Acid-resistant Lactobacillus rhamnosus strain AFY02 and its application

Abstract

The present invention provides an acid-resistant Lactobacillus rhamnosus AFY02 and its application, belonging to the field of microbial technology. The acid-resistant Lactobacillus rhamnosus AFY02 provided by the present invention is classified and named Lacticaseibacillus rhamnosus, and the preservation unit is the General Microbiology Center of the China Microbiological Culture Collection Administration Committee. The preservation date is May 17, 2023, the preservation number is CGMCC No. 27363, and the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The acid-resistant Lactobacillus rhamnosus AFY02 of the present invention can survive normally in an artificial gastric fluid environment, improve the weight loss, increased organ index, shortened colon and increased intestinal index caused by colon cancer, regulate NF-κB and apoptosis pathways, effectively alleviate the process of intestinal inflammation, intervene in the development of intestinal tumors, and prevent / treat colon cancer.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an acid-resistant Lactobacillus rhamnosus AFY02 and an application thereof. Background Art

[0002] Colon cancer is a common malignant tumor of the digestive tract that occurs in the colon, and its incidence ranks third among gastrointestinal tumors. The pathophysiological mechanism of colon cancer 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 colon cancer, affecting the initiation, progression and metastasis of the tumor. Current evidence shows that NF-κB, as the main link between inflammation and cancer, plays a key role in the occurrence and development of colon cancer by mediated transcription. Activation of the NF-κB signaling pathway promotes the establishment of a pro-inflammatory tumor microenvironment in colon cancer, 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-x L ), it will also inactivate the expression of pro-apoptotic proteins (Bid, Bax, Bak), reduce the activity of caspases, and thus inhibit the apoptosis of colon cancer cells.

[0003] At present, the treatment drugs for colon cancer mainly include chemotherapy drugs, targeted therapy drugs and immunotherapy drugs, but these drugs are often accompanied by certain side effects. 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. Xinjiang, my country relies on its unique geographical, environmental and climatic conditions. The diverse animal husbandry and abundant raw milk provide sufficient raw materials for herders to manually process various dairy products, especially handmade yogurt, which has distinct regional characteristics and unique flavor. These fermented dairy products are rich in lactic acid bacteria, which are a natural source of probiotic strains and have great research and application value. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an acid-resistant Lactobacillus rhamnosus AFY02 and an application thereof. The acid-resistant Lactobacillus rhamnosus AFY02 provided by the present invention can survive normally in artificial gastric juice, effectively alleviate the process of intestinal inflammation, and intervene in the development of intestinal tumors.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A strain of acid-resistant rhamnosus Lactobacillus AFY02, wherein the acid-resistant rhamnosus Lactobacillus AFY02 is classified and named Lacticaseibacillus rhamnosus, the deposit unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is May 17, 2023, the deposit number is CGMCC No.27363, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] In certain embodiments, the 16S rDNA of the acid-tolerant Lactobacillus rhamnosus AFY02 has a nucleotide sequence as shown in SEQ ID NO.1.

[0008] In certain embodiments, the acid-tolerant Lactobacillus rhamnosus AFY02 is isolated from naturally fermented yogurt in the homes of herders in the Altay region of Xinjiang.

[0009] The present invention also provides a use of the acid-resistant Lactobacillus rhamnosus AFY02 described in the above technical solution in the preparation of a product for preventing and / or treating colon cancer.

[0010] In certain embodiments, the product is a microbial preparation, a food, or a pharmaceutical product.

[0011] In certain embodiments, the raw material of the product includes acid-tolerant Lactobacillus rhamnosus AFY02 or a lysate, extract, and / or metabolite thereof.

[0012] In certain embodiments, the acid-tolerant Lactobacillus rhamnosus AFY02 can improve weight loss, increased organ index, shortened colon, and increased intestinal index caused by colon cancer.

[0013] Beneficial technical effects: The present invention provides an acid-resistant Lactobacillus rhamnosus AFY02, the acid-resistant Lactobacillus rhamnosus AFY02 is classified and named Lacticaseibacillus rhamnosus, the deposit unit is the General Microbiology Center of the China Microbiological Culture Collection Administration, the deposit date is May 17, 2023, the deposit number is CGMCC No. 27363, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The acid-resistant Lactobacillus rhamnosus AFY02 of the present invention can survive normally in an artificial gastric fluid environment, improve the weight loss, increased organ index, shortened colon and increased intestinal index caused by colon cancer, regulate NF-κB and apoptosis pathways, effectively alleviate the process of intestinal inflammation, intervene in the development of intestinal tumors, and prevent / treat colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a Gram staining microscopic examination of Lactobacillus rhamnosus AFY02;

[0015] Figure 2 The effect of LR-AFY02 on the body weight of mice; different lowercase letters indicate significant differences (P<0.05);

[0016] Figure 3 The effect of LR-AFY02 on the colon tissue of mice; A is the colon length; B is the visceral (colon) index; C is the colon coefficient; D is the number of colon tumors; different lowercase letters indicate significant differences (P < 0.05);

[0017] Figure 4 The effect of LR-AFY02 on colon histopathology in mice;

[0018] Figure 5 The effect of LR-AFY02 on the levels of serum inflammatory cytokines in mice; A is IL-1β; B is IL-6; C is TNF-α; D is NF-κB; E is iNOS; different lowercase letters indicate significant differences (P<0.05);

[0019] Figure 6 The effect of LR-AFY02 on the levels of inflammatory cytokines in the mouse colon; A is IL-1β; B is IL-6; C is TNF-α; D is NF-κB; E is iNOS; different lowercase letters indicate significant differences (P<0.05);

[0020] Figure 7 The effect of LR-AFY02 on the relative expression of inflammatory factors mRNA in mouse colon tissue; A is the relative expression of IκBβ; B is the relative expression of p65; C is the relative expression of p50; D is the relative expression of p52; different lowercase letters indicate significant differences (P < 0.05);

[0021] Figure 8 The effect of LR-AFY02 on the relative expression of mRNA of apoptosis-related factors in mouse colon tissue cells; A is the relative expression of Bid; B is the relative expression of Bcl-2; C is the relative expression of Bcl-x L Relative expression; D is the relative expression of caspase-8; different lowercase letters indicate significant differences (P<0.05). DETAILED DESCRIPTION

[0022] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0023] Example 1 Isolation and Identification of Lactobacillus rhamnosus AFY02

[0024] 1.1 Test materials

[0025] 40 mL of naturally fermented yogurt was collected from the Altay region of Xinjiang. The yogurt was placed in a sterile centrifuge tube, placed in a food sampling box, and stored in a 4°C refrigerator in the laboratory for later use.

[0026] 1.2 Separation and purification

[0027] Take 1 mL of naturally fermented 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, cultured at 37℃ for 24h-48h, and the colony morphology was observed and recorded. Colonies with different morphologies on the plate were picked for streaking separation, cultured at 37℃ for 48h, and single colonies with different morphologies on the plate were picked for streaking separation again. This was repeated 2-3 times until pure single colonies with consistent morphology were obtained.

[0028] 1.2 Identification

[0029] The DNA of the target strain was extracted, its 16S rDNA was amplified, and sent to Shenzhen BGI Co., Ltd. for sequencing. The successfully sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI. Figure 1 ), the strain is Lactobacillus rhamnosus, named Lactobacillus rhamnosus AFY02. Lactobacillus rhamnosus AFY02 was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on May 17, 2023, with the deposit number CGMCC No.27363, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0030] The specific nucleotide sequence of 16S rDNA of Lactobacillus rhamnosus AFY02 is as follows:

[0031]

[0032] Example 2 0.3% bile salt resistance test

[0033] Porcine bile salt was added to MRS-THIO medium (MRS broth containing 0.2% sodium thioglycolate) to make the concentration 0.3%, and sterilized at 121℃ for 15min. 5mL of activated bacteria were inoculated into MRS-THIO medium without bile salt (0.0%) and MRS-THIO medium containing 0.3% bile salt at a 2% (v / v) inoculation volume, respectively. Blank medium (MRS-THIO medium without bacteria) was used as control. After culturing at 37℃ for 24h, the OD values ​​of the above different concentrations of culture medium were measured respectively. 600 The bile salt tolerance of Lactobacillus rhamnosus AFY02 was calculated according to the following formula:

[0034]

[0035] The results showed that the survival rate of Lactobacillus rhamnosus AFY02 in 0.3% bile salt was 7.85%.

[0036] Example 3 Artificial Gastric Juice Tolerance Test

[0037] Preparation of artificial gastric juice: composed of 0.2% NaCl and 0.35% pepsin, weigh the NaCl and pepsin required for the test according to the corresponding mass volume ratio, adjust the pH of the prepared artificial gastric juice to 3.0 with 1 mol / L HCl, and then filter and sterilize with a 0.22 μm filter membrane for use.

[0038] In the clean bench, 5 mL of the cultured bacterial culture was taken into a 10 mL sterile centrifuge tube, centrifuged at 3000 r / min for 10 min, the upper culture medium was discarded and the bacteria were collected, and an equal volume (5 mL) of sterile physiological saline was added and mixed to make a bacterial suspension, and then 1 mL of the bacterial suspension was mixed with 9 mL of artificial gastric juice with pH = 3.0, and 1 mL of the above mixture was taken as a sample of artificial gastric juice treatment for 0 h, and the remaining 9 mL of the mixture was placed in a constant temperature water bath shaker (37 ° C, 150 r / min) and cultured for 3 h. The samples of 0 h and 3 h were diluted by 10 times gradient, and the number of viable bacteria was determined by plate coating method with appropriate gradients, and cultured on MRS solid medium at 37 ° C for 48 h, and the survival rate (%) was calculated according to the following formula.

[0039]

[0040] The test showed that the survival rate of Lactobacillus rhamnosus AFY02 in artificial gastric juice was 144.14%, proving that the strain can survive and reproduce well in an artificial gastric juice environment with a pH of 3 and has good acid resistance.

[0041] Example 4 Animal Test

[0042] 4.1 Materials and methods

[0043] 4.1.1 Materials and reagents

[0044] (1) Activation of Lactobacillus rhamnosus AFY02

[0045] Lactobacillus rhamnosus AFY02 was activated twice in MRS liquid culture medium at an inoculation rate of 2%, cultured at 37°C for 14-18 hours, centrifuged at 5000 rpm for 5 minutes, washed twice with physiological saline, collected, and centrifuged again at 5000 rpm for 5 minutes for use.

[0046] Inoculate P2 generation Lactobacillus rhamnosus AFY02 into 5 mL MRS medium at a 2% inoculum volume (100 μL), culture at 36°C for 16 h, perform gradient dilutions, calculate the stock solution concentration, and then dilute to 1×10 8 CFU / / mL (LR-AFY02 low concentration, LR-AFY02L) and 1×10 9 CFU / / mL (LR-AFY02 high concentration, LR-AFY02H), calculated according to the weight of mice and then gavage was performed.

[0047] (2) Azoxymethane, Sigma-Aldrich (Shanghai) Trading Co., Ltd.; dextran sulfate sodium, MP Biomedicals LLC, USA; enteric-coated aspirin tablets, Bayer HealthCare Co., Ltd.; interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), NF-κB, and inducible NO synthase (iNOS) ELISA kits, Shanghai ELISA Biotechnology Co., Ltd.; RNase-Free water, Beijing Solebow Technology Co., Ltd.; qPCR SYBR Green Master Mix, Shanghai Yishen Biotechnology Co., Ltd.; TRIzol Reagent and RevertAid First Strand cDNA Synthesis Kit, Thermo Fisher Scientific, USA. All other reagents were domestically produced or analytically pure.

[0048] 4.1.2 Instruments and equipment

[0049] 6D45415 upright microscope, Olympus Instrument Co., Ltd., Japan; Bioprep-24 biological sample homogenizer, Nano-300 micro-spectrophotometer, Hangzhou Aosheng Instrument Co., Ltd.; A200 gradient PCR instrument, Hangzhou Langji Scientific Instrument Co., Ltd.; VLBL0TD1 multi-function microplate reader, StepOnePlus Real-Time PCR System, Thermo Fisher Scientific (Suzhou) Co., Ltd.

[0050] 4.1.3 Methods

[0051] (1) Animal experiment design

[0052] C57BL / 6 mice (SPF grade, male, 6 weeks old) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (Animal Qualification License No.: SCXK (Xiang) 2019-0004) and randomly divided into 5 groups after 7 days of adaptive feeding in a constant temperature and humidity environment: normal group, model group, aspirin group, LR-AFY02 low concentration (LR-AFY02L) group and LR-AFY02 high concentration (LR-AFY02H) 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 LR-AFY02 group were intraperitoneally injected with AOM at a concentration of 10 mg / kg, and fed with 2.5% DSS aqueous solution at 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 67 mg / kg aspirin solution. The mice in the LR-AFY02L group and the LR-AFY02H group were gavaged with 1×10 8 CFU / kg and 1×10 9 The LR-AFY02 bacterial suspension was gavaged 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 mice was weighed and recorded weekly. After the model was established, blood was collected from the eyeballs and the mice were killed by spinal dissection, and the colon tissue of the mice was dissected for testing.

[0053] (2) Determination of organ index and colon coefficient

[0054] The colon tissue of the experimental mice was weighed, and the mouse organ index was calculated by the following formula: organ index = visceral weight (g) / mouse body weight (g) × 100. At the same time, the length of the mouse colon was measured and the occurrence of intestinal tumors was observed, and the mouse colon coefficient was calculated: colon coefficient = colon weight (mg) / colon length (cm).

[0055] (3) Colon histopathological observation

[0056] The colon tissue with a length of about 0.5 cm was immersed in a 4% paraformaldehyde fixative solution for 48 hours, dehydrated with ethanol and embedded in paraffin, cut into slices of about 2 μm to 3 μm with a microtome, stained with hematoxylin and eosin (H&E) and fixed on a slide to make mouse colon pathological tissue sections, and the morphological changes were observed under an upright microscope.

[0057] (4) Determination of levels of inflammatory-related factors in mouse serum and colon tissue

[0058] The whole blood samples of mice were collected and centrifuged at 4000r / min for 10min at 4°C to obtain serum. According to the instructions of the enzyme linked immunosorbent assay (ELISA) kit, the levels of IL-6, IL-1β, TNF-α, NF-κB and iNOS in mouse serum and colon tissue were determined.

[0059] (5) Determination of expression levels of genes related to inflammation and apoptosis pathways in mouse colon tissue

[0060] 50 mg of mouse colon tissue was weighed, total RNA was extracted using TRIzol reagent, and RNA was reverse transcribed into cDNA according to the instructions provided in the cDNA synthesis kit. The concentration and purity of RNA and cDNA were determined using a microphotometer. Amplification was performed using the StepOnePlus Real-Time PCR system under the following conditions: 95°C, 60 s; 95°C, 15 s, 40 cycles; 55°C, 30 s; 72°C, 35 s; 95°C, 30 s; 55°C, 35 s. Eef2 was used as the internal reference gene. The relative expression intensity of each gene was calculated by the method. Table 1 shows the primer sequences used in this experiment.

[0061] Table 1 Primer sequences

[0062]

[0063]

[0064] (5) Data analysis

[0065] All data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA in IBM SPSS27.0 was used for significance analysis, and GraphPad 9.3.1 was used for drawing. Different letters ad in the graphs indicate that the differences were statistically significant (P < 0.05) after Duncan's multi-range test.

[0066] 4.2 Results and Analysis

[0067] 4.2.1 Effect of LR-AFY02 on mouse body weight

[0068] The colon cancer mouse model constructed using the chemical inducer AOM / DSS can reliably reproduce the stages of intestinal tumor occurrence and development, and highly simulate the pathological conditions of human colon cancer. This model can lead to weight loss in cancer-bearing mice. During the modeling process, it was observed that the mice in the normal group were in good health, had normal eating and drinking, and had no diarrhea or blood in the stool. However, the other four groups of mice showed varying degrees of loose stools, blood in the stool, and even rectal prolapse, as well as decreased mobility, sparse and dull fur, and weight loss. Figure 2 As shown in the data, 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 had significant differences (P<0.05). After intervention with aspirin and LR-AFY02, the trend of pathological weight loss in mice was significantly alleviated.

[0069] 4.2.2 Effects of LR-AFY02 on colon length, visceral index, colon coefficient and intestinal tumors in mice

[0070] Colon length is one of the indicators used to characterize the degree of inflammation in DSS-induced colitis. Figure 3 A), the colon length of mice in the normal group was 6.65±0.31cm, that of the model group was 4.87±0.37cm, that of the aspirin group was 5.77±0.29cm, and that of the LR-AFY02L and LR-AFY02H groups were 2.12±0.25cm and 5.69±0.28cm, respectively. Among them, the colon length of mice in the model group was significantly shorter than that of the normal group (P<0.05), while LR-AFY02 intervention could alleviate the shortening of the colon of mice, and the results were significantly different (P<0.05), and its effect was close to that of the aspirin group. At the same time, Figure 3 B and 3C indicate that the visceral (colon) index and colon coefficient of the normal group, aspirin group, LR-AFY02H group, and LR-AFY02L group increased in turn, and were significantly lower than those of the model group (P<0.05). The organ coefficient is one of the basic indicators of biomedical research and an important basis for research. Inflammation and edema can lead to an increase in organ weight and organ coefficient. In addition, the AOM used in this experiment can not only induce tumors, but can even promote tumor activity. In this experiment, no intestinal tumors were found in the normal group of mice, and several tumors of different sizes were observed in the colon segments of the model group, aspirin group, and LR-AFY02 group. The model group had the largest number of tumors (10±1.92), while the aspirin group, LR-AFY02L group, and LR-AFY02H group had significantly fewer tumors, which were 3±0.99, 4±1.31, and 2±1.06, respectively ( Figure 3D). It can be seen that LR-AFY02 can effectively alleviate the shortening of the colon caused by intestinal inflammation and edema, reduce the colon coefficient of mice, reduce the visceral index and reduce the occurrence of intestinal tumors in mice.

[0071] 4.2.3 Effect of LR-AFY02 on colon histopathology in mice

[0072] 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 formation and development of intestinal adenomas. H&E staining analysis shows that ( Figure 4 ), the colon 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 LR-AFY02 group, the crypt structure was relatively intact. Compared with low concentrations of LR-AFY02, high concentrations of LR-AFY02 improved the pathological damage of colon tissue caused by AOM / DSS to a greater extent.

[0073] 4.2.4 Effects of LR-AFY02 on the levels of inflammatory cytokines in mouse serum and colon tissue

[0074] Cytokines are the main signaling molecules released by inflammatory cells and are involved in multiple functions. The main proinflammatory cytokines include IL-1β, IL-6, and TNF-α. Chronic inflammation involving proinflammatory cytokines is a promoting factor of inflammation-related colorectal cancer. Studies have shown that chronic inflammation leading to tumorigenesis is mainly related to various inflammatory cytokines and their inflammatory-related pathways. This study evaluated whether the anti-tumor activity of LR-AFY02 is related to the expression of its proinflammatory cytokines. The expression levels of IL-1β, IL-6, TNF-α, NF-κB and iNOS in mouse serum and colon tissue were analyzed by ELISA experiments. 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, immune regulation and 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. The results showed ( Figure 5AC, 6A-C), after AOM / DSS modeling, the levels of IL-1β, IL-6 and TNF-α in the serum and colon of the model group mice were significantly increased, and aspirin and LR-AFY02 could effectively reduce their expression, and the levels of serum IL-1β, IL-6 and TNF-α in the LR-AFY02H group were significantly lower than those in the LR-AFY02L group (P<0.05), indicating that high concentrations of LR-AFY02 can more significantly reduce the levels of pro-inflammatory cytokines in the serum of mice with colon cancer. At the same time, recent reports also stated that Lactobacillus plantarum L15 can significantly reduce the level of cytokine IL-1β, which indirectly provides evidence that Lactobacillus plantarum can reduce the level of inflammatory factors. 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 in cancer. Studies have shown that changes in the level of this factor can reflect the condition of patients with colon cancer.

[0075] In this study, the NF-κB concentration in the serum and colon tissue of mice in the model group was the highest ( Figure 5 D and 6D), the NF-κB concentrations in the serum of mice in the normal group, aspirin group and LR-AFY02 group were lower than those in the model group, and there were significant differences (P<0.05). Consistent with the research results of Yang Xiaodong et al., NF-κB in the lesion tissue of patients with colon cancer was higher than that in the non-lesion tissue. In addition, when NF-κB is activated, an increase in iNOS levels can be observed. At the same time, TNF-α can also activate iNOS in inflammatory responses. Studies have shown that iNOS is related to the occurrence and development of tumors and is a new target for tumor prevention and treatment. The experimental results show ( Figure 5 E and 6E), iNOS was highly expressed in the serum and colon tissue of mice in the model group, and the iNOS concentrations in the serum of mice in the normal group, aspirin group, and LR-AFY02 group were all lower than those in the model group, and there were significant differences (P<0.05). The data of this study revealed that AOM / DSS treatment can induce high expression of proinflammatory cytokines and nitric oxide synthase in mice, and activate the NF-κB inflammatory pathway, causing a sustained inflammatory response in the body. Intragastric administration of the experimental strain LR-AFY02 can effectively reduce the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in mice with colon cancer, downregulate the expression of NF-κB and iNOS, and alleviate the pathological state of inflammation. The results in mouse serum and colon tissue were consistent.

[0076] 4.2.5 Effect of LR-AFY02 on mRNA expression levels of inflammatory pathway-related factors in mouse colon tissue

[0077] Studies have shown that under AOM / DSS induction, the NF-κB signaling pathway in mice is activated, leading to increased levels of inflammation. Therefore, by inhibiting the NF-κB signaling pathway, mice can be protected from inflammatory stimulation and can intervene in the occurrence and development of colon cancer. 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, and 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, among which the phosphorylated subunit p65 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, are important biomarkers for the diagnosis and prognosis of colon cancer, while the dimer of p52 and RelB can promote the transcription of inflammation-related genes through nuclear translocation. In this study, real-time fluorescence quantitative PCR was used to analyze the mRNA expression levels of IκBβ, p65, p50 and p52 in mouse colon tissues, and the results showed that ( Figure 7 ), the mRNA expression levels of IκBβ, p65, p50 and p52 in the colon tissue of mice in the model group were the highest, which were significantly reduced in the aspirin group and the LR-AFY02 group (P<0.05), and the effect of the high-concentration LR-AFY02 gavage group was close to that of aspirin.

[0078] 4.2.6 Effect of LR-AFY02 on mRNA expression levels of apoptosis pathway-related factors in mouse colon tissue

[0079] It has been reported that NF-κB signaling can promote the establishment of an inflammatory tumor microenvironment in colon cancer, 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 (Bcl-2) and B-cell lymphoma-extra-large (Bcl-x). L ). Bid is a pro-apoptotic member of the Bcl-2 family and can induce mitochondrial apoptosis in colorectal cancer cells. LThe expression of Bid can inhibit the normal apoptosis process of tumor cells and promote their survival and development. Therefore, targeting apoptosis-related factors to intervene in colon cancer is an effective way. The experimental results showed that compared with the normal group, the pro-apoptotic factor Bid was lowly expressed in the model group, and the relative expression levels in the aspirin group and the LR-AFY02 group increased ( Figure 8 A), the difference was significant (P<0.05). L Highly expressed in the model group ( Figure 8 B and 8C), while the expression in the aspirin group and LR-AFY02 group was significantly reduced (P<0.05). In addition, as an indispensable part in the regulation and initiation of programmed cell death activation mediated by death receptors, caspase-8 is highly associated with intrinsic and extrinsic apoptosis pathways. It has been reported that dysfunction of caspase-8 may contribute to the development of malignant tumors in mice and humans. The experimental results showed that ( Figure 8 D), compared with the normal group, the relative expression level of caspase-8 mRNA in the colon tissue of the model group mice was significantly reduced under the induction of AOM / DSS (P<0.05), while the intervention of aspirin and LR-AFY02 significantly improved the low expression of caspase-8 mRNA caused by cancer in mice, and compared with the model group, the caspase-8 mRNA levels in the high and low concentration groups of LR-AFY02 were significantly different (P<0.05). The current experimental results show that LR-AFY02 can significantly upregulate the expression levels of pro-apoptotic factors Bid and caspase-8, while reducing the anti-apoptotic factors Bcl-2 and Bcl-x L expression.

[0080] In summary, this experiment used the AOM / DSS-induced mouse inflammation-related colon cancer model to study the effect and mechanism of LR-AFY02 isolated from Xinjiang fermented yogurt in intervening inflammation-related colon cancer from five aspects: mouse body weight and organ index, colon index and intestinal tumor number, colon tissue pathological analysis, serum and colon tissue inflammation-related cytokine analysis, colon tissue NF-κB and apoptosis signaling pathway related gene expression analysis. The experimental results showed that LR-AFY02 significantly improved the symptoms of weight loss, increased organ index, shortened colon and increased intestinal index caused by cancer in mice, effectively reduced the incidence of intestinal tumors in cancer-bearing mice, and improved colon tissue pathological damage (P<0.05). At the same time, the intervention of LR-AFY02 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, LR-AFY02 can also significantly downregulate the expression of pro-inflammatory factors IκBβ, p65, p50, p52 and anti-apoptotic factors Bcl-2 and Bcl-x in colon tissue at the gene level. L This study first suggested that LR-AFY02 has a significant effect in intervening in the development of inflammation-related colon cancer in mice, and revealed its specific mechanism of slowing down the development of colon cancer by improving intestinal inflammation and promoting apoptosis of intestinal tumor cells.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of acid-resistant rhamnosus Lactobacillus AFY02 in the preparation of a product for treating colon cancer; the acid-resistant rhamnosus Lactobacillus AFY02 is classified and named Lacticaseibacillus rhamnosus, the preservation unit is the General Microbiology Center of the China Microbiological Culture Collection Administration, the preservation date is May 17, 2023, the preservation number is CGMCC No. 27363, and the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the 16S rDNA of the acid-resistant rhamnosus Lactobacillus AFY02 has the nucleotide sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The product is a microbial preparation or a medicine.

3. The use according to claim 1, characterized in that: The raw materials of the product include acid-resistant Lactobacillus rhamnosus AFY02.

4. The use according to claim 1, characterized in that: The acid-resistant Lactobacillus rhamnosus AFY02 can improve the weight loss, increased organ index, shortened colon and increased intestinal index caused by colon cancer, and regulate NF-κB and cell apoptosis pathways.

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