Application of Mycobacterium finschii in preparing medicine for inhibiting acute graft-versus-host disease

Abstract

The present invention relates to the field of biomedicine, and in particular to the use of Mycobacterium finschii in the preparation of a drug for treating acute graft-versus-host disease. The present invention clarifies a specific strain of Mycobacterium finschii that has the effect of inhibiting acute graft-versus-host disease. By obtaining a single strain of bacteria with an inhibitory effect, it is helpful to prepare drugs in a standardized and streamlined manner, thereby obtaining drugs with clear ingredients, strict quality testing, and direct clinical application, so that the same standard drug can be widely used in patients with acute graft-versus-host disease, thereby improving the universality of the drug.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to the application of Mycobacterium finschii in preparing medicine for treating acute graft-versus-host disease. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective means to treat malignant blood tumors such as leukemia. However, acute graft-versus-host disease (aGVHD) after transplantation is the most common complication and the main cause of death in allo-HSCT. Therefore, the prevention and treatment of aGVHD is a key issue to improve the efficacy of allo-HSCT.

[0003] Currently, the treatment of gastrointestinal aGVHD through fecal microbiota transplantation is a hot topic in the field. However, the effect of this method on aGVHD caused by inflammatory responses in other organs such as the skin and liver is unclear. In addition, the intestinal flora contains too many bacterial species, which are difficult to obtain through simple culture and difficult to detect the adulteration of foreign bacteria, which is not conducive to large-scale production and application. Therefore, it is of great significance to study the therapeutic effects of specific bacterial species in the intestinal flora on aGVHD. Summary of the invention

[0004] The purpose of the present invention is to provide a specific bacterial species capable of inhibiting acute graft-versus-host disease.

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

[0006] Application of Mycobacterium finschii in the preparation of drugs for inhibiting acute graft-versus-host disease.

[0007] Optionally, the drug contains Mycobacterium finschii as an active ingredient.

[0008] Alternatively, M. finochium inhibits acute graft-versus-host disease by suppressing post-transplant Th1 cell responses.

[0009] Alternatively, M. finochium suppresses acute graft-versus-host disease by inhibiting post-transplantation Tc1 cell responses.

[0010] Alternatively, M. finhoei promotes the expression of tight junction proteins in intestinal tissue.

[0011] Alternatively, Mycobacterium finchenii inhibits acute graft-versus-host disease by promoting intestinal epithelial barrier repair.

[0012] Optionally, the drug is administered orally.

[0013] Optionally, the drug is administered daily, and the duration of administration is any value between 10 days and 30 days.

[0014] Optionally, the dosage of Mycobacterium finschii is 1×10 7 CFU / g~1×10 8 Any value in CFU / g.

[0015] The beneficial effect of the present invention is that a specific bacterial species, Mycobacterium finschii, which has the effect of inhibiting acute graft-versus-host disease is identified. By obtaining a single bacterial species with an inhibitory effect, it is helpful to prepare drugs in a standardized and streamlined manner, thereby obtaining drugs with clear ingredients, strict quality inspections, and direct clinical applications, so that the same standard drug can be widely used in patients with acute graft-versus-host disease, thereby improving the universality of the drug.

[0016] Furthermore, the mechanism by which Mycobacterium finschii inhibits acute graft-versus-host disease was explored, which will help in the subsequent exploration of adding ingredients that have synergistic effects with it into drugs to improve their efficacy.

[0017] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a statistical graph of the cumulative incidence of aGVHD in the high-A. spp. abundance group and the low-A. spp. abundance group in the present invention;

[0019] Figure 2 A statistical diagram illustrating the relationship between the abundance of Mycobacterium finschii and Mycobacterium shahiii and the cumulative incidence of aGVHD in the present invention;

[0020] Figure 3 This is a statistical graph of the survival rates of mice in the AF group and the control group shown in Example 1 of the present invention;

[0021] Figure 4 This is a schematic diagram of the scores and weight changes of mice in the AF group and the control group shown in Example 1 of the present invention;

[0022] Figure 5 HE staining images of the intestinal tract, liver and lung tissues of mice in the AF group and the control group shown in Example 1 of the present invention;

[0023] Figure 6 This is a comparison diagram of the number of Th1 cells and Tc1 cells in the spleen tissue of mice in the AF group and the control group shown in Example 1 of the present invention;

[0024] Figure 7This is a schematic diagram of the expression of various tight junction protein genes in the small intestinal tissues of mice in the AF group and the control group shown in Example 1 of the present invention;

[0025] Figure 8 This is a schematic diagram of the expression of various tight junction protein genes in the large intestine tissue of mice in the AF group and the control group shown in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0028] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0029] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Alistipes finegoldii (AF) belongs to the genus Alistipes, phylum Bacteroidetes, gram-negative bacteria, and is a relatively new genus of bacteria, mainly isolated from medical clinical samples. At present, there are relatively few studies on this genus, and it may be a potential bacterium that regulates inflammatory emotional responses, and it is worth studying its correlation with aGVHD.

[0031] In the present invention, 48 patients were included in the study as research subjects. The patients were between 18 and 65 years old, all suffering from hematological malignancies, and underwent allogeneic hematopoietic stem cell transplantation at the First Affiliated Hospital of Soochow University from 2020 to 2022. Serum and stool samples were collected from each patient three weeks after the transplantation, the clinical baseline characteristics of the patients were recorded, and the patients were followed up for 100 days to observe the occurrence of aGVHD. The samples were divided into two groups according to whether aGVHD occurred in the corresponding patients within 100 days, namely, the aGVHD group (I to IV degree aGVHD) and the non-aGVHD group (no aGVHD).

[0032] 16S rDNA high-throughput sequencing was performed on 48 stool samples to observe the correlation between the abundance of Alistipes in stool and aGVHD. The median relative abundance of Alistipes in the corresponding samples was used as the critical value (Median = 0.25%), and the patients were divided into a high Alistipes abundance group and a low Alistipes abundance group. Please refer to Figure 1 The cumulative incidence of aGVHD and patient survival rates of patients in the highAlistipes group and lowAlistipes group at different time points after surgery are presented in the form of a line graph. It can be seen that the cumulative incidence of aGVHD in the highAlistipes group has a decreasing trend over time compared with the lowAlistipes group (P=0.05), but has no significant effect on the overall survival outcome. Further analysis was conducted on two common species of Alistipes, Alistipes finegoldii and Alistipes shahii. See Figure 2The patients were divided into high Alistipesfinegoldii+ group and low Alistipesfinegoldii- group with the median relative abundance of Alistipesfinegoldii in the corresponding samples as the critical value. The cumulative incidence of aGVHD in the two patients over time after surgery was calculated and drawn as a line graph. The patients were then divided into high Alistipesshahiii+ group and low Alistipesshahii- group by a similar method. The cumulative incidence of aGVHD in the two patients over time after surgery was calculated and drawn as a line graph. The results showed that the cumulative incidence of aGVHD in the Alistipesfinegoldii+ group was significantly lower than that in the Alistipesfinegoldii- group (P=0.02), while there was no significant difference between the two groups divided by the abundance of Alistipesshahii. The above results suggest that Alistipesfinegoldii may affect the occurrence and development of aGVHD.

[0033] In the present invention, further in vivo experiments in mice verified that Alistipes finegoldii has the effect of inhibiting the occurrence of aGVHD.

[0034] The invention provides the use of Mycobacterium finschii in preparing a medicine for inhibiting acute graft-versus-host disease.

[0035] In some embodiments, the drug uses Mycobacterium finschii as an active ingredient and can use commonly used phosphate buffered saline (PBS) as a carrier for easy storage and administration.

[0036] In some embodiments, Mycobacterium finchenii inhibits acute graft-versus-host disease by suppressing post-transplant Th1 cell responses.

[0037] In some embodiments, Mycobacterium finchenii inhibits acute graft-versus-host disease by suppressing post-transplantation Tc1 cell responses.

[0038] In some embodiments, M. finchenii promotes the expression of tight junction proteins in intestinal tissues, including Claudin1, Claudin2, Claudin3, Claudin4, occludin, zo-1, JAM, and muc-1.

[0039] In some embodiments, Mycobacterium finchenii inhibits acute graft-versus-host disease by promoting intestinal epithelial barrier repair.

[0040] In some embodiments, the drug is administered orally. Oral administration is a common administration method, because Mycobacterium finschii is found in the human intestine and is adapted to the environment of the human digestive system. Oral administration is convenient for administering Mycobacterium finschii.

[0041] In some embodiments, the drug is administered daily, and the duration of administration is any value between 10 and 30 days, for example, any value between 10, 15, 20, 25 and 30 days.

[0042] In some embodiments, the dosage of Mycobacterium finschii is 1×10 7 CFU / g~1×10 8 CFU / g, for example, 1×10 7 CFU / g, 3×10 7 CFU / g, 5×10 7 CFU / g, 7×10 7 CFU / g, 9×10 7 CFU / g and 1×10 8 Any value in CFU / g.

[0043] Please refer to the following examples for details.

[0044] Embodiment 1:

[0045] BALB / c (H-2d) female mice were used as recipient animals and were evenly divided into two groups, the control group and the AF group, according to the weight of the mice, with 15 mice in each group. The recipient mice received 6.25Gry systemic irradiation at the irradiation center to construct a leukemia model. At the same time, 6-8 week-old C57BL / 6 (H-2b) female mice were used as donor animals. The donor mice were killed by dislocation of the neck. The spleen of the mice was removed in a sterile environment, ground into tissue-free blocks and filtered with a 200-mesh filter. At the same time, the femur and iliac bone of the donor mouse were taken, the bone marrow cells were flushed out and filtered with a 200-mesh filter. After the two were mixed, the red blood cells were lysed with ammonium chloride buffer to prepare a mixed cell suspension containing bone marrow hematopoietic stem cells (BM cells) and spleen cells of the donor mice. 4h after the end of irradiation of the recipient mice, 0.3mL of the mixed cell suspension was injected into the tail vein. The mixed cell suspension contained 4×10 6 spleen cells from donor mice and 1×10 7 BM cells of donor mice were used to simulate hematopoietic stem cell transplantation. The cultured Alistipes finegoldii was washed and resuspended with PBS to obtain a bacterial solution. The content of Alistipes finegoldii in the bacterial solution was 1.0×10 9CFU / 100μL. The mice in the AF group injected with the mixed cell suspension were intragastrically administered with 100μL of bacterial solution per mouse, and the control group was intragastrically administered with an equal amount of placebo, i.e., PBS. Both groups of mice were administered with medication at regular intervals every day.

[0046] The survival rates of the two groups of mice were counted and recorded starting from the day of allogeneic transplantation. Figure 3 It can be seen that after the 20th day after allogeneic transplantation, the survival rate of the control group was 0, and the administration of Alistipes finegoldii significantly delayed the survival time of the mice.

[0047] After the injection of the mixed cell suspension, the recipient mice were observed once a day, and the changes in the physical signs of the mice were observed and scored, and the average score of the scores of each mouse in the group was used as the score of the two groups of mice on that day. The weight of each mouse was measured once every two days, and the average weight of the weight of each mouse in the group was used as the weight of the two groups of mice on that day. The scoring criteria are shown in Table 2 below.

[0048] Table 1:

[0049]

[0050] See also Figure 4 The scores and weight changes of mice in the AF group and the control group over time were statistically analyzed, and it can be seen that the physical sign recovery of mice in the AF group was significantly better than that in the control group.

[0051] The donor mice and recipient mice were selected for model construction and drug administration in the same manner. After 14 days of continuous drug administration, the recipient mice were killed by cervical dislocation, and the intestinal, liver, lung and spleen tissues were collected. The intestinal, liver and lung tissues were stained with hematoxylin-eosin (HE). The specific steps of HE staining are as follows:

[0052] 1. Dewax paraffin sections to water: Soak tissue sections in xylene for 10 minutes, repeat twice, soak in anhydrous ethanol for 5 minutes, repeat twice, soak in 95% alcohol for 5 minutes, soak in 90% alcohol for 5 minutes, soak in 80% alcohol for 5 minutes, soak in 70% alcohol for 5 minutes, and wash once with distilled water.

[0053] 2. Hematoxylin staining of cell nuclei: Place tissue sections in hematoxylin staining solution for 3 to 8 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, turn blue with 0.6% ammonia solution, and rinse with tap water.

[0054] 3. Eosin staining of cytoplasm: Place tissue sections in eosin staining solution for 1 to 3 minutes and rinse with tap water.

[0055] 4. Dehydration and sealing: Soak the tissue sections in 95% alcohol for 5 minutes, repeat twice, soak in anhydrous ethanol for 5 minutes, repeat twice, soak in xylene for 5 minutes, repeat twice, and dehydrate. After dehydration and transparency, take the sections out of xylene and dry them slightly, and seal them with neutral gum.

[0056] See also Figure 5 It can be seen that oral administration of Alistipesfinegoldii can significantly reduce the degree of damage to target organs and has a good therapeutic effect on aGVHD.

[0057] Take the spleen tissue of the recipient mouse, grind it until no obvious tissue blocks are visible, and filter it with a 200-mesh cell sieve to obtain a cell suspension. Centrifuge the cell suspension at 1300 rpm for 8 minutes at 4°C, discard the supernatant, and add 5 mL of red blood cell lysis solution to the cell pellet. After all the cells are fully mixed, place them at room temperature for 5 minutes, add 30 mL of RPMI-1640 medium to terminate the reaction, and then centrifuge at 1300 rpm for 6 minutes at 4°C, discard the supernatant, resuspend the cells with 1 mL of RPMI-1640 medium, obtain a resuspension, count and set aside.

[0058] Take 1×10 6 The cell suspension of 10 cells was added to the flow tube, CD16 / CD32 antibody was added to block at 4°C for 20 minutes, and centrifuged at 1300 rpm for 5 minutes. After fully discarding the supernatant, the cells were resuspended with 100 μL of PBS containing 1% fetal bovine serum (FBS), and different components of flow antibodies were added according to the instructions of the flow cytometer, and incubated at 4°C in the dark for 30 minutes. After the incubation, centrifuge at 1300 rpm for 5 minutes, discard the supernatant, wash the cell pellet twice with PBS, and finally add 200 μL of PBS to resuspend the cells and detect them using a flow cytometer. Set a single positive tube for adjustment compensation, and distinguish the negative and positive groups through the negative tube gate, and analyze the number of Th1 cells and Tc1 cells using the Flowjo software.

[0059] See also Figure 6 Compared with the control group, Alistipesfinegoldii gavage significantly reduced the number of Th1 cells and Tc1 cells in the spleen. The experimental results suggest that Alistipesfinegoldii can alleviate aGVHD by reducing the number of Th1 cells and Tc1 cells in target organs.

[0060] The small intestine tissue and large intestine tissue of mice were taken for qPCR analysis. The tissue was cut into small pieces, and the total RNA in the intestinal tissue was extracted by the Trizol kit, and the extracted total RNA was reverse transcribed into cDNA by the reverse transcription kit for subsequent real-time quantitative PCR detection. A variety of tight junction protein genes were selected as target genes, including Claudin1, Claudin2, Claudin3, Claudin4, occludin, zo-1, JAM and muc-1, and β-actin was used as the internal reference gene. The primers and corresponding probes of each target gene and internal reference gene were designed and synthesized, and each primer and probe were evenly mixed with cDNA and enzymes were added, and amplified by QuantStudio3 real-time quantitative PCR instrument. The amplification conditions are: 95°C, 3 minutes, 1 cycle; 95°C, 10 seconds, 60°C, 1 minute, 40 cycles. Using QuantStudio TM Design&Analysis software (Applied Biosystems) was used to analyze the Ct value. The expression of the internal reference gene β-actin was used as a reference to calculate the expression level ΔCt of each target gene in any sample in the AF group and the control group compared with the internal reference gene. The relative expression level ΔΔCt of any target gene in the AF group and the same target gene in the control group was then analyzed to evaluate the relative expression level of each target gene.

[0061] See also Figure 7 and Figure 8 , using box plots to compare the expression levels of various tight junction protein genes in the AF group and the control group, it can be seen that Alistipesfinegoldii significantly upregulated the expression of tight junction proteins in small and large intestinal cells. Tight junction proteins are important components of the intestinal epithelial barrier. They prevent harmful substances and pathogens from entering the intestinal tissue by closing the gaps between cells. When the intestinal epithelial barrier is damaged, the expression and distribution of tight junction proteins will change, resulting in increased intestinal permeability, making it easier for harmful substances and pathogens to enter the intestinal tissue, triggering inflammatory and immune responses. Therefore, repairing tight junction proteins is the key to restoring intestinal epithelial barrier function. Alistipesfinegoldii significantly upregulated the expression of tight junction proteins, indicating that it alleviates aGVHD by promoting intestinal epithelial barrier repair.

[0062] In summary, it can be seen that Alistipes finegoldii has the effect of inhibiting acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation. The mechanism of inhibition lies in its ability to directly inhibit post-transplantation Th1 cell response and post-transplantation Tc1 cell response, and can also promote intestinal epithelial barrier repair and maintain the integrity of epithelial cells.

[0063] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0064] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of Mycobacterium finschii in the preparation of drugs for inhibiting acute graft-versus-host disease.

2. The use according to claim 1, characterized in that The medicine uses Mycobacterium finschii as an effective ingredient.

3. The use according to claim 2, characterized in that Mycobacterium finschii inhibits acute graft-versus-host disease by suppressing Th1 cell responses after transplantation.

4. The use according to claim 2, characterized in that Mycobacterium finschii suppresses acute graft-versus-host disease by inhibiting Tc1 cell responses after transplantation.

5. The use according to claim 2, characterized in that Mycobacterium finschii promotes the expression of tight junction proteins in intestinal tissue.

6. The use according to claim 5, characterized in that Mycobacterium finschii inhibits acute graft-versus-host disease by promoting intestinal epithelial barrier repair.

7. The use according to claim 2, characterized in that The drug is administered orally.

8. The use according to claim 2, characterized in that The drug is administered daily, and the duration of administration is any value between 10 days and 30 days.

9. The use according to claim 2, characterized in that The dosage of Mycobacterium finschii was 1×10 7 CFU / g~1×10 8 Any value in CFU / g.

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