Application of GDF15 in preparation of medicine for treating DON poisoning and medicine thereof

Abstract

The invention provides application of GDF15 in preparation of a medicine for treating or preventing hematopoietic dysfunction caused by DON poisoning. The research finds that the GDF15 has the effects of treating or preventing bone marrow total cell reduction, hematopoietic stem progenitor cell damage, erythroid progenitor cell damage, erythroid end developmental stage damage, reticulocyte number reduction and peripheral blood mature erythrocyte damage caused by DON poisoning, and the exogenous GDF15 can relieve inhibition of DON poisoning on cell growth and development through research. And a GDF15-beta-catenin-Myc signal channel is activated, so that the downstream Myc, p27 and p21 are normally expressed. The invention discloses a new application of GDF15 in treatment of DON poisoning.

Description

Technical Field

[0001] The invention relates to the technical field of molecular medicine, in particular to application of GDF15 in preparing medicine for treating DON poisoning and the medicine. Background Art

[0002] Growth differentiation factor 15 (GDF15) is a cytokine that can affect systemic energy metabolism through its anorectic effects. GFRAL is the only receptor for GDF15, expressed in hindbrain neurons, and activation of the GDF15-GFRAL pathway is associated with increased sympathetic outflow and may also be associated with activation of the hypothalamus-pituitary-adrenal (HPA) stress axis. The expression and secretion of GDF15 can be induced under various stress conditions, resulting in highly variable levels of circulating GDF15. Chinese patent document CN112912395A discloses an anti-GDF15 antibody, composition, and method of use, which indicates that GDF15 expression is upregulated during inflammation and malignant tumors, resulting in significantly increased circulating concentrations of GDF15 in cancer, heart failure, chronic kidney disease, sarcopenia, and chronic obstructive pulmonary disease. Therefore, it is believed that weight loss caused by or associated with cachexia is mediated by or associated with increased GDF15 expression levels. This document discloses that the toxicity of a subject can be reduced or inhibited by using a GDF15 antibody, and that a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to GDF15 can be administered to achieve a therapeutic effect for infectious or non-infectious stimuli, diseases or syndromes including mycotoxins.

[0003] Deoxynivalenol (DON), a type of trichothecenes (i.e., mycotoxins), commonly known as "vomiting toxin", is a toxic compound produced by certain types of fungi that contaminates food crops under certain conditions. It is commonly found in grain products and is currently classified as a Class III carcinogen. DON remains chemically stable from growth, harvesting to processing, allowing it to enter the human body through the food chain, causing vomiting, anemia, immune system disorders, and neurological diseases.

[0004] At present, in the individual research level of experimental animals, exposure to DON for 23 days leads to a decrease in hemoglobin (HGB), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) values ​​in rainbow trout. Pigs can induce a decrease in their own red blood cell (RBC) and HGB content after exposure to DON. RBC and HGB levels are also considered as relevant biomarkers for toxicity detection in chickens after DON poisoning. Depending on the amount of DON intake, RBC and HGB reductions can be observed to varying degrees. At the cellular level, DON has been shown to disrupt cell signaling, proliferation, differentiation, and macromolecular synthesis, thereby affecting gastrointestinal homeostasis, growth, neuroendocrine function, and immunity. The pro-apoptotic properties of DON have been extensively verified in cancer cells of humans and animals, as well as normal cells of the digestive and immune systems. At the molecular level, DON binds to ribosomes, destroys the functional structure of ribosomes by breaking peptide bonds and peptide chains, interferes with the active center of peptidyl transferase located on the 60S subunit of the ribosome, and ultimately inhibits protein synthesis. In addition, DON also affects the alternative splicing of precursor messenger RNA (pre-mRNA), thereby affecting protein synthesis.

[0005] Anemia usually occurs simultaneously with the development of DON poisoning and is the most common hematological manifestation of DON poisoning. At the same time, rapid changes in hematological indicators are also the most obvious indication after DON poisoning, which will affect the recovery of DON poisoning and may also cause various complications that seriously endanger human health. Therefore, the severity of anemia can be used as an indicator of the degree of DON poisoning. At present, there is a lack of effective therapeutic drugs for hematopoietic disorders induced by DON poisoning in clinical practice.

[0006] In view of this, it is urgent to fully understand the molecular basis of hematopoietic disorders caused by DON poisoning and to find effective and feasible therapeutic targets. Summary of the invention

[0007] The purpose of the present invention is to provide application of GDF15 in preparing medicine for treating DON poisoning and the medicine.

[0008] The present invention provides the following technical solutions:

[0009] Application of GDF15 in the preparation of medicine for treating or preventing hematopoietic dysfunction caused by DON poisoning.

[0010] Furthermore, the hematopoietic dysfunction includes a decrease in total bone marrow cells, damage to hematopoietic stem cells, damage to erythroid progenitor cells, damage to the terminal development stage of erythroid cells, a decrease in the number of reticulocytes, and damage to mature red blood cells in peripheral blood.

[0011] Furthermore, the drug comprises an effective amount of GDF15 and pharmaceutically acceptable excipients.

[0012] Furthermore, the GDF15 has an amino acid sequence as shown in SEQ ID NO.1;

[0013] The SEQ ID NO.1 has the following sequence:

[0014] ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.

[0015] The present invention provides a drug for treating or preventing hematopoietic dysfunction caused by DON poisoning, wherein GDF15 is used as an active ingredient.

[0016] Furthermore, the drug also includes excipients; the GDF15 and the excipients are prepared into a clinically acceptable preparation through a preparation process, wherein the clinically acceptable preparation at least includes an injection.

[0017] It should be noted that the mechanism of action of GDF15 with hematopoiesis and related diseases is relatively complex and is not yet clear. Hematopoiesis is a complex process regulated by a variety of factors, including EPO, G-CSF, and iron homeostasis. Hematopoietic dysfunction is only one of the causes of anemia. There are many types of anemia, which can be caused by a variety of diseases. In addition to hematopoietic dysfunction, it also includes a variety of diseases such as chronic renal failure, chemotherapy, myelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation. For example, U.S. Patent US2019292252A1 discloses a method for using a GDF15 regulator and a composition containing a GDF15 regulator for the treatment of kidney disease or renal dysfunction, especially chronic kidney disease, end-stage renal failure, glomerulonephritis, anemia, diabetes, insulin resistance, etc. By reducing or inhibiting the activity of GDF15, kidney disease or renal dysfunction including anemia or reduced erythropoietin production can be improved. In addition, many literatures have verified that ineffective erythropoiesis is the main cause of iron overload. During the process of amplified erythropoiesis, the apoptosis of primitive erythrocytes will induce the upregulation of GDF15, and the upregulation of GDF15 will inhibit the secretion of hepcidin in liver cells, thereby increasing intestinal iron absorption, causing iron overload, and causing a series of pathological changes in important tissues and organs of the body. It can be seen that the effects of GDF15 on different types of anemia are complex and highly targeted. At present, there is no research on the mechanism of action of GDF15 and hematopoietic dysfunction caused by DON poisoning.

[0018] The present invention utilizes mouse DON poisoning model, human CD34 +The method combining in vitro cell culture system and proteomic analysis has clarified that DON can induce severe downregulation of GDF15 protein expression by changing the post-transcriptional translation process of erythroid progenitor cells, thereby inhibiting cell proliferation and differentiation. It has been verified that appropriate supplementation of GDF15 can help alleviate the inhibition of DON on cell growth and development, activate the GDF15-β-catenin-Myc signaling pathway, and normalize the expression of downstream Myc, p27, and p21. GDF15 can be used as a potential molecular target to improve hematopoietic disorders after DON poisoning, effectively treat or prevent hematopoietic dysfunction caused by DON poisoning, promote the restoration of hematopoietic balance and promote erythropoiesis, and promote the rapid recovery of hematopoietic function after food poisoning, thereby achieving the goal of treating DON poisoning. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 Schematic diagram of the detection time during the construction of the short-term DON poisoning model in mice;

[0021] Figure 2 This is a dynamic diagram of the blood routine test results of mice with short-term DON poisoning;

[0022] Figure 3 This is a comparison of the total number of bone marrow cells in mice in the PBS group and the DON poisoning group;

[0023] Figure 4 Comparison of HE staining of bone marrow sections of mice in the PBS group and the DON poisoning group;

[0024] Figure 5 Lin in the bone marrow of mice in the PBS group and the DON poisoning group - Comparison of the number of cells and erythroid progenitor cells;

[0025] Figure 6 This is a comparison of the number of BFU-E and CFU-E of mouse erythroid progenitor cells in the PBS group and the DON poisoning group;

[0026] Figure 7 This is a graph showing the dynamic changes in the number of nucleated erythrocytes and reticulocytes at the final stage of erythroid development in mice with short-term DON poisoning.

[0027] Figure 8 CD34 cells were treated with DON (0, 50, 100 ng / mL) from day 2 in the in vitro culture system.+ Growth curve of post-erythroid cells;

[0028] Fig. 9 Representative flow cytometric analysis of human erythroid progenitor cells BFU-E and CFU-E treated with PBS and different concentrations of DON on days 5 and 6 in an in vitro culture system;

[0029] Fig.10 CD34 in PBS group and DON treatment group + Comparison of BFU-E and CFU-E cell generation on days 5 and 6 of cell culture;

[0030] Fig.11 CD34 in PBS group and DON treatment group + Flow cytometric analysis and quantitative statistics of protein synthesis rate of cell-derived erythroid progenitor BFU-E;

[0031] Fig.12 CD34 in PBS group and DON treatment group + Flow cytometric analysis and quantitative statistics of protein synthesis rate of cell-derived erythroid progenitor CFU-E;

[0032] Fig.13 CD34 in PBS group and DON treatment group + Analysis plots of the first principal component and the second principal component (PCA) of the cell-derived erythroid progenitor proteomic sample;

[0033] Fig.14 This is the heat map of differentially expressed proteins between the PBS group and the DON-treated group;

[0034] Fig.15 This is the differential protein volcano map between the PBS group and the DON-treated group, in which the Top1 downregulated molecule is GDF15;

[0035] Fig.16 CD34 in PBS group and DON treatment group + Western blots of β-catenin, Myc, GDF15, p21, and p27 in erythroid progenitor cells of cell origin;

[0036] Fig.17 CD34 in PBS group and DON treatment group + Analysis of the expression of β-catenin, Myc, GDF15, p21, and p27 in cell-derived erythroid progenitor cells;

[0037] Fig.18Starting from the second day in the in vitro culture system, CD34 cells were treated with GDF15 (50 ng / mL), DON (100 ng / mL), and GDF15 and DON combined. + Growth curve of erythroid cells after cells;

[0038] Fig.19 CD34 cells were treated with GDF15 (50 ng / mL), DON (100 ng / mL), or a combination of GDF15 and DON. + The percentage of BFU-E and CFU-E cells on the 6th day of cell post-culture;

[0039] Fig. 20 CD34 cells were treated with GDF15 (50 ng / mL), DON (100 ng / mL), or a combination of GDF15 and DON. + Western blot of post-erythroid progenitors;

[0040] Fig.21 CD34 cells were treated with PBS, GDF15 (50 ng / mL), DON (100 ng / mL), and GDF15 and DON combined. + Quantitative analysis of β-catenin, Myc, p27, and p21 protein expression in cell-derived erythroid progenitor cells;

[0041] Fig. 22 Schematic diagram of the mouse model treated with GDF15 after short-term DON poisoning;

[0042] Fig.23 This is a comparison of the total number of bone marrow cells in mice in the PBS group, DON poisoning group, and GDF15 treatment group;

[0043] Fig.24 This is a comparison of the number of mouse erythroid progenitor cells in the PBS group, DON poisoning group, and GDF15 treatment group;

[0044] Fig.25 This is a comparison chart of the number of nucleated erythrocytes and reticulocytes in the terminal stage of erythroid development in mice in the PBS group, DON poisoning group, and GDF15 treatment group. DETAILED DESCRIPTION

[0045] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. In the embodiments of the present invention, if specific conditions are not specified, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially, and the raw materials of different manufacturers and models do not affect the implementation of the technical solution of the present invention and the realization of the technical effect.

[0046] Example

[0047] The drug for treating or preventing hematopoietic dysfunction caused by DON poisoning of this embodiment has GDF15 as an active ingredient and also includes auxiliary materials; the GDF15 and the auxiliary materials are prepared into an injection through a preparation process.

[0048] It should be noted that the GDF15 can be obtained by preparing recombinant GDF15 protein by genetic engineering methods, or can be obtained by purchasing. In this embodiment, the GDF15 lyophilized powder obtained by purchase is, for example, Human GDF-15 / MIC-1 Recombinant Protein,

[0049] The excipients may be osmotic pressure regulators (such as sodium chloride and other substances), stabilizers, antioxidants, buffers, etc., or other clinically acceptable pharmaceutical excipients, and those skilled in the art may select them according to actual conditions.

[0050] The preparation process can be a conventional preparation process, for example, it can be a conventional preparation process for dosage forms such as lyophilized powder injection, emulsion injection, solution injection, etc.

[0051] The drug for treating or preventing hematopoietic dysfunction caused by DON poisoning described in this embodiment is obtained by the following method:

[0052] The GDF15 lyophilized powder was taken and dissolved in ultrapure water to make the concentration of GDF15 0.4 μg / ml to obtain an injection.

[0053] Experimental example

[0054] Below, the following experiments are carried out to verify the technical effects of the present invention:

[0055] 1. Construction of a short-term DON poisoning model in mice

[0056] Take 8-week-old wild-type C57BL / 6 mice, weighing 20±2g. The mice were randomly divided into a control group and a DON poisoning group. A short-term poisoning model was constructed by injecting mice with a single injection of 10 mg / kg of DON, and the mice were tested on the 1st, 3rd, 4th, 5th and 7th days after poisoning. The animals in the control group were injected with the same volume of PBS (recorded as the PBS group). After acute DON poisoning, the animals in the GDF15 treatment group were intraperitoneally injected with the injection obtained in the example at a dose of 150 μg / kg.

[0057] All mice were housed in the experimental animal room of the School of Life Sciences of Zhengzhou University, which was SPF level. The indoor temperature was 25°C, the humidity was 60%, the animal cages were equipped with individual ventilation devices, and the light was on for 12 hours day and night.60 Irradiated sterilized mouse feed.

[0058] 2. Routine blood test of mice

[0059] (1) Place the mouse in the anesthesia device in the fume hood, use a pipette to draw 500 μL of isoflurane and add it. After anesthesia for 40-60 seconds (the anesthesia time depends on the weight of the mouse and the specific anesthesia conditions), the mouse enters a deep anesthesia state;

[0060] (2) The mouse was quickly removed from the anesthesia device and placed on the operating table with the abdomen facing upward. A 1 mL sterile syringe containing a trace amount of EDTA was inserted through the skin below the cartilage at the bottom of the thorax to draw blood from the heart.

[0061] (3) Rapidly transfer the peripheral blood to a 1.5 mL centrifuge tube, add EDTA at a ratio of peripheral blood: EDTA = 18:1, and gently flick the tube upside down to mix well to prevent uneven mixing of EDTA and the formation of clots;

[0062] (4) Turn on the computer and host of the fully automatic blood routine tester, wait for the system to automatically start and flush, and ensure that all indicators detected by the instrument are normal.

[0063] (5) Draw 200 μl of peripheral blood that has been mixed with EDTA into a new 1.5 mL EP tube, edit the sample test date, name, dilution factor, and species on the computer, and then load the sample.

[0064] (6) After the instrument test is completed, record the results, rinse the machine and turn it off.

[0065] 3. Preparation of Mouse Bone Marrow Single Cell Suspension

[0066] (1) Immobilize adult mice and quickly kill them by dislocating their necks and disinfecting their surfaces;

[0067] (2) Use scissors to cut the skin and muscles of the legs, peel off the femur and tibia, remove the remaining muscle tissue, and place them in a clean dish containing buffer on ice;

[0068] (3) Use a 10 mL syringe to extract the buffer solution to flush out the bone marrow from the femur and tibia, collect it in a 15 mL centrifuge tube, and use a 1 mL pipette to gently blow the bone marrow apart (no clumps);

[0069] (4) Extract the dispersed bone marrow and filter it through a 70 μm single-cell filter into a sterile 50 mL centrifuge tube. Add buffer solution to make up to 15 mL, invert and mix, and store in an ice box away from light.

[0070] (5) Take 380 μL of buffer in a 1.5 mL centrifuge tube, add 20 μL of bone marrow single cell suspension, vortex to mix, and obtain a 20-fold diluted single cell suspension. Then take 50 μL of 2× trypan blue dye in a new sterile 1.5 mL centrifuge tube, add 50 μL of the 20-fold diluted bone marrow single cell suspension, and vortex to obtain a 40-fold diluted bone marrow single cell suspension.

[0071] (6) Add 10 μL of 40-fold diluted single-cell suspension to the upper and lower sides of a clean counting plate for counting, and calculate the density of the bone marrow single-cell suspension and the total number of bone marrow cells based on the counting results.

[0072] 4. Flow Cytometry Detection of Mouse Erythroid Progenitor Cells

[0073] 1. Isolation method of mouse bone marrow erythroid progenitor cells BFU-E and CFU-E:

[0074] (1) According to the counting results, the required mouse bone marrow cell suspension was extracted into a 50 mL centrifuge tube, the centrifuge was set to 300 g, the sample was centrifuged for 10 min, and the supernatant was discarded to collect the cells;

[0075] (2) Prepare the antibody mixture according to the system in the table below and mix the cells. Place the centrifuge tube on a horizontal shaker in a 4°C refrigerator at 150 rpm and incubate for 30 minutes in the dark.

[0076] Table 1 Screening of erythroid stem cell staining system (5×10 7 cells / 500μL)

[0077] Antibody Name Conjugate volume Gr1 Biotin 1.25μL CD11b Biotin 1.25μL B220 Biotin 2.5μL CD3e Biotin 1.5μL Ter119 Biotin 5μL

[0078] (3) Add 10 mL of buffer, set the centrifuge to 300 g, centrifuge for 10 min to wash the cells, and discard the supernatant;

[0079] (4) Resuspend the cells in 180 μL of buffer, add 50 μL of anti-Biotin Microbeads, mix well, and place on a horizontal shaker in a 4°C refrigerator, incubate at 150 rpm in the dark for 15 min;

[0080] (5) Add 10 mL of buffer, set the centrifuge to 300 g, centrifuge the sample for 10 min, discard the supernatant and collect the cells;

[0081] (6) Resuspend the cells in 500 μL of buffer, add the suspension to the rinsed magnetic column (MS column), and collect the lineage flowing out of the magnetic column. -After the cell suspension has completely flowed out, add 500 μL of buffer to the magnetic column using a Pasteur pipette to wash the magnetic column, and repeat the operation twice. Finally, a total of 1.5 mL of suspension was collected and the cells were counted using the above method.

[0082] 2. Flow cytometry of mouse bone marrow erythroid progenitor cells BFU-E and CFU-E

[0083] (1) Based on the counting results, extract the BFU-E and CFU-E cell suspensions isolated from the required mouse fetal liver cells or adult mouse bone marrow cells into a 1.5 mL EP tube;

[0084] (2) Prepare the antibody mixture according to the system in the table below, mix the cells and incubate at 22°C in the dark for 30 min;

[0085] Table 2 Erythroid progenitor cell staining system (2×10 5 cells / 50μL)

[0086] Antibody Name Conjugate volume CD16 / 32 BV421 1.2μL CD41 BV421 1.2μL CD34 APC 1μL Sca-1 APC 0.2μL CD117 APC-Cy7 0.2μL CD71 BV750 0.4μL Streptavidin PercP 0.5μL

[0087] (3) Add 1 mL of buffer, set the centrifuge to 300 g, and centrifuge for 10 min to wash away unbound antibodies;

[0088] (4) Resuspend the cells in Buffer and transfer them to a flow cytometer. Add 7AAD at 1 / 100 volume of the buffer in the tube and analyze the cells using a flow cytometer. The analysis results are processed using FlowJo software.

[0089] 5. Mouse terminal red blood cell flow cytometry

[0090] (1) According to the counting results, extract the required mouse fetal liver and bone marrow cell suspension into a 1.5 mL EP tube. Set the centrifugal force of the centrifuge to 300 g, centrifuge the sample for 10 min, discard the supernatant and collect the cells;

[0091] (2) Prepare the antibody mixture according to the system in the table below and mix the cells. Incubate on ice for 30 min in the dark, tap the tube wall every 10 min to prevent cell sedimentation.

[0092] Table 3 Terminal erythroid cell staining system (1×10 6 cells / 50μL)

[0093]

[0094]

[0095] 3) After the antibody incubation is completed, add Buffer to the EP tube, set the centrifugal force of the centrifuge to 300g, centrifuge the sample for 10 minutes, and discard the unbound antibody in the supernatant;

[0096] 4) Resuspend the cells in Buffer and extract them into a flow tube, add 7AAD with 1 / 100 volume of the buffer in the tube, and analyze the cells using a flow cytometer. The analysis results are processed using FlowJo software.

[0097] 6. HE staining

[0098] Mouse leg bones were taken and embedded in paraffin, and mouse bone slices were cut from the paraffin-embedded tissue. After decalcification, they were stained with HE dye to detect the morphological changes of the bone marrow. The images were observed and photographed under an optical microscope at a magnification of 400×.

[0099] VII. CD34 + Cell culture

[0100] Isolation of CD34 from human umbilical cord blood using magnetic bead separation system + Cells. The cells were cultured in Iscove's modified Dulbecco's medium (IMDM) containing 8% heat-inactivated fetal bovine serum (FBS), 2% plasma (PB), 10 μg / mL insulin, 3 Iu / mL heparin, 10 μg / mL SCF, 1 μg / mL IL3, 3 Iu / mL EPO, 200 μg / mL TF and 1% PS in a humidified incubator (37°C, 5% CO2). The cell culture process includes 3 stages. In the first stage (D0 to D7), the cell density in the culture medium was 10 5 cells / mL. In the second stage (D7 to D11), IL-3 was not added to the culture medium. In the third stage (D11 to D15), SCF and IL-3 were not added to the culture medium.

[0101] 8. CD34 + Flow cytometry detection of early cell differentiation

[0102] (1) Count D5 and D6 cultured in vitro, taking 1×10 5 The cells were centrifuged at 4°C, 300 g for 5 min, and the supernatant was discarded; the cells were washed with 1 mL of Buffer, centrifuged at 4°C, 300 g for 5 min, and the supernatant was discarded.

[0103] (2) Resuspend the cells in 20 μL of buffer and add the following antibodies to the cell suspension:

[0104] Table 4CD34 + Cell early differentiation staining system (1×105 cells / 25μL)

[0105]

[0106]

[0107] (3) First add PE-CD34 and mix thoroughly. Incubate at room temperature in the dark for 10 min. Then add the remaining antibodies and incubate at room temperature in the dark for 15 min.

[0108] (4) After the antibody incubation is completed, add buffer to the EP tube, set the centrifugal force of the centrifuge to 300g, centrifuge the sample for 10 minutes, and discard the unbound antibody in the supernatant;

[0109] (5) Resuspend the cells in Buffer and transfer them to a flow cytometer. Add 7AAD at 1 / 100 volume of the buffer in the tube and analyze the cells using a flow cytometer. The analysis results are processed using FlowJo software.

[0110] IX. Western Blotting

[0111] 1. Extraction of total cell protein and determination of protein concentration by BCA method

[0112] (1) Mix the strong RIPA protein lysis buffer, protease inhibitor cocktail, and protein phosphatase inhibitor cocktail in proportion, dilute to working concentration, and pre-cool on ice for later use;

[0113] (2) According to the calculation of 1×10 6 Add 10 μL of prepared protein lysis buffer to the cells and suspend them. Set the vortexer to level 6 and shake the cells every 5 minutes for 30 minutes to fully lyse the cells and release the proteins. All operations were performed on ice to avoid protein degradation.

[0114] (3) After lysis, set the centrifuge speed to 14,000 rpm at 4°C and centrifuge the sample for 10 min;

[0115] (4) Gently draw the supernatant into a new tube without blowing up the precipitate, and mark the tube wall;

[0116] (5) When using 4 mg / mL BSA standard to prepare the standard curve, the protein standard solution needs to be set to 8 concentrations in sequence: 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL and 0.015625 mg / mL. Use ultrapure water to perform gradient dilution of the standard solution to ensure that the final solution volume in each well is 30 μL;

[0117] (6) Add the sample to be tested to a new well and dilute it with ultrapure water to ensure that the final solution volume is 30 μL;

[0118] (7) Mix BCA reagent and Cu 2+ The reagents were prepared and mixed according to the working concentration, kept away from light, and prepared before use;

[0119] (8) Add 200 μL of the mixed Cu 2+ Color development solution, ensuring a total volume of 230 μL per well, was placed in a 37°C incubator away from light for 30 min;

[0120] (9) Adjust the detection wavelength of the microplate reader to 562 nm and perform sample concentration detection. Draw a standard curve based on the absorbance value of the protein standard and calculate the sample protein concentration;

[0121] (10) Add loading buffer to the extracted sample, mix well, boil the protein to denature it, cool it down and then load it or store it in a -80°C refrigerator.

[0122] 2. Western Blotting

[0123] (1) Take a clean and dry 10 mm glass plate and comb, fix them on the gel-making rack to check their sealing condition, and avoid protein contamination during the whole process;

[0124] (2) Prepare gel using 10% CFAS PAGE rapid gel preparation kit, insert a comb that fits the gel plate, and let stand on a flat laboratory bench for 30 min;

[0125] (3) Assemble the gel running device, place it in a dry and clean electrophoresis tank, and add protein electrophoresis buffer according to the number of gel plates;

[0126] (4) Pull out the comb vertically along the wall of the gel plate to avoid deformation or damage of the gel holes. Add protein markers and samples to the gel holes in sequence. Keep the electrodes of the electrophoresis device, electrophoresis tank cover and electrophoresis instrument matched, turn on the power supply, and perform electrophoresis at 80V for 30 minutes to concentrate the sample, and at 100V for 90 minutes to separate the protein;

[0127] (5) After the electrophoresis is completed, remove the gel plate, cut off the concentrated gel and the protein-free portion, cut a PVDF membrane of the same size as the gel block and activate it by immersing it in methanol;

[0128] (6) Place the transfer clamp with the black cathode side facing downwards, and place two layers of transfer solution-soaked sponge, filter paper, activated PVDF membrane, glue block, filter paper and sponge on top in sequence, and remove the bubbles in each layer.

[0129] (7) Place the assembled transfer clip into the transfer tank, pour pre-cooled transfer solution into the tank and add ice packs. Confirm that the transfer clip, transfer tank, electrophoresis tank cover and the electrode orientation of the electrophoresis instrument match;

[0130] (8) Turn on the power supply and transfer the membrane at 100 V for 45-60 min depending on the size of the target protein band;

[0131] (9) After the transfer, stain the protein bands on the PVDF membrane with Ponceau solution. After confirming that the protein bands are clear and in the correct position, wash off the staining solution in time and cut the target bands. Place the membrane in 5% skimmed milk powder prepared with PBST, shake it slowly at 50 rpm on a horizontal shaker, and block it at room temperature for two hours;

[0132] (10) Use blocking solution to dilute the antibody of the target protein to the working concentration. After blocking, put the PVDF membrane and antibody into ziplock bags respectively, remove the bubbles, seal the ziplock bags, fix them on a flip shaker in a 4°C refrigerator, and incubate overnight;

[0133] (11) Take out the PVDF membrane and place it on a horizontal shaker at room temperature. Add PBST and wash it three times at 100 rpm for 15 min each time.

[0134] (12) Use blocking solution to dilute the secondary antibody to the working concentration. After the primary antibody is washed, add the diluted secondary antibody and incubate at room temperature for 2 h on a horizontal shaker at 50 rpm;

[0135] (13) Take out the PVDF membrane and place it on a horizontal shaker at room temperature. Add PBST and wash it three times at 100 rpm for 15 min each time.

[0136] (14) Under light-proof conditions, mix appropriate amounts of ECL super-sensitive luminescent liquid A and liquid B in a 1:1 ratio. Use a pipette to draw an appropriate amount of the mixed liquid and evenly drip it onto the PVDF membrane. Use filter paper to absorb the excess luminescent liquid. Use a multifunctional molecular imaging system to take pictures, and use ImageJ to process and quantitatively analyze the obtained pictures.

[0137] 10. qRT-PCR

[0138] 1. Extraction of Cellular RNA

[0139] (1) Count the cells and take the required cells. Resuspend the cells in 350 μL of Buffer RL and pipette repeatedly.

[0140] (2) Let stand for 3-5 minutes, centrifuge at 12000 rpm for 2 minutes, and remove the supernatant with a negative pressure pump.

[0141] (3) Add 350 μL of 70% ethanol and mix by pipetting repeatedly.

[0142] (4) Transfer it to a nucleic acid adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0143] (5) Add 350 μL of Buffer RW1, centrifuge at 12000 rpm for 2 min, and discard the waste liquid.

[0144] (6) Prepare DNase I digestion solution: 70 μL RNase-Free Water, 8 μL 10× Reaction Buffer, and 2 μL DNase I, a total of 80 μL. Add DNase I digestion solution and treat for 25 min.

[0145] (7) Add 200 μL of Buffer RW1, centrifuge at 12000 rpm for 2 min, and discard the waste liquid.

[0146] (8) Buffer RW2 (500 μL), centrifuge at 13000 rpm, centrifuge for 3 min.

[0147] (9) Repeat (8).

[0148] (10) Empty the centrifuge tube for 1.5 min, centrifuge at 12,000 rpm, and discard the waste liquid. Leave the adsorption column open for 4 min to remove the ethanol.

[0149] (11) Add 30 μL of enzyme-free water for elution, centrifuge at 12,000 rpm, and centrifuge for 2 min at room temperature. Determine the concentration and store in a -80 °C refrigerator.

[0150] 2. Reverse transcription of RNA into cDNA

[0151] (1) Thaw the RNA and each reagent in the reverse transcription kit on ice.

[0152] (2) Add reagents and mix them for PCR. The reverse transcription system is shown in the following table:

[0153] Table 5 Reverse transcription PCR system

[0154]

[0155]

[0156] (3) After adding all the above reaction reagents, mix well.

[0157] (4) Set the PCR reaction program: 42°C for 40 min, 85°C for 5 min, and 4°C for cooling. When the PCR reaction temperature drops to 4°C, take it out and store it in a -20°C refrigerator.

[0158] 3. Fluorescence real-time quantitative qRT-PCR

[0159] (1) Design primers for qRT-PCR, submit primer information to a primer synthesis company for synthesis, and dilute the synthesized primer powder with sterile water to 10 μM.

[0160] (2) The qRT-PCR reaction system is shown in the following table:

[0161] Table 6 qRT-PCR system

[0162] Reagents Usage Forward primer 1.0μL Reverse primer 1.0μL Master Mix 10.0μL cDNA 200ng-2.0μg water Make up to 20.0 μL

[0163] Mix well in the dark, set up the qRT-PCR program, and pre-denature at 95°C for 10 min.

[0164] Experimental Results

[0165] Based on the above experiments, the following conclusions are obtained:

[0166] 1. DON induces rapid changes in peripheral blood indicators in mice, destroys the directional differentiation of mouse bone marrow erythroid progenitor cells, and blocks the terminal stage of erythroid development

[0167] According to Experiment 1 and Experiment 2, in the construction of the short-term DON poisoning model in mice, routine blood tests were performed on the 1st, 3rd, 4th, 5th and 7th days after poisoning. Figure 1 The following is a schematic diagram of the detection time during the construction of the short-term DON poisoning model in mice. The dynamic changes of peripheral blood parameters in mice after short-term DON poisoning are shown in Figure 2. Figure 2 As shown. Mice poisoned by DON showed anemia, and peripheral red blood cell (RBC) indexes decreased rapidly. The number of peripheral blood red blood cells (RBC), hemoglobin (HGB), and hematocrit (HCT) dropped to the lowest level on the first day, and then began to recover from the first to third days after DON poisoning. In addition, the peripheral blood reticulocyte count (Retic), which reflects the activity of bone marrow erythropoiesis, also decreased from the first to the third day.

[0168] like Figure 3 As shown in the figure, by further studying the effect of DON on the homeostasis and directional differentiation of bone marrow hematopoietic cells, it was found that the total number of bone marrow cells in the DON-treated mice was significantly reduced compared with that in the control mice (e.g. Figure 3 ). According to Experiment 8, Figure 4 As shown, HE staining of bone marrow sections also confirmed the reduction in cell number.

[0169] Erythroid progenitor cells have a strong proliferation capacity for self-renewal and downward differentiation. The erythroid progenitor cell development stage is the core stage of erythropoiesis. According to Experiment 5, by using flow cytometry, Lin - CD16 / 32 - CD41 - CD34 - Sca1 - CD117 + Phenotype is a characteristic marker of erythroid progenitor cells. Figure 5 As shown, after intraperitoneal injection of DON in adult mice, Lin - The number of cells decreased significantly. Figure 6 As shown in Figure 2, quantitative statistics showed that the number of erythroid progenitor cells (including BFU-E and CFU-E) was significantly reduced. The expression of Ter119 and CD44 was used to characterize the terminal stage erythroid cells in the bone marrow by flow cytometry, as shown in Figure 2. Figure 7 As shown in the figure, on the first day after DON poisoning, the number of nucleated erythrocytes and reticulocytes in the terminal stage of the bone marrow erythroid lineage was significantly reduced, indicating that the erythroid development process in the bone marrow was impaired.

[0170] It can be seen that DON induces rapid changes in peripheral blood indicators of mice, destroys the directional differentiation of erythroid progenitor cells in the mouse bone marrow, and blocks the terminal stage development process of erythroid cells.

[0171] 2. DON disrupts the proliferation and differentiation process of human erythroid progenitor cells

[0172] To analyze the specific effects of DON on erythropoiesis in the absence of an in vivo hematopoietic microenvironment, human umbilical cord blood CD34 + On the second day of culture, DON was added to the culture medium and cell growth was examined. Figure 8 The growth curves shown indicate that DON significantly inhibited cell proliferation in a dose-dependent manner. Fig. 9 As shown, BFU-E and CFU-E cultured in vitro were characterized by flow cytometry using the expression of IL3R, GPA, CD34 and CD36 as markers. Fig.10 As shown, compared with the PBS group, the BFU-E% of the DON-treated group significantly accumulated on days 5 and 6, while the CFU-E% was significantly decreased, indicating that the differentiation at the erythroid progenitor stage was blocked.

[0173] It can be seen that DON destroys the proliferation and differentiation process of human erythroid progenitor cells.

[0174] 3. DON strongly inhibits GDF15 protein expression in erythroid progenitor cells and inhibits the GDF15-β-catenin-Myc signaling pathway

[0175] Studies have shown that ribosome damage is a common consequence of DON poisoning. DON may affect protein synthesis through post-transcriptional regulation. TM Plus OPP Alexa Fluor TM 488 Protein Synthesis Assay Kit Instructions OP-Puro incorporation assays were performed to assess protein synthesis rates in cultured erythroid progenitor cells. Figure 11-12 As shown in Figure 3, protein synthesis rates of BFU-E and CFU-E in the DON-treated group were significantly reduced. Erythroid progenitor cells treated with PBS and DON were collected for proteomic analysis. PCA confirmed the reproducibility of the three biological samples in each group and highlighted the different expression profiles between the groups, such as Fig.13 As shown in Figure 5, erythroid progenitor cells exposed to DON exhibited a unique expression signature. Fig.14 As shown in Figure 2, based on the total number of proteins identified in the two groups, we screened differentially expressed proteins (DEPs) with a fold change of at least 1.2 and a significance threshold of P < 0.05. GDF15 was a protein downregulated by Top1, as shown in Figure 2. Fig.15 Subsequent experiments further confirmed the reduction of GDF15 protein levels in erythroid progenitor cells after DON poisoning, and the changes in cell cycle and proliferation-related genes were also detected, such as Fig.16 Western blot analysis results are shown. Fig.17 As shown in Figure 3, DON treatment resulted in a two-fold decrease in β-catenin and Myc expression and an increase in p21 and p27 expression. These results further suggest that the effect of DON on erythroid progenitor cell growth may be due to post-transcriptional and post-translational changes in the protein expression levels of cell cycle-related genes.

[0176] It can be seen that DON strongly inhibited the expression of GDF15 protein and suppressed the GDF15-β-catenin-Myc signaling pathway.

[0177] 4. GDF15 can restore hematopoietic dysfunction induced by DON poisoning

[0178] like Fig.18 As shown, in the in vitro CD34 culture system, 50 ng / ml of GDF15 was added simultaneously with DON from the second day of culture to perform a hematopoietic function recovery experiment, and the erythroid cells were counted and found to increase gradually. Fig.19 As shown in Figure 2, by day 6, the percentage of BFU-E in the GDF15-treated group was reduced. Figure 20-21 As shown in Figure 2, GDF15 effectively normalizes the overexpression of β-catenin, Myc, p27, and p21, indicating that it has a significant regulatory effect on key proliferation and differentiation pathways of erythroid progenitors. Figure 22-25As shown in the short-term DON poisoning model in mice, supplementing mice with 150 μg / kg of GDF15 significantly increased the total number of bone marrow cells and restored them to normal levels, and the number of erythroid progenitor cells, terminal nucleated erythrocytes, and reticulocytes was significantly restored. Among them, the number of reticulocytes increased, and as a precursor of mature erythrocytes, they were released into the periphery to replenish peripheral mature erythrocytes. These results highlight the powerful effect of GDF15 in restoring hematopoietic balance and promoting erythropoiesis under stress conditions.

[0179] It can be seen that GDF15 can restore the hematopoietic dysfunction induced by DON poisoning.

[0180] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are encompassed by the present invention.

Claims

1. Application of GDF15 in the preparation of drugs for treating or preventing hematopoietic dysfunction caused by DON poisoning.

2. The use according to claim 1, characterized in that: The hematopoietic dysfunction includes one or more of a decrease in total bone marrow cells, damage to hematopoietic stem cells, damage to erythroid progenitor cells, damage to the terminal development stage of erythroid cells, a decrease in the number of reticulocytes, and damage to mature erythrocytes in peripheral blood.

3. The use according to claim 2, characterized in that: The drug comprises an effective amount of GDF15 and pharmaceutically acceptable excipients.

4. A drug for treating or preventing hematopoietic dysfunction caused by DON poisoning, characterized in that: The active ingredient is GDF15.

5. The drug according to claim 4, characterized in that It also includes auxiliary materials; the GDF15 and the auxiliary materials are prepared into a clinically acceptable preparation through a preparation process.

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