Application of lactobacillus paracasei in preparation of medicine for repairing intestinal barrier function and treating AML (acute myeloid leukemia)

Abstract

The invention belongs to the field of medicine, and discloses application of lactobacillus paracasei in preparation of medicine for repairing intestinal barrier function and treating AML (acute myeloid leukemia). Generally, gastrointestinal mucosal barriers, immune systems and nutrient absorption of AML patients receiving induced chemotherapy are severely damaged, and the factors severely destroy intestinal microbial balance, so that the progress of the disease course is promoted, and the occurrence rate of complications is increased. The invention finds that the lactobacillus paracasei has a remarkable effect in preparing the medicine for repairing the intestinal barrier function and treating AML (acute myeloid leukemia), and particularly, the lactobacillus paracasei can repair the intestinal barrier function and enhance the curative effect of cytarabine.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to application of Lactobacillus paracasei in preparing a medicine for repairing intestinal barrier function and treating AML. Background Art

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous and complex malignant invasive tumor. It is the most typical type of leukemia in adults. It is characterized by excessive proliferation, differentiation failure and apoptosis disorder of bone marrow primitive cells, which leads to abnormal aggregation of primitive cells in the bone marrow and peripheral blood. AML is the most common type of acute leukemia in adults, accounting for about 70% of adult leukemia. Its incidence is closely related to age, and the incidence rate of patients over 60 years old increases significantly. Although the continuous improvement of treatment regimens and the development of new drugs have improved the prognosis and survival rate of AML patients, drug resistance and relapse after treatment are still the main problems faced by most AML patients. Therefore, finding new biomarkers or therapeutic targets to judge prognosis and optimize individualized treatment strategies is of great significance for the treatment of AML.

[0003] AML patients generally have intestinal microbial imbalance. Studies have found that the intestinal microbial composition of newly diagnosed AML patients has changed significantly compared with the healthy control group, and its diversity has been significantly reduced. Wang et al. found that at the phylum level, the Bacteroidetes were enriched in the feces of AML patients, and the abundance of Firmicutes was significantly reduced. At the genus level, the Bacteroidetes genus was enriched, and the abundance of Faecalibacterium, Roseburia, and Bifidobacterium was significantly reduced. Xu et al. found through linear discriminant analysis that Collinsella and Red Stinkbugs can be used as biomarkers for AML patients. In addition, AML patients generally need to receive multiple antibiotics during treatment, which will further seriously disrupt the balance of intestinal microbiota. Wang et al. confirmed in animal models that intestinal microbiota imbalance caused by antibiotic use will accelerate the progression of AML in mice. The above research results all indicate that intestinal microbiota may play an important role in the occurrence and development of AML. In addition, the researchers also found that in the feces of AML patients, the butyrate produced by intestinal microorganisms was significantly reduced; further studies found that the damaged intestinal barrier of AML model mice accelerated the entry of lipopolysaccharide (LPS) into the blood, and the increased LPS content aggravated the progression of AML both in vivo and in vitro; butyrate can repair intestinal barrier damage and inhibit the absorption of LPS by AML mice, delaying the progression of AML. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide application of Lactobacillus paracasei in preparing a drug for repairing intestinal barrier function and treating AML.

[0005] To achieve the purpose of the present invention, the present invention provides the following technical solutions: The invention provides application of Lactobacillus paracasei in preparing a medicine for repairing intestinal barrier function.

[0006] Furthermore, in some embodiments of the present invention, the active ingredient in the drug for repairing intestinal barrier function is Lactobacillus paracasei.

[0007] The invention also provides the use of Lactobacillus paracasei in preparing a medicine for treating AML in patients.

[0008] Furthermore, in some embodiments of the present invention, the patient has a damaged intestinal barrier.

[0009] Cytarabine (pyrimidine nucleoside analogue) is an antimetabolite antitumor drug that inhibits the synthesis of deoxyribonucleic acid. It specifically acts on the S phase of the cell cycle, interfering with cell proliferation, thereby achieving an anti-tumor effect. Continuous infusion of cytarabine (Ara-C) plus anthracycline drugs is the main means of induction therapy for AML. Cytarabine can interfere with DNA synthesis and is also an antimetabolite.

[0010] AML patients who receive induction chemotherapy usually have severely damaged gastrointestinal mucosal barriers, immune systems, and nutrient absorption. These factors seriously disrupt the balance of intestinal microorganisms, thereby promoting disease progression and increasing the incidence of complications. In addition, RASHIDI A et al. found that even after discharge, the damaged intestinal microbiota of AML patients who received chemotherapy during hospitalization could not be restored to their original state. MALARD F et al. found through research that the microbiota imbalance that occurred in AML patients after receiving intensive chemotherapy and antibiotic treatment could be effectively restored after autologous FMT treatment.

[0011] After anticancer chemotherapy and radiotherapy, epithelial cell division is inhibited and the renewal capacity is lost, leading to intestinal mucosal damage. Intestinal mucosal damage is one of the common side effects of cytarabine chemotherapy, but there are few effective drugs for intestinal mucosal damage.

[0012] Furthermore, in some embodiments of the present invention, the patient is an AML patient who has received chemotherapy.

[0013] Furthermore, in some embodiments of the present invention, the patient has taken antimetabolite anti-tumor drugs.

[0014] Furthermore, in some embodiments of the present invention, the antimetabolite antitumor drug is cytarabine. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1This is a schematic diagram of the in vitro experimental results for verifying the probiotic properties of Lactobacillus paracasei in Example 1 of the present invention; Figure 2 The results of gastrointestinal toxicity of mice in different experimental groups in Example 2 of the present invention and the experimental results of Lactobacillus paracasei alleviating intestinal barrier damage caused by cytarabine treatment; Figure 3 The following are the results of RT-qPCR analysis of representative common bacterial genera selected in Example 2 of the present invention. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. Additional aspects and advantages of the present invention will be given in part in the following description, and part will become apparent from the following description, or will be understood through the practice of the present invention. It should be understood that the following description is only used to explain the present invention and is not intended to limit the present invention.

[0017] As used herein, the terms "comprises," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0018] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0019] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0020] Singular forms include plural references unless the context clearly indicates otherwise. "Optional" or "either" means that the subsequently described event or incident may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0021] Approximate terms in the specification and claims are used to modify quantities, indicating that the present invention is not limited to the specific quantity, but also includes acceptable and modified parts close to the quantity without causing changes in the relevant basic functions. Accordingly, the use of "about", "approximately", etc. to modify a numerical value means that the present invention is not limited to the exact numerical value. In some examples, the approximate terms may correspond to the accuracy of the instrument for measuring the numerical value. In the specification and claims of this application, range limitations can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0022] The indefinite articles "a" and "an" before the elements or components of the present invention have no limitation on the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the quantity obviously refers to the singular form only.

[0023] In addition, the descriptions of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" described below mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0024] Example 1 In vitro experiments: verifying the probiotic properties of Lactobacillus paracasei 1. Growth curve of Lactobacillus paracasei The growth curve was determined by turbidimetry. Since the concentration of the bacterial suspension is proportional to the turbidity, the optical density of the bacterial suspension can be measured by a spectrophotometer to infer the concentration of the bacterial solution. Lactobacillus paracasei was activated overnight, 1 mL was inoculated into 100 mL of TSB liquid culture medium, and cultured anaerobically at 37°C. The optical density was measured at a wavelength of 600 nm at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h. A growth curve was drawn with time as the horizontal axis and optical density as the vertical axis.

[0025] 2. Acid resistance test Adjust the pH of PBS buffer to 2, 3, 4, 5, and 7 respectively. Prepare MRS solid culture medium. Sterilize and pour the plate. Seal the plate with sealing film and place it in a 4℃ refrigerator. Take 100 μL of Lactobacillus paracasei liquid and add 5 mL of MRS liquid culture medium. Culture in a 37℃ incubator overnight.

[0026] Take 100 μL of Lactobacillus paracasei liquid, centrifuge at 2500 rpm for 4 min, and discard the supernatant. Add PBS buffer with pH values ​​of 2, 3, 4, 5, and 7, and place in a 37°C incubator for 4 hours. Use PBS buffer to dilute 10 1 , 10 3 , 10 5 Multiple times (take 100 μL and add it to 900 μL PBS buffer, then take 10 μL and add it to 990 μL PBS, then take 10 μL and add it to 990 μL PBS) 10 EP tubes for each gradient. Mix well and take 10 μL to plate, then culture in a 37℃ incubator overnight. Count the viable bacteria and keep good experimental records.

[0027] 3. Bile salt tolerance test (1) MRS liquid culture medium with bile salts. No. 1 (without bile salts), No. 2 (with 0.1% bile salts), No. 3 (with 0.2% bile salts), No. 4 (with 0.3% bile salts), and sterilized MRS solid culture medium.

[0028] (2) After sterilization, pour the plates, seal them with sealing film and place them in a 4℃ refrigerator.

[0029] (3) Take 100 μL of Lactobacillus paracasei liquid and add it to 5 mL of MRS liquid culture medium with different bile salt concentrations, and culture it in a 37°C incubator overnight.

[0030] (4) In the morning, take out the cultured bacterial solution, mix it well, and apply 10 μL on the solid culture medium. Incubate in a 37℃ incubator for 12-48 hours and count the live bacteria. Keep good experimental records.

[0031] 4. Antibacterial Experiment Lactobacillus paracasei was inoculated and cultured in MRS medium for 16 h, and the supernatant was removed after centrifugation at 8000 rpm for 10 min. Another 20 μL of 7 pathogenic bacteria (Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7, Shigella dysenteriae, Staphylococcus aureus, Salmonella enteritidis, Shigella flexneri, and hemolytic Streptococcus) cultured overnight were spread on LB solid medium plates, placed on Oxford cups, and 250 μL of bacterial supernatant was drawn into the Oxford cups. After incubation at 37°C for 8 h, the diameter of the inhibition zone was measured.

[0032] 5. Antibiotic resistance test The antibiotic susceptibility of probiotics to penicillin (10 U), erythromycin (15 μg), chloramphenicol (30 μg), cefazolin (30 μg), norfloxacin (10 μg), meropenem (10 μg), vancomycin (30 μg), gentamicin (10 μg), and kanamycin (30 μg) was determined by disk diffusion method.

[0033] The concentration of the activated bacterial solution was adjusted to 10 6 CFU / mL; Use sterile cotton swabs to evenly spread the bacterial solution on the surface of MRS plates; Place the plates at room temperature for 10 minutes to dry; Use sterile tweezers to pick up drug-sensitive paper and evenly place it on the surface of the plates; Incubate the plates in a 37°C constant temperature incubator for 24 hours, and measure and record the diameter of the inhibition zone of each drug-sensitive paper. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as quality control strains. The test results were determined according to the standards established by the Clinical and Laboratory Standards Institute (CLSI) of the United States to determine the drug sensitivity of the strains. The results were expressed as sensitive (S), intermediate (I) and resistant (R).

[0034] 6. Antioxidant test (1) Determination of DPPH free radical scavenging ability: Add 2 mL (0.2 mmol / L) DPPH methanol solution to 2 mL bacterial culture supernatant, react at room temperature in the dark for 30 min, take the supernatant to measure OD at 517 nm, and use 2 mL deionized water plus DPPH methanol solution as a control: DPPH free radical scavenging rate = [1-A517 (sample) / A517 (blank)] × 100% (2) Determination of hydroxyl radical scavenging ability: Take 1 mL of 2 mmol / L FeSO4 solution, 1 mL of 6 mmol / L H2O2, and 1 mL of 6 mmol / L salicylic acid, add 1 mL of bacterial culture supernatant, let stand at room temperature for 30 min, use deionized water as blank control, measure the absorbance at 510 nm, and calculate the scavenging rate of hydroxyl free radicals: Hydroxyl radical scavenging rate = [1-A510 (sample) / A510 (blank)] × 100% (3) Determination of superoxide free radical scavenging ability: Add 2 mL of 150 mmol / L Tris-HCl solution (pH=8.0) and 1 mL of 1.2 mmol / L pyrogallol solution to 0.5 mL of bacterial culture solution, react at room temperature for 30 min, and measure the absorbance at 330 nm: Superoxide radical scavenging rate = [1-(A11-A10) / (A01-A00) × 100% A00: does not contain sample and pyrogallol; A01: does not contain sample and contains pyrogallol A10: Contains sample but not pyrogallol; A11: Contains sample and pyrogallol (4) Fe 2+ Determination of chelating capacity: Add 0.1 mL of 0.4% ferrous sulfate solution to 0.5 mL of sample solution, gently invert and mix, then add 0.1 mL of 1% VC and 1 mL of 0.2 mol / L NaOH solution, react at room temperature for 20 min, then add 1 mL of 10% trichloroacetic acid, centrifuge at 6,000 rpm at 4°C for 10 min to remove protein, take 0.4 mL of the above solution and add 4 mL of 0.1% o-phenanthroline, react at room temperature for 10 min, and measure the absorbance at 536 nm: Fe 2+ Chelating capacity = [A blank - A sample / A blank] × 100% (5) Determination of total reducing power Take 1 mL of bacterial culture, add 1 mL of phosphate buffer (pH 6.6) and 1 mL of 1% K3[Fe(CN)6] solution, mix thoroughly by inverting, keep warm at 50 °C in a constant temperature box for 2 min, add 1 mL of 10% trichloroacetic acid solution, shake and mix, take 1 mL of the mixed solution, add 4 mL of deionized water and 0.4 mL of 0.1% FeCl3 solution, and let stand for 10 min. Then use deionized water as a blank and measure the absorbance at 700 nm. The larger the absorbance value, the stronger the reducing power of the analyte.

[0035] In the above experiment, Lactobacillus paracasei reached the logarithmic growth phase within 6 hours. At 18 hours, its colony abundance reached a peak, indicating that it has good growth ability and is suitable for further experimental applications ( Figure 1 E, F). In the acid and bile salt tolerance experiments, as the solution acidity or bile salt concentration increased, the Lactobacillus paracasei bacterial colonies remained active. Specifically, at pH 1.0, there were still 10 4 CFU can survive, while at 0.3% bile salt concentration, 10 3 CFU survival ( Figure 1 GI). Antioxidant experiments showed that Lactobacillus paracasei showed significant scavenging ability, reaching 57.67% DPPH free radical scavenging rate, 63.47% hydroxyl free radical (OH) scavenging rate, and 88.17% superoxide anion free radical (O2 - ) removed, 15.77% of ferrous ions (Fe2 + ) chelation, the total reduction capacity OD value is 0.89 ( Figure 1 J). In the antibacterial test, it was found that Lactobacillus paracasei was sensitive to a variety of pathogens, including Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7, Shigella dysenteriae, Staphylococcus aureus, Salmonella enteritidis, Shigella flexneri and hemolytic Streptococcus, with inhibition zone diameters of 1.99 cm, 2.03 cm, 1.89 cm, 1.77 cm, 2.02 cm, 1.81 cm, 1.94 cm and 1.63 cm, respectively. In addition, in the antibiotic resistance assessment, Lactobacillus paracasei showed sensitivity to penicillin, erythromycin, chloramphenicol, ampicillin, co-trimoxazole, ceftriaxone and tetracycline, showing inhibition zones with diameters of 2.30 cm, 3.35 cm, 2.68 cm, 2.80 cm, 2.21 cm, 2.59 cm and 1.72 cm ( Figure 1 L, M). Taken together, these findings suggest that Lactobacillus paracasei has promising probiotic properties.

[0036] Example 2 (I) Study on the disruption of intestinal flora homeostasis and intestinal barrier by cytarabine chemotherapy in AML mice 1. Model selection: acute myeloid leukemia model.

[0037] Female C57BL / 6 mice were purchased from Corresponding, housed in specific pathogen-free microisolators, and used as recipients for human cell transplantation at 6–8 weeks of age.

[0038] Add the C1498-LUC cell line to DMEM culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and culture in a 37°C, 5% CO2 cell culture incubator. Replace the culture medium every 2 days, and subculture after the C1498 cells have grown to 90%. Transfer all the cells in the culture flask to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 1 mL of culture medium to the centrifuge tube and gently blow to resuspend the cells. Transfer the cell suspension to a new culture flask and subculture at a ratio of 1:2. Count the number of live cells with 2% trypan blue and adjust the density to 1 × 10 with PBS. 6 / mL.

[0039] Inject 200 μL of 1.0 × 10 6 C1498-LUC cells resuspended in phosphate-buffered saline (PBS) were added to C57BL / 6 mice and the leukocyte levels were measured two weeks later.

[0040] 2. Animal grouping: Thirty-six 6-8 week old SPF female C57BL / 6 mice were selected and divided into three groups according to the random number table method, with 12 mice in each group. The mice were housed at 6 mice per cage and fed with corresponding diets.

[0041] (1) Group C: Healthy control group (2) Group M: Model group (tail vein injection of C1498 cells) (3) MA group: model + cytarabine group (tail vein injection of C1498 cells + cytarabine treatment) 3. Cytarabine administration strategy: (1) Cytarabine administration strategy: In C57BL / 6 mice, 1X10 6 / mL murine leukemia C1498 cells were injected into the tail vein to establish the C1498 leukemia model. C1498 cells were injected on day 0, and cytarabine (100 mg / kg) was injected into the leukemia mice by intraperitoneal injection for five consecutive days starting from day 21. The peripheral blood, bone marrow, liver and spleen organs were analyzed and compared, and the survival time was evaluated. The mice were killed on day 28 and the blood, bone marrow, spleen, liver and colon of the mice were analyzed.

[0042] 4. Experimental content: (1) Compare the general conditions of each group of mice: observe the diet, spirit, hair, body weight and survival status of each group of mice, and draw a survival curve after the experiment.

[0043] (2) Comparison of disease progression in each group of mice: a. Blood and bone marrow smears and staining to examine tumor cell infiltration.

[0044] b. Measure the size and weight of the spleen, and observe the infiltration and proliferation of tumor cells in the liver and spleen by H&E staining.

[0045] c. Ki67 staining was used to detect the proliferation of mouse spleen tumor cells.

[0046] d. In vivo imaging of mice to detect tumor burden.

[0047] e. WB was used to detect the expression levels of apoptotic proteins such as BCL-2 and BAX in the spleen of mice.

[0048] (3) Comparison of intestinal flora differences among the groups of mice: 16S rRNA sequencing was used to analyze the intestinal flora differences among the groups of mice, and their alpha diversity, beta diversity, and species abundance were analyzed.

[0049] (4) Comparison of intestinal barrier conditions of mice in each group: a. The length of colon in each group of mice was recorded.

[0050] b. H&E staining was used to detect the mouse colon tissue structure.

[0051] c. FITC-dextran was used to detect intestinal epithelial permeability.

[0052] d. WB detection of tight junction proteins claudin-1 and occludin levels in mouse colon.

[0053] e. Immunofluorescence detection of tight junction protein ZO-1 levels in mouse colon.

[0054] f. ELISA was used to detect the concentration of LPS in mouse feces, colon and blood.

[0055] g. WB was used to detect the expression levels of LPS downstream signaling pathway proteins TLR4, Myd88, p65, p-p65, and IκBα in the colon of mice.

[0056] h. Q-PCR was used to measure the levels of inflammatory factors IL-6, IL-1β, and TNF-α in mouse colon tissue.

[0057] The results showed that the cytarabine treatment group significantly reduced the tumor burden in acute myeloid mice compared with the untreated acute myeloid group (2.38E+09 vs. 1.73E+07, p<0.0001). In addition, we observed a significant decrease in spleen size and weight in treated mice (acute myeloid group vs. chemotherapy group = 0.22 vs. 0.14, p<0. 01). We performed H&E staining of spleen and liver tissues to evaluate leukemic cell infiltration. In addition, we evaluated the proliferation of leukemic cells in the spleen using Ki67 immunohistochemical staining. Consistently, treatment with cytarabine resulted in a significant decrease in leukemic cell infiltration and proliferation.

[0058] In addition, some clinical trial reports showed that most patients with acute myeloid leukemia exhibited gastrointestinal adverse reactions when receiving cytarabine chemotherapy. We used kaolin experiments to verify its effect on vomiting reactions. The results showed that the intake of kaolin on the first day in the chemotherapy group was significantly higher than that in the disease group (acute myeloid leukemia group vs. chemotherapy group = 1.77% vs. 7.91%, p < 0.0001). In addition, we comprehensively evaluated the intestinal barrier function of the acute myeloid leukemia mouse model treated with cytarabine. Our results showed that the colon length of acute myeloid leukemia mice treated with cytarabine was significantly reduced compared with the untreated acute myeloid leukemia group (7.14 vs. 6.07, p < 0.001), suggesting a potential association between cytarabine treatment and gastrointestinal dysfunction. After chemical intervention, H&E staining and AB-PAS histological examination staining showed that the intestinal barrier of acute myeloid leukemia mice was significantly damaged, and the abundance of goblet cells was significantly reduced. To further understand the changes in intestinal permeability, we evaluated the expression levels of key tight junction proteins in the intestine. The results showed that the expression of these tight junction protective proteins was significantly downregulated in the chemotherapy group compared with the acute myeloid leukemia group, including ZO-1, Occludin (p < 0.01), and Claudin-1 (p < 0.05). In addition, we used RT-qPCR analysis to evaluate the colonic inflammation of mice, and we observed significantly increased levels of inflammatory cytokines such as IL-1β, IL-6, TNF-α, and IL-6 in the chemotherapy group compared with the acute myeloid leukemia group (p < 0.05). Previous studies have confirmed that increased levels of lipopolysaccharide in the blood of mice promote the progression of acute myeloid leukemia after intestinal barrier damage. As a result, we observed a significant increase in lipopolysaccharide after cytarabine treatment compared with untreated acute myeloid leukemia mice (p < 0.01). Then, we found that inflammatory factors such as IL-1β, IL-6, and TNF-α (p < 0.01) were significantly increased in the peripheral blood of mice after cytarabine treatment. These findings suggest that although cytarabine exerts antitumor effects, it also damages the intestinal barrier and causes intestinal inflammation, potentially impairing its antitumor efficacy.

[0059] Next, we analyzed the intestinal microbial composition of mice in different groups and found that there was no significant difference in the a diversity of intestinal microbial communities between these groups (p>0.05). The three groups of samples showed obvious clustering, indicating that there were differences in microbial diversity between the control group and the acute myeloid group and chemotherapy group. In addition, the use of Venn diagrams showed that the microbial composition of acute myeloid mice after chemotherapy was significantly changed compared with the control group. The results of LEfSe analysis further confirmed that the intestinal microbial composition of mice in the disease group was different from that in the chemotherapy group. To determine the specific changes in bacteria, we examined the relative abundance of these classifications. At the phylum level, after cytarabine treatment, the abundance of Firmicutes in the chemotherapy group was lower than that in the acute myeloid group (61.88% vs. 37.20%, p>0.05), and the abundance of Proteobacteria was significantly higher than that in the acute myeloid group (0.32% vs. 10.69%, p<0.01). However, no significant statistical differences were observed in the Bacteroidetes phylum (p>0.05). At the genus level, cytarabine treatment led to a decrease in the abundance of probiotic bacteria, such as Lactobacillus (p < 0.05), and an increase in the abundance of bacteria that are harmful to the human body, such as Parasutterella (p>0.05), indicating that the administration of cytarabine aggravated the imbalance of intestinal microbiota in acute myeloid mice.

[0060] 2. Proof that Lactobacillus paracasei repairs intestinal barrier function and enhances the efficacy of cytarabine 1. Model selection: acute myeloid leukemia model.

[0061] 2. Animal grouping: Forty-eight 6-8 week old SPF female C57BL / 6 mice were selected and divided into four groups according to the random number table method, with 12 mice in each group. The mice were housed in a cage with 6 mice and fed with corresponding diets.

[0062] (1) Group C: Healthy control group (2) Group A: Model group (tail vein injection of C1498 cells) (3) AA group: model + cytarabine group (tail vein injection of C1498 cells + cytarabine treatment + saline treatment) (4) AAL group: model + cytarabine + Lactobacillus paracasei group (tail vein injection of C1498 cells + cytarabine treatment + Lactobacillus paracasei treatment) 3. Cytarabine administration and single bacterial transplantation strategy: (1) Cytarabine administration strategy: In C57BL / 6 mice, 1X10 6 / mL murine leukemia C1498 cells were injected into the tail vein to establish the C1498 leukemia model. C1498 cells were injected on day 0, and the number of white blood cells was detected on day 14 to predict the progression of the disease in mice. Starting from day 21, cytarabine (100 mg / kg) was injected into leukemia mice by intraperitoneal injection for five consecutive days, and the analysis and comparison of indicators such as white blood cells, bone marrow, liver and spleen organs and the evaluation of survival time were performed. The mice were killed on day 28 and the blood, bone marrow, spleen, liver and colon of the mice were analyzed.

[0063] (2) Single bacteria transplantation strategy: Lactobacillus paracasei suspension (100 μL per mouse) was transplanted once every two days by oral gavage. After the model was established, the mice were killed and their blood, bone marrow, spleen, liver, and colon were analyzed.

[0064] 4. Experimental content: (1) Compare the general conditions of each group of mice: observe the diet, spirit, hair, body weight and survival status of each group of mice, and draw a survival curve after the experiment.

[0065] (2) Comparison of intestinal flora differences among the groups of mice: Q-PCR was used to measure the mRNA expression levels of seven common bacterial genera (Clostridium, Bacteroides, Lactobacillus, Akkermansia, Bifidobacterium, Enterococcus, and Enterobacter) in the feces of the mice in each group.

[0066] (3) Comparison of disease progression in each group of mice: a. Blood and bone marrow smears and staining to examine tumor cell infiltration.

[0067] b. Measure the size and weight of the spleen, and observe the infiltration and proliferation of tumor cells in the liver and spleen by H&E staining.

[0068] c. Ki67 staining was used to detect the proliferation of mouse spleen tumor cells.

[0069] d. In vivo imaging of mice to detect tumor burden.

[0070] e. WB was used to detect the expression levels of apoptotic proteins such as BCL-2 and BAX in the spleen of mice.

[0071] (4) Comparison of intestinal barrier conditions of mice in each group: a. The length of colon in each group of mice was recorded.

[0072] b. H&E staining was used to detect the mouse colon tissue structure.

[0073] c. FITC-dextran was used to detect intestinal epithelial permeability.

[0074] d. WB detection of tight junction proteins claudin-1 and occludin levels in mouse colon.

[0075] e. Immunofluorescence detection of tight junction protein ZO-1 levels in mouse colon.

[0076] f. ELISA was used to detect the concentration of LPS in mouse feces, colon and blood.

[0077] g. WB was used to detect the expression levels of LPS downstream signaling pathway proteins TLR4, Myd88, p65, p-p65, and IκBα in the colon of mice.

[0078] h. Q-PCR was used to measure the levels of inflammatory factors IL-6, IL-1β, and TNF-α in mouse colon tissue.

[0079] Since the abundance of lactobacilli in the feces of acute myeloid mice was significantly reduced after cytarabine treatment, and considering the significant improvement of chemotherapy efficacy by Lactobacillus paracasei, we attempted to investigate the effect of Lactobacillus paracasei on the outcome of cytarabine treatment. The results showed that the tumor burden in the probiotic group was significantly reduced compared with the acute myeloid and chemotherapy groups (chemotherapy group vs. probiotic group = 2.71E+07 vs. 2.47E+06, p > 0.05). In addition, the spleen size and weight of mice in the probiotic group were reduced (chemotherapy group vs. probiotic group = 0.16 vs. 0.10, p < 0.05). To evaluate the infiltration of leukemic cells in the spleen and liver of mice, we used H&E staining. In addition, Ki67 immunohistochemical staining and TUNEL immunofluorescence staining were used to evaluate the proliferation and apoptosis of leukemic cells in the spleen. Consistent with our hypothesis, the intervention of Lactobacillus paracasei reduced leukemic cell infiltration and proliferation and increased cell apoptosis. These findings suggest that Lactobacillus paracasei enhances the efficacy of cytarabine in the treatment of acute myeloid leukemia.

[0080] Subsequently, we carefully examined the gastrointestinal toxicity of mice in different experimental groups. Notably, the results showed that Lactobacillus paracasei had a significant alleviating effect on nausea and vomiting after chemotherapy, and the kaolin intake on the first day in the probiotic group was significantly lower than that in the chemotherapy group (8.72% vs. 4.64%, p < 0.01) ( Figure 2 A). We also further evaluated the intestinal barrier integrity of acute myeloid mice. Our findings showed that the length of the large intestine in the probiotic group was significantly longer than that in the chemotherapy group (5.51 vs. 7.14, p < 0.01) ( Figure 2 B, C). In addition, histological analysis using H&E and AB-PAS staining showed that Lactobacillus paracasei alleviated the intestinal barrier damage induced by cytarabine treatment, as manifested by a significant increase in the number of goblet cells ( Figure 2D). In addition, we observed that Lactobacillus paracasei significantly restored the expression of key tight junction proteins in the mouse intestine, including Occludin (p < 0.001), Claudin-1 (p < 0.001), and ZO-1, indicating that Lactobacillus paracasei has a restorative effect on the intestinal barrier function of chemotherapy mice ( Figure 2 EH).

[0081] The levels of lipopolysaccharide in the blood of mice in each group were further studied, and it was found that the levels of peripheral blood lipopolysaccharide in the probiotic group were significantly lower than those in the chemotherapy group (p < 0.01). Since lipopolysaccharide is an agonist of TLR4, we hypothesized that Lactobacillus paracasei may act through the TLR4 / NF-kB inflammatory pathway. Therefore, Western blot further identified the key proteins of the colon TLR4 / NF-kB inflammatory pathway. The results showed that the expression of TLR4 (p < 0.01), MyD88 (p < 0.05), and p-NF-kB (p < 0.01) in the colon of mice in the probiotic group was significantly increased compared with the chemotherapy group. In addition, Lactobacillus paracasei significantly reduced the levels of inflammatory cytokines in the colon and peripheral blood, such as IL-1β, IL 6, TNF-α, and TNF-α (p < 0.05). These results indicate that Lactobacillus paracasei inhibits colon inflammation caused by cytarabine through the TLR 4 / NF-kB inflammatory pathway.

[0082] Finally, we selected some representative common bacterial genera for RT-qPCR analysis. The results showed that Lactobacillus paracasei could reverse the imbalance of microbiota caused by chemotherapy drugs. The abundance of Lactobacillus (p < 0.05) in the feces of mice in the probiotic group was significantly increased, while the abundance of Bacteroides (p < 0.001), Enterococcus (p < 0.1) and Clostridium (p < 0.5) was significantly decreased (Figure 3A-E).

[0083] Those skilled in the art will easily understand that the above descriptions are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of Lactobacillus paracasei in preparing a drug for repairing intestinal barrier function, characterized in that: The active ingredient in the drug for repairing intestinal barrier function is Lactobacillus paracasei.

2. The application of Lactobacillus paracasei in preparing a drug for treating AML in patients, characterized in that: The patient has a damaged intestinal barrier.

3. the application of Lactobacillus paracasei in preparing patient treatment AML medicine according to claim 2, is characterized in that, The patient is an AML patient who has received chemotherapy.

4. The application of Lactobacillus paracasei in preparing a patient treatment AML medicine according to claim 3, is characterized in that, The patient had taken antimetabolite anti-tumor drugs.

5. The application of Lactobacillus paracasei in preparing a patient treatment AML medicine according to claim 4, characterized in that, The antimetabolite antitumor drug is cytarabine.

6. Application of Lactobacillus paracasei in preparing a drug for repairing intestinal barrier function and treating AML in patients, characterized in that: The patient is an AML patient who has received chemotherapy.

7. The application of Lactobacillus paracasei in preparing a drug for repairing intestinal barrier function and treating AML in patients according to claim 6, characterized in that, The patient had taken antimetabolite anti-tumor drugs.

8. The application of Lactobacillus paracasei in preparing a drug for repairing intestinal barrier function and treating AML in patients according to claim 7, characterized in that, The antimetabolite antitumor drug is cytarabine.

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