prevotella copris possesses the core antitumor activity of inhibiting malignant tumor cell proliferation, providing important evidence for its antitumor research.

[0054] 3.10 Effect of IF assay on protein expression of Prevotella copris in HCT116 cells The effect of Prevotella copris on the expression of tumor-associated protein p53, intestinal barrier protein ZO-1, and PI3K, AKT, and mTOR pathway-related proteins in HCT116 cells was detected by IF assay. The results are as follows: Figure 11 Prevotella copris intervention in HCT116 cells significantly upregulated p53 and ZO-1 protein levels and reduced PI3K, AKT, and mTOR protein expression. This study revealed the intrinsic pathways of Prevotella copris' direct antitumor activity and regulation of cell phenotype at the cellular and molecular level, thus establishing molecular evidence for its antitumor effects in vitro.

[0055] 3.11 Non-targeted metabolomics detection of Prevotella copri metabolites Non-targeted metabolomics analysis of Prevotella copri metabolites yielded the following results: Figure 12 As shown, cholylcitrulline is significantly enriched in the metabolites of Prevotella copri. Prevotella copri possesses an enzyme system that efficiently converts and modifies bile acid and amino acid metabolic pathways, and can synthesize and secrete these conjugated bile acid derivatives during normal metabolic and symbiotic interactions. This experiment precisely traced the characteristic material basis of the antitumor and metabolic reprogramming effects of this strain, demonstrating that cholylcitrulline is the core effector molecule mediating cross-boundary signal transduction between Prevotella copri and host cells, and synergistically regulating downstream antitumor and growth-promoting pathways. This provides crucial metabolomics evidence for elucidating the pharmacodynamic material basis of this in vivo biopharmaceutical.

[0056] 3.12 Effects of Cholylcitrulline on the intestinal tract of mice with AOM / DSS colorectal cancer A mouse model of colorectal cancer induced by AOM / DSS was constructed, and choylcitrulline was used for intervention. The results are as follows: Figure 13 As shown, Cholylcitrulline can reduce the incidence of colorectal cancer tumors and alleviate intestinal shortening. As a core enriched metabolite of Prevotella copri, Cholylcitrulline can repair the intestinal mucosa and reduce intestinal damage and atrophy caused by pathological conditions. This study directly confirms that Cholylcitrulline is the direct effector molecule through which this strain exerts its antitumor and intestinal protective effects in vivo, clarifying the pharmacodynamic basis of Cholylcitrulline as a candidate drug for colorectal cancer.

[0057] 3.12 Effects of Cholylcitrulline on mRNA expression of PI3K / AKT / mTOR signaling pathway-related genes in colorectal tissue of AOM / DSS colorectal cancer mice The expression of tumor-related genes p53, intestinal barrier gene ZO-1, and PI3K / AKT / mTOR signaling pathway-related genes in colorectal tissue of AOM / DSS mice was detected by qRT-PCR. The results are as follows: Figure 14 As shown, compared with the AOM / DSS group, Cholylcitrulline intervention significantly increased the expression levels of p53 and ZO-1 gene mRNA in colorectal tissue, while significantly inhibiting the expression levels of PI3K, AKT, and mTOR gene mRNA (p<0.01). Cholylcitrulline can restore barrier homeostasis by activating the tumor suppressor factor p53 and repairing tight junction proteins, while simultaneously blocking the PI3K / AKT / mTOR signaling pathway to exert its anti-tumor effect. This experiment reveals the pathway by which Cholylcitrulline exerts its anti-tumor and intestinal protective effects at the molecular level, providing solid genomic evidence for its potential as a candidate monomeric drug for colorectal cancer.

Claims

1. The use of Prevotella copri and / or its metabolite Cholylcitrulline in the preparation of a drug for treating colorectal cancer.

2. The application as described in claim 1, characterized in that: The metabolite Cholylcitrulline is a bile acid derivative, cholycitrulline.

3. The application as described in claim 1, characterized in that: The drug includes Prevotella copri and / or its metabolite Cholylcitrulline.

4. The application as described in claim 3, characterized in that: The effective concentration of live Prevotella bacteria in the drug is 1×10⁻⁶. 6 CFU / ml ~ 1×10 14 CFU / ml; the effective dosage of cholylcitrulline is 0.1 mg / d to 5000 mg / d.

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