Probiotics for preventing, treating or assisting in treating colorectal cancer and use thereof

Abstract

The present invention provides a probiotic for preventing, treating or assisting in the treatment of colorectal cancer and its application, and relates to the technical field of microorganisms. Lactobacillus plantarum , specifically Lactobacillus plantarum KSFY202401, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee, and the deposit number is CGMCC NO.30984. The Lactobacillus plantarum KSFY202401 provided by the present invention has a significant effect in intervening in the occurrence and development of colorectal cancer, and it slows down the development of colorectal cancer by improving intestinal inflammation and promoting intestinal tumor cell apoptosis. It provides scientific evidence for the healthy functionality of Lactobacillus plantarum KSFY202401 on the intestine, and provides more scientific basis for the prevention and auxiliary treatment of colorectal cancer from the dietary aspect.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and specifically relates to a probiotic for preventing, treating or assisting in treating colorectal cancer and an application thereof. Background Art

[0002] In order to explore the pathological mechanism of colorectal cancer (CRC) and simulate the pathological process of human CRC, a variety of CRC experimental animal models have been developed in current research. Among them, the azoxymethane (AOM) / dextran sodium sulfate (DSS) model is a CRC animal model with simple methods and high reproducibility. As a carcinogen, AOM can induce tumor formation, while DSS is a heparin-like polysaccharide that can damage colon epithelial cells, leading to intestinal inflammation. The main features of the AOM / DSS model are its high modeling accuracy and short cycle. The development of intestinal tumors can occur in as short as 10 weeks. In addition, the tumor tissue pathology induced by AOM / DSS can highly simulate the occurrence and development of human CRC.

[0003] The pathophysiological mechanism of CRC is complex, and its development is a multi-stage process. The nuclear factor κB (NF-κB) signaling pathway plays a key role in the pathophysiology of CRC, affecting the initiation, progression and metastasis of tumors. Current evidence shows that NF-κB, as the main link between inflammation and cancer, plays a key role in the occurrence and development of CRC through its mediated transcription. Activation of the NF-κB signaling pathway promotes the establishment of a pro-inflammatory tumor microenvironment in CRC and regulates cell proliferation, apoptosis, metastasis, angiogenesis, drug resistance and the expression of inflammation-related target genes. At the same time, NF-κB is considered to be a major anti-apoptotic factor that can not only activate anti-apoptotic proteins (Bcl-2, Bcl-xL), but also inactivate the expression of pro-apoptotic proteins (Bid, Bax, Bak), reduce the activity of caspases, and thus inhibit the apoptosis of CRC tumor cells.

[0004] At present, commonly used drugs for CRC patients include fluorouracil, irinotecan, oxaliplatin and raltitrexed, but they are usually accompanied by certain side effects. Compared with drug treatments with multiple side effects, intervention in the occurrence and development of CRC from a dietary perspective has become a hot topic. Among them, isolating and screening probiotic strains from fermented dairy products to develop functional foods that are more effective for CRC is of great significance.

[0005] Studies have shown that probiotics can maintain the intestinal health of the host and slow down the occurrence of digestive tract cancer to a certain extent. Fermented dairy products are rich in lactic acid bacteria, which are a natural source of probiotic strains and have great research and application value. As a major category of lactic acid bacteria, Lactobacillus plantarum is widely present in fermented dairy products, meat and vegetables. At the same time, as a probiotic group in the human gastrointestinal tract, it has a great promoting effect on human health. As an edible lactic acid bacteria species, Lactobacillus plantarum exists in the human body and the environment. While affecting the occurrence and development of CRC, it provides a certain protective effect on normal cells and organisms. Lactobacillus plantarum can directly intervene in the occurrence and development of CRC by regulating immunity, regulating cell stress response, inhibiting inflammation, and regulating cell proliferation, apoptosis, pyroptosis and metastasis.

[0006] The research on probiotics in CRC has shown initial results. At present, there is still a need to develop more candidate strains for probiotic functional foods targeting CRC and provide scientific basis. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a probiotic for preventing, treating or assisting in the treatment of colorectal cancer and its application, which relates to the field of microbial technology. Lactobacillus plantarum , specifically Lactobacillus plantarum KSFY202401, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee, and the deposit number is CGMCC NO.30984. The Lactobacillus plantarum KSFY202401 provided by the present invention has a significant effect in intervening in the occurrence and development of colorectal cancer, and it slows down the development of colorectal cancer by improving intestinal inflammation and promoting intestinal tumor cell apoptosis. It provides scientific evidence for the healthy functionality of Lactobacillus plantarum KSFY202401 on the intestine, and provides more scientific basis for the prevention and auxiliary treatment of colorectal cancer from the dietary aspect.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a Lactobacillus plantarum, characterized in that the Lactobacillus plantarum is Lactobacillus plantarum KSFY202401, with a preservation number of CGMCC NO.30984.

[0010] In a second aspect, the present invention provides a method for culturing the above-mentioned Lactobacillus plantarum, which comprises inoculating Lactobacillus plantarum KSFY202401 into a culture medium.

[0011] Specifically, the culture medium includes, but is not limited to, solid culture medium, semi-solid culture medium or liquid culture medium.

[0012] Preferably, the culture medium includes, but is not limited to: MRS culture medium, MC culture medium or BCP culture medium.

[0013] In a third aspect, the present invention provides a probiotic preparation, comprising one or more of a culture, a culture extract, a bacterial cell, a fermentation broth, and a freeze-dried powder of Lactobacillus plantarum, wherein the preservation number of the Lactobacillus plantarum is CGMCC NO.30984.

[0014] In a fourth aspect, the present invention provides a functional food, wherein the functional food comprises the above-mentioned Lactobacillus plantarum.

[0015] Specifically, the functional food contains ≥10 8 CFU / g of Lactobacillus plantarum KSFY202401.

[0016] Preferably, the functional food contains 10 8 -10 9 CFU / g of Lactobacillus plantarum KSFY202401.

[0017] More preferably, the functional food contains 10 8 CFU / g or 10 9 CFU / g of Lactobacillus plantarum KSFY202401.

[0018] Specifically, the functional food includes but is not limited to candy flakes, soy milk, yogurt, canned food, biscuits, chocolate, cakes, cream, cheese, milk powder, ice cream, popsicles, jam, puree, candied fruit, preserved fruit, bread, egg rolls, protein drinks, solid drinks, lactic acid bacteria drinks, plant protein drinks, carbonated drinks, coffee, puffed foods or health foods.

[0019] Preferably, the health food includes but is not limited to tea products, granules, medicinal wine, capsules, fruit juice, fruit vinegar, granules, fermented milk products, fermented cereal products, fermented bean products, powders, honey pastes or meal replacement powders.

[0020] Preferably, the functional food further comprises food-acceptable auxiliary materials.

[0021] Further preferably, the food-acceptable auxiliary materials include but are not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids or nutritional enhancers.

[0022] In a fifth aspect, the present invention provides the use of the above-mentioned Lactobacillus plantarum, probiotic preparations or functional foods in the preparation of drugs for preventing, treating or assisting in the treatment of colorectal cancer.

[0023] In a sixth aspect, the present invention provides a drug for preventing, treating or assisting in the treatment of colorectal cancer, wherein the drug comprises the above-mentioned Lactobacillus plantarum, microecological preparation or functional food.

[0024] Specifically, the drug contains ≥10 8 CFU / g of Lactobacillus plantarum KSFY202401.

[0025] Preferably, the drug contains 10 8 -10 9 CFU / g of Lactobacillus plantarum KSFY202401.

[0026] More preferably, the drug contains 10 8 CFU / g or 10 9 CFU / g of Lactobacillus plantarum KSFY202401.

[0027] Specifically, the drug plays a role in preventing, treating or assisting in the treatment of colorectal cancer by improving intestinal inflammation and promoting apoptosis of intestinal tumor cells.

[0028] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0029] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, adhesives, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0030] Specifically, the dosage form of the drug includes, but is not limited to, a gastrointestinal dosage form and a parenteral dosage form according to the administration method.

[0031] Preferably, the dosage forms for administration via the gastrointestinal tract include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0032] Preferably, the gastrointestinal administration dosage form includes but is not limited to an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form and a cavity administration dosage form.

[0033] The beneficial effects of the present invention are:

[0034] The Lactobacillus plantarum KSFY202401 provided by the present invention has a significant effect in intervening in the occurrence and development of colorectal cancer, and slows down the development of colorectal cancer by improving intestinal inflammation and promoting apoptosis of intestinal tumor cells. It provides scientific evidence for the healthy functionality of Lactobacillus plantarum KSFY202401 on the intestine, and provides more scientific basis for the prevention and adjuvant treatment of colorectal cancer from the perspective of diet.

[0035] Collection Instructions

[0036] Biomaterial name: KSFY202401;

[0037] Classification name: Lactobacillus plantarum Lactobacillus plantarum ;

[0038] Deposit date: June 11, 2024;

[0039] Deposit number: CGMCC NO.30984;

[0040] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0041] Abbreviation of depository unit: CGMCC;

[0042] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Figure 2 shows the effects of LP-KSFY202401 on the body weight and organ indexes of mice; A in the figure shows the effects of LP-KSFY202401 on the body weight of mice; B shows the spleen index; C shows the liver index; different lowercase letters in the figure indicate significant differences (P ﹤0.05).

[0044] Figure 2 The figure shows the effect of LP-KSFY202401 on the colorectal-related indicators and intestinal tumor incidence of mice; A in the figure is the colorectal length; B is the colorectal index (weight / length) in units of (mg / cm); C is the number of colorectal tumors in units of (pieces); D is a colorectal picture; different lowercase letters in the figure indicate significant differences (P ﹤ 0.05).

[0045] Figure 3 The effect of LP-KSFY202401 on rectal tissue pathology in mice.

[0046] Figure 4 The effect of LP-KSFY202401 on the serum concentration of inflammatory factors in mice; different lowercase letters in the figure indicate significant differences (P ﹤ 0.05).

[0047] Figure 5The effect of LP-KSFY202401 on the colon concentration of inflammatory-related factors in mice; different lowercase letters in the figure indicate significant differences (P ﹤ 0.05).

[0048] Figure 6 The effect of LP-KSFY202401 on the relative mRNA expression of inflammatory factors in mouse colon tissue; different lowercase letters in the figure indicate significant differences (P ﹤ 0.05).

[0049] Figure 7 The figure shows the effect of LP-KSFY202401 on the mRNA expression levels of factors related to the apoptosis pathway in mouse colon tissue; different lowercase letters in the figure indicate significant differences (P ﹤ 0.05). DETAILED DESCRIPTION

[0050] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0051] 1. The experimental materials and reagents used in the present invention are shown in Table 1:

[0052] Table 1 Experimental materials and reagents

[0053]

[0054] 2. The experimental instruments used in the present invention are shown in Table 2:

[0055] Table 2 Experimental instruments

[0056]

[0057] 3. Experimental animals

[0058] The present invention uses 6-week-old SPF-grade healthy male C57BL / 6 mice, which were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number: SCXK (Xiang) 2019-0004.

[0059] 4. Statistical analysis

[0060] All data in the present invention are expressed as mean ± standard deviation (SD), and the results are compared between groups using one-way analysis of variance (ANOVA) and Duncan's multiple range test. The same lowercase letters in ae indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P < 0.05.

[0061] Example 1 Lactobacillus plantarum KSFY202401

[0062] The Lactobacillus plantarum KSFY202401 used in the present invention Lactiplantibacillus plantarun KSFY202401, LP-KSFY202401) was isolated from naturally fermented yogurt in herdsmen's homes in Kashgar, Xinjiang Uygur Autonomous Region by the Collaborative Innovation Center for Children's Nutrition and Healthy Development. On June 11, 2024, it was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC NO.30984.

[0063] Example 2 Effect of Lactobacillus plantarum KSFY202401 on colorectal cancer

[0064] 2.1 Experimental groups and test samples

[0065] After 7 days of adaptive feeding in a constant temperature and humidity environment, C57BL / 6 mice were randomly divided into 5 groups: normal group, model group, aspirin group, LP-KSFY202401 low concentration (LP-KSFY202401L) group and LP-KSFY202401 high concentration (LP-KSFY202401H) group, with 8 mice in each group. During the experiment, mice were free to eat and drink water. On the first day of the experimental modeling, the model group, aspirin group, and LP-KSFY202401 group were intraperitoneally injected with AOM at 10 mg / kg, and were fed with 2.5% DSS aqueous solution in the 2nd, 5th, and 8th weeks, respectively. The mice in the normal group were not treated with special treatment and were gavaged with sterile saline every day. The mice in the model group were gavaged with sterile saline every day. The mice in the aspirin group were gavaged with aspirin solution at 67 mg / kg. The mice in the LP-KSFY202401L group and the LP-KSFY202401H group were gavaged with 1×10 8 CFU / kg and 1×10 9 The mice were gavaged with LP-KSFY202401 suspension at a dose of CFU / kg for 10 weeks. The status of mice in each group was observed during the experiment, and the weight of the mice was weighed and recorded every week. After the model was established, the mice were euthanized, and the large intestine, spleen, and liver tissues of the mice were removed for testing.

[0066] 2.2 Effects of LP-KSFY202401 on body weight and organ indexes of mice

[0067] The large intestine, spleen and liver of the mice were removed by dissection, the tissues around the organs were removed, the organs were washed with physiological saline and then dried with filter paper, and they were weighed respectively. The mouse organ index was calculated according to formula (1).

[0068]

[0069] like Figure 1 As shown in A, the body weight of mice in the normal group showed an increasing trend, while the body weight of mice in the other four groups showed a decreasing trend after each feeding of DSS aqueous solution, and the body weight of mice in each group showed significant differences (P<0.05). After intervention with aspirin and LP-KSFY202401, the trend of pathological weight loss in mice was significantly alleviated, among which the effect of the high-concentration LP-KSFY202401 group was significantly better than that of the low-concentration group (P<0.05), which is similar to the effect of drug treatment.

[0070] Mouse spleen index and liver index results are as follows Figure 1 B and Figure 1 As shown in Figure C, the spleen index and liver index of the model group mice were significantly increased compared with the normal group (P<0.05). Compared with the model group, the spleen index and liver index of the aspirin group and LP-KSFY202401 group were significantly decreased (P<0.05), indicating that both the drug aspirin and the strain LP-KSFY202401 can improve the symptoms of spleen and liver inflammation and edema in colorectal cancer mice.

[0071] 2.3 Effects of LP-KSFY202401 on colorectal-related indicators and intestinal tumorigenesis in mice

[0072] By visually observing the tumors on the mouse colon and counting the masses, it was determined that the model group mice had the largest number of colon tumors. After KSFY202401 intervention, the number of tumors decreased, and high concentrations of KSFY202401 were able to better reduce the number of tumors.

[0073] like Figure 2 As shown in the figure, the colorectal length of the mice in the normal group was the longest among the five groups. After AOM / DSS modeling, the colorectal length of the mice was affected. Among them, the colorectal length of the mice in the model group was significantly shortened compared with the normal group, and the colorectal weight and length ratio was also significantly increased (P<0.05). LP-KSFY202401 intervention can alleviate the shortening of the colorectal colon of mice and restore the colorectal weight and length ratio. The results were significantly different from those in the model group (P<0.05). Among them, the effect of high concentration of LP-KSFY01 in restoring intestinal weight / length was significantly better than that of low concentration (P<0.05).

[0074] 2.4 Effect of LP-KSFY202401 on rectal histopathology in mice

[0075] Under the influence of inflammation and cancer, all organs of mice are affected to varying degrees, among which intestinal pathology is the most significant, including abnormal crypt structure, the generation and development of intestinal adenomas. The present invention uses H&E staining analysis to clearly observe the pathological changes of rectal tissue in CRC mice. Rectal tissue with a length of no more than 0.5 cm was immersed in 4% paraformaldehyde fixative solution for 48 h, dehydrated with ethanol and embedded in paraffin, cut into slices of about 2-3 μm with a slicer, stained with H&E and fixed on a slide, that is, a mouse rectal pathological tissue section was made, and the morphological changes were observed under an upright microscope.

[0076] H&E staining analysis showed that ( Figure 3 ), the rectal mucosal epithelial cells of the normal group mice were intact, the crypts were normal, the glands were neatly arranged, and there was no ulcer; 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 structure damage; although slight inflammatory infiltration was observed in the LP-KSFY202401 group, the crypt structure was relatively intact. Compared with low concentrations of LP-KSFY202401, high concentrations of LP-KSFY202401 improved the pathological damage of the rectal tissue caused by AOM / DSS to a greater extent.

[0077] 2.5 Determination of levels of inflammatory factors in mouse serum and colon tissue

[0078] Cytokines are the main signaling molecules released by inflammatory cells and participate in multiple functions. The main proinflammatory cytokines include IL-1β, IL-6 and TNF-α. Chronic inflammation involving proinflammatory cytokines is a promoting factor for inflammation-related colorectal cancer. Studies have shown that chronic inflammation leading to tumorigenesis is mainly related to various inflammatory cytokines and their inflammation-related pathways. The proinflammatory cytokine IL-6 can activate the NF-κB signaling pathway and promote the expression of intercellular adhesion molecules. At the same time, as the most representative member of the interleukin family, it participates in inflammatory response and immune regulation to mediate cancer. IL-1β, which is also an inflammatory cytokine, stimulates inflammatory cells to enter the intestine through autocrine and paracrine mechanisms, damages intestinal tissue and causes inflammation. In addition, TNF-α, as a key inflammatory factor regulating the activation of the classic inflammatory pathway NF-κB, is not only an activator of NF-κB, but also a product of its activation. Therefore, TNF-α and NF-κB are usually studied simultaneously. As a key mediator of inflammatory response, NF-κB is one of the most common regulatory factors of cancer. Studies have shown that changes in the level of this factor can reflect the condition of CRC patients. In addition, an increase in iNOS levels can be observed when NF-κB is activated, and TNF-α can also activate nitric oxide synthase (iNOS) in inflammatory responses.

[0079] The whole blood of mice was collected and allowed to stand at room temperature for 30 min, and then centrifuged at 4°C and 4000 r / min for 15 min. The upper serum was aspirated, and appropriate amounts were dispensed and stored at -80°C for later use. Serum and colon tissue samples were prepared according to the instructions of the enzyme linked immunosorbent assay (ELISA) kit to determine the levels of IL-6, IL-1β, TNF-α, NF-κB and iNOS in mouse serum, and the levels of IL-6, IL-1β, TNF-α and iNOS in colon tissue.

[0080] The results of the test are as follows Figure 4-Figure 5 As shown in the figure, after AOM / DSS modeling, the levels of IL-1β, IL-6 and TNF-α in the serum and colon of mice in the model group were significantly increased, and aspirin and LP-KSFY202401 could effectively reduce their expression. The NF-κB concentration in the serum of mice in the model group was the highest, and the NF-κB concentration in the serum of mice in the normal group, aspirin group and LP-KSFY202401 group was lower than that in the model group, and there was a significant difference (P<0.05). At the same time, the serum of mice in the model group contained a high concentration of iNOS, and the iNOS concentration in the serum of mice in the normal group, aspirin group and LP-KSFY202401 group was lower than that in the model group, and there was a significant difference (P<0.05). Among them, the effect of high concentration of LP-KSFY202401 in reducing inflammatory-related factors was significantly better than that of low concentration of LP-KSFY202401L (P<0.05), indicating that high concentration of LP-KSFY202401 can exert anti-inflammatory effects by reducing the concentrations of serum and colon inflammatory factors.

[0081] 2.6 Determination of the expression levels of genes related to inflammation and apoptosis pathways in mouse colon tissue

[0082] Weigh an appropriate amount of mouse colon tissue, add TRIzol for homogenization to extract total RNA from the tissue, then use a spectrophotometer to measure the concentration and purity of the RNA extract, and reverse transcribe the RNA extract into cDNA according to the instructions of the reverse transcription kit. After reverse transcription, prepare the cDNA reaction system in an eight-tube strip, namely 10 μL of Master mix, 1 μL of upstream and downstream primers, 1 μL of template cDNA, and 7 μL of sterile enzyme-free water. After the reaction system is prepared, place it on the StepOnePlus Real-Time PCR System for amplification detection. Amplification conditions: pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and the whole process is repeated 40 times. After the detection, the internal reference gene Eef2 is used as the benchmark and the formula is used. Calculate the relative expression of target gene mRNA. The primer sequences used in this experiment are shown in Table 3.

[0083] Table 3 Primer sequences

[0084]

[0085] Note: "F" in the table represents the upstream primer, and "R" in the table represents the downstream primer.

[0086] (1) Effect of LP-KSFY202401 on the mRNA expression levels of inflammatory pathway-related factors in mouse colon tissue

[0087] Under AOM / DSS induction, the NF-κB signaling pathway in mice was activated, leading to increased levels of inflammation. Therefore, by inhibiting the NF-κB signaling pathway, mice can be protected from inflammatory stimulation and the occurrence and development of CRC can be intervened. NF-κB is composed of five transcription factors, including RelA / p65, RelB, c-Rel, NF-κB1 (p50 / p105) and NF-κB2 (p52 / p100). These transcription factors regulate the expression of various genes involved in a variety of biological processes, including inflammation, cell development and differentiation, cell cycle progression and cell migration. As a member of the IκB protein, IκBβ has a dual regulatory effect on NF-κB. It prefers to bind to p65:cRel to form an IκBβ:p65:cRel complex, which can promote the transcription of target genes and lead to the sustained activation of the NF-κB pathway. Free NF-κB plays an important regulatory role between the cytoplasm and the nucleus, in which the phosphorylated subunit p65 It plays an important role by triggering the transcription of certain genes, such as TNF-α, IL-1β and IL-6. Subsequently, these cytokines will further promote the activation of TLR4-mediated pathways, thereby further enhancing and amplifying the inflammatory response. At the same time, studies have shown that p65 and p50 As two important subunits of NF-κB, they are important biomarkers for the diagnosis and prognosis of colorectal cancer. p52 The dimer with RelB can promote the transcription of inflammation-related genes through nuclear translocation. IκBβ, p65, p50 and p52 mRNA expression levels.

[0088] Real-time fluorescence quantitative PCR was used to analyze the IκBβ, p65, p50 and p52 The mRNA expression level of Figure 6 ), in the colon tissue of the model group mice IκBβ, p65, p50 and p52The mRNA expression level of α-KSFY202401 was the highest, and it was reduced to varying degrees in the aspirin group and LP-KSFY202401 group, and the effect of the high-concentration LP-KSFY202401 group was more significant.

[0089] (2) Effect of LP-KSFY202401 on the mRNA expression levels of apoptosis pathway-related factors in mouse colon tissue

[0090] NF-κB signaling can promote the establishment of an inflammatory tumor microenvironment in CRC, which can serve as a source of multiple tumor markers such as increased cell survival, proliferation, metastasis, and angiogenesis. In the tumor microenvironment, NF-κB upregulates the expression of anti-apoptotic proteins, such as B-cell lymphoma 2 (B-cell lymphoma 2), Bcl-2 ) and B-cell lymphoma-extra-large protein (B-celllymphoma-extra-large, Bcl-xL ). Bid yes Bcl-2 Pro-apoptotic members of the family can induce mitochondrial apoptosis in colorectal cancer cells. Bcl-2 and Bcl-xL The expression of can inhibit the normal apoptosis program of tumor cells and promote their survival and development. In addition, as an indispensable part in the regulation and initiation of programmed cell death activation mediated by death receptors, caspase- 8 Highly related to the intrinsic and extrinsic apoptosis pathways, it has been reported that Caspase-8 Dysfunction of apoptosis may contribute to the development of malignant tumors in mice and humans. Therefore, targeting apoptosis-related factors to intervene in colorectal cancer is an effective approach.

[0091] The experimental results show that ( Figure 7 ), compared with the normal group, the pro-apoptotic factor Bid It was lowly expressed in the model group, but its relative expression levels increased in the aspirin group and LP-KSFY202401 group, with significant differences (P<0.05). Bcl-2 and Bcl-xL It was highly expressed in the model group, but significantly decreased in the aspirin group and LP-KSFY202401 group (P<0.05). Compared with the normal group, the colon tissue of the model group mice was significantly Caspase-8 The relative mRNA expression level of KSFY202401 was significantly decreased (P<0.05), while the intervention of aspirin and LP-KSFY202401 could significantly improve the colorectal cancer-induced Caspase-8 mRNA expression was low, and compared with the model group, the LP-KSFY202401 high and low concentration groups Caspase-8The mRNA levels were significantly different (P<0.05). The current experimental results show that LP-KSFY202401 can significantly upregulate pro-apoptotic factors Bid and Caspase-8 The expression level of anti-apoptotic factors Bcl-2 and Bcl-xL was reduced.

[0092] In summary, LP-KSFY202401 can significantly improve the symptoms of weight loss, increased organ index, colon shortening and increased intestinal index caused by cancer in mice, effectively reduce the incidence of intestinal tumors in cancer-bearing mice, and improve rectal tissue pathological damage (P<0.05). At the same time, the intervention of LP-KSFY202401 also significantly reduced the expression levels of inflammatory cytokines IL-1β, IL-6, TNF-α, NF-κB and iNOS in mouse serum and colon tissue (P<0.05). In addition, LP-KSFY202401 can also significantly downregulate pro-inflammatory factors in colon tissue at the genetic level. IκBβ, p65, p50, p52 and anti-apoptotic factors Bcl-2 and Bcl-xL mRNA expression of pro-apoptotic factors Bid and Caspase-8 mRNA expression of glial cells (P<0.05).

[0093] Example 3 Comparison with Lactobacillus rhamnosus GG

[0094] In order to compare the plant lactobacillus KSFY202401 of the present invention with the strains in the prior art, in this example, the rhamnosus lactobacillus GG ( Lactobacillus rhamnosus The therapeutic effect of Lactobacillus rhamnosus GG, LGG) on colorectal cancer mice. The Lactobacillus rhamnosus GG was purchased from Shanghai Collection Biotechnology Center, strain number: SHBBC D24676.

[0095] 3.1 Experimental groups and test samples

[0096] After 7 days of adaptive feeding in a constant temperature and humidity environment, C57BL / 6 mice were randomly divided into 4 groups: normal group, model group, LP-KSFY202401 group and Lactobacillus rhamnosus GG (LGG group), 8 mice in each group. During the experiment, mice were free to eat and drink water. On the first day of the experimental modeling, the model group, aspirin group, and LP-KSFY202401 group were intraperitoneally injected with AOM at 10 mg / kg, and fed with 2.5% DSS aqueous solution in the second, fifth, and eighth weeks, respectively. The mice in the normal group were not specially treated and were gavaged with sterile saline every day; the mice in the model group were gavaged with sterile saline every day; the mice in the LP-KSFY202401 group and the LGG group were gavaged with 1×10 9The mice were intragastrically gavaged with LP-KSFY202401 and LGG bacterial suspensions at a dose of 1.5 CFU / kg for 10 weeks.

[0097] 3.2 Determination of levels of inflammation-related factors in mouse serum

[0098] The whole blood of mice was collected and allowed to stand at room temperature for 30 min, and then centrifuged at 4°C and 4000 r / min for 15 min. The upper serum was aspirated, and appropriate amounts were dispensed and stored at -80°C for later use. Serum and colon tissue samples were prepared according to the instructions of the enzyme linked immunosorbent assay (ELISA) kit, and the levels of IL-6, IL-1β, TNF-α, and iNOS in mouse serum were determined. The results are shown in Table 4.

[0099] Table 4 Comparison of the effects of LP-KSFY202401 and LGG on serum concentrations of inflammatory-related factors in mice

[0100]

[0101] Comparison of the levels of IL-1β, IL-6, TNF-α and iNOS in the serum of mice in the LP-KSFY202401 group and the LGG group showed that after AOM / DSS modeling, the levels of IL-1β, IL-6 and TNF-α in the serum of mice in the model group increased significantly, and LP-KSFY202401 and LGG could effectively reduce their expression. At the same time, the serum of mice in the model group contained a high concentration of iNOS, and the iNOS concentrations in the serum of mice in the normal group, LP-KSFY202401 group and LGG group were all lower than those in the model group. The effect of LP-KSFY202401 on reducing inflammatory-related factors was significantly better than that of LGG, indicating that LP-KSFY202401 can exert anti-inflammatory effects by reducing the concentration of serum inflammatory factors, and the effect is better than that of the commercially available LGG strain.

[0102] 3.3 Determination of the expression levels of genes related to inflammation and apoptosis pathways in mouse colon tissue

[0103] The expression levels of genes related to the inflammation and apoptosis pathways were determined according to the method described in “2.6 Determination of the expression levels of genes related to the inflammation and apoptosis pathways in mouse colon tissue” in Example 2.

[0104] (1) The mRNA expression levels of inflammatory pathway-related factors in mouse colon tissue are shown in Table 5:

[0105] Table 5 Comparison of mRNA expression of factors related to inflammatory pathways between LP-KSFY202401 and LGG

[0106]

[0107] Colon tissue of model group mice IκBβ , p65 , p50 and p52 The mRNA expression level was the highest in the LP-KSFY202401 and LGG groups, and the relief effect of LP-KSFY202401 was about twice that of LGG.

[0108] (2) The mRNA expression levels of factors related to the apoptosis pathway in mouse colon tissue are shown in Table 6:

[0109] Table 6 Comparison of mRNA expression of factors related to apoptosis pathway between LP-KSFY202401 and LGG

[0110]

[0111] Compared with the normal group, the pro-apoptotic factor Bid The expression of anti-apoptotic factor was low in the model group, and the relative expression levels in the LP-KSFY202401 and LGG groups were increased. Bcl-2 and Bcl-x L It was highly expressed in the model group, but significantly decreased in the LP-KSFY202401 and LGG groups. Compared with the normal group, the colon tissue of the model group mice was induced by AOM / DSS. Caspase-8 The relative expression level of caspase-8 mRNA was reduced in the model group, while the intervention of LP-KSFY202401 and LGG could significantly improve the low expression of caspase-8 mRNA caused by colorectal cancer in mice, and compared with the model group, the LP-KSFY202401 and LGG groups Caspase-8 The current experimental results show that LP-KSFY202401 can significantly upregulate pro-apoptotic factors Bid and Caspase-8 The expression level of anti-apoptotic factors Bcl-2 and Bcl-x L expression, and its regulatory ability is greater than that of LGG.

[0112] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A Lactobacillus plantarum ( Lactobacillus plantarum ), characterized in that, The plant lactobacillus is plant lactobacillus KSFY202401, and its preservation number is CGMCC NO.30984.

2. The method for cultivating plant lactobacillus according to claim 1, wherein The culture method comprises inoculating plant lactobacillus KSFY202401 into a culture medium.

3. A microecological preparation, characterized in that: The microecological preparation comprises one or more of the culture, bacterial cells and freeze-dried powder of Lactobacillus plantarum, and the preservation number of the Lactobacillus plantarum is CGMCC NO.30984.

4. Use of the plant lactobacillus according to claim 1 or the probiotic preparation according to claim 3 in the preparation of a medicament for preventing, treating or assisting in the treatment of colorectal cancer.

5. A drug for preventing, treating or assisting in the treatment of colorectal cancer, characterized in that: The medicine comprises the plant lactobacillus according to claim 1 or the microecological preparation according to claim 3.

6. The drug according to claim 5, characterized in that The drug contains ≥10 8 CFU / g of Lactobacillus plantarum KSFY202401.

7. The drug according to claim 5, characterized in that The drug plays a role in preventing, treating or assisting in the treatment of colorectal cancer by improving intestinal inflammation and promoting apoptosis of intestinal tumor cells.

8. The drug according to claim 5, characterized in that The medicine also includes pharmaceutically acceptable excipients.

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