Lactobacillus plantarum KSFY202409 and its use in colorectal cancer

Abstract

The present invention discloses Lactobacillus plantarum KSFY202409 and its use for colorectal cancer, and relates to the technical field of microorganisms. Lactiplantibacillus plantarum ), with a deposit number of CGMCC No. 31103. The present invention also provides the use of the above-mentioned plant lactobacillus in the preparation of a product for preventing or treating colorectal cancer. The plant lactobacillus of the present invention can effectively reduce the incidence of tumors, improve rectal tissue pathological damage, reduce the level of inflammatory factors, down-regulate the expression of anti-apoptotic factors, and up-regulate the expression of pro-apoptotic factors. It has a significant effect in intervening in the occurrence and development of inflammation-related colorectal cancer in mice, and can be used as a potential strain for preventing and assisting in the treatment of inflammation-related colorectal cancer, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Lactobacillus plantarum KSFY202409 and use thereof for colorectal cancer. Background Art

[0002] Colorectal cancer is a malignant tumor of the digestive system that is extremely harmful. The occurrence of colorectal cancer is affected by genetic and environmental factors. Early colorectal cancer patients have no obvious symptoms. Typical symptoms of colorectal cancer are painless blood in the stool, accompanied by changes in bowel habits, thinner stools or indentations. Left-sided colorectal cancer is prone to intestinal obstruction and intestinal perforation, while right-sided colorectal cancer is more likely to cause systemic symptoms. Colorectal cancer mainly spreads through the lymphatic and blood routes, and can also be transferred by implantation.

[0003] The main treatment strategies for colorectal cancer include surgical resection, radiotherapy, chemotherapy and biological drug therapy. Complete surgical resection of the primary tumor and metastasis is an ideal treatment method, but there are certain difficulties in implementing surgical treatment for advanced patients. For patients with unresectable tumor lesions and those who cannot tolerate surgery, radiotherapy and chemotherapy are the most important measures, which can shrink the tumor and inhibit tumor progression and metastasis. Studies have shown that long-term use of aspirin can reduce the incidence of colorectal cancer, but when using aspirin, there will be certain side effects (such as indigestion, gastrointestinal bleeding, stomach pain, etc.). Therefore, providing a probiotic with tumor inhibition effect is of great significance to reduce the mortality rate of colorectal cancer.

[0004] Chinese patent CN107629987A discloses a plant lactobacillus with the function of inhibiting the occurrence of colorectal cancer and its use. The plant lactobacillus CCFM164 provided by the invention can tolerate gastric acid and bile salts, can significantly reduce the inflammatory response in the colorectal part of the colorectal cancer model mouse, and can reduce the number of tumors in the colon and rectum of the model mouse by regulating the expression of Notch1, Notch2 signaling pathways and VEGFR2 molecules in colorectal tissue. In addition, plant lactobacillus CCFM164 can also improve the intestinal flora structure and the level of short-chain fatty acids in the intestine.

[0005] Azomethane (AOM) is a chemical mutagen that induces carcinogenesis by causing DNA alkylation. Dextran sulfate sodium (DSS) is a heparin-like polysaccharide that can damage epithelial integrity, thereby increasing intestinal permeability, which may further lead to immune cell infiltration and intestinal inflammation. The AOM / DSS model is considered to be stable and reproducible, and the pathogenesis of this model is similar to that of human colorectal tumors, and these tumors are rarely invasive. The inflammation-driven colon tumors of the first injection of AOM followed by three seven-day cycles of DSS within 10 cycles contain infiltrating inflammatory cells, proliferating cancer cells, blood vessels, tumor stroma, and various related tissue cells that constitute the tissue microenvironment. The tumors induced by this model are very similar to those occurring in human colorectal cancer at the level of histological and molecular changes, providing a very valuable model for the study of tumorigenesis and prevention of colorectal cancer diseases. Therefore, the AOM / DSS combined spontaneous tumor model induced in mice was used to simulate the course of human colorectal cancer.

[0006] Probiotics are regarded as a new potential drug alternative because of their safety, high efficiency and no toxic side effects. Currently, there are few studies on probiotics that can prevent colorectal cancer. There is an urgent need to provide a probiotic that is safe, efficient and effective in preventing and treating colorectal cancer. Summary of the invention

[0007] The purpose of the present invention is to disclose Lactobacillus plantarum KSFY202409 and its use in colorectal cancer.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum ), the deposit number is CGMCC No. 31103.

[0010] Specifically, the Lactobacillus plantarum is Lactobacillus plantarum KSFY202409.

[0011] Specifically, the colony characteristics of the Lactobacillus plantarum are that the color is mostly white or milky white, the shape is round, the edges are neat, and the surface is moist and smooth.

[0012] In another aspect, the present invention provides a method for culturing Lactobacillus plantarum, wherein the culturing method comprises inoculating Lactobacillus plantarum into a culture medium for culturing.

[0013] Specifically, the culture medium includes but is not limited to MRS medium, M17 medium, PDA medium, LB medium, TB medium, SOB medium, TPY medium, improved TJA medium, improved MC medium, and YCFA medium.

[0014] Specifically, the culture obtained by the culture method includes Lactobacillus plantarum fermentation liquid, Lactobacillus plantarum fermentation liquid precipitation, and Lactobacillus plantarum fermentation liquid supernatant.

[0015] Specifically, the fermentation liquid refers to the liquid obtained by inoculating the bacteria into the culture medium and culturing the culture medium.

[0016] More specifically, the fermentation broth supernatant refers to the clarified liquid on the upper layer of the fermentation broth after centrifugation; it contains abundant metabolites and some bacterial fragments during the bacterial growth and reproduction process.

[0017] More specifically, the fermentation liquid precipitation refers to the liquid precipitation after centrifugation, including free protein, residual bacteria, broken cells, and residues of the culture matrix.

[0018] In another aspect, the present invention provides a probiotic preparation, which comprises the above-mentioned Lactobacillus plantarum or a culture obtained by culturing the above-mentioned culturing method.

[0019] Specifically, the probiotic preparation is a powder or a liquid preparation.

[0020] Furthermore, the bacterial agent may be solid, liquid, semisolid or any physical form that can be obtained by a person skilled in the art through current or future technologies and contains the necessary active ingredients for achieving any application, function or effect described in the present invention.

[0021] In some embodiments, the bacterial agent is in solid form, and can be freeze-dried powder.

[0022] Specifically, the preparation method of the lyophilized powder can adopt the method currently conventionally used in the art, or other methods that may appear in the future to obtain a solid form product of a microorganism or microbial culture. It should be clarified that the preparation method of the lyophilized powder should not become a condition limiting the scope of protection of the present invention.

[0023] In another aspect, the present invention provides use of the aforementioned Lactobacillus plantarum or the culture obtained by the aforementioned culturing method or the aforementioned probiotic preparation in the preparation of a product for preventing or treating colorectal cancer.

[0024] Specifically, the colon cancer is inflammatory colon cancer.

[0025] Specifically, the colon cancer includes left-side colon cancer and right-side colon cancer.

[0026] Furthermore, the left colon cancer includes left 1 / 3 transverse colon cancer, splenic flexure colon cancer, descending colon cancer, sigmoid colon cancer and rectal cancer.

[0027] Furthermore, the right colon cancer includes cecal cancer, appendix cancer, ascending colon cancer, hepatic flexure colon cancer, and right 2 / 3 transverse colon cancer.

[0028] Specifically, the number of viable bacteria of Lactobacillus plantarum in the product is 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g, such as 1×10 3 CFU / ML (CFU / g), 1×10 4 CFU / ML (CFU / g), 1×10 5 CFU / ML (CFU / g), 1×10 6 CFU / ML (CFU / g), 1×10 7 CFU / ML (CFU / g), 1×10 8 CFU / ML (CFU / g), 1×10 9 CFU / ML (CFU / g), 1×10 10 CFU / ML (CFU / g), 1×10 11 CFU / ML (CFU / g), 1×10 12 CFU / ML (CFU / g), 1×10 13 CFU / ML (CFU / g), 1×10 14 CFU / ML (CFU / g), etc. Other point values ​​within this numerical range can be selected.

[0029] Furthermore, the number of viable Lactobacillus plantarum in the product is 1×10 8 -1×10 9 CFU / mL or 1×10 8 -1×10 9 CFU / g.

[0030] Furthermore, the number of viable Lactobacillus plantarum in the product is 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0031] Specifically, the product is a medicine.

[0032] Furthermore, the medicine includes a pharmaceutically acceptable carrier.

[0033] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of an excipient, a stabilizer, a diluent, a binder, a preservative, and a lubricant.

[0034] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose and mannitol.

[0035] Specifically, the stabilizer is selected from at least one of agar, alginic acid, cellulose ether, and carboxymethyl chitosan.

[0036] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0037] Specifically, the binder is selected from at least one of ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinyl pyrrolidone.

[0038] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric cyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0039] Specifically, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, and poloxamer.

[0040] 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.

[0041] Furthermore, the dosage forms for administration via the gastrointestinal tract include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0042] Furthermore, the non-gastrointestinal dosage forms include, but are not limited to, injection dosage forms, respiratory tract dosage forms, skin dosage forms, mucosal dosage forms and cavity dosage forms.

[0043] Furthermore, the injectable dosage forms include but are not limited to intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0044] Furthermore, the respiratory tract administration dosage forms include but are not limited to sprays, aerosols and powder sprays.

[0045] Furthermore, the skin administration dosage forms include but are not limited to lotions, ointments, external solutions, hardeners, pastes, and patches.

[0046] Furthermore, the mucosal administration dosage forms include but are not limited to eye drops, nasal drops, eye ointments, sublingual tablets, and patches.

[0047] Furthermore, the cavity administration dosage forms include but are not limited to suppositories, aerosols, effervescent tablets, drops and pills.

[0048] Specifically, the product has the following effects:

[0049] (1) Increase the patient's weight;

[0050] (2) Reduce spleen and liver indexes;

[0051] (3) reduce colon shortening and bowel index;

[0052] (4) Reduce the number of tumors;

[0053] (5) Reduce the expression levels of inflammatory cytokines IL-1β, IL-6, TNF-α, and iNOS in serum and colon tissue;

[0054] (6) Down-regulation of colon tissue Bcl-2 , Bcl-x L and Bcl-w mRNA expression, up-regulated Bax mRNA expression.

[0055] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating colorectal cancer, wherein the pharmaceutical composition comprises the aforementioned Lactobacillus plantarum or the culture obtained by the aforementioned culture method or the aforementioned probiotic preparation.

[0056] Specifically, the number of viable Lactobacillus plantarum in the pharmaceutical composition is 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g, such as 1×10 3 CFU / ML (CFU / g), 1×10 4 CFU / ML (CFU / g), 1×10 5 CFU / ML (CFU / g), 1×10 6 CFU / ML (CFU / g), 1×10 7 CFU / ML (CFU / g), 1×10 8 CFU / ML (CFU / g), 1×10 9 CFU / ML (CFU / g), 1×10 10 CFU / ML (CFU / g), 1×10 11 CFU / ML (CFU / g), 1×10 12CFU / ML (CFU / g), 1×10 13 CFU / ML (CFU / g), 1×10 14 CFU / ML (CFU / g), etc. Other point values ​​within this numerical range can be selected.

[0057] Furthermore, the number of viable Lactobacillus plantarum in the pharmaceutical composition is 1×10 8 -1×10 9 CFU / mL or 1×10 8 -1×10 9 CFU / g.

[0058] Furthermore, the number of viable Lactobacillus plantarum in the pharmaceutical composition is 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0059] Specifically, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0060] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of an excipient, a stabilizer, a diluent, a binder, a preservative, and a lubricant.

[0061] The beneficial effects of the present invention are:

[0062] (1) Lactobacillus plantarum KSFY202409 significantly improved the symptoms of colorectal cancer-induced weight loss, increased organ index, colon shortening and increased intestinal index in mice, effectively reduced the incidence of intestinal tumors in cancer-bearing mice, and improved rectal tissue pathological damage.

[0063] (2) Intervention with Lactobacillus plantarum KSFY202409 also significantly reduced the expression levels of inflammatory cytokines IL-1β, IL-6, TNF-α and messenger molecule iNOS in mouse serum and colon tissue.

[0064] (3) Lactobacillus plantarum KSFY202409 can also significantly downregulate anti-apoptotic factors in colon tissue at the genetic level Bcl-2 , Bcl-x L and Bcl-w mRNA expression of pro-apoptotic factors Bax mRNA expression.

[0065] The present invention first suggests the significant effect of Lactobacillus plantarum KSFY202409 in intervening the occurrence and development of inflammation-related colorectal cancer in mice, reveals the anti-inflammatory and anti-tumor effects on inflammation-related colorectal cancer, and can be used as a potential strain for the prevention and adjuvant treatment of inflammation-related colorectal cancer. It provides scientific evidence for the healthy functionality of KSFY202409 on the intestine.

[0066] Collection Instructions

[0067] Biomaterial: KSFY202409

[0068] Deposit number: CGMCC No. 31103;

[0069] Classification name: Lactobacillus plantarum Lactiplantibacillus plantarum ;

[0070] Preservation time: June 27, 2024;

[0071] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0072] Abbreviation of depository unit: CGMCC;

[0073] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 The figure shows the weight changes of mice in each group. 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.

[0075] Figure 2 is the spleen index of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0076] Figure 3 is the liver index of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0077] Figure 4 is the length of the colon and rectum of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0078] Figure 5is the colorectal weight / length of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0079] Figure 6 is the number of colorectal tumors in each group of mice. The same lowercase letters in ac indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0080] Figure 7 The following are pictures of the colorectum of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups. P <0.05.

[0081] Figure 8 These are the pathological images of rectal tissues of mice in each group.

[0082] Fig. 9 is the serum IL-6 concentration of mice in each group. 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.

[0083] Fig.10 is the serum IL-1β concentration of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0084] Fig.11 is the serum TNF-α concentration of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0085] Fig.12 is the serum iNOS concentration of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0086] Fig.13 is the colon IL-6 concentration of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0087] Fig.14 is the colon IL-1β concentration of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0088] Fig.15 is the colon TNF-α concentration of mice in each group. The same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0089] Fig.16 is the colon iNOS concentration of mice in each group. The same lowercase letters in ac indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0090] Fig.17 For each group of mice Bcl-2 mRNA relative expression, the same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0091] Fig.18 For each group of mice Bcl-xL mRNA relative expression, the same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0092] Fig.19 For each group of mice Bcl-w mRNA relative expression, the same lowercase letters in ad indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0093] Fig. 20 For each group of mice Bax Relative mRNA expression, the same lowercase letters ae indicate no significant difference between the two groups, different lowercase letters indicate significant difference between the two groups P <0.05. DETAILED DESCRIPTION

[0094] 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.

[0095] Basic Example Isolation and Identification of Lactobacillus plantarum

[0096] Isolation: The plant lactobacillus of the present invention is isolated from naturally fermented yogurt. Take 1 mL of yogurt sample and dilute it 10 times to 10 with sterile saline. -6 , then take 10 -4 , 10 -5 , 10 -6 100 μL of 3 gradient bacterial solutions were spread on the plate and cultured at 37℃ for 24-48 h. The colony morphology was observed and recorded. The colonies with different morphologies on the plate were picked for streaking separation. After culturing at 37℃ for 48 h, the single colonies with different morphologies on the plate were picked for streaking separation again. This was repeated 2 to 3 times until pure single colonies with consistent morphology were obtained. The pure colonies on the plate were picked and inoculated into 5 mL of MRS liquid culture medium and cultured at 37℃ for 24 h. 1 mL of the above bacterial culture medium was taken into a sterile centrifuge tube, centrifuged at 4000 r / min for 10 min, and then the upper culture medium was discarded. The purified suspected target strain was inoculated into MRS broth, cultured at 37℃ for 18-24 h, and DNA was extracted using a bacterial genomic DNA extraction kit. The extracted DNA was subjected to PCR amplification, including 1 μL of upstream primer 27F (5'-AGAGTT TGATCCTGGCTCAG-3', SEQ ID NO:12), 1 μL of downstream primer 1495R (5'-CTACGGCTACCTTGTTAC GA-3', SEQ ID NO:13), 12.5 μL of 2 × Taq plus Buffer, and 1 μL of template DNA. The system was supplemented to 25 μL with sterile double distilled water (dd H2O). Sterile ultrapure water was used to replace template DNA as a negative control. The amplification conditions were: 94℃ 5 min; 94℃ 30 s, 55℃ 30 s, 72℃ 1 min, a total of 29 cycles, and finally 72℃ extension for 5 min. Then 5 μL of the amplified product was taken for agarose gel electrophoresis detection, the agarose concentration was 1.5%, and the electrophoresis conditions were 110 V, 45 min. The successfully detected PCR products were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for sequencing, and the successfully sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0097] The 16S sequencing result is shown in SEQ ID NO:11.

[0098] SEQ ID NO:11:

[0099]

[0100] Example 1

[0101] Table 1 Experimental materials and reagents

[0102]

[0103] Table 2 Instruments and equipment

[0104]

[0105] 1 Experimental methods

[0106] 1.1 Animal Experiment Design

[0107] A total of 40 SPF-grade 4-week-old male C57BL / 6 mice were purchased from Chongqing Labite Biotechnology Co., Ltd. and fed with standard feed with free access to drinking water under a 12 h / 12 ​​h light-dark cycle, temperature of 22°C-25°C, and humidity of 50% ± 10%. After 1 week of adaptation, colorectal cancer modeling was performed.

[0108] Mice were randomly divided into 5 groups, including normal group, model group, aspirin group, low-concentration KSFY202409 group (KSFY202409L), and high-concentration Lactobacillus plantarum KSFY202409 group (KSFY202409H), with 8 mice in each group. Lactobacillus plantarum KSFY202409 was administered in the form of resuscitated culture. The experimental modeling period was 10 weeks. Except for the normal group, the other four groups were intraperitoneally injected with 10 mg / kg AOM on the first day of the experimental period, and then fed with 2.5% DSS aqueous solution in the second, fifth, and eighth weeks, respectively. The gavage treatment lasted for 10 weeks. The normal group and the model group were given sterile saline daily, the aspirin group was given 67 mg / kg aspirin solution daily, and the KSFY202409L group was gavaged at a concentration of 1×10 8 CFU / g, oral administration concentration of KSFY202409H group was 1×10 9 CFU / g. The mice were allowed to drink water freely at all times except for the DSS cycle. The changes in the state of the mice during the experiment were closely observed, and the weight of the experimental mice was recorded regularly and analyzed. After the end of the 10th week, all mice were fasted for 16-24 h, weighed and dissected.

[0109] 1.2 Determination of organ indicators

[0110] The large intestine, spleen and liver of the mouse were removed by dissection, the tissues around the organs were removed, and the organs were washed with physiological saline and then dried with filter paper. They were weighed separately, and the mouse organ index was calculated according to the following formula.

[0111]

[0112] 1.3 Pathological observation of mouse rectal tissue

[0113] Rectal tissue with a length not exceeding 0.5 cm was immersed in 4% paraformaldehyde fixative solution for 48 h, dehydrated with ethanol and embedded in paraffin, cut into slices of approximately 2-3 μm with a microtome, stained with H&E and fixed on a slide to prepare mouse rectal pathological tissue sections, and the morphological changes were observed under an upright microscope.

[0114] 1.4 Determination of levels of inflammatory factors in mouse serum and colon tissue

[0115] 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, NF-κB, and iNOS in mouse serum and colon tissue were determined.

[0116] 1.5 Determination of expression levels of genes related to apoptosis pathway in mouse colon tissue

[0117] 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 premixed reagent (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. Eef2 was used as the internal reference gene and the expression of cDNA was measured at 2 μL. -△△Ct The relative expression intensity of each gene was calculated by the primer sequence shown in Table 3.

[0118] Table 3 Primer sequences

[0119]

[0120] 1.6 Data processing and statistical analysis

[0121] All data are expressed as mean ± standard deviation (SD), and one-way analysis of variance (ANOVA) and Duncan's multiple range test were used for comparison between groups. 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.

[0122] 2 Experimental Results

[0123] 2.1 Effects of KSFY202409 on body weight and organ indexes of mice

[0124] like Figure 1 As shown in the figure, during the period of DSS administration (weeks 2, 5, and 8), the weight of mice in each modeling group showed a downward trend. After the DSS administration was stopped, the weight of mice in each group showed a trend of gradual increase and recovery. After the experimental modeling period, the weight of mice in the model group was significantly lower than that in the normal group, while the weight of mice in the aspirin and KSFY202409 intervention groups showed different degrees of recovery, and the difference was significant compared with the model group ( P <0.05). Among them, the body weight of KSFY202409H mice was heavier than that of KSFY202409L group, and there was no significant difference between the KSFY202409H group and the aspirin group. The results showed that AOM / DSS caused the mice to lose weight, and aspirin and KSFY202409 intervention could alleviate the trend of weight loss in mice to a certain extent.

[0125] Organ index is one of the basic indicators of biomedical research and an important basis for research. Figure 2 and Figure 3 As shown, compared with the normal group, the spleen index and liver index of the model group mice increased significantly ( P <0.05, suggesting that DSS treatment led to organ enlargement and inflammation in mice. Compared with the model group, the organ indexes of the aspirin group, KSFY202409L group, and KSFY202409H group were significantly reduced ( P <0.05). This indicates that KSFY202409 can effectively alleviate the increase of spleen and liver indexes caused by AOM / DSS.

[0126] 2.2 Effects of KSFY202409 on colorectal-related indicators and intestinal tumor incidence in mice

[0127] When the colorectum of mice is inflamed, the length of the colorectum shrinks and swells to a certain extent. Figure 4 and Figure 5 As shown, the ratio of colorectal weight to colorectal length of mice in the model group was significantly different from that in the normal group ( P<0.05). The length of the colon and rectum of mice in the healthy normal group was the longest among the five groups. After AOM / DSS modeling, the length of the colon and rectum of mice was significantly affected. KSFY202409 intervention alleviated the shortening of the colon and rectum of mice and restored the ratio of colon and rectum weight to length. The effect of high concentration of KSFY202409 was significantly better than that of low concentration ( P <0.05). At the same time, the number of intestinal tumors produced in the model group ( Figure 6 ) indicated that AOM / DSS successfully induced colorectal cancer in experimental mice. Compared with the control group, the number of intestinal tumors in mice was significantly reduced after intervention with aspirin and KSFY202409 ( P <0.05), among which, the number of intestinal tumors in the aspirin group was similar to that in the high-concentration KSFY202409 group, and the effect of the high-concentration KSFY202409 group was significantly better than that of the low-concentration KSFY202409 group ( P <0.05), colorectal images such as Figure 7 As shown. This shows that the intervention of KSFY202409 can effectively reduce the number of intestinal tumors in mice.

[0128] 2.3 Effect of KSFY202409 on rectal histopathology in mice

[0129] H&E staining analysis showed that ( Figure 8 ), 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; in the model group, severe erosion of the rectal mucosa was observed, the crypts were highly damaged, the goblet cells were sharply reduced, there was no clear glandular structure, there were a large number of inflammatory cell infiltrations, and tumors occupying the entire intestinal cavity were visible. After intervention with aspirin and KSFY202409, the pathological state of the rectum of mice was significantly improved, the crypts were relatively intact, clear glandular structures were visible, and they were neatly arranged, the number of goblet cells increased, and the structural integrity of the intestinal mucosa was effectively maintained. Among them, the aspirin group had a lower degree of inflammatory cell infiltration and less crypt structure damage; although mild inflammatory infiltration was observed in the KSFY202409 group, the crypt structure was relatively intact. Compared with low concentrations of KSFY202409, high concentrations of KSFY202409 improved the pathological damage of rectal tissue caused by AOM / DSS to a greater extent.

[0130] 2.4 Effect of KSFY202409 on serum inflammatory cytokine levels in mice

[0131] The present invention analyzed the effect of KSFY202409 on the concentration of serum inflammatory cytokines (IL-1β, IL-6, TNF-α and iNOS) in mice with inflammation-related colorectal cancer (CRC). The results showed that ( Figure 9-12), after AOM / DSS modeling, the levels of serum IL-1β, IL-6 and TNF-α in the model group mice were significantly increased, and aspirin and KSFY202409 could effectively reduce their expression ( P <0.05). The serum of mice in the model group contained high concentrations of iNOS. The serum iNOS concentrations of mice in the normal group, aspirin group, and KSFY202409 group were all lower than those in the model group, and there were significant differences ( P <0.05). Among them, the effect of high concentration of KSFY202409 in reducing inflammatory related factors was significantly better than that of low concentration of KSFY202409L ( P <0.05), indicating that high concentrations of KSFY202409 can exert anti-inflammatory effects by reducing the concentrations of serum inflammatory factors.

[0132] Effects of KSFY202409 on the levels of inflammatory factors in the colon of mice

[0133] The present invention also simultaneously analyzed the changes in the concentrations of IL-1β, IL-6, TNF-α and iNOS in the colon tissue of mice ( Figure 13-Figure 16 ), induced by AOM / DSS, the concentrations of inflammatory cytokines IL-1β, IL-6, TNF-α and iNOS in the colon of mice in the model group were significantly increased compared with those in the normal group ( P <0.05). After oral administration of KSFY202409, the concentrations of IL-1β, IL-6, TNF-α and iNOS in the colon tissues of mice in each group decreased to varying degrees, which was significantly different from that in the model group. In addition, the effect of high concentration of KSFY202409 in reducing inflammatory cytokines was significantly better than that of low concentration ( P <0.05). This result is consistent with the trend of inflammatory cytokines in mouse serum measured in the previous study, further indicating that KSFY202409 can effectively reduce the level of inflammation in mice.

[0134] 2.6 Effect of KSFY202409 on the mRNA expression levels of apoptosis-related factors in mouse colon tissue

[0135] In the present invention, real-time fluorescence quantitative PCR was used to analyze Bcl-2 , Bcl-x L , Bcl-w and Bax The mRNA expression level of Figure 17-Figure 20 ), compared with the normal group, the colon tissue of the model group mice Bcl- 2 , Bcl-x L and Bcl-wThe relative mRNA expression of Bax The mRNA expression level of Bcl-2 , Bcl-x L and Bcl-w The expression level of Bax The mRNA expression level of P <0.05). Compared with low concentration of KSFY202409, the effect of high concentration of gavage was more significant.

[0136] The above results indicate that oral administration of KSFY202409 can effectively reduce the increase in spleen and liver indexes caused by AOM / DSS ( P <0.05), improved the inflammation and edema symptoms in the spleen and liver of mice with colorectal cancer. In addition, colon length can be used to characterize the degree of inflammation in DSS-induced colitis. Intervention with the experimental strain KSFY202409 can effectively alleviate the shortening of colorectal length caused by DSS in mice, and the effect of the high-concentration group is significantly better than that of the low-concentration group. Intervention with KSFY202409 significantly reduced the number of tumors in the colorectal segments of CRC mice. It can be seen that KSFY202409 can effectively alleviate the shortening of the colorectum caused by intestinal inflammation and edema, reduce the ratio of colorectal weight and length in mice, and reduce the incidence of intestinal tumors in mice.

[0137] The present invention utilizes H&E staining analysis to clearly observe the pathological changes in rectal tissue of colorectal cancer (CRC) mice. The results show that treatment with KSFY202409 can significantly improve the pathological damage of rectal tissue caused by AOM / DSS, and high concentrations of KSFY202409 have better protective effects on rectal tissue than low concentrations of KSFY202409.

[0138] The results of the present invention show that KSFY202409 can significantly reduce the concentrations of inflammatory cytokines IL-1β, IL-6 and TNF-α in the serum and colon of mice with colorectal cancer (CRC). Compared with the normal group, the serum and colon of the AOM / DSS-induced model group mice contained high concentrations of iNOS, and after oral gavage intervention with aspirin and KSFY202409, the serum and colon concentrations of iNOS were significantly reduced, indicating that KSFY202409 has the possibility of intervening in the development of colorectal cancer based on reducing inflammatory-related factors.

[0139] At the same time, the results of the present invention show that KSFY202409 can upregulate pro-apoptotic factors Bax mRNA expression levels, and down-regulation of anti-apoptotic factors Bcl-2 , Bcl-x L and Bcl-wThe mRNA expression of KSFY202409 plays a role in promoting cell apoptosis. This reveals the potential intervention effect of KSFY202409 on colorectal cancer mice based on the cell apoptosis pathway.

[0140] Comparative Example 1

[0141] 1.1 Animal Experiment Design

[0142] A total of 40 SPF-grade 4-week-old male C57BL / 6 mice were purchased from Chongqing Labit Biotechnology Co., Ltd., with a 12 h / 12 ​​h light-dark cycle, a temperature of 22℃-25℃, and a humidity of 50% ± 10%. They were fed with standard feed and had free access to water. After 1 week of adaptation, colorectal cancer modeling was performed. The mice were randomly divided into 4 groups: normal group, model group, KSFY202409 group (KSFY202409), and 5-aminosalicylic acid. The experimental modeling cycle was 10 weeks. Except for the normal group, the other three groups were intraperitoneally injected with AOM at a concentration of 10 mg / kg on the first day of the experimental cycle, and then fed with 2.5% DSS aqueous solution in the second, fifth, and eighth weeks, respectively. The gavage treatment lasted for 10 weeks. The normal group and the model group were given sterile saline every day, and the KSFY202409 group was gavaged with a concentration of 1×10 9 CFU / g, the 5-aminosalicylic acid group was intragastrically administered with 75 mg / kg 5-aminosalicylic acid aqueous suspension daily. Water was freely available at all times except for the DSS cycle. After the end of the 10th week, all mice were fasted for 16-24 h, weighed, and then dissected.

[0143] 1.2 Determination of levels of inflammatory factors in mouse serum

[0144] The experimental process is the same as that of Example 1 above.

[0145] 1.3 Determination of the expression levels of genes related to inflammation and apoptosis pathways in mouse colon tissue

[0146] The experimental process is the same as that of Example 1 above.

[0147] 2 Experimental Results

[0148] 2.1 Levels of inflammatory cytokines in mouse serum tissues

[0149] Comparison of the levels of IL-1β, IL-6, TNF-α, and iNOS in the serum of mice in the KSFY202409 group and the 5-aminosalicylic acid 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 were significantly increased, and KSFY202409 and 5-aminosalicylic acid could effectively reduce their expression (Table 4). 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, KSFY202409 group, and 5-aminosalicylic acid group were all lower than those in the model group. The effect of KSFY202409 on reducing inflammatory-related factors was slightly better than that of 5-aminosalicylic acid, indicating that KSFY202409 can exert anti-inflammatory effects by reducing the concentration of serum inflammatory factors, and the effect reaches the level of drug 5-aminosalicylic acid.

[0150] Table 4 Comparison of the effects of KSFY202409 and 5-aminosalicylic acid on serum concentrations of inflammatory-related factors in mice

[0151]

[0152] Remission degree (%) = (model group - sample group) / (model group - normal group) × 100.

[0153] 2.2 mRNA expression levels of inflammatory pathway-related factors in mouse colon tissue

[0154] The results showed (Table 5) that the colon tissue of the model group mice Bcl-2 , Bcl-x L and Bcl-w The mRNA expression level of KSFY202409 and 5-aminosalicylic acid groups were the highest. Bcl-2 , Bcl-x L and Bcl-w At the same time, the mRNA expression of Bax The mRNA expression level of KSFY202409 and 5-aminosalicylic acid groups were both able to increase the expression of Bax The mRNA expression of KSFY202409 was significantly reduced, and the relief effect of KSFY202409 was close to that of 5-aminosalicylic acid.

[0155] Table 5 Comparison of the mRNA expression of inflammatory pathway-related factors in mouse colon tissues by KSFY202409 and 5-aminosalicylic acid

[0156]

[0157] Remission degree (%) = (model group - sample group) / (model group - normal group) × 100.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ), characterized in that, The deposit number is CGMCC No. 31103.

2. The method for culturing plant lactobacillus according to claim 1, characterized in that: The culture method comprises the steps of inoculating plant lactobacillus into a culture medium for culture.

3. A probiotic preparation, characterized in that: The probiotic preparation comprises the plant lactobacillus according to claim 1 or the culture obtained by culturing according to the culturing method according to claim 2.

4. Use of the plant lactobacillus according to claim 1, the culture obtained by the culture method according to claim 2, or the probiotic preparation according to claim 3 in the preparation of a medicament for preventing or treating colorectal cancer.

5. The use according to claim 4, characterized in that: The colon cancer is inflammatory colon cancer.

6. The use according to claim 4, characterized in that: The viable count of Lactobacillus plantarum in the drug is 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g.

7. The use according to claim 6, characterized in that: The viable count of Lactobacillus plantarum in the drug is 1×10 8 -1×10 9 CFU / mL or 1×10 8 -1×10 9 CFU / g.

8. The use according to claim 4, characterized in that: The medicine includes a pharmaceutically acceptable carrier.

9. A pharmaceutical composition for preventing or treating colorectal cancer, characterized in that: The pharmaceutical composition comprises the Lactobacillus plantarum according to claim 1 or the culture obtained by the cultivation method according to claim 2 or the probiotic preparation according to claim 3.

10. The pharmaceutical composition according to claim 9, characterized in that The number of viable Lactobacillus plantarum in the pharmaceutical composition is 1×10 8 -1×10 9 CFU / mL or 1×10 8 -1×10 9 CFU / g.

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