Application of metabolite tauroursodeoxycholic acid of parabacteroides faecalis flora in preparation of medicine for treating graft-versus-host disease

Abstract

The invention relates to application of metabolite taurine ursodeoxycholic acid of paracteroides faecalis in preparation of a medicine for treating graft-versus-host disease. The parabacteroides faecalis is used as a single strain for coprophilous fungus transplantation to effectively treat intestinal GVHD, the key metabolite taurine ursodeoxycholic acid can have the effect of inhibiting allogeneic T cell proliferation and inflammatory response in vitro and in an animal model, and the survival rate and intestinal microenvironment of GVHD mice are effectively improved. Therefore, a brand new treatment possibility is provided for GVHD treatment in transplanted leukemia patients. Compared with the traditional FMT, the metastatic therapy has the advantages of definite components, standardization, low risk and the like, and shows a good application prospect.

Description

Technical Field

[0001] The present invention relates to the application of Parabacteroides faecalis metabolites, and in particular to the application of tauroursodeoxycholic acid, a metabolite of Parabacteroides faecalis flora, in the preparation of a drug for treating graft-versus-host disease. Background Art

[0002] Acute leukemia is a major disease that seriously endangers human life and health, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the main method to cure acute leukemia. Graft-versus-host disease (GVHD) is a common complication after allo-HSCT, and it is also the reason that hinders the widespread application of allo-HSCT and causes non-relapse death after transplantation. Among the organs commonly affected by acute GVHD (aGVHD), intestinal GVHD seriously affects the quality of life and survival rate of transplant patients. Clinically, patients with hormone-refractory intestinal GVHD are often difficult to cure and have a poor prognosis, with an overall survival rate of only 5-30%.

[0003] Studies in recent years have shown that intestinal flora plays an important role in the occurrence and development of intestinal GVHD. Pretreatment methods such as radiotherapy and chemotherapy before transplantation can seriously disrupt the balance of intestinal flora, reduce its diversity, and thus aggravate GVHD symptoms. Fecal microbiota transplantation (FMT) restores the balance of intestinal flora by introducing fecal flora from healthy donors into the patient's intestines. A number of international clinical studies, including the inventor's previous studies, have shown that the reduction in intestinal flora diversity in patients after Allo-HSCT is significantly associated with the severity of intestinal GVHD. The applicant's team has found that fecal microbiota transplantation (FMT) has a good effect in anti-GVHD treatment. In addition to treating hormone-refractory intestinal acute GVHD, it has also shown efficacy in patients with hormone-refractory chronic GVHD. At the same time, the side effects related to treatment are low, and no treatment-related deaths occur. It is a safe and effective emerging anti-GVHD treatment. However, FMT treatment currently faces some technical challenges. First, the flora composition in FMT is complex and difficult to standardize, resulting in inconsistent treatment effects. Secondly, FMT may introduce pathogens or induce immune rejection reactions, which poses a safety hazard to the treatment method. Finally, differences in the source of healthy donors will affect the treatment effect. Postbiotics, as a new treatment method, can effectively overcome these technical challenges. Postbiotics are metabolic end products produced by the flora during metabolism and are used to treat diseases. Postbiotics have many significant advantages in treatment. First, postbiotics do not contain live microorganisms, so there is no risk of introducing pathogens or inducing immune rejection reactions, thereby improving the safety of treatment. Secondly, the specific ingredients and mechanisms of action of postbiotics are relatively clear, which can more effectively and specifically solve patients' clinical problems, regulate the intestinal microenvironment, and treat GVHD. Finally, postbiotics can be manufactured through precisely controlled production processes, and their ingredients can be standardized to ensure consistency in treatment effects. Summary of the invention

[0004] The present invention aims to provide a new application of tauroursodeoxycholic acid, a metabolite of Parabacteroides merdae (P.merdae) flora.

[0005] Specifically, the present invention provides the use of tauroursodeoxycholic acid, a metabolite of the Parabacteroides faecalis flora, in a drug for treating graft-versus-host disease.

[0006] The present invention proposes that the secondary conjugated bile acid tauroursodeoxycholic acid (TUDCA) of the Parabacteroides Merdae flora is used as a postbiotic for the treatment of GVHD. Studies have shown that fecal microbiota transplantation of Parabacteroides Merdae as a single strain is effective in the treatment of intestinal GVHD, among which the key metabolite TUDCA can inhibit allogeneic T cell proliferation and inflammatory response both in vitro and in animal models, and effectively improve the survival rate and intestinal microenvironment of GVHD mice. This provides a new treatment possibility for the treatment of GVHD in patients with post-transplant leukemia. Compared with traditional FMT, postbiotic therapy has the advantages of clear ingredients, standardization, and low risk, and shows good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 This is the identification and comparison result of 16s RNA sequence of P.merdae.

[0008] Figure 2 This is a comparison chart of P.merdae bacteria in the treatment of acute GVHD transplantation model.

[0009] Figure 3 This is a comparison chart of P.merdae bacteria in treating chronic GVHD transplantation model.

[0010] Figure 4 P.merdae improves the symptoms of intestinal GVHD by promoting the balance between Th1 and Treg, inhibiting the proliferation and activation of T cells.

[0011] Figure 5 Results of non-targeted metabolic analysis of feces from mice with GVHD treated with P. Merdae bacteria;

[0012] (A) OPLS-DA results; (B) Metabolite volcano plot results; (C) KEGG pathway enrichment analysis results of differential metabolites; (D) Heat map of bile acid-related metabolites; (E) Schematic diagram of the main metabolic pathways of bile acid; (F) Statistical results of bile acid metabolites.

[0013] Figure 6 TUDCA, a bile acid metabolite, inhibits macrophage mitochondrial ROS levels and reduces allogeneic T cell activation;

[0014] (A) Direct effects of various bile acid metabolites on mouse T cells; (B) Indirect effects of various bile acid metabolites on mouse T cells in MLR; (C) Effects of various bile acids on mouse bone marrow macrophages; (D) TUDCA inhibits macrophage activation of allogeneic T cells in vitro.

[0015] Figure 7 TUDCA, a bile acid metabolite, inhibits macrophage mitochondrial ROS levels and reduces allogeneic T cell activation;

[0016] (A) Direct effects of various bile acid metabolites on mouse T cells; (B) Indirect effects of various bile acid metabolites on mouse T cells in MLR; (C) Effects of various bile acids on mouse bone marrow macrophages; (D) TUDCA inhibits macrophage activation of allogeneic T cells in vitro. DETAILED DESCRIPTION

[0017] 1. Isolation, preservation and in vitro culture methods of Parabacteroides faecalis

[0018] This strain was isolated and purified from the feces of healthy people. It is a Gram-negative bacterium with short rod-shaped bodies, often arranged singly or in pairs, and strictly anaerobic. It showed positive reactions in indole, hydrogen sulfide, methyl red, Voges-Proskauer, nitrate reduction and other tests; it showed negative reactions in catalase, oxidase, citrate utilization, gelatin liquefaction, urease and other tests. After 48 hours of culture on Mianyang blood agar medium, the colonies were round, smooth, with neat edges, about 1-2 mm in diameter, and milky white to light yellow.

[0019] The preparation method of Parabacteroides faecalis comprises the following steps in sequence:

[0020] (1) Separation

[0021] Take a stool sample from a healthy person, suspend the stool sample in sterile saline, and mix at a ratio of 1:10. After shaking and mixing, let it stand, take the supernatant and perform 10-fold serial dilutions, spread the dilutions on Mianyang blood agar plates, and culture anaerobically at 37°C for 48-72 hours.

[0022] (2) Purification

[0023] The initially isolated colonies were picked and purified by the plate streak method. Single colonies were picked and inoculated on new BHI agar plates, and culture and selection were repeated until a pure strain was obtained. The strain was stored on a slant made from Mianyang blood agar plates and placed in a 4°C refrigerator for later use. After high-throughput sequencing of 16S ribosomal DNA identification and comparison with the NCBI database, the strain was finally determined to be Parabacteroides merdae, see Figure 1 .

[0024] (3) Preservation method of Parabacteroides faecalis

[0025] The BHI medium for the preservation of the Parabacterium faecalis strains is composed of: 37.0 g of brain heart infusion, 10.0 g of peptone, 2.0 g of glucose, 5.0 g of sodium chloride, 2.5 g of potassium dihydrogen phosphate, 1000 ml of distilled water, and a pH value of pH 7.4. The glycerol preservation method (25% glycerol to medium ratio) is used and the temperature is minus 80 degrees.

[0026] (4) In vitro amplification and culture method of Parabacteroides faecalis

[0027] The frozen Parabacteroides faecalis strain was cultured in thioglycollate medium at 37°C for 24-48 hours to restore its activity. Then, the activated bacterial solution was transferred to deoxygenated BHI medium and shaken well, and the bacterial solution was dipped with a disposable sterile inoculation loop and streaked on a blood agar plate. The blood agar plate was placed in an anaerobic incubator and cultured anaerobically at 37°C for 48-72 hours using a deoxygenated bag. The morphology, color and size of the colonies on the plate were observed. Using fluorescent real-time quantitative polymerase chain reaction (qPCR), the purity of the bacterial flora was dynamically monitored using primer pairs F: AGGGTGCGTAGGTGGTGAT and R: TTCACCGCTACACCACGC. Thermo Fisher's PowerUp SYBR TM Green Master Mix Kit. In a 20 μL qPCR reaction system, prepare the following components: 10 μL 2X PowerUp SYBR TM Green Master Mix, 0.5 μM forward primer (F), 0.5 μM reverse primer (R), 2 ng template DNA, add deionized water to the total reaction volume. qPCR amplification conditions are as follows:

[0028] step temperature time Cycle times Pre-denaturation 95℃ 2 minutes 1 transsexual 95℃ 15 seconds 40 Annealing / Extension 60℃ 1 minute 40 Melting curve analysis 95℃ 15 seconds 1 60℃ 1 minute 1

[0029] The colony with the strongest qPCR signal on the plate was selected and inoculated into deoxygenated BHI medium for anaerobically culture, and the medium was changed by centrifugation every 48-72 hours. The number of viable bacteria was detected by flow cytometry using SYT09 and propidium iodide staining.

[0030] 2. Isolation of Parabacteroides faecalis for graft-versus-leukemia effect and graft-versus-host disease

[0031] Experimental methods:

[0032] The following experiments were conducted using TUDCA standard substances.

[0033] 1. Construction of GVHD model: Balb / c (H-2d) mice and FVB (H-2q) mice were used as donors, and C57BL / 6 (H-2b) mice were used as recipients. After whole-body irradiation (800-1000 cGy), bone marrow and T cells from the donor were infused back through the tail vein to induce GVHD. In the chronic GVHD (cGVHD) model, the transplantation operation is similar to aGVHD, but the occurrence of chronic GVHD is induced by reducing the amount of initial T cell input. During the experiment, mice were randomly divided into PBS control group and P.Merdae treatment group for intervention, and the severity of GVHD was scored by symptoms such as skin, weight, hair, and activity of the recipient mice.

[0034] 2. Mesenteric lymph node bacterial culture method: Lymph nodes were aseptically isolated from the mesentery of mice and made into a suspension in PBS. The lymph nodes were evenly ground using a tissue grinder, and then the suspension was spread on an LB agar plate (purchased from Solebaugh) and cultured at 37°C for 24-48 hours. After culture, the colony forming units (CFU) on each culture plate were counted, and the number of bacterial colonies was calculated based on the dilution multiple. The results can be used to evaluate the intestinal barrier function and bacterial translocation, and then analyze the protective effect of the treatment on the intestinal barrier.

[0035] 3. HE staining to evaluate tissue pathology: The tissue is fixed in 4% paraformaldehyde, dehydrated and paraffin-embedded before sectioning. The sections are first dewaxed in xylene, then hydrated through gradient alcohol, stained with Hematoxylin for 5-10 minutes, rinsed and blued in ammonia. Then stain with Eosin for 30 seconds to 1 minute, dehydrated and transparentized, and finally sealed in neutral gum. The stained sections are observed under a microscope. Hematoxylin stains the nucleus blue, and Eosin stains the cytoplasm and matrix pink, which is used to evaluate histopathological changes.

[0036] 4. Observe the superficial surface of the eye with fluorescein sodium staining: drip 0.5% fluorescein sodium eye drops into the lower eyelid to ensure that the dye evenly covers the surface of the cornea and conjunctiva. The dye will adhere to the damaged area on the front surface of the eye. After closing the eyes for a while, use a slit lamp microscope and a blue light filter to observe. The damaged area will show green fluorescent spots or stripes, thereby evaluating the degree of damage to the cornea and conjunctiva.

[0037] 5. Tear measurement: Place a standard Schirmer test paper (purchased from Tianjin Yinuo Xinkang Medical Equipment Co., Ltd.) on the inner side of the mouse's lower eyelid for 5 minutes. The length of the test paper soaked represents the amount of tear secretion.

[0038] 6. CFSE staining and flow cytometry of donor T cells: The donor T cells were sorted with magnetic bead antibodies, and 5 μM CFSE was added to them. After incubation at room temperature for 10 minutes, the staining was terminated with serum-containing culture medium, and then the cells were washed twice with PBS. The CFSE-labeled donor T cells were reinfused into C57 mice that had undergone radiotherapy and myeloablation through the tail vein. One week later, the tissues and organs were collected and ground into cell suspensions. The cell suspension was resuspended with PBS, and then surface antibodies pre-labeled with fluorescent dyes (such as antibodies against CD4, CD8 or other markers) were added, incubated at 4°C for 30 minutes, and protected from light. After incubation, the cells were washed with PBS to remove unbound antibodies. If intranuclear antibodies are required, Foxp3 antibodies are added after the membrane is broken. After incubation, the cells are washed with PBS to remove unbound antibodies. Finally, the fluorescence intensity of the cell surface antibody markers was detected using a flow cytometer to analyze the expression of specific molecules or cell populations.

[0039] 7. Identification of non-target metabolites in feces: After collecting fresh fecal samples, they were immediately placed in liquid nitrogen for rapid freezing and stored at -80°C to prevent metabolite degradation. Take about 100 mg of fecal sample, add 1 ml of pre-cooled 80% methanol aqueous solution, place on ice and vortex mix for 5 minutes, and then centrifuge at 12000g for 10 minutes at 4°C to obtain the supernatant. Transfer the supernatant to a new centrifuge tube and concentrate and dry. The dried sample is resuspended in 200 microliters of 50% methanol solution, mixed evenly, and needs to be transferred to the injection bottle and detected on an ultra-high performance liquid chromatography-mass spectrometry instrument. The analysis conditions are: using a C18 reverse phase column, the mobile phase is water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), gradient elution mode, flow rate of 0.3mL / min, and column temperature set to 40°C. The sample injection volume is 5 microliters. The mass spectrometry detection mode selected the positive and negative ion alternating scanning mode, and the scanning range was 100-1000 m / z. After the LC-MS run, MetaboAnalyst was used for data processing to identify, quantify and normalize the metabolite peaks.

[0040] 8. Mixed lymphocyte reaction: The mice were killed and the spleens were removed aseptically and placed in pre-cooled PBS. The spleens were ground and the cell suspension was collected in a 15 mL centrifuge tube. 3 mL of red blood cell lysis buffer was added and gently mixed. After lysis at room temperature for 3 minutes, PBS was immediately added to terminate the reaction. Centrifuge again at 300g for 5 minutes and discard the supernatant. The donor T cells were positively selected with magnetic beads and the recipient T cells were eliminated. The T cells of the allogeneic donor and recipient mice and the spleen cells with T eliminated were mixed for reaction. After 4 days of culture, they were centrifuged at 300g for 10 minutes, the supernatant was carefully discarded, and the corresponding flow cytometry antibodies were added to each well for detection.

[0041] 9.MitoSOX immunofluorescence staining: Cells were seeded on coverslips or culture dishes and cultured to 50-70% confluence. Cells were washed once with pre-warmed PBS buffer to remove residual culture medium. 5 μM MitoSOX red working solution was prepared and added to the culture dish to ensure complete coverage of the cells. Incubate at 37°C in the dark for 10-20 minutes. After incubation, cells were quickly and gently washed three times with pre-warmed PBS to remove unbound dye. To fix the cells, 4% paraformaldehyde was added and fixed at room temperature for 10-15 minutes. Subsequently, the cells were washed three times with PBS for 5 minutes each. After fixation, cells were permeabilized and incubated in 0.1% Triton X-100 in PBS for 5 minutes to increase cell membrane permeability. After washing three times with PBS, 5% BSA blocking solution was added and blocked at room temperature for 1 hour to block non-specific binding. After blocking, primary antibody was added at a dilution of 1:200 in 1% BSA and incubated at 37°C for 1 hour. Then, the cells were washed three times with PBS for 5 minutes each. Add fluorescently labeled secondary antibody, dilute 1:500 in 1% BSA, and incubate in the dark for 30 minutes. Wash again with PBS three times, 5 minutes each time. To stain the nucleus, add DAPI solution and incubate in the dark for 5 minutes, rinse with PBS to remove excess dye. Finally, mount the slide with anti-fluorescence quenching mounting medium and gently cover with a coverslip. Place the prepared sample under a fluorescence microscope, select appropriate excitation and emission wavelengths for observation, and record the fluorescence signal.

[0042] 10. Detection of cellular reactive oxygen species (ROS): Wash mouse bone marrow cells twice with pre-warmed PBS buffer and centrifuge at 300g for 5 minutes each time. Resuspend the cells in 500μL pre-warmed serum-free medium, add ROS fluorescent probe DCFH-DA at a final concentration of 10μM, mix gently and incubate at 37℃ in the dark for 30 minutes. During this period, mix gently every 5-10 minutes to ensure that the dye penetrates the cells evenly. After incubation, centrifuge at 300g for 5 minutes and discard the supernatant. Wash the cells three times with pre-warmed PBS buffer to remove unbound dye, and discard the supernatant after each centrifugation. Resuspend the stained cells in 500μL PBS and filter through a 40-micron cell strainer to remove cell clumps and ensure single cell suspension. Detect the ROS fluorescence signal FITC channel (488nm excitation, 525nm emission) using a flow cytometer.

[0043] 11. TUDCA treatment of GVHD model: Balb / c (H-2d) mice and FVB (H-2q) mice were used as donors, and C57BL / 6 (H-2b) mice were used as recipients. After whole-body irradiation (800-1000 cGy), bone marrow and T cells from the donor were reinfused through the tail vein to induce GVHD. In the chronic GVHD (cGVHD) model, the transplantation operation is similar to aGVHD, but the occurrence of chronic GVHD is induced by reducing the amount of initial T cell input. During the experiment, mice were randomly divided into a PBS control group and a TUDCA treatment group (purchased from Aladdin) for gavage treatment, and the severity of GVHD was scored by symptoms such as the skin, weight, hair, and activity of the recipient mice.

[0044] Experimental Results

[0045] After a large number of experimental studies, the inventors found that the Parabacteroides merdae (P.merdae) strain has the functions of efficient repair and inhibition of inflammation, especially has a significant inhibitory effect on intestinal inflammation caused by GVHD, can effectively improve systemic GVHD symptoms, including acute GVHD and chronic GVHD, can improve post-transplant survival rate, and enhance the graft-versus-leukemia effect.

[0046] After using this strain, in the aGVHD transplantation mouse model with C57 as donor and Balb / c as recipient, the survival rates on days 20, 30, 40, and 80 were increased by 21.0%, 44.7%, 52.0%, and 52.0%, respectively, compared with the control group. Figure 2 A and B; P<0.05). To exclude species differences, the inventors also verified the strain in an aGVHD transplant mouse model with FVB as the donor and C57 as the recipient. The results showed that the survival rates on days 20, 30, 40, and 80 were still improved by 25.6%, 42.6%, 48.0%, and 51.2% compared with the control group ( Figure 2 C; P<0.05). Pathological observation of intestinal target organs in the aGVHD model showed that the length of intestinal organs after P.Merdae treatment was significantly longer than that of the control group, indicating that organ inflammation was mild and there was no obvious contracture. The bacterial colony of mesenteric lymph nodes was measured and it was found that the number of intestinal flora invading mesenteric lymph nodes was significantly reduced after P.Merdae treatment ( Figure 2 D, P.Merdae group: mean bacterial colony value 5 CFU / plate vs control group: 35 CFU / plate; P<0.05). HE pathological staining of GVHD target organ sections showed that lymphocyte infiltration in multiple target organs decreased after P.Merdae treatment, especially in the intestine, where the structure of the small intestine and colon was significantly restored ( Figure 2E). In addition, in the cGVHD model with C57 as donor and Balb / c as recipient, P.Merdae treatment can alleviate cGVHD symptoms, with clinical symptoms reduced by 1, 2, and 3 points at 30, 40, and 50 days, respectively, compared with the control group (P<0.05). In the fundus and tear gland function tests, it was found that P.Merdae reduced the appearance of fundus neovascularization and significantly improved the secretion function of the tear gland ( Figure 3 AC, mean tear fluid content of the left and right eyes in the P.Merdae group: 2mm vs. control group: 1mm; P<0.05). The above results indicate that the strain plays an important and positive role in improving acute GVHD and chronic GVHD, and has a significant effect on improving the survival rate after transplantation. The P.Merdae bacteria treatment GVHD model was analyzed by flow cytometry, and the results showed that P.Merdae bacteria significantly inhibited the proliferation and activation of T cells, regulated the balance between Th1 cells and Treg cells, and reduced the attack of immune cells on intestinal epithelial cells ( Figure 4 ), which promoted the improvement of GVHD symptoms in recipients.

[0047] Based on the previous discovery that P.Merdae strains have a protective effect on GVHD mice, in order to clarify the specific microbial-related metabolites of P.Merdae bacteria that play a protective role, the inventors collected feces from mice treated with P.Merdae bacteria and the control group for non-targeted metabolite analysis. The inventors found that after P.Merdae bacteria treatment, orthogonal partial least squares discriminant analysis (OPLS-DA) can significantly distinguish the control group from the P.Merdae bacteria treatment intervention group, indicating that the metabolite composition of the two groups is significantly different ( Figure 5 A). The distribution of differentially expressed metabolites was explored through the volcano plot (Valcano), and inter-group metabolite screening and KEGG pathway enrichment analysis were performed. It was found that the bile acid metabolic pathway and related products were significantly increased after P. Merdae treatment ( Figure 5 BC), suggesting that P. Merdae bacteria may play an important role in anti-GVHD through bile acid metabolism. A variety of primary and secondary bile acids increased after P. Merdae bacteria treatment, among which the levels of a variety of secondary conjugated bile acids represented by taurine deoxycholic acid (TDCA) and taurine ursodeoxycholic acid (TUDCA) were significantly increased ( Figure 5 DF). The results of bile acid targeted metabolism showed that TUDCA was significantly increased after P. Merdae treatment ( Figure 5 G). In the CD3 / CD28 antibody coated T cell stimulation in vitro system, TUDCA has no direct effect on T cells ( Figure 6 A), and in vitro mixed lymphocyte reaction experiments found that TUDCA could reduce the activation of allogeneic T cells, suggesting that TUDCA inhibits the activation of allogeneic T cells by regulating antigen presenting cells ( Figure 6 B). In vitro experiments have shown that TUDCA can inhibit the levels of cytokines and reactive oxygen species in mouse bone marrow-derived macrophages ( Figure 6 C and D), thereby reducing the ability of M1 macrophages to activate allogeneic CD4+ T cells ( Figure 6 E). The above results show that P.Merdae regulates the activation of allogeneic donor T cells by donor macrophages through TUDCA, reduces GVHD inflammation, restores recipient intestinal immune homeostasis, protects intestinal integrity, and improves transplant survival prognosis by promoting the generation of donor CD4+Treg. Finally, in the aGVHD transplant mouse model with C57 as donor and Balb / c as recipient, the survival rate on the 30th, 40th, and 50th days of TUDCA gavage treatment was increased by 20.8%, 38.7%, and 34.8% respectively compared with the control group ( Figure 7 ; P<0.05).

[0048] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and product of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. Application of tauroursodeoxycholic acid, a metabolite of Parabacteroides faecalis, in the preparation of drugs for the treatment of graft-versus-host disease.

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