Lactobacillus paracasei DACN623 and application thereof in prevention and treatment of colon cancer

Abstract

The invention discloses a strain of Lactobacillus paracasei DACN623 and an application of the Lactobacillus paracasei DACN623 in prevention and treatment of colon cancer, and particularly discloses a strain of Lactobacillus paracasei DACN623 and a preparation method of the Lactobacillus paracasei DACN623. The Lactobacillus paracasei DACN623 separated from Inner Mongolian mare's milk has the characteristics of acid resistance and bile salt resistance, a strain hemolytic evaluation experiment shows that the Lactobacillus paracasei DACN623 has safety, and the experiment proves that the Lactobacillus paracasei DACN623 has good anti-colon cancer activity; furthermore, in-vivo experiments show that the relieving effect of the compound on colon cancer mice is achieved, and it is proved that the compound has the excellent colon cancer resisting effect. The lactobacillus paracasei DACN623 provided by the invention can obviously inhibit cell proliferation, can be developed into an anti-tumor probiotic preparation, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to a strain of Lactobacillus paracasei DACN623 and application thereof in preventing and treating colon cancer. Background Art

[0002] Colon cancer is the third most common cancer in the world and one of the malignant tumors with high morbidity and mortality rates worldwide. According to statistics, the incidence of colon cancer in 2020 was 10%, and the mortality rate caused by colon cancer was 9.4%. Most colon cancers occur due to lifestyle and aging, and only a few cases are related to underlying genetic diseases. Other factors such as eating habits and intestinal microorganisms are also associated with the development of colon cancer. Conventional treatment strategies for colon cancer include surgery, chemotherapy, and radiotherapy. Surgery is suitable for early patients with lesions confined to the colon. After surgical resection, patients have the potential for complete recovery and are an effective choice for localized cancerous growth. Chemotherapy is the main treatment option for patients with metastatic colon cancer, while radiotherapy uses ionizing radiation to control the proliferation of malignant cells and can be used to treat most common types of cancer. At present, immunotherapy, as a new treatment strategy for colon cancer, has shown benefits for patients with colon cancer in clinical practice, especially those with high microsatellite instability. However, these treatments can produce some adverse side effects, so it is necessary to find new alternative strategies to prevent and treat colon cancer.

[0003] Probiotics are nutritional supplements produced by live non-pathogenic microorganisms that produce health benefits for the host. Increasing evidence supports the potential role of probiotics in preventing colon cancer. Probiotics such as Pediococcus pentosaceus GS4, Propionibacterium pentosaceus SP2, and Lactobacillus paracasei SP5 inhibit the growth of colon cancer cells in vitro. Gavage of probiotics to mice with colon cancer can reduce the incidence of tumors. Probiotics can also improve intestinal symptoms and quality of life in patients with colon cancer. In addition, the metabolites and bacterial components of probiotics also have anticancer activity. The supernatant of probiotics has anti-metastatic and anti-proliferative activities on HT-29 cells and promotes cell apoptosis. Probiotic exopolysaccharides and surface proteins also have inhibitory effects on colon cancer. Therefore, screening lactic acid bacteria strains that inhibit colon cancer cells has important practical significance for developing functional health products, enriching the types of fermented products, and establishing a lactic acid bacteria strain resource library. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a strain of Lactobacillus paracasei DACN623 and its application in preventing and treating colon cancer.

[0005] In order to achieve its purpose, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a strain of Lactobacillus paracasei

[0007] DACN623 is deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M20242060.

[0008] The second aspect of the present invention provides a bacterial agent, the active ingredient of which comprises the above-mentioned Lactobacillus paracasei DACN623 or its fermentation liquid.

[0009] Preferably, the bacterial agent is a freeze-dried powder.

[0010] The third aspect of the present invention provides a method for preparing the above-mentioned bacterial agent, which is obtained by inoculating the above-mentioned Lactobacillus paracasei DACN623 into a culture medium for cultivation.

[0011] The preparation method of the fermentation liquid of DACN623 is as follows: after activating the strain of Lactobacillus paracasei DACN623, the strain is cultured in a liquid culture medium at 35-37° C. for 18-24 hours to obtain the fermentation liquid of Lactobacillus paracasei DACN623; preferably, the liquid culture medium is an MRS liquid culture medium.

[0012] The fourth aspect of the present invention provides the use of the above-mentioned Lactobacillus paracasei DACN623 or the above-mentioned bacterial agent in a product for any of the following purposes:

[0013] (1) Inhibit the proliferation of colon cancer cells;

[0014] (2) Promote apoptosis of colon cancer cells;

[0015] (3) Restoration of colon length;

[0016] (4) Alleviate weight loss in patients with colon cancer;

[0017] (5) reduce the levels of pro-inflammatory factors in patients with colon cancer;

[0018] (6) Reduce the expression of tumor markers in patients with colon cancer.

[0019] In the above application technology scheme, Lactobacillus paracasei DACN623 or the above bacterial agent acts through the following mechanism:

[0020] Reduce the intracellular mitochondrial membrane potential, induce cell apoptosis, induce apoptosis of colon cancer cells; increase the ROS level in colon cancer cells, causing cell apoptosis.

[0021] The fifth aspect of the present invention provides the use of the above-mentioned Lactobacillus paracasei DACN623 or the above-mentioned bacterial agent in the preparation of a drug for the prevention, treatment, auxiliary treatment and / or prognosis care of colon cancer.

[0022] The sixth aspect of the present invention provides a product for the prevention, treatment, adjuvant treatment and / or prognosis care of colon cancer, comprising the above-mentioned Lactobacillus paracasei DACN623 or the above-mentioned bacterial agent.

[0023] Preferably, the product is selected from food, food additives, health products, feed, feed additives, reagents or medicines.

[0024] The beneficial effects of the present invention are:

[0025] The Lactobacillus paracasei DACN623 isolated from Inner Mongolia sour mare's milk of the present invention has the characteristics of acid and bile salt resistance, and the strain hemolytic evaluation experiment shows that it has safety, and the good anti-colon cancer activity is confirmed by experiments; further in vivo experiments characterize its alleviating effect on colon cancer mice, confirming that it has excellent anti-colon cancer effect. The Lactobacillus paracasei DACN623 provided by the present invention can significantly inhibit cell proliferation, can be developed into an anti-tumor probiotic preparation, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The colony morphology (A) and Gram staining results (B) of Lactobacillus paracasei DACN623.

[0027] Figure 2 The hemolytic test results of Staphylococcus aureus (A) and Lactobacillus paracasei DACN623 (B).

[0028] Figure 3 The effect of Lactobacillus paracasei DACN623 on apoptosis of colon cancer cells.

[0029] Figure 4 The effect of Lactobacillus paracasei DACN623 on the mitochondrial membrane potential apoptosis of colon cancer cells.

[0030] Figure 5 The effect of Lactobacillus paracasei DACN623 on ROS in colon cancer cells.

[0031] Figure 6 The effect of Lactobacillus paracasei DACN623 on the weight change trend (A) and final weight (B) of mice.

[0032] Figure 7 The effect of Lactobacillus paracasei DACN623 on the colon length of mice.

[0033] Figure 8 This is the effect of Lactobacillus paracasei DACN623 on serum inflammatory factors in mice.

[0034] Fig. 9 The effect of Lactobacillus paracasei DACN623 on serum cancer markers in mice.

[0035] All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation. IBM SPSS Statistics24 and GraphPadPrism 9.5 software were used for data analysis and plotting. Differences between groups were estimated by one-way ANOVA, followed by post hoc multiple comparisons. Values ​​were statistically significant at p < 0.05. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0038] Example 1 Isolation and Identification of Lactobacillus paracasei

[0039] 1 Experimental Materials

[0040] The strains used in the experiment were from the strain resource bank of Suo Huayi Laboratory of Southwest University.

[0041] 2 Experimental methods

[0042] 2.1 Isolation and purification of lactic acid bacteria

[0043] The collected samples were diluted 10-fold in a gradient manner to 10 -7 . Select 4 appropriate dilution gradients (10 -4 , 10 -5 , 10 -6 , 10 -7 ) 100 μL was spread on MRS solid plates, and after culturing at 37°C for 48 h, single colonies with different morphologies were selected to separate strains by plate streaking. The above steps were repeated until purified strains were obtained, and morphological observation was performed by Gram staining.

[0044] 2.2 Morphological observation of strains

[0045] The strains preserved in the strain resource bank were activated by culturing in MRS liquid medium at 37°C for 24 h. The bacterial liquid after two generations of activation was picked up with an inoculation loop and streaked on MRS solid medium plates for separation. After culturing at 37°C for 48 h, the colony morphology was observed and a single colony was picked for morphological observation by Gram staining.

[0046] 2.3 PCR amplification of 16S rDNA sequences

[0047] The DNA of the purified strain was extracted using a bacterial genomic DNA extraction kit. PCR amplification was performed using a 25 μL reaction system, and agarose gel electrophoresis was used for detection after the reaction. Qualified samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were analyzed for homology comparison using the BLAST program in NCBI.

[0048] 2.4 Determination of the survival rate of strains in artificial gastric fluid at pH 3.00

[0049] The isolated strain was cultured at 37°C for 18 h, and the cells were collected by centrifugation at 3000 r / min for 15 min. The cells were washed with sterile saline and resuspended into a bacterial suspension. The obtained bacterial suspension was mixed with artificial gastric juice (0.2% NaCl, 0.35% pepsin 1:10000, pH adjusted to 3.00 with 1 mol / L HCl) at a volume ratio of 1:9. After culture at 37°C for 3 h, the number of viable cells at 0 h and 3 h was determined by the plate coating method, and the survival rate of the strain in artificial gastric juice at pH 3.00 was calculated according to formula (1).

[0050]

[0051] In formula (1):

[0052] c——survival rate, %;

[0053] m1——3h viable bacteria count, CFU / mL;

[0054] m2——0h viable bacteria count, CFU / mL.

[0055] 2.5 Determination of growth rate of strains in 0.30% bile salt

[0056] The isolated strain was cultured at 37°C for 18 h, and inoculated at an inoculum size of 2% into MRS-THIO medium containing 0.00% and 0.30% ox bile salts, respectively. After culture at 37°C for 4 h, its growth rate was measured. The liquid culture medium without inoculation of bacterial liquid was used as a blank control, and the growth rate of the strain in bile salts was calculated according to formula (2).

[0057]

[0058] In formula (2):

[0059] c——growth rate, %;

[0060] A0——blank control OD 600nm value;

[0061] A1——medium containing 0.00% bile salts OD 600nm value;

[0062] A2——medium containing 0.30% bile salts OD 600nm value.

[0063] 2.6 Safety evaluation of strains

[0064] 2.6.1 Evaluation of hemolytic activity of strains

[0065] Lactobacillus paracasei DACN623 was activated in MRS medium at 37℃ for 24h, and hemolytic Staphylococcus aureus was activated in LB medium at 37℃ for 24h as a positive control for hemolysis. The culture solution of the strain was streaked on a Columbia blood agar medium plate containing 5% sterile defibrinated sheep blood using a sterilized inoculation loop, and cultured at 37℃ for 48h. The hemolysis zone was observed and photographed for record.

[0066] 2.6.2 Evaluation of strain antimicrobial susceptibility

[0067] Adjust the concentration of activated bacterial solution to 1×10 7 CFU / mL, take 100 μL in a 5mL sterile centrifuge tube, add 4mL MRS agar semi-solid medium (containing 0.75% agar) and mix well, and quickly pour it on the MRS agar solid plate. After solidification, use sterile tweezers to pick up 20 different drug-sensitive paper discs (Bickman Biology, BKMAM) and place them on the plate, with three drug-sensitive paper discs (triangular) on each plate. In order to minimize the error, the measuring instrument uses a vernier caliper, with the center of the inhibition zone as the center of the circle, and measures the distance between two points on the circumference of the circle passing through the center of the inhibition zone, which is used as the diameter of the inhibition zone, and the diameter of the inhibition zone is used to represent the size of the inhibition zone.

[0068] 2.7 Statistical analysis

[0069] Each experiment was performed three times in parallel. The experimental data were expressed as "mean ± standard deviation" and variance analysis was performed using SPSS20. P < 0.05 indicated statistical significance.

[0070] 3 Experimental results and analysis

[0071] 3.1 Colony morphology and cell morphology of isolated strains

[0072] After the strain was isolated and purified, it formed a single colony in MRS medium. The colony was round, white, and had a smooth and moist surface. After Gram staining, purple cell morphology was observed under a microscope and it was determined to be Gram-positive bacteria (G+). The colony morphology and Gram staining results of the strain are shown in Figure 1 .

[0073] 3.2 Analysis of strain 16S rDNA sequences

[0074] The 16S rDNA gene sequencing result sequence was compared and identified in the GenBank database by NCBI. The analysis found that the homology with the known Lactobacillus paracasei in the database was 98.99%, and the strain was identified as Lactobacillus paracasei DACN623. The bidirectional sequence of the 16S rDNA gene amplification product of Lactobacillus paracasei DACN623 is shown in SEQ ID NO.1.

[0075] The bidirectional sequence of the 16S rDNA gene amplification product (SEQ ID NO.1) is as follows:

[0076]

[0077] 3.3 Evaluation of strain tolerance to artificial gastric juice

[0078] The digestive tract is the body's natural barrier against foreign substances. The stomach is an important digestive organ in the human body. The strong acid environment of pepsin and gastric juice becomes an obstacle for most microorganisms to enter the intestine. Under normal circumstances, the pH of the human stomach is usually around 3.00 after eating, and the digestion time is 1 to 3 hours. Therefore, the artificial gastric juice pH of 3.00 and the action time of 3 hours were used as screening conditions. According to the document "List of Bacteria that Can Be Used in Food" issued by the Health Bureau Supervision

[2010] No. 65, the tolerance of Lactobacillus casei to artificial gastric juice was evaluated. The results showed that the strain had a certain tolerance to artificial gastric juice, and the survival rate was 126.28% ± 1.34%. It shows that the strain continues to grow and reproduce in the artificial gastric juice environment of pH 3.00, which increases the number of viable bacteria and results in a survival rate of > 100%. This shows that the strain has good acid resistance.

[0079] 3.4 Evaluation of strain tolerance to bile salts

[0080] The strain's tolerance to bile salts in the small intestine is one of the basic criteria for screening lactic acid bacteria. The mass concentration of bile salts in the human body is about 0.03% to 0.30%. The strain's growth rate at a bile salt concentration of 0.30% was measured to evaluate the strain's tolerance to bile salts. The results showed that the strain's growth rate at a bile salt concentration of 0.30% was 49.95% ± 1.68%. This shows that the strain has good bile salt tolerance.

[0081] 3.5 Evaluation of hemolytic activity of strains

[0082] Detect whether Lactobacillus paracasei is hemolytic. Figure 2 It can be observed that Lactobacillus paracasei DACN623 ( Figure 2 B) No transparent hemolytic ring was produced on the blood plate, indicating that the strain was non-hemolytic. On the contrary, the positive control hemolytic Staphylococcus aureus ( Figure 2 A) An obvious transparent hemolytic ring appeared, which preliminarily determined that Lactobacillus paracasei DACN623 was safe.

[0083] 3.6 Antibiotic resistance experiment

[0084] This experiment uses the drug sensitivity paper method to detect the sensitivity of Lactobacillus paracasei to 20 antibiotics, which is the most widely used drug sensitivity test method in clinical practice. The drug sensitivity of probiotic strains to multiple antibiotics is judged by referring to the latest NCCLS standards provided by WHO. If a probiotic is sensitive to a certain antibiotic, it is represented by S; if it is resistant, it is represented by R; if it is intermediate, it is represented by I. By comparing the average value of the inhibition zone diameter produced by the resistance of Lactobacillus paracasei to these 20 antibiotics determined in the experiment with the NCCLS drug sensitivity test standard, the drug sensitivity results of this Lactobacillus paracasei to these 20 antibiotics are shown in Table 1:

[0085] Table 1 Results of resistance of Lactobacillus paracasei DACN623 to 20 antibiotics

[0086]

[0087]

[0088] *Note: In Table 1, “—” indicates insensitive, S indicates sensitive, R indicates resistant, and I indicates moderately sensitive.

[0089] The standard for evaluating whether a probiotic can be safely used in food is whether it has drug resistance. Although the strain does not produce inhibition zones to antibiotics such as vancomycin and polymyxin, and has a certain degree of resistance to antibiotics such as kanamycin and streptomycin, it is sensitive to 12 of the most commonly used antibiotics in clinical practice, including ceftazidime, minocycline, tetracycline, erythromycin, doxycycline, cefacililin, cefoperazone, cefuroxime sodium, penicillin, piperacillin, ceftriaxone, and ampicillin, and is moderately sensitive to lincomycin, cephalexin, gentamicin, amikacin, etc., and there is no risk of transmitting resistance genes of commonly used clinical antibiotics to intestinal microorganisms.

[0090] The deposit information of Lactobacillus paracasei DACN623 is as follows:

[0091] The strain DACN623 was sent to the China Center for Type Culture Collection (CCTCC) for preservation in September 2024. The address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation date is September 24, 2024; the preservation number is CCTCC NO: M 20242060; the classification name is Lactobacillus paracasei DACN623.

[0092] Example 2 Inhibitory effect of Lactobacillus paracasei DACN623 on colon cancer cells Caco-2

[0093] 1 Experimental Materials

[0094] The experimental strain is Lactobacillus paracasei DACN623, and the preservation number is CCTCC NO: M 20242060.

[0095] 2 Experimental methods

[0096] 2.1 In vitro antiproliferative activity of colon cancer cells

[0097] Preparation of bacterial suspension: Inoculate the purified strain at a ratio of 2% (v / v) (i.e. 200 μL) into 10 mL MRS liquid culture medium and place it in a 37°C constant temperature incubator for 18 h. After the strain is activated, take 100 μL of the bacterial solution and add it to a 1.5 mL EP tube containing 900 μL 0.9% saline, pipette and mix, then take another 100 μL of the bacterial solution and add it to a 1.5 mL EP tube containing 900 μL 0.9% saline, pipette and mix, and do this in sequence to dilute the bacterial solution in a gradient manner until the concentration is 10 -6 . Take 10 -4 , 10 -5 , 10 -6 10 μL of three concentrations of bacterial solution were added to the MRS solid plate, and three parallels were selected for each concentration gradient. After the coating was completed, the plate was inverted and placed in a 37°C constant temperature incubator for 48 hours. After the incubation was completed, the colonies on the plate were counted, and only the plates with colony counts between 30 and 300 were counted, and then the bacterial concentration in the original bacterial solution was calculated. The bacterial concentration in the original bacterial solution (CFU / mL) = [(A1+A2+A3) / 3]×10 n (dilution factor) × 100. After counting, the bacterial solution was centrifuged (8000 g, 10 min, 4 °C) to collect the bacterial solution precipitate, and washed three times with phosphate buffered saline (PBS, pH 7.2) to collect the above bacterial solution precipitate, and the strain was resuspended in a double antibody-free serum-free cell culture medium, and the bacterial concentration was adjusted to 2 × 10 8 , 2×10 9 CFU / mL.

[0098] After counting, the concentration of the cultured cell suspension was adjusted to 1×10 5 100 μL of bacterial suspension (final concentration 1×10 ) was added to each well of the 96-well plate. 100 μL of cell culture medium (no cells) was added as a blank group. Six replicates were set up for each culture well and placed in a constant temperature incubator at 37°C and 5% CO2 for overnight culture until the cells adhered to the wall and converged. The cell adhesion status of each well was observed and photographed. 100 μL of bacterial suspension (final concentration 1×10 ) was added to each well. 8 , 1×10 9CFU / mL), and 100μL 5-FU was added as a positive control (final concentration of 100μg / mL). Six duplicate wells were set for each culture well, and 100μL of serum-free culture medium was added as a control. The 96-well plate was placed in a constant temperature incubator at 37°C and 5% CO2 for 24h / 48h. The cell adhesion state of each well was observed and photographed for record. The supernatant was discarded, washed twice with PBS, 90μL of serum-free culture medium and 10μL of 5mg / mL MTT solution (final concentration of 0.5mg / mL) were added, and the 96-well plate with MTT added was placed in a constant temperature incubator at 37°C and 5% CO2 for 4h. After the incubation time was over, the supernatant was discarded, and 150μL DMSO was added to each well. After sufficient oscillation and mixing, the absorbance value of each well was detected at a wavelength of 490nm using an enzyme marker. The inhibition rate of the strain on the cells was calculated according to the cell growth inhibition rate (%) = 1-(experimental group OD-blank group OD) / (control group OD-blank group OD)×100.

[0099] 2.2 Adhesion of strains to human colon cancer cells

[0100] After the frozen strain of Lactobacillus paracasei DACN was revived and inoculated into MRS broth medium, the culture was cultured at 37°C for 24 hours. After the culture was completed, the suspension was centrifuged at -4°C and 5000 r / min for 10 minutes and washed several times with sterile PBS buffer. The concentration of the bacterial suspension was adjusted to 1×10 6 CFU / mL and set aside. Resuscitate colon cancer cells, inoculate them into six-well cell culture dishes, add DMEM complete medium and culture at 37°C, 5% CO2, and change the culture medium every two days. When the cell adhesion state reaches 80%, use 0.25% trypsin-EDTA to digest and subculture. After the culture is completed, count them with a cell blood cell counting plate and adjust the cell concentration to 5×10 6 / mL. Add 1mL of cell suspension to one of the culture wells of a six-well cell culture dish and place it in an incubator for culture. When the cells in the culture plate grow to a monolayer, discard the DMEM culture medium and rinse each well 3 times with sterile PBS. Add 1mL of the prepared bacterial suspension to the cell well, shake the cell culture plate slightly, and draw a small amount of bacterial liquid from the well for plate counting. The result is used as the initial number of viable bacteria in the bacterial suspension. Incubate the cell plate at 37℃ for 2h, discard the culture medium and wash 3 times with sterile PBS buffer. Use 0.7mL of 0.25% trypsin-EDTA to digest the cells for 10min. After the cells are completely detached, add 0.3mL of DMEM culture medium to terminate the digestion. Collect the culture medium after the adhesion experiment for plate counting, and the result is used as the number of viable adherent bacteria. Adhesion rate (%) = number of lactic acid bacteria at the end of the period / initial number of lactic acid bacteria inoculated × 100%.

[0101] 2.3 Inhibitory effect of fermentation supernatant on colon cancer cell growth

[0102] Preparation of fermentation supernatant: After Lactobacillus paracasei was revived, it was inoculated into MRS broth medium and cultured at 37°C for 24 h until the concentration of bacterial suspension reached 1×10 9 After the culture is completed, centrifuge at 4°C and 5000 r / min for 10 min, collect the fermentation supernatant, filter it with a bacterial filter membrane and set aside.

[0103] Cytotoxicity experiment: CCK-8 kit (Cell Counting Kit-8 cell counting reagent) was used to determine the effect of the fermentation supernatant of the strain on the growth of colon cancer cells. The cells in the logarithmic growth period were digested and centrifuged and evenly spread into a 96-well plate. After 12 hours of culture, the prepared 4% (volume ratio of fermentation supernatant: cell culture medium) fermentation supernatant was added after the cells were stably attached to the wall. A control (4% culture medium) was set up, and 3 replicates were set up for each treatment. After 24 and 48 hours of culture, CCK-8 kit detection was performed, and the cells were incubated in the cell culture incubator for 1.5 hours. The absorbance was measured at 450nm using an enzyme marker.

[0104] 2.4 Effects of strains on apoptosis of colon cancer cells

[0105] When Caco-2 cells grew to 70-80% in 6-well plates, 9 Treat cells with strains with CFU / mL for 24h, and the untreated group serves as a blank control. Remove the old culture medium from the 6-well plate. Wash twice with pre-cooled PBS, digest the cells with trypsin, and collect the cells into a centrifuge tube;

[0106] Mix the collected culture medium and cells, centrifuge at 300g, 4℃ for 5min, and discard the supernatant. Wash the cells twice with pre-cooled PBS, centrifuge at 4℃ for 5min each time; add 100μL 1×Binding Buffer to resuspend the cells; add 5μL Annexin-V and 10μLPI, mix gently; incubate at room temperature in the dark for 10-15min; add 400μL 1×Binding Buffer, mix well, place on ice, and detect with flow cytometer as soon as possible (within 1h).

[0107] Setting of control tubes: Blank tube: normal cells without dye, used to adjust voltage; single-stained tube: two single-stained tubes with only PI and fluorescent-labeled Annexin-V are needed to adjust compensation; experimental tube: cells induced with apoptosis are added with all fluorescent dyes of this experiment; negative tube: normal cultured cells are added with dye to confirm the normal state of cells and exclude false positives.

[0108] Effects of 2.5 strains on mitochondrial membrane potential of colon cancer cells

[0109] When Caco-2 cells grew to 70-80% in 6-well plates, 9 CFU / mL of the strain was treated for 24 h, and the untreated group was used as a blank control. Remove the old culture medium in the 6-well plate and wash it once with pre-cooled PBS;

[0110] Positive control setting: CCCP (10mM) provided in the kit was added to the cell culture medium at a recommended ratio of 1:1000, diluted to 10μM, and treated the cells for 20 minutes. Then JC-1 was loaded according to the following method to detect mitochondrial membrane potential.

[0111] Add 1 mL of JC-1 staining working solution, mix thoroughly, and incubate in a cell culture incubator away from light for 20 min. After incubation, discard the supernatant and add 1 mL of JC-1 staining buffer (1×) to wash the cells twice. Finally, add 2 mL of cell culture medium and observe under a fluorescence microscope. Obtain two pictures of red fluorescence and green fluorescence for each area, and calculate the average red / green fluorescence intensity ratio using Image-J software.

[0112] 2.6 Effects of strains on ROS in colon cancer cells

[0113] (1) Cells in logarithmic growth phase were inoculated into 6-well plates;

[0114] (2) When Caco-2 cells grew to 70-80% in a 6-well plate, 10 9 CFU / mL of the strain was treated with cells for 24 hours. When collecting cells, the culture medium was aspirated into a centrifuge tube, the cells were washed once with PBS, the cells were digested with trypsin, and collected in a centrifuge tube.

[0115] (3) Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L, add 1 mL of the diluted DCFH-DA, and incubate in a cell culture incubator in the dark for 20 min;

[0116] (4) Wash the cells three times with serum-free culture medium to fully remove DCFH-DA that has not entered the cells;

[0117] (5) Add 1 mL of serum-free culture medium, mix well, place on ice, and detect using flow cytometry as soon as possible.

[0118] 2.7 Statistical analysis

[0119] Each experiment was conducted in parallel for 3 times. The experimental data were expressed as "mean ± standard deviation". SPSS20 was used for variance analysis. P < 0.05 indicated statistical significance.

[0120] 3 Experimental results and analysis

[0121] 3.1 In vitro anti-proliferation activity of colon cancer cells

[0122] The results are shown in Table 2. When the strain concentration was 1×10 8 CFU / mL, the inhibition rate of cells was about 38.41±1.30% and 52.56±1.83% after 24h and 48h of action on cells; 9 CFU / mL, the inhibition rate of cells was about 95.87±0.50% and 99.01±0.28% after 24h and 48h of action on cells, indicating that Lactobacillus paracasei DACN623 can effectively inhibit the proliferation activity of colon cancer cells, and it is concentration- and time-dependent.

[0123] Table 2 Inhibition rate of strains on Caco-2 cells

[0124]

[0125] 3.2 Adhesion of strains to colon cancer cells

[0126] The results are shown in Table 3. It can be seen that Lactobacillus paracasei DACN623 has good adhesion to colon cancer cells, with an average adhesion rate of 46.36%, which is conducive to the performance of the probiotic effect.

[0127] Table 3 Adhesion rate of strains to colon cancer cells (%)

[0128] strain Repeat 1 Repeat 2 Repeat 3 Mean(%) DACN623 38.49 45.70 54.88 46.36±6.71

[0129] 3.3 Inhibitory effect of fermentation supernatant on colon cancer cells

[0130] The results are shown in Table 4. 4% Lactobacillus paracasei DACN623 fermentation supernatant showed a significant inhibitory effect on the growth of colon cancer cells. The colon cancer cell density after 48 hours of culture was 50.43% of that of the control, indicating that the fermentation supernatant of Lactobacillus paracasei DACN623 had a significant inhibitory effect on the growth of colon cancer cells.

[0131] Table 4 Effect of fermentation supernatant of strains on the growth of colon cancer cells (absorbance value)

[0132] Experimental Group 24h 48h Comparison 0.490±0.003 0.692±0.036 DACN623 0.299±0.015 0.349±0.123

[0133] 3.4 Effects of strains on apoptosis of colon cancer cells

[0134] In order to explore whether the strains rely on inducing apoptosis to inhibit colon cancer cells, PI / Annexin V-FITC double staining method was used to detect apoptosis by flow cytometry. Figure 3As shown in the figure, compared with the untreated cells, the apoptosis rate of the positive control group (5-FU) and the strain-treated group increased significantly. 5-FU administration increased the apoptosis rate (early / late apoptosis rate) from 16.62% to 34.9% 24 hours later, while 10 9 CFU / mL of the strain treated cells for 24 hours, the early / late apoptosis rate increased to 99.26%. The results showed that Lactobacillus paracasei DACN623 can induce apoptosis of colon cancer cells.

[0135] Effects of 3.5 strains on mitochondrial membrane potential of colon cancer cells

[0136] The effects of the strains on the mitochondrial membrane potential of colon cancer cells were detected by JC-1 staining and quantification of fluorescence intensity. Figure 4 As shown in the figure, compared with untreated cells, the ratio of red / green fluorescence intensity was significantly reduced after strain treatment, indicating that the red fluorescence intensity was reduced, the green fluorescence intensity was enhanced, and the mitochondrial membrane potential was reduced, while there was no significant difference between the 5-FU group and the control group. The results showed that Lactobacillus paracasei DACN623 caused the mitochondrial membrane potential in cells to decrease and induced cell apoptosis.

[0137] 3.6 Effects of strains on ROS in colon cancer cells

[0138] In order to explore the effect of strains on intracellular ROS levels, flow cytometry was used to detect the expression level of cellular ROS. Figure 5 As shown, compared with untreated cells, the intracellular ROS levels increased significantly after positive control (5-FU) and strain treatment. The results showed that Lactobacillus paracasei DACN623 increased the ROS level in colon cancer cells to a certain extent, thereby adversely affecting the dynamic balance of the redox system in cancer cells and causing cell apoptosis.

[0139] Example 3: The alleviating effect of Lactobacillus paracasei DACN623 on colon cancer in mice

[0140] 1 Experimental Materials

[0141] The experimental strain was Lactobacillus paracasei DACN623.

[0142] The experimental animals were 6-7 week old male C57BL / 6J mice purchased from Chongqing Enswell Biotechnology Co., Ltd. They were kept in a standardized laboratory with a room temperature of 25±2°C, a relative humidity of 50±5%, and 12h light / 12h dark. The experiment began after one week of adaptive feeding.

[0143] 2 Experimental methods

[0144] 2.1 Experimental Animal Grouping and Treatment

[0145] Thirty 6-7 week old male C57BL / 6J mice were randomly divided into three groups: normal group (NC), model group (AOM / DSS), and Lactobacillus paracasei DACN623 group (AOM / DSS+DACN623), with 10 mice in each group. The NC group received a single intraperitoneal injection of normal saline, and was gavaged with 0.9% normal saline every day, and had normal drinking water; the model group received a single intraperitoneal injection of AOM (10 mg / (kg·bw)) on day 0 of the experiment, had normal drinking water for 1 week, and continuously drank 2% DSS (w / v) solution for 1 week, then resumed normal drinking water for 2 weeks, and repeated 3 cycles; the modeling scheme was the same as the model group, and after the injection of AOM, 0.2 mL of 1×10 9 CFU / kg·BW of probiotic solution (dissolved in 0.9% saline). The mice were weighed weekly until the end of the experiment, and their disease conditions were observed.

[0146] 2.2 Collection of Animal Tissues

[0147] After the experiment, all mice were fasted and deprived of water for 24 h, and blood was collected. Serum was collected by centrifugation at 4°C and 3000 r / min for 10 min and stored at -80°C for later use. After blood collection, the mice were killed by dislocating the cervical vertebrae, and the colon tissue was quickly dissected out. The liver was rinsed with saline, and the colon length was recorded.

[0148] 2.3 Serum biochemical assay

[0149] The levels of TNF-α, IL-1β, IL-6, CEA and CA19-9 in mouse serum were measured according to the kit instructions.

[0150] 2.4 Statistical analysis

[0151] Each experiment was conducted in parallel for 3 times. The experimental data were expressed as "mean ± standard deviation". SPSS20 was used for variance analysis. P < 0.05 indicated statistical significance.

[0152] 3 Experimental results and analysis

[0153] 3.1 Effect of Lactobacillus paracasei DACN623 on body weight of mice with colon cancer

[0154] To evaluate the effect of Lactobacillus paracasei DACN623 on the body weight of mice bearing AOM / DSS-induced colon cancer, the body weight changes of mice were recorded weekly. The results showed that Figure 6As shown in (A), the weight of mice in the NC group showed a steady increase. Within one week of drinking DSS, the weight of mice in the AOM / DSS group and the AOM / DSS+DACN623 group began to decrease, and their weight resumed increasing after switching to normal drinking water. At the end of the experimental period, the weight of mice in the AOM / DSS group was significantly reduced compared with the NC group. After being treated with Lactobacillus paracasei DACN623, the weight loss of mice was alleviated. Compared with the AOM / DSS group, the weight of mice in the AOM / DSS+DACN623 group was significantly increased. The final weight of mice is shown in Figure 2. Figure 6 As shown in (B), compared with the control group (NC), the body weight of mice in the AOM / DSS group was significantly reduced, while the AOM / DSS+DACN623 group had an alleviating effect on weight loss. The results showed that Lactobacillus paracasei DACN623 had a significant effect on protecting against AOM / DSS-induced weight loss.

[0155] 3.2 Effect of Lactobacillus paracasei DACN623 on colon length in mice with colon cancer

[0156] To evaluate the effect of Lactobacillus paracasei DACN623 on the colon of mice with AOM / DSS-induced colon cancer, the length of the mice's colon was measured, and the results showed that Figure 7 As shown, compared with the NC group (7.24±0.80cm), the colon length of the AOM / DSS group (5.50±0.48cm) was significantly decreased, and after treatment with Lactobacillus paracasei DACN623, the decrease in colon length was alleviated. Compared with the AOM / DSS group, the colon length of the AOM / DSS+DACN623 group (6.90±0.79cm) was significantly increased. The results showed that Lactobacillus paracasei DACN623 improved AOM / DSS-induced colon cancer by restoring colon length.

[0157] 3.3 Effects of Lactobacillus paracasei DACN623 on serum inflammatory factors in mice with colon cancer

[0158] To evaluate the effect of Lactobacillus paracasei DACN623 on inflammation, we detected the levels of proinflammatory cytokines IL-6, IL-1β, and TNF-α in mouse serum by enzyme-linked immunosorbent assay. The results showed that Figure 8 As shown, AOM / DSS induced high expression of IL-6, IL-1β, and TNF-α compared with the NC group, but their expression was significantly reduced after treatment with Lactobacillus paracasei DACN623. The results showed that Lactobacillus paracasei DACN623 significantly reduced AOM / DSS-induced inflammation by reducing the levels of pro-inflammatory factors in vivo.

[0159] 3.4 Effects of Lactobacillus paracasei DACN623 on serum cancer markers in mice with colon cancer

[0160] In order to evaluate the effect of Lactobacillus paracasei DACN623 on tumor markers, we detected the levels of tumor markers CEA and CA19-9 in mouse serum by enzyme-linked immunosorbent assay. The results showed that Fig. 9 As shown, AOM / DSS induced high expression of CEA and CA19-9 compared with the NC group, but their expression was significantly reduced after treatment with Lactobacillus paracasei DACN623. The results showed that Lactobacillus paracasei DACN623 improved AOM / DSS-induced colon cancer by reducing the levels of tumor markers in vivo.

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A strain of Lactobacillus paracasei DACN623, which was deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242060.

2. A bacterial agent, wherein the active ingredient comprises the Lactobacillus paracasei DACN623 or its fermentation liquid according to claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent is freeze-dried powder.

4. A method for preparing the bacterial agent according to claim 2 or 3, characterized in that: The Lactobacillus paracasei DACN623 according to claim 1 is inoculated into a culture medium and cultured to obtain the product.

5. The preparation method according to claim 4, characterized in that: The preparation method of the fermentation liquid of DACN623 is as follows: after activating the strain of Lactobacillus paracasei DACN623, the strain is cultured in a liquid culture medium at 35-37° C. for 18-24 hours to obtain the fermentation liquid of Lactobacillus paracasei DACN623; preferably, the liquid culture medium is an MRS liquid culture medium.

6. Use of the Lactobacillus paracasei DACN623 according to claim 1 or the bacterial agent according to claim 2 in a product for any of the following purposes: (1) Inhibit the proliferation of colon cancer cells; (2) Promote apoptosis of colon cancer cells; (3) Restoration of colon length; (4) Alleviate weight loss in patients with colon cancer; (5) reduce the levels of pro-inflammatory factors in patients with colon cancer; (6) Reduce the expression of tumor markers in patients with colon cancer.

7. The use according to claim 6, characterized in that: Lactobacillus paracasei DACN623 or the bacterial agent described in claim 2 acts through the following mechanism: Reduce the intracellular mitochondrial membrane potential, induce cell apoptosis, induce apoptosis of colon cancer cells; increase the ROS level in colon cancer cells, causing cell apoptosis.

8. Use of the Lactobacillus paracasei DACN623 described in claim 1 or the bacterial agent described in claim 2 in the preparation of a medicine for preventing, treating, adjuvant therapy and / or prognosis care of colon cancer.

9. A product for the prevention, treatment, adjuvant treatment and / or prognostic care of colon cancer, characterized in that: It includes the Lactobacillus paracasei DACN623 described in claim 1 or the bacterial agent described in claim 2.

10. The product according to claim 9, characterized in that: The product is selected from food, food additives, health products, feed, feed additives, reagents or medicines.

Images