Use of lactobacillus rhamnosus AFY01 and product thereof in inflammatory colon cancer

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to the use of Lactobacillus rhamnosus AFY01 and a product thereof in inflammatory colon cancer. Provided is a strain of Lactobacillus rhamnosus AFY01 with the deposit number of CGMCC No. 27362. The AFY01 can effectively alleviate weight loss in mice with colon cancer and colon shortening caused by intestinal inflammation and edema, reduce the colon coefficient of mice, reduce the visceral index and reduce the occurrence of intestinal tumors in mice, alleviate intestinal inflammation, and promote intestinal tumor cell apoptosis to slow down the development of colon cancer. By means of the present invention, the strain resources are expanded, thus providing technical support for the prevention and treatment of intestinal cancers.

Description

Application of Lactobacillus rhamnosus AFY01 and its products in inflammatory colon cancer Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to application of Lactobacillus rhamnosus AFY01 and products thereof in inflammatory colon cancer. Background Art

[0002] Colon cancer is a common digestive tract malignancy that originates in the colon. The pathogenesis of colon cancer is complex and diverse, involving environmental and dietary factors, personal habits, familial and hereditary factors. Chronic inflammation is also recognized as a significant risk factor for various cancers. The degree of chronic intestinal inflammation is positively correlated with the development of intestinal tumors, promoting the development of inflammation-related colon cancer. To investigate the pathological mechanisms of colon cancer and mimic the pathological progression of human colon cancer, various experimental animal models have been developed. Among them, the azoxymethane (AOM) / dextran sodium sulfate (DSS) model offers a simple and highly reproducible approach. AOM, a carcinogen, can induce tumor formation, while DSS, a heparin-like polysaccharide, damages colon epithelial cells, leading to intestinal inflammation. The key features of the AOM / DSS model are its high modeling accuracy and rapid timeline; intestinal tumor development can occur in as little as 10 weeks. Furthermore, the histopathology of AOM / DSS-induced tumors closely mimics the development and progression of human colon cancer.

[0003] The pathophysiology of colon cancer is complex, and its development is a multistage process. The nuclear factor κB (NF-κB) signaling pathway plays a key role in the pathophysiology of colon cancer, influencing tumor initiation, progression, and metastasis. Current evidence suggests that NF-κB, as a major link between inflammation and cancer, mediates transcription and plays a key role in the development and progression of colon cancer. Activation of the NF-κB signaling pathway promotes the establishment of a proinflammatory tumor microenvironment in colon cancer and regulates cell proliferation, apoptosis, metastasis, angiogenesis, drug resistance, and the expression of target genes associated with inflammation. NF-κB is also considered a major anti-apoptotic factor, not only activating anti-apoptotic proteins (Bcl-2 and Bcl-xL) but also inactivating the expression of pro-apoptotic proteins (Bid, Bax, and Bak), reducing the activity of caspases, and thereby inhibiting apoptosis in colon cancer cells.

[0004] Currently, commonly used treatments for colon cancer patients include fluorouracil, irinotecan, oxaliplatin, and raltitrexed, but these drugs are often accompanied by certain side effects. A large body of evidence shows that aspirin has a preventive effect on cancer, especially colon cancer. However, clinical studies have found that aspirin can cause a series of gastrointestinal side effects, especially damage to the upper digestive tract, including indigestion, peptic ulcer bleeding, and even death. Compared to drug treatments with multiple side effects.

[0005] Lactobacillus plantarum, a major type of lactic acid bacteria, is widely found in fermented dairy products, meat, and vegetables. It also serves as a probiotic in the human gastrointestinal tract, significantly promoting human health. Lactobacillus rhamnosus, an edible lactic acid bacteria species, is present in the human body and the environment. While influencing the development and progression of colon cancer, it also provides a certain degree of protection for normal cells and the body. Lactobacillus rhamnosus can directly intervene in the development and progression of colon cancer by regulating immunity, modulating cellular stress responses, inhibiting inflammation, and regulating cell proliferation, apoptosis, pyroptosis, and metastasis.

[0006] Patent CN117264814A provides a Lactobacillus rhamnosus strain with preventative and therapeutic effects against digestive tract diseases. Its deposit number is CGMCC No. 27362. This strain is tolerant to gastric acid and bile salts and can alleviate antibiotic-induced diarrhea in mice. It also has preventative effects against DSS-induced colitis in mice. It also alleviates AOM / DSS-induced colon cancer in mice.

[0007] Given the high morbidity and mortality of colon cancer, the development of more safer and more effective probiotics has great clinical application significance.

[0008] Summary of the Invention

[0009] In order to solve the above problems, the present invention provides a strain of Lactobacillus rhamnosus AFY01. The AFY01 can effectively intervene in the chemical inducer AOM / DSS to establish an inflammation-related colon cancer model in mice, has a good therapeutic effect on colon cancer, and provides a screening candidate strain for the development of probiotics for colon cancer.

[0010] In one aspect, the present invention provides a strain of Lactobacillus rhamnosus AFY01, with a deposit number of CGMCC No.27362.

[0011] In another aspect, the present invention provides a method for culturing the aforementioned Lactobacillus rhamnosus AFY01, comprising inoculating Lactobacillus rhamnosus AFY01 on a culture medium for culturing; the culture medium includes but is not limited to MRS culture medium.

[0012] In another aspect, the present invention provides use of the aforementioned Lactobacillus rhamnosus AFY01 in the preparation of products for preventing, treating and / or assisting in the treatment of intestinal cancer.

[0013] Specifically, the products include but are not limited to: fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Lactobacillus rhamnosus AFY01.

[0014] More specifically, the fermentation liquid refers to the liquid obtained by inoculating the bacteria into the culture medium and culturing for a period of time.

[0015] More specifically, the fermentation broth supernatant refers to the clarified liquid at the top of the fermentation broth after centrifugation; it contains abundant metabolic products from the bacterial growth and reproduction process and some bacterial fragments. The acidic substances and bacteriocins secreted by the bacteria have antagonistic and killing effects on harmful bacteria; the amino acids and synthesized vitamins after the bacteria decompose food are all in the culture medium, as well as enzymes secreted by the bacteria that are useful to the human body; and some bacterial components also have an immune-promoting effect on the human body.

[0016] More specifically, the fermentation liquid precipitate refers to the liquid precipitate obtained by centrifugation, including free protein, residual bacteria, broken cells, and residues of the culture matrix, mainly protein and intracellular matrix.

[0017] More specifically, the live bacteria, also known as active flora, can colonize and multiply in the intestine, which is beneficial to increasing the number of beneficial bacteria.

[0018] More specifically, the dead bacteria are microorganisms that have lost their vitality and are unable to grow and reproduce. The probiotics lose their vitality due to the production process, such as high temperature treatment or excessive drying.

[0019] Specifically, the intestinal cancer is small intestine cancer, colon cancer and / or rectal cancer.

[0020] Preferably, the intestinal cancer is colon cancer.

[0021] In another aspect, the present invention provides a product for preventing, treating and / or assisting in the treatment of intestinal cancer, wherein the product comprises the aforementioned Lactobacillus rhamnosus AFY01.

[0022] Specifically, the product is a medicine, which includes the fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Lactobacillus rhamnosus AFY01.

[0023] More specifically, the drug includes live bacteria of Lactobacillus rhamnosus AFY01; the number of live bacteria is not less than 1×10 8 CFU / kg.

[0024] Preferably, the viable cell count of Lactobacillus rhamnosus AFY01 in the drug is 1×10 8 CFU / kg-1×10 12 CFU / kg.

[0025] More preferably, the viable cell count of Lactobacillus rhamnosus AFY01 in the drug is 1×10 8 CFU / kg-1×10 9 CFU / kg, such as 1×10 8 CFU / kg, 2×10 8 CFU / kg, 3×10 8 CFU / kg, 4×10 8 CFU / kg, 5×10 8 CFU / kg, 6×10 8 CFU / kg, 7×10 8 CFU / kg, 8×10 8 CFU / kg, 9×10 8 CFU / kg, etc. Other point values ​​within this numerical range can be selected.

[0026] More specifically, the medicine further includes pharmaceutically acceptable excipients.

[0027] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0028] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0029] More specifically, the dosage form of the drug can be powder, tablet, capsule or pill.

[0030] Specifically, the product is a food, which includes the fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Lactobacillus rhamnosus AFY01.

[0031] More specifically, the food includes food-acceptable excipients.

[0032] More specifically, the food-scientifically acceptable excipient is selected from one or a combination of two or more of a wetting agent, an emulsifier, a suspension stabilizer, an excipient, a diluent, a lubricant, a preservative, a sweetener and a flavoring.

[0033] Preferably, the food-scientifically acceptable excipient is at least one selected from lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0034] More specifically, the food includes but is not limited to: tea, juice, biscuits, drinks, coffee, soda, beverages, cakes, bread, moon cakes, dairy products, milk products, candy and / or chocolate.

[0035] Specifically, the product is a health product, and the food includes fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Lactobacillus rhamnosus AFY01.

[0036] More specifically, the health care product includes excipients acceptable in health care products.

[0037] More specifically, the auxiliary material is selected from one or a combination of two or more of fillers, capsule shell materials, solvents, stabilizers, flavoring agents, sweeteners, and pigments.

[0038] Preferably, the filler is selected from at least one of starch, corn flour, and grapes;

[0039] The capsule shell material is selected from at least one of gelatin, hydroxypropyl methylcellulose, and polyvinyl alcohol;

[0040] The solvent is selected from at least one of water, alcohol, glycerin and ethanol;

[0041] The stabilizer is selected from at least one of an antioxidant and a preservative;

[0042] The flavoring agent is selected from at least one of natural flavors and artificial flavors;

[0043] The sweetener is selected from at least one of natural sweeteners and artificial sweeteners;

[0044] The pigment is selected from at least one of natural pigments and artificial pigments.

[0045] The technical effects achieved by the present invention are:

[0046] (1) LR-AFY01 can effectively alleviate the pathological weight loss in mice.

[0047] (2) LR-AFY01 can effectively alleviate the shortening of the colon caused by intestinal inflammation and edema, reduce the colon coefficient of mice, reduce the visceral index and reduce the occurrence of intestinal tumors in mice.

[0048] (3) LR-AFY01 can effectively improve the pathological damage of colon tissue caused by AOM / DSS.

[0049] (4) LR-AFY01 can significantly reduce the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in colon cancer mice, downregulate the expression of NF-κB and iNOS, and alleviate the pathological state of inflammation.

[0050] (5) LR-AFY01 can significantly downregulate the pro-inflammatory factors IκBβ, p65, p50, p52 and the anti-apoptotic factors Bcl-2 and Bcl-x in colon tissue L It also up-regulates the mRNA expression of pro-apoptotic factors Bid and caspase-8.

[0051] Preservation Instructions:

[0052] Strain name: AFY01;

[0053] Deposit number: CGMCC No.27362;

[0054] Classification name: Lacticaseibacillus rhamnosus;

[0055] Deposit date: May 17, 2023;

[0056] Depository: General Microbiology Center, China Culture Collection Administration;

[0057] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 shows the gel electrophoresis results of the amplified products of the genomic DNA of Lactobacillus rhamnosus AFY01, where M is 5000 bp, 3000 bp, 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp from top to bottom.

[0059] FIG2 is a Gram staining microscopic image of Lactobacillus rhamnosus AFY01.

[0060] FIG3 shows the effect of Lactobacillus rhamnosus AFY01 on the body weight of mice.

[0061] Figure 4 shows the effects of Lactobacillus rhamnosus AFY01 on mouse colon tissue, where (A) is the colon length; (B) is the visceral (colon) index; (C) is the colon coefficient; and (D) is the number of colon tumors. Different lowercase letters indicate significant differences (P<0.05).

[0062] FIG5 shows the effect of Lactobacillus rhamnosus AFY01 on the colon histopathology of mice.

[0063] Figure 6 shows the effects of Lactobacillus rhamnosus AFY01 on the levels of inflammatory cytokines in mouse serum, including (A) IL-1β, (B) IL-6, (C) TNF-α, (D) NF-κB, and (E) iNOS.

[0064] Figure 7 shows the effects of Lactobacillus rhamnosus AFY01 on the levels of inflammatory cytokines in mouse colon, where (A) is IL-1β; (B) is IL-6; (C) is TNF-α; (D) is NF-κB; and (E) is iNOS.

[0065] Figure 8 shows the effect of Lactobacillus rhamnosus AFY01 on the relative expression of inflammatory factors mRNA in mouse colon tissue, where (A) is the relative expression of IκBβ; (B) is the relative expression of p65; (C) is the relative expression of p50; and (D) is the relative expression of p52. Different lowercase letters indicate significant differences (P<0.05).

[0066] Figure 9 shows the effect of Lactobacillus rhamnosus AFY01 on the relative expression of apoptosis-related factors mRNA in mouse colon tissue cells, where A is the relative expression of Bid; B is the relative expression of Bcl-2; C is the relative expression of Bcl-xL; D is the relative expression of caspase-8; different lowercase letters indicate significant differences (P<0.05). DETAILED DESCRIPTION

[0067] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0068] The Lactobacillus rhamnosus AFY01 of the present invention is referred to as LR-AFY01 or AFY01 for short.

[0069] Example 1 Isolation, purification and identification of Lactobacillus rhamnosus AFY01

[0070] Naturally fermented yogurt was collected from the Altay region of Xinjiang. 40 mL of naturally fermented yogurt was taken and placed in a sterile centrifuge tube, which was placed in a food sampling box and stored in a laboratory refrigerator at 4°C for later use.

[0071] 1.1 Isolation and identification

[0072] 1.1.1 Separation and purification

[0073] Take 1 mL of naturally fermented yogurt sample and dilute it 10-fold to 10 -6 , then take 10 -4 , 10 -5 , 10 -6 Spread 100 μL of the three gradient bacterial solutions on plates (MRS medium) and incubate at 37°C for 24-48 hours. Observe and record colony morphology. Pick colonies of different morphologies from the plates and streak them apart. After incubating at 37°C for 48 hours, pick single colonies of different morphologies from the plates and streak them apart again. Repeat this process 2-3 times until pure single colonies with consistent morphology are obtained.

[0074] The formula of the MRS medium is: peptone 10.0 g / L, beef extract 10.0 g / L, yeast 5.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, Tween-80 1.0 mL / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate heptahydrate 0.05 g / L, and the rest is distilled water.

[0075] 1.1.2 DNA extraction

[0076] Inoculate the purified suspected target strain into MRS broth and incubate at 37°C for 18-24 hours. Extract DNA using a bacterial genomic DNA extraction kit. Number the extracted DNA and store it in a -20°C freezer until needed.

[0077] 1.1.3 PCR amplification and agarose gel electrophoresis of genomic DNA

[0078] Extracted DNA was subjected to PCR amplification using 1 μL of upstream primer 27F (SEQ ID NO. 1: 5'-AGA GTT TGATCCTGGCTC AG-3'), 1 μL of downstream primer 1495R (SEQ ID NO. 2: 5'-CTA CGGCTA CCTTGTTACGA-3'), 12.5 μL of 2× Taq plus buffer, and 1 μL of template DNA. The volume was made up to 25 μL with sterile ddH2O. Sterile ultrapure water was used instead of template DNA as a negative control. Amplification conditions were: 94°C for 5 min, followed by 29 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 5 min.

[0079] 5 μL of the amplified product was then subjected to agarose gel electrophoresis at 1.5% agarose concentration and 110 V for 45 min. Successful PCR products were sent to Shenzhen BGI Genomics Co., Ltd. for sequencing. Successful sequences were aligned using the BLAST (Basic Local Alignment Search Tool) program from NCBI.

[0080] 1.2 In vitro resistance screening

[0081] 1.2.1 Ability to tolerate 0.3% bile salts

[0082] Pig bile salt was added to MRS-THIO medium (MRS broth containing 0.2% sodium thioglycolate) to make the concentration 0.3%, and sterilized at 121℃ for 15min. 5mL of activated bacteria were inoculated into MRS-THIO medium without bile salt (0.0%) and MRS-THIO medium containing 0.3% bile salt at a 2% (v / v) inoculum, respectively. Blank medium (MRS-THIO medium without bacteria) was used as a control. After culturing at 37℃ for 24h, the OD values ​​of the above different concentrations of culture medium were measured. 600nm The tolerance of the strain to bile salts was calculated according to formula (1):

[0083] 1.2.2 Artificial gastric juice tolerance test

[0084] Preparation of artificial gastric juice: Artificial gastric juice consists of 0.2% NaCl and 0.35% pepsin. The NaCl and pepsin required for the experiment were weighed according to the corresponding mass-to-volume ratio for preparation. The pH of the prepared artificial gastric juice was adjusted to 3.0 with 1 mol / L HCl and then filtered through a 0.22 μm filter membrane for sterilization.

[0085] In a clean bench, 5 mL of the cultured bacterial culture was transferred to a 10 mL sterile centrifuge tube and centrifuged at 3000 r / min for 10 min. The supernatant culture medium was discarded and the bacteria were collected. An equal volume (5 mL) of sterile saline was added and mixed to prepare a bacterial suspension. 1 mL of the bacterial suspension was then mixed with 9 mL of artificial gastric juice at pH 3.0. At this point, 1 mL of the mixture was taken as the artificial gastric juice treatment sample for 0 h. The remaining 9 mL of the mixture was placed in a constant temperature water bath shaker (37°C, 150 r / min) and incubated for 3 h. The samples at 0 h and 3 h were diluted 10-fold, and the viable count was determined by plate coating using the appropriate gradient. The samples were incubated at 37°C on MRS solid medium for 48 h, and the survival rate (%) was calculated according to Formula 2.

[0086] 1.3 Results and Analysis

[0087] 1.3.1 PCR amplification product sequencing and gel electrophoresis results

[0088] The results of agarose gel electrophoresis are shown in Figure 1.

[0089] 1.3.2 Species of strains analyzed by Gene Bank

[0090] BLAST program comparison analysis showed that the isolated and purified strain was identified as Lacticaseibacillus rhamnosus.

[0091] 1.3.3 In vitro resistance results of Lactobacillus

[0092] As shown in Table 1, Lactobacillus fermentum AFY01 has a higher survival rate in artificial gastric juice and a stronger ability to tolerate bile salts.

[0093] Table 1 Survival rate of Lactobacillus fermentum AFY01 in pH 3.0 artificial gastric juice and 0.3% bile salt

[0094] Example 2

[0095] 2.1 Materials and reagents

[0096] It was isolated from naturally fermented yogurt in the homes of herders in the Altay region of Xinjiang and deposited in the General Microbiology Center of the China Culture Collection Committee with the deposit number CGMCC No.27362.

[0097] Azoxymethane (AOM) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; dextran sulfate sodium (DSS) was purchased from MP Biomedicals LLC. Aspirin enteric-coated tablets (approval number: HJ20160685, barcode: 6924147659034) were purchased from Bayer HealthCare Co., Ltd. ELISA kits for interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), NF-κB, and inducible nitric oxide synthase (iNOS) were purchased from Shanghai ELISA Biotechnology Co., Ltd. RNase-free water was purchased from Beijing Solebao Technology Co., Ltd. qPCR SYBR Green Master Mix was purchased from Shanghai Yisheng Biotechnology Co., Ltd. TRIzol Reagent and RevertAid First Strand cDNA Synthesis Kit were purchased from Thermo Fisher Scientific. All other reagents were domestically produced or of analytical grade.

[0098] 2.2 Instruments and Equipment

[0099] 6D45415 upright microscope, Olympus Instruments Co., Ltd., Japan; Bioprep-24 biological sample homogenizer, Nano-300 micro-volume spectrophotometer, Hangzhou Aosheng Instrument Co., Ltd.; A200 gradient PCR instrument, Hangzhou Longji Scientific Instrument Co., Ltd.; VLBL0TD1 multi-function microplate reader, StepOnePlus Real-Time PCR System, Thermo Fisher Scientific (Suzhou) Co., Ltd.

[0100] 2.3 Experimental methods

[0101] 2.3.1 Animal Experiment Design

[0102] C57BL / 6 mice (SPF, male, 6 weeks old) were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. [Animal Qualification License No.: SCXK(Xiang)2019-0004]. After acclimation for 7 days in a constant temperature and humidity environment, they were randomly divided into five groups: normal group, model group, aspirin group, low-concentration LR-AFY01 (LR-AFY01L) group, and high-concentration LR-AFY01 (LR-AFY01H) group. During the experiment, mice had free access to food and water. On the first day of the experimental modeling, the model, aspirin, and LR-AFY01 groups received an intraperitoneal injection of 10 mg / kg azoxymethane (AOM). They were then fed with a 2.5% DSS aqueous solution at weeks 2, 5, and 8, respectively. The mice in the normal group received no special treatment and were gavaged with sterile saline daily. The mice in the model group received gavage with sterile saline daily. The mice in the aspirin group received gavage with 67 mg / kg aspirin solution. The mice in the LR-AFY01L group and the LR-AFY01H group received 1×10 8 CFU / kg and 1×10 9 LR-AFY01 bacterial suspension was administered orally at a dose of 10 CFU / kg for 10 weeks. The mice in each group were observed throughout the experiment, and their weights were weighed and recorded weekly. After modeling, blood was drawn from the eyeballs and the mice were sacrificed by spinal dissection. Colon tissue was then dissected and analyzed.

[0103] The bacterial suspension was prepared as follows: 2% inoculum (100 μL) of P2 bacteria was inoculated into 5 mL of MRS medium, cultured at 36°C for 16 hours, and then gradient dilution was performed. The concentration of the stock solution was calculated and then diluted to 1×10 8 CFU / kg and 1×10 9 CFU / kg dose, the diluent used for dilution is distilled water.

[0104] 2.3.2 Determination of organ index and colon coefficient

[0105] Colon tissue from experimental mice was weighed, and the mouse organ index was calculated using the following formula: Organ index = visceral weight (g) / mouse body weight (g) × 100. Colon length was also measured and intestinal tumor development was observed. The colon coefficient was calculated as: colon coefficient = colon weight (mg) / colon length (cm).

[0106] 2.3.3 Colonic histopathological observation

[0107] Colon tissue approximately 0.5 cm in length was immersed in a 4% paraformaldehyde fixative solution for 48 h, dehydrated with ethanol, embedded in paraffin, and cut into approximately 2-3 μm sections using a microtome. The sections were stained with hematoxylin and eosin (H&E) and fixed on glass slides to prepare mouse colon pathological tissue sections. Morphological changes were observed under an upright microscope.

[0108] 2.3.4 Determination of inflammatory-related factor levels in mouse serum and colon tissue

[0109] Whole blood samples were collected from mice and centrifuged at 4000 rpm for 10 minutes at 4°C to obtain serum. IL-6, IL-1β, TNF-α, NF-κB, and iNOS levels in mouse serum and colon tissue were measured using enzyme-linked immunosorbent assay (ELISA) kits.

[0110] 2.3.5 Determination of expression levels of genes related to inflammation and apoptosis pathways in mouse colon tissue

[0111] 50 mg of mouse colon tissue was weighed and total RNA was extracted using TRIzol reagent. RNA was reverse transcribed into cDNA according to the instructions provided in the cDNA synthesis kit. The concentration and purity of RNA and cDNA were determined using a microphotometer. Amplification was performed using the StepOnePlus Real-Time PCR system for 40 cycles under the following conditions: 95°C for 15 seconds, 60°C for 30 seconds, 95°C for 15 seconds, 60°C for 60 seconds, and 95°C for 15 seconds. Eef2 was used as the internal reference gene and the expression was determined according to 2 -△△Ct Table 2 shows the primer sequences used in this experiment.

[0112] Table 2 Primer sequences

[0113] 2.4 Data Analysis

[0114] All data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for significance analysis in IBM SPSS 27.0, and GraphPad 9.3.1 was used for plotting. Different letters in the graphs indicate statistically significant differences (P < 0.05) as determined by Duncan's multivariate test.

[0115] 2.5 Results and Analysis

[0116] 2.5.1 Effect of LR-AFY01 on mouse body weight

[0117] During the modeling process of the present invention, mice in the normal group were observed to be healthy, eating and drinking normally, and without diarrhea or bloody stools. However, mice in the other four groups showed varying degrees of symptoms such as loose stools, bloody stools, and even rectal prolapse. They also showed decreased mobility, sparse and dull fur, and weight loss. As shown in Figure 3, the weight of mice in the normal group showed an increasing trend, while the weight of mice in the other four groups showed a decreasing trend after each feeding of the DSS aqueous solution. The weight of mice in each group showed significant differences (P<0.05). After intervention with aspirin and LR-AFY01, the trend of pathological weight loss in mice was significantly alleviated.

[0118] 2.5.2 Effects of LR-AFY01 on colon length, visceral index, colon coefficient, and intestinal tumors in mice

[0119] As shown in Figure 4 (A), the colon length of mice in the normal group was 6.65±0.31 cm, that in the model group was 4.87±0.37 cm, that in the aspirin group was 5.77±0.29 cm, and that in the LR-AFY01L and LR-AFY01H groups was 5.22±0.27 cm and 5.79±0.37 cm, respectively. The colon length of mice in the model group was significantly shorter than that in the normal group (P<0.05). LR-AFY01 treatment significantly alleviated this shortening (P<0.05), demonstrating an effect comparable to that of the aspirin group. Figures 4 (B) and (C) show that the visceral (colon) index and colon coefficient increased in the normal group, aspirin group, LR-AFY01H group, and LR-AFY01L group, respectively, and were all significantly lower than those in the model group (P<0.05).

[0120] As shown in Figure 4 (D), no intestinal tumors were observed in the normal group mice. However, several tumors of varying sizes were observed in the colon segments of the model, aspirin, and LR-AFY01 groups. The model group had the highest number of tumors (9.5±0.85), while the aspirin, LR-AFY01L, and LR-AFY01H groups had significantly fewer tumors (3.4±0.52, 6.9±0.88, and 3.5±0.53, respectively). This suggests that LR-AFY01 can effectively alleviate colon shortening caused by intestinal inflammation and edema, reduce the colon coefficient, visceral index, and the incidence of intestinal tumors in mice.

[0121] 2.5.3 Effects of LR-AFY01 on colonic histopathology in mice

[0122] H&E staining analysis showed (Figure 5) that the normal group mice had intact colonic mucosal epithelial cells, normal crypts, neatly arranged glands, and no ulcers. The intestines of the model group mice were infiltrated by a large number of inflammatory cells, with multiple necrotic lesions and crypt abscesses. The aspirin group had a lower degree of inflammatory cell infiltration and less crypt structural damage. Although mild inflammatory infiltration was observed in the LR-AFY01 group, the crypt structure was relatively intact. Compared with low-concentration LR-AFY01, high-concentration LR-AFY01 significantly improved the pathological damage of colon tissue caused by AOM / DSS.

[0123] 2.5.4 Effects of LR-AFY01 on inflammatory cytokine levels in mouse serum and colon tissue

[0124] The present invention evaluated whether the anti-tumor activity of LR-AFY01 is associated with its proinflammatory cytokine expression. ELISA assays were used to analyze the expression levels of IL-1β, IL-6, TNF-α, NF-κB, and iNOS in mouse serum and colon tissue. Figures 6(A)-(C) and 7(A)-(C) show that after AOM / DSS modeling, IL-1β, IL-6, and TNF-α levels were significantly elevated in the serum and colon of model mice. Aspirin and LR-AFY01 effectively reduced their expression. Furthermore, serum IL-1β, IL-6, and TNF-α levels in the LR-AFY01H group were significantly lower than those in the LR-AFY01L group (P<0.05), indicating that high concentrations of LR-AFY01 can more significantly reduce proinflammatory cytokine levels in the serum of mice with colon cancer.

[0125] Figures 6(D) and 7(D) show that the NF-κB concentration in the serum and colon tissue of mice in the model group was the highest. The NF-κB concentration in the serum of mice in the normal group, aspirin group, and LR-AFY01 group was significantly lower than that in the model group (P < 0.05). Figures 6(E) and 7(E) show that iNOS was highly expressed in the serum and colon tissue of mice in the model group. The iNOS concentration in the serum of mice in the normal group, aspirin group, and LR-AFY01 group was significantly lower than that in the model group (P < 0.05). The data of the present invention reveal that AOM / DSS treatment can induce high expression of proinflammatory cytokines and nitric oxide synthase in mice, while activating the NF-κB inflammatory pathway and causing a sustained inflammatory response in the body. Oral administration of the experimental strain LR-AFY01 can effectively reduce the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in colon cancer mice, downregulate the expression of NF-κB and iNOS, and alleviate the pathological state of inflammation. The results in mouse serum and colon tissue are consistent.

[0126] 2.5.5 Effect of LR-AFY01 on mRNA expression levels of inflammatory pathway-related factors in mouse colon tissue

[0127] In the present invention, real-time fluorescence quantitative PCR was used to analyze the mRNA expression levels of IκBβ, p65, p50, and p52 in the colon tissue of mice. The results showed (Figure 8) that the mRNA expression levels of IκBβ, p65, p50, and p52 in the colon tissue of mice in the model group were the highest, and were significantly decreased in both the aspirin group and the LR-AFY01 group (P<0.05). The effect of the high-concentration LR-AFY01 gavage group was close to that of aspirin.

[0128] 2.5.6 Effect of LR-AFY01 on mRNA expression levels of apoptosis pathway-related factors in mouse colon tissue

[0129] As shown in Figure 9 (A), compared with the normal group, the pro-apoptotic factor Bid was lowly expressed in the model group, but its relative expression increased in the aspirin group and LR-AFY01 group, with significant differences (P<0.05). At the same time, Figure 9 (B) and (C) showed that the anti-apoptotic factors Bcl-2 and Bcl-x L It was highly expressed in the model group, while its expression was significantly reduced in the aspirin group and LR-AFY01 group (P<0.05). Figure 9 (D) shows that compared with the normal group, the relative expression level of caspase-8 mRNA in the colon tissue of the model group mice was significantly reduced under the induction of AOM / DSS (P<0.05), while the intervention of aspirin and LR-AFY01 significantly improved the low expression of caspase-8 mRNA caused by cancer in mice. Compared with the model group, the caspase-8 mRNA levels in the high and low concentration groups of LR-AFY01 were significantly different (P<0.05). The current experimental results show that LR-AFY01 can significantly upregulate the expression levels of pro-apoptotic factors Bid and caspase-8, while reducing the anti-apoptotic factors Bcl-2 and Bcl-x. L expression.

[0130] 2.6 Conclusion

[0131] This study investigated the effects and mechanisms of LR-AFY01, isolated from traditional fermented yogurt from Xinjiang, on inflammation-related colon cancer in mice induced by AOM / DSS. These effects were assessed through five key aspects: body weight and organ index, colon index and number of intestinal tumors, colon histopathological analysis, serum and colon tissue analysis of inflammatory cytokines, and expression of genes involved in the NF-κB and apoptosis signaling pathways in colon tissue. The results showed that LR-AFY01 significantly ameliorated cancer-related symptoms in mice, including weight loss, increased organ index, colon shortening, and increased intestinal index. It also effectively reduced the incidence of intestinal tumors and ameliorated colon histopathological damage (P<0.05). Furthermore, LR-AFY01 treatment significantly reduced the expression of inflammatory cytokines IL-1β, IL-6, TNF-α, NF-κB, and iNOS in serum and colon tissue (P<0.05). In addition, LR-AFY01 can significantly downregulate the expression of pro-inflammatory factors IκBβ, p65, p50, p52 and anti-apoptotic factors Bcl-2 and Bcl-x in colon tissue at the gene level. L This study demonstrated for the first time that LR-AFY01 significantly reduces the mRNA expression of IL-61 and upregulates the mRNA expression of the pro-apoptotic factors Bid and caspase-8 (P<0.05). This study demonstrates for the first time the significant effect of LR-AFY01 in intervening in the development and progression of inflammation-associated colon cancer in mice, revealing its specific mechanism of action in slowing colon cancer progression by ameliorating intestinal inflammation and promoting apoptosis of intestinal tumor cells. This provides scientific evidence for the functional benefits of LR-AFY01 on intestinal health, further supporting the dietary prevention and adjuvant treatment of colon cancer, and warrants further clinical research in the future.

[0132] Comparative Example

[0133] The apoptosis pathway-related genes Bcl-2 and Bcl-x in mouse colon tissue were determined by referring to the experimental method in 2.3 of Example 2. L The expression level of the α-aminobutyric acid was determined by setting the following comparative example. The difference between the comparative example and Example 2 and the test results are shown in Table 3 below:

[0134] Table 3

[0135] The above results showed that the apoptosis factors Bcl-2 and Bcl-x in the colon tissue of mice after intervention with Lactobacillus rhamnosus AFY05 (CGMCC No.27365) L The degree of downregulation of the relative expression of mRNA of Bcl-2 and Bcl-x was not as good as that of the LR-AFY01 high concentration (LR-AFY01H) group of the present invention, and the L The relative mRNA expression levels of the LR-AFY01 and LR-AFY01L groups were significantly higher than those of the LR-AFY01 low concentration group (LR-AFY01L) of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus AFY01, characterized in that: The deposit number is CGMCC No.27362.

2. The method for culturing Lactobacillus rhamnosus AFY01 according to claim 1, wherein The method comprises inoculating rhamnosus lactobacillus AFY01 on a culture medium for culturing; the culture medium comprises an MRS culture medium.

3. Use of the Lactobacillus rhamnosus AFY01 according to claim 1 in the preparation of a product for preventing, treating and / or assisting in the treatment of inflammatory colon cancer; the product comprises a fermentation broth and / or live bacteria of Lactobacillus rhamnosus AFY01.

4. A product for preventing, treating and / or assisting in the treatment of inflammatory colon cancer, characterized in that: The product comprises the Lactobacillus rhamnosus AFY01 according to claim 1.

5. The product according to claim 4, characterized in that The product is a medicine, and the viable count of Lactobacillus rhamnosus AFY01 in the medicine is not less than 1×10 8 CFU / kg.

6. The product according to claim 5, characterized in that The viable bacterial count of Lactobacillus rhamnosus AFY01 in the drug is 1×10 8 CFU / kg—1×10 12 CFU / kg.

7. The product according to any one of claims 4 to 6, characterized in that The medicine also includes pharmaceutically acceptable excipients.

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