Application of taurodeoxycholic acid in preparation of medicine for recovering damaged hematopoietic stem cells

Abstract

The invention discloses an application of taurodeoxycholic acid in preparation of a medicine for recovering damaged hematopoietic stem cells, taurodeoxycholic acid TDCA is formed by combining deoxycholic acid Deoxycholic acid, DCA and taurine, belongs to secondary bile acid, contains two hydroxyls 3 alpha and 12 alpha, and is relatively high in hydrophobicity. The TDCA can promote lipid absorption and help to emulsify fat and fat-soluble vitamins. The TDCA is mainly used for anti-inflammation and liver protection, can relieve liver inflammation and fibrosis such as non-alcoholic fatty liver disease and cholestasis through an FXR pathway and can be potentially used for treating inflammatory bowel disease IBD, and it is found that the TDCA can recover the number and functions of hematopoietic cells of mice with hematopoietic damage caused by long-term drinking of high-temperature water besides the functions.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of tauroursodeoxycholic acid in preparation of a drug for restoring damaged hematopoietic stem cells. BACKGROUND

[0002] Hematopoietic stem cells (HSCs) are a type of adult stem cells with self-renewal and multipotent differentiation capabilities, mainly existing in bone marrow and a small amount of distribution in umbilical cord blood and peripheral blood. They are the origin of all blood cells and immune cells, responsible for maintaining the body's lifelong hematopoietic function. Among them, self-renewal mainly refers to maintaining the stability of the stem cell pool through asymmetric division to avoid hematopoietic failure; multipotent differentiation refers to that HSCs can differentiate into myeloid progenitor cells and lymphoid progenitor cells, and then generate all mature blood cells, including red blood cells (oxygen carrying), platelets (hemostasis), granulocytes (anti-infection), monocytes / macrophages, B cells (antibody production), T cells (cellular immunity), and NK cells (natural killer).

[0003] The main functions of HSCs include: 1. Maintaining lifelong hematopoiesis: about 100 billion blood cells are generated daily to replace aging or damaged blood cells (such as red blood cells with a lifespan of about 120 days), regulated by cytokines (such as SCF, TPO) and bone marrow niche. 2. Immune system regeneration: continuously generating immune cells to maintain adaptive immunity (such as B / T cells) and innate immunity (such as neutrophils), accelerating hematopoiesis to meet the demand in the case of infection or inflammation.

[0004] HSCs are the core cells that maintain lifelong hematopoiesis and the immune system. Once damaged, it will lead to a variety of serious consequences, the specific impact depends on the extent and range of damage. HSCs damage will cause severe hematopoietic and immune system dysfunction, including bone marrow failure, immune deficiency, increased risk of leukemia, etc., which seriously affects the quality of life of patients and even endangers life. At present, the treatment methods for HSCs damage mainly include hematopoietic stem cell transplantation (HSCT), gene therapy, immune regulation and cytokine support, etc., but still face many challenges, such as donor matching difficulty, graft rejection, safety and efficiency of gene editing, etc.

[0005] Taurine deoxycholic acid (TDCA) is a secondary bile acid, and its main functions include promoting lipid absorption, regulating FXR / TGR5 receptor-mediated metabolism and anti-inflammatory signaling pathways. In the hematopoietic system, TDCA may affect the microenvironment of HSCs by regulating the bile acid-immune axis: 1, inhibiting excessive inflammatory response and protecting HSCs from oxidative stress damage; 2, regulating the balance of self-renewal and differentiation of HSCs through the FXR receptor; 3, its metabolites may be involved in the formation of the bone marrow adipose microenvironment, indirectly affecting hematopoietic homeostasis. Existing studies have shown that bile acid metabolism disorder is related to bone marrow aplastic disease, but the specific mechanism of TDCA in hematopoietic regulation still needs further exploration. Based on the above scientific basis, the present application first proposes that taurine deoxycholic acid (TDCA) can be used to prepare a drug for restoring the function of damaged HSCs caused by long-term drinking of high-temperature water, which is expected to fill the gap in existing treatment drugs. SUMMARY

[0006] The technical problems solved are:

[0007] The present application solves the problems of donor matching difficulty, graft rejection, low safety and efficiency of gene editing, and lack of effective methods for damaged hematopoietic stem cells caused by long-term drinking of high-temperature water in the prior art, and provides an application of taurine deoxycholic acid in the preparation of a drug for restoring damaged hematopoietic stem cells, which verifies the effect of TDCA from the number and function.

[0008] Technical scheme

[0009] An application of taurine deoxycholic acid in the preparation of a drug for restoring damaged hematopoietic stem cells.

[0010] Preferably, the drug comprises taurine deoxycholic acid and a pharmaceutically acceptable excipient.

[0011] Preferably, the excipient is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant.

[0012] Preferably, the drug is an oral or non-oral drug, and the drug is a tablet, a capsule, a powder, a pill, a granule, a solution, a suspension, a syrup, an injection, a suppository, an adsorbent or a spray.

[0013] Beneficial effects

[0014] 1. TDCA can effectively increase the proportion and number of hematopoietic stem cells in the bone marrow cells of mice that have long-term drunk high-temperature water;

[0015] 2. TDCA can effectively reduce the apoptosis rate of hematopoietic stem cells in the bone marrow cells of mice that have long-term drunk high-temperature water;

[0016] 3. TDCA can effectively increase the in vitro reconstruction ability of hematopoietic cells in the bone marrow cells of mice long-term drinking high-temperature water;

[0017] 4. TDCA is used to restore the function and number of damaged hematopoietic cells due to long-term drinking of high-temperature water, which can be further promoted to repair bone marrow suppression after chemotherapy, aplastic anemia, and aging-related hematopoietic function decline. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A proportion diagram of mouse bone marrow hematopoietic stem cells for the high-temperature drinking mouse model constructed in the present application;

[0019] Figure 2 A graph of the absolute number of mouse bone marrow hematopoietic cells for the high-temperature drinking mouse model constructed in the present application;

[0020] Figure 3 A representative flow cytometry graph of hematopoietic stem cells in mouse bone marrow cells after adding TDCA;

[0021] Figure 4 A proportion diagram of hematopoietic stem cells in mouse bone marrow cells after adding TDCA;

[0022] Figure 5 A graph of the absolute number of hematopoietic stem cells in mouse bone marrow cells after adding TDCA;

[0023] Figure 6 A representative flow cytometry graph of hematopoietic stem cell apoptosis in mouse bone marrow cells after adding TDCA;

[0024] Figure 7 A graph of the apoptosis rate of hematopoietic stem cells after adding TDCA;

[0025] Figure 8 A graph of the number of colonies formed in the in vitro cloning experiment of hematopoietic cells after adding TDCA. DETAILED DESCRIPTION

[0026] The present application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made. These all belong to the protection scope of the present application.

[0027] Example 1

[0028] The application of a kind of tauroursodeoxycholic acid in the preparation of drug for restoring damaged hematopoietic stem cells, mouse high-temperature processing model and hematopoietic stem cell detection, specific steps are as follows:

[0029] The purchased normal C57BL / 6J (CD45.2) mice were randomly divided into two groups (5 mice in each group), the control group was given 100 μL of sterile water by gavage, and the high temperature treatment group was given 70°C sterile 100 μL, once a day. At the same time, the high temperature treatment group changed the normal temperature drinking water to 70°C high temperature water. The model time period was 8 weeks.

[0030] After eight weeks, the mice were euthanized, and their bone marrow cells were taken, and the function of the mouse hematopoietic system was detected by flow cytometry analysis. The specific way is as follows:

[0031] First step: take the hind legs of the mouse and put them in the alcohol in the six-well plate;

[0032] Second step: put the thigh bone in the six-well plate containing 2% FBS PBS, and cut off both ends;

[0033] Third step: use a 3 mL syringe to suck the 2% FBS PBS to gently blow out the bone marrow, and use a 1 mL pipette to blow the cells;

[0034] Fourth step: filter through a 70 μm filter screen;

[0035] Fifth step: centrifuge at 4°C, 1500 rpm, 5 minutes, and discard the supernatant;

[0036] Sixth step: after lysing for 3 minutes (3 mL per mouse), blow the cells evenly;

[0037] Seventh step: add twice the volume of 2% FBS PBS (6 mL per mouse) and mix well to stop the lysis;

[0038] Eighth step: filter through a 70 μm filter screen;

[0039] Ninth step: centrifuge at 4°C, 400g, 5 minutes, and discard the supernatant;

[0040] Tenth step: resuspend the cells with 1 mL of 2% FBS PBS, mix well, and take 1 μL for cell counting;

[0041] Eleventh step: after obtaining the mouse bone marrow mononuclear cells, add the corresponding antibodies, incubate at 4°C in the dark for 30-45 minutes; resuspend the cells with 1 mL of 2% FBS PBS, centrifuge at 4°C, 400g, 5 minutes, and discard the supernatant; finally, resuspend the cells with 200 μL of 2% FBS PBS, filter through a 70 μm filter screen, and detect on the machine. HSCs staining: take 5e6 cells, add 7 μL of antibody combination (Lineage, Sca1, c-Kit, CD150, CD48); Lineage is composed of CD3e, CD4, CD8a, CD45R, IgM, Mac-1, Gr-1, Ter-119, etc.

[0042] The specific steps for apoptosis detection are as follows:

[0043] Step 1: The cells are first stained (HSC antibody combination), then 1 mL of apoptosis buffer is added to resuspend the cells, and the cells are centrifuged at 400g at 4℃ and the supernatant is discarded.

[0044] Step 2: Resuspend cells in 200 μL apoptosis buffer, add 5 μL Annexin V per 5e6 cells, and stain at 4°C in the dark for 30 minutes.

[0045] Step 3: Resuspend the cells in 1 mL of apoptosis buffer, centrifuge at 400 g at 4°C and discard the supernatant;

[0046] Step 4: Resuspend the cells in 200 μL buffer, filter through a 70 μm filter, and analyze the cells. Apoptotic cells were identified as Annexin V positive cells.

[0047] like Figure 1 and Figure 2 As shown, Figure 1 A diagram showing the proportion of bone marrow hematopoietic stem cells in a mouse model constructed for this application involving high-temperature drinking water. Figure 2 The absolute number of hematopoietic cells in the bone marrow of the mouse model constructed for this application under high-temperature drinking water conditions was plotted using... Figure 1 and Figure 2 This study demonstrates that long-term consumption of hot water can damage the hematopoietic system, specifically manifested as a decrease in the proportion and number of hematopoietic stem cells in the bone marrow of mice; high-temperature treatment damages hematopoietic stem cells in the bone marrow of mice.

[0048] Example 2

[0049] The application of taurine deoxycholic acid in the preparation of a drug for restoring damaged hematopoietic stem cells, bone marrow cell culture and TDCA complementation experiment, the specific steps are as follows:

[0050] Step a: After cell counting as described in Example 1, cells were cultured in a U-bottom 96-well plate, with 1.5e5 cells and 100 μL of HSC cell culture medium added to each well. The HSC cell culture medium consisted of 40 mL of StemSpan SFEM culture medium, 40 μL of mouse SCF cytokine (10 μg / mL), 40 μL of mouse TPO cytokine (20 μg / mL), and 40 μL of heparin (10 mg / mL). The medium was stored at 4°C and prepared fresh for immediate use.

[0051] Step b: After culturing for 1-2 days, add an additional 100 μL of HSC culture medium; and during the culturing process, every three days, aspirate half of the liquid (100 μL) from the top layer of the culture medium and replace it with 100 μL of fresh HSC culture medium, and mix the cells well.

[0052] Step c: After one week of culture, the cells were passaged. After mixing the cells, 100 μL of the cell suspension was taken and 100 μL of fresh HSC cell culture solution was added;

[0053] Step d: After 1-2 days of culture, the relevant bile acid TDCA (concentration: 10 ng / mL) was added to the 96-well plate together with another 100 μL of HSC cell culture solution in step b, and the subsequent process was consistent with the original process.

[0054] Step e: After 7 days of culture, the proportion, number and apoptosis rate of hematopoietic stem cells and hematopoietic progenitor cells were analyzed in the above manner.

[0055] As shown in Figure 3 , it is a representative flow chart of hematopoietic stem cells in mouse bone marrow cells after the addition of TDCA. After the replenishment of TDCA, the proportion of mouse hematopoietic stem cells is restored. Figure 3 As shown in

[0056] , it is a graph of the proportion and absolute number of hematopoietic stem cells in mouse bone marrow cells after the addition of TDCA. After the replenishment of TDCA, the proportion and number of mouse hematopoietic stem cells increase. Figure 4 Figure 4 As shown in , it is a representative flow chart of apoptosis of hematopoietic stem cells in mouse bone marrow cells after the addition of TDCA. After the replenishment of TDCA, the apoptosis of mouse hematopoietic stem cells is reduced.

[0057] Figure 5 As shown in Figure 5 and , it is a representative flow chart of apoptosis of hematopoietic stem cells in mouse bone marrow cells after the addition of TDCA;

[0058] Figure 6 , it is a graph of the apoptosis rate of hematopoietic stem cells after the addition of TDCA. After the replenishment of TDCA, the apoptosis of mouse hematopoietic stem cells is reduced. Figure 7 Figure 6 As shown in Figure 7 , it is a representative flow chart of apoptosis of hematopoietic stem cells in mouse bone marrow cells after the addition of TDCA;

[0059] Example 3

[0060] The application of a kind of taurodeoxycholic acid in the preparation of drug for restoring damaged hematopoietic stem cells is verified by hematopoietic cell in vitro colony formation experiment, and the specific steps are as follows:

[0061] S1: take 5000 bone marrow cells from different treated mice, resuspend in 200 μL of HSC culture solution, and add to 1 mL of M3434 semi-solid culture medium, mix the cells and culture medium thoroughly, and then culture in a six-well plate.

[0062] S2: After 8 days of culture, observe the colony formation under a light microscope and count the total number of colonies formed.

[0063] As shown in​​Figure 8 As shown, Figure 8 Figure 15 is a graph showing the number of colonies formed by hematopoietic cells in vitro after the addition of TDCA. The number of colonies formed by mouse hematopoietic stem cells was restored after complementation with TDCA.

Claims

1. The application of taurine deoxycholic acid in the preparation of a drug for restoring damaged hematopoietic stem cells.

2. The application of taurine deoxycholic acid according to claim 1 in the preparation of a drug for restoring damaged hematopoietic stem cells, characterized in that: The drug includes taurine deoxycholic acid and pharmaceutically acceptable excipients.

3. The application of taurine deoxycholic acid according to claim 2 in the preparation of a drug for restoring damaged hematopoietic stem cells, characterized in that: The excipients are one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorbent carrier, and lubricant.

4. The application of taurine deoxycholic acid according to claim 2 in the preparation of a drug for restoring damaged hematopoietic stem cells, characterized in that: The drug may be an oral or non-oral formulation.

5. The application of taurine deoxycholic acid according to claim 2 in the preparation of a drug for restoring damaged hematopoietic stem cells, characterized in that: The drug is in the form of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, adsorbents, or sprays.

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