CN113797345B - Application of glucocorticoid and glycolytic demodulator in preparation of medicines for treating acute graft-versus-host disease - Google Patents

Abstract

The invention discloses application of glucocorticoid and glycolytic demodulator in preparing medicines for treating acute graft-versus-host disease. The invention protects the application of glucocorticoid and glycolytic demodulator in preparing medicines; the medicament is a medicament for treating acute graft versus host disease. The invention also protects the application of the glycolysis regulator in preparing products; the function of the product is to treat and/or prevent glucocorticoid resistance in patients with acute graft versus host disease. The invention discovers that the T cell function can be improved by regulating and controlling the glucose metabolism level of the T cell, so that the glucocorticoid and the glycolytic demodulator are used for cooperatively treating the transplanted aGVHD patient, and the invention has very important significance for clinical targeted treatment.

Description

Application of glucocorticoids and glycolysis regulators in the preparation of drugs for acute graft-versus-host disease

technical field

In the field of biomedicine, the present invention relates to the application of glucocorticoids and glycolysis regulators in the preparation of medicines for acute graft-versus-host disease, and specifically relates to the synergistic treatment of glucocorticoids and glycolysis regulators for acute graft-versus-host disease sick.

Background technique

Acute graft-versus-host disease (aGVHD) is an important complication of allogeneic hematopoietic stem cell transplantation. aGVHD is generally considered to be an immune-mediated disease. Specifically, aGVHD is stimulated by "cytokine storm" to enhance the immune response to recipient antigens, and the main targets of aGVHD are the recipient's skin, liver and intestinal tract. Cytotoxic attack is characteristic, resulting in increased associated mortality and patient medical costs.

Glucocorticoids are currently the first-line treatment for aGVHD, but 40-60% of patients still develop steroid resistance, and the current second-line treatment is not effective. Therefore, the in-depth elucidation of the pathogenesis of aGVHD and the establishment of new treatment strategies are important clinical scientific issues that need to be resolved urgently.

Contents of the invention

The purpose of the present invention is to provide the application of glucocorticoids and glycolysis regulators in the preparation of medicines for acute graft-versus-host disease, specifically related to the synergistic treatment of glucocorticoids and glycolysis regulators for acute graft-versus-host disease .

The invention protects the application of glucocorticoids and glycolysis regulators in the preparation of medicines; the medicines are medicines for acute graft-versus-host disease. When the medicine is applied, the glucocorticoid and the glycolysis regulator exert a curative effect against acute graft-versus-host disease through a synergistic effect.

Specifically, the glycolysis regulator is 3PO.

Specifically, the glycolysis regulator is MTX.

Specifically, the glucocorticoid is MP.

Specifically, the molar ratio of MP and 3PO may be 1:2.5-20.

Specifically, the molar ratio of MP and 3PO is 1:10.

Specifically, the mass ratio of MP and 3PO is 2:25.

Specifically, the molar ratio of MP and MTX is 1:0.25-2.

Specifically, the molar ratio of MP and MTX is 1:0.25.

Specifically, the mass ratio of MP and MTX is 2:1.

The invention also protects a medicine whose active ingredients are glucocorticoid and glycolysis regulator; the medicine is a medicine for acute graft-versus-host disease. When the medicine is applied, the glucocorticoid and the glycolysis regulator exert a curative effect against acute graft-versus-host disease through a synergistic effect.

Specifically, the glycolysis regulator is 3PO.

Specifically, the glycolysis regulator is MTX.

Specifically, the glucocorticoid is MP.

Specifically, the molar ratio of MP and 3PO may be 1:2.5-20.

Specifically, the molar ratio of MP and 3PO is 1:10.

Specifically, the mass ratio of MP and 3PO is 2:25.

Specifically, the molar ratio of MP and MTX is 1:0.25-2.

Specifically, the molar ratio of MP and MTX is 1:0.25.

Specifically, the mass ratio of MP and MTX is 2:1.

The invention also protects the application of the glycolysis regulator in the preparation of medicine; the function of the medicine is to treat and/or prevent glucocorticoid resistance in patients with acute graft-versus-host disease. The glycolysis regulator can be used to treat and/or prevent glucocorticoid resistance in patients with acute graft-versus-host disease, so that the glucocorticoid and the glycolysis regulator exert a curative effect against acute graft-versus-host disease through a synergistic effect.

Specifically, the glycolysis regulator is 3PO.

Specifically, the glycolysis regulator is MTX.

Specifically, the glucocorticoid is MP.

The invention also protects a medicine whose active ingredient is a glycolysis regulator; the function of the medicine is to treat and/or prevent glucocorticoid resistance in patients with acute graft-versus-host disease. The glycolysis regulator can be used to treat and/or prevent glucocorticoid resistance in patients with acute graft-versus-host disease, so that the glucocorticoid and the glycolysis regulator exert a curative effect against acute graft-versus-host disease through a synergistic effect.

Specifically, the glycolysis regulator is 3PO.

Specifically, the glycolysis regulator is MTX.

Specifically, the glucocorticoid is MP.

The patient with acute graft-versus-host disease may be a patient with acute graft-versus-host disease after hematopoietic stem cell transplantation.

Metabolic reactions in T cells control cell proliferation, differentiation, activation, and apoptosis, and the imbalance of cell metabolism and immune disorders are mutually causal. Therefore, an in-depth understanding of T cell metabolism and its dynamic regulation will provide effective molecular targets and new methods for potential clinical treatment for the prevention and treatment of immune-related diseases. Through animal models, it was found that in the development of graft-versus-host disease, glycolysis becomes the main energy source of effector T cells. In a transplanted mouse model of major histocompatibility complex incompatibility, it was found that donor alloreactive T cells obtain the substances required for activation and proliferation through increased glycolysis and oxidative phosphorylation, while the cells metabolize key enzymes The level of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, PFKFB3) was significantly increased. Drug blocking of PFKFB3 and inhibition of glycolysis can reduce the generation of alloreactive T cells, thereby reducing the severity of acute graft-versus-host disease.

At present, whether there are abnormalities in the metabolic state of T cells in patients with aGVHD after transplantation remains to be further studied. The present invention finds that the function of T cells can be improved by regulating the glucose metabolism level of T cells, so that the synergistic treatment of aGVHD patients after transplantation with glucocorticoids and glycolysis regulators has very important significance for clinical targeted therapy.

Description of drawings

Fig. 1 is the result figure of embodiment 1.

Fig. 2 is the result graph of embodiment 2.

Fig. 3 is the result graph of embodiment 3.

FIG. 4 is the result of survival rate in Example 4.

Fig. 5 is the scoring result in embodiment 4.

FIG. 6 is the result of detecting PFKFB3 and GLUT1 by flow cytometry in Example 4.

FIG. 7 is the results of flow cytometry detection of the ratio of CD4 ⁺ and CD8 ⁺ cells in Example 4.

FIG. 8 is the result of HE staining in Example 4.

FIG. 9 is the result of in vivo bioluminescence imaging in Example 4. FIG.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Methylprednisolone (MP), white crystalline powder, CAS accession number is 82-43-2. Methotrexate (MTX), yellow crystalline powder, CAS accession number 59-05-2. 3PO, full name 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one : Sigma, St.Louis, MO, USA, the product number is SML1343, and the chemical structure is C ₁₃ H ₁₀ N ₂ O. MP is a glucocorticoid. MTX is a glycolysis regulator. 3PO is a glycolysis regulator.

CD3 magnetic beads: Miltenyi Biotec (Bergisch Gladbach, Germany), Cat. No. 130-050-101. MiniMACS ^™ Separator with MS separation column: Miltenyi Biotec (Bergisch Gladbach, Germany), Cat. No. 130-090-312.

The Th1 cell phenotype was CD3 ⁺ and CD8 ^- and IFN-γ ⁺ . The Th2 cell phenotype was CD3 ⁺ and ^CD8- and IL-4 ⁺ . Th17 cells were phenotyped as CD3 ⁺ and ^CD8- and IL-17A ⁺ . Treg cell phenotypes were CD3 ⁺ and CD8 ^- and CD25 ⁺ and Foxp3 ⁺ . The phenotype of Tc1 cells was CD3 ⁺ and CD8 ⁺ and IFN-γ ⁺ . Tc2 cells were phenotyped as CD3 ⁺ and CD8 ⁺ and IL-4 ⁺ . Antibodies in flow cytometry are as follows: CD8 antibody (BD, catalog number: 560347), IFN-γ antibody (Biolegend, catalog number: 502536), IL-4 antibody (BD, catalog number: 559333), IL-17A antibody (Biolegend, Cat. No.: 512304), CD25 Antibody (BD Cat. No.: 335807), Foxp3 Antibody (BD, Cat. No.: 560045). DMEM high glucose medium: gibco, product number: 11965092. CD3/CD28 antibody-coupled magnetic beads (CD3/CD28 monoclonal antibody beads): gibco, catalog number: 11131D.

Example 1. A prospective clinical cohort study found that the glycolysis level of peripheral T cells in patients with aGVHD was abnormally elevated

Research objects: aGVHD patients (indicated by GVHD) and non-aGVHD patients (indicated by non-GVHD) after allogeneic hematopoietic stem cell transplantation (allogeneic hematopoietic stem cell transplantation, allo-HSCT). The basic characteristics of aGVHD patients and non-aGVHD patients (such as gender, age, pre-transplantation underlying diseases, chemotherapy times, risk assessment, conditioning regimen, source of transplanted stem cells, total nucleated cell dose, detection time after transplantation, cytomegalovirus infection history) and so on.

Through a prospective clinical cohort study, it was found that the glycolysis level of peripheral T cells in patients with aGVHD was abnormally elevated.

The inventor detected the distribution of peripheral T cell subsets in patients with aGVHD and found that compared with non-aGVHD patients, T cells in aGVHD patients differentiated towards Th1 and Tc1, and Th17 cells in aGVHD patients increased, and the ratio of Th17 to Treg cells was out of balance, showing a proinflammatory tendency. Imbalanced state of T cell differentiation in phenotypic direction.

The inventors further evaluated whether there is a difference in the level of glycolysis in the peripheral T cells of aGVHD patients and non-aGVHD patients. The abundance of PFKFB3 in peripheral blood CD3 ⁺ T cells of the subjects was detected by flow cytometry, and compared with non-aGVHD patients, the abundance of PFKFB3 in aGVHD patients was increased (Figure 1A). The abundance of PFKFB3 in the peripheral blood total protein of the subjects was detected by Wseternblot. Compared with non-aGVHD patients, the abundance of PFKFB3 in aGVHD patients was increased (Figure 1D). Seahorse cell energy metabolism analysis, lactate assay kit, and glucose assay kit were used to detect the glucose metabolism level of T cells in aGVHD patients; compared with non-aGVHD patients, T cells in aGVHD patients showed higher ECAR values (Figure 1 E and F ), higher glucose consumption rate (Figure 1 I) and higher lactate production rate (Figure 1 J). However, there was no significant change in OCR of T cells in aGVHD patients compared with non-aGVHD patients (Figure 1G and H). As detected by flow cytometry, compared with non-aGVHD patients, the PFKFB3 protein level of T cells in aGVHD patients, especially CD4 ⁺ naive T cells, was significantly increased (Fig. 1B). Further real-time fluorescent quantitative PCR found that compared with non-aGVHD patients, the mRNA levels of metabolic enzymes of the glycolytic pathway were increased in aGVHD patients, including hexokinase 3 (HK3), PFKFB3 and lactate dehydrogenase B (LDHB) mRNA levels were significantly elevated (Figure 1C).

Taken together, these results suggest a PFKFB3-mediated increase in glycolysis in T cells from patients with aGVHD.

Example 2, PFKFB3 modulators inhibit the proliferation and activation of peripheral T cells in patients with aGVHD by inhibiting glycolysis

Patients with aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were confirmed patients at Peking University People's Hospital, and they gave informed consent to relevant experiments. The peripheral blood of the aGVHD patient was collected, the peripheral blood mononuclear cells were separated, and then the CD3 ⁺ cells were separated from the peripheral blood mononuclear cells, which were peripheral CD3 ⁺ T cells.

Peripheral CD3 ⁺ T cells were collected and divided into two groups, one group was treated with 3PO (this group is represented by GVHD+3PO), and the other group was used as a control group (this group was represented by GVHD).

The abundance of PFKFB3 in the cells was detected by flow cytometry, and the results are shown in A of FIG. 2 . The glucose consumption of the cells was detected by a glucose assay kit, and the results are shown in Figure 2B. The lactic acid production of the cells was detected with a lactic acid assay kit, and the results are shown in Figure 2C. The expression of pro-inflammatory cytokines in T cells was detected by flow cytometry, and the ratio of Th1 cells, Tc1 cells and Th17 cells were obtained. The results are shown in D, E and F of Figure 2. The abundance of T cell inflammatory transcription factors (T-bet and RORγT) in the cells was detected by flow cytometry, and the results are shown in G and H in Figure 2. The abundance of GATA3 and Foxp3 in the cells was detected by flow cytometry, and the results are shown in I and J of Figure 2. The proliferative ability and apoptosis of T cells in the cells were detected by flow cytometry, and the results are shown in K and L of FIG. 2 .

The results show that the application of 3PO can significantly reduce the expression of PFKFB3 in T cells of patients with aGVHD, reduce the rate of glucose consumption, and significantly reduce the rate of lactic acid production; 3PO can reduce the expression of proinflammatory cytokines in T cells of patients with aGVHD, Th1 cells, Tc1 cells and The proportion of Th17 cells decreased; 3PO decreased the expression of inflammatory transcription factors, T-bet and RORγT in T cells of patients with aGVHD; 3PO had no significant effect on the expression of transcription factors GATA3 and Foxp3; 3PO decreased the proliferation ability of T cells in patients with aGVHD, without affecting the apoptosis of T cells.

In summary, 3PO inhibits the activation and proliferation of peripheral T cells in patients with aGVHD by inhibiting glycolysis.

Example 3, glucocorticoids and glycolysis regulators synergistically treat aGVHD

1. Acquisition of CD3 ⁺ T cells from aGVHD patients

All aGVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were all confirmed patients at Peking University People's Hospital, and they gave informed consent to the relevant experiments.

1. Take the peripheral blood of the patient and separate the mononuclear cells.

2. Use CD3 magnetic beads and MiniMACS ^TM separator with MS separation column to separate CD3 ⁺ cells from the mononuclear cells obtained in step 1, which are CD3 ⁺ T cells.

2. Fluorescence microscope observation

The test cells are: CD3 ⁺ T cells prepared in Step 1.

1. Take a 96-well plate and coat it with 1× recombinant human fibronectin (sigma, product number: ECM001) for 24 hours in advance.

2. After completing step 1, inoculate 2×10 ⁵ test cells per well, and culture in 100-200 μl DMEM high-glucose medium containing CD3/CD28 antibody-coupled magnetic beads and 10% FBS for 24 hours. The ratio of the number of CD3/CD28 antibody-coupled magnetic beads to the number of cells is 1:1.

3. After step 2 is completed, the grouping process is as follows:

The first group: add MP and 3PO, then cultivate for 48 hours; the concentration of MP in the system is set to 1 μM, and the concentration of 3PO in the system is set to 2.5 μM, 5 μM, 10 μM or 20 μM respectively (the treatment without adding 3PO is set, as 0 concentration control of 3PO).

The second group: add MP and MTX, then cultivate for 48 hours; the concentration of MP in the system is 1 μM, and the concentration of MTX in the system is set to 0.25 μM, 0.5 μM, 1 μM or 2 μM respectively (the treatment without adding MTX is set, as 0 concentration control of MTX).

4. After completing step 3, discard the supernatant, add 5 μl DAPI to each well and incubate at room temperature for 10 minutes, then wash with PBS buffer, and then use a fluorescent microscope to excite the green light and blue light to observe and count.

The results are shown in Figure 3 I.

3. Detection of other indicators

1. Group processing

(1) Take a 96-well plate, inoculate 8× ¹⁰⁴ CD3 ⁺ T cells prepared in Step 1 in each well, and culture them in 100-200 μl DMEM high-glucose medium containing CD3/CD28 antibody-coupled magnetic beads and 10% FBS for 24 Hour. The ratio of the number of CD3/CD28 antibody-coupled magnetic beads to the number of cells is 1:1.

(2) After completing step (1), the grouping process is as follows:

The first group (GVHD+3PO group): add 3PO, and then cultivate; the concentration of 3PO in the system is 10 μM;

The second group (GVHD+MP group): add MP, and then cultivate; the concentration of MP in the system is 1 μM;

The third group (GVHD+MTX group): add MTX, and then cultivate; the concentration of MTX in the system is 0.25 μM;

The fourth group (GVHD+MP+3PO group): add MP and 3PO, and then cultivate; the concentration of MP in the system is 1 μM, and the concentration of 3PO in the system is 10 μM;

The fifth group (GVHD+MP+MTX group): add MP and MTX, and then cultivate; the concentration of MP in the system is 1 μM, and the concentration of MTX in the system is 0.25 μM;

The sixth group (GVHD group): without any treatment.

The incubation time was 48 hours.

2. Detection of PFKFB3 and GLUT1 by flow cytometry

(1) Take the cells treated in step 1 to the flow detection tube.

(2) Add 5 μl CD3 ⁺ cell surface flow cytometry antibody CD3 to the flow detection tube, and incubate at room temperature for 15 minutes in the dark.

(3) Add 2ml of PBS buffer and mix well, centrifuge at 1500rpm for 5 minutes; then, discard the supernatant, add 100μl of fixed medium A (invitrogen, product number: GAS004) in the FIX&PERM ^TM cell permeabilization kit, mix well, and keep in the dark at room temperature Incubate for 15min.

(4) Add 2ml of PBS buffer and mix well, centrifuge at 1500rpm for 5 minutes; then, discard the supernatant, add 100μl of permeabilization medium B (invitrogen, product number: GAS004) in the FIX&PERM ^TM cell permeabilization kit, mix well, add 1μl For intracellular flow cytometry antibodies, incubate at room temperature for 15 minutes in the dark. The intracellular flow cytometry antibodies were: intracellular flow cytometry antibody PFKFB3 (Cell Signaling Technology, Danvers, MA, USA) or intracellular flow cytometry antibody GLUT1 (Cell Signaling Technology, Danvers, MA, USA).

(5) Add 2ml of PBS buffer and mix well, and centrifuge at 1500rpm for 5 minutes; then, discard the supernatant, add 100μl of the permeabilization medium B in the FIX&PERM ^TM cell permeabilization kit, mix well, and add rabbit anti-mouse secondary antibody (Cell Signaling Technology , Danvers, MA, USA), and incubated at room temperature for 15 min in the dark.

(6) Add 2ml of PBS buffer and mix well, centrifuge at 1500rpm for 5 minutes; then, discard the supernatant, add 200μl of PBS buffer to resuspend the cells, and test on the machine within 4 hours. Flow image analysis uses Diva 7.0 software (BD, USA).

The results are shown in Figure 3, F and G.

3. Detection of glucose consumption

(1) Take a 96-well plate, inoculate 2×10 ⁵ cells treated in step 1 into each well, and culture in 100-200 μl DMEM high-glucose medium containing CD3/CD28 antibody-coupled magnetic beads and 10% FBS for 24 hours. The ratio of the number of CD3/CD28 antibody-coupled magnetic beads to the number of cells is 1:1.

(2) After completing step (1), centrifuge at 1500 rpm for 5 minutes, and collect the supernatant.

(3) Take the supernatant obtained in step (2), use a glucose assay kit (Nanjing Jiancheng Bioengineering Research Institute, article number: 361510) to detect the glucose content, and calculate the glucose consumption rate.

The results are shown in Figure 3D.

4. Detection of lactic acid production

(1) Take a 96-well plate, inoculate 2×10 ⁵ cells treated in step 1 into each well, and culture in 100-200 μl DMEM high-glucose medium containing CD3/CD28 antibody-coupled magnetic beads and 10% FBS for 24 hours. The ratio of the number of CD3/CD28 antibody-coupled magnetic beads to the number of cells is 1:1.

(2) After completing step (1), centrifuge at 1500 rpm for 5 minutes, and collect the supernatant.

(3) Take the supernatant obtained in step (2), use a lactic acid assay kit (Nanjing Jiancheng Bioengineering Research Institute, article number: A019-2-1) to detect the lactic acid content, and calculate the lactic acid production rate.

The results are shown in Figure 3, E.

5. Detection of the expression of pro-inflammatory cytokines in T cells

(1) Take a 96-well plate, inoculate each well with 2× ¹⁰⁵ cells treated in step 1, add 100 μl of 1 ng/μl PMA aqueous solution, 2 μl of 1 μg/μl ionomycin aqueous solution, and 0.7 μl of Golgistop aqueous solution, shake and mix well, Incubate in a 37°C incubator for 4-6 hours.

(2) Centrifuge at 1500rpm for 5min, discard the supernatant, pop the cells, add surface flow cytometry antibodies (CD3 antibody, CD8 antibody and CD25 antibody), and incubate at room temperature for 15min.

(3) Add 2ml of PBS buffer, centrifuge at 1500rpm for 5min, discard the supernatant and pop the cells.

(4) Add 1ml of fixative solution prepared by ^{eBioscienceTM} Foxp3/transcription factor fixation concentrate and diluent buffer at a ratio of 1:3, vortex and mix well, and incubate at 4°C for 30min.

(5) Add 2ml of nucleation membrane-breaking working solution (invitrogen, ^{eBioscienceTM} Foxp3/transcription factor staining buffer kit, catalog number: 00-5523-00) prepared by wash buffer and deionized water at a ratio of 1:9, and centrifuge at 2000rpm for 5min , discard the supernatant.

(6) Add intracellular antibodies (IFN-γ, IL-4, IL-17 and Foxp3), vortex and mix well, and incubate at 4°C for 30min.

(7) Add 2ml of nuclear membrane-breaking working solution, 2000rpm×5min, discard the supernatant, add 200μl of nuclear-breaking working solution, and pop the cells.

(8) On-board testing within 24 hours. Flow image analysis was performed using Diva 7.0 software.

The results are shown in Figure 3, A and B.

6. CCK-8 detection of T cell proliferation ability

(1) Take a 96-well plate, inoculate 100 μl of 1×10 ⁵ /ml cells treated in Step 1 into each well, and culture for 24 hours.

(2) Add 10 μl CCK-8 solution to each well and incubate in the incubator for 2 hours.

(3) Measure the absorbance at 450 nm with a microplate reader.

The results are shown in Figure 3C.

7. Detection of NF-κB p65

(1) Take the cells treated in step 1 to the flow detection tube.

(2) Add 5 μl CD3 ⁺ cell surface flow cytometry antibody CD3 to the flow detection tube, and incubate at room temperature for 15 minutes in the dark.

(3) Add 2ml of PBS buffer and mix well, centrifuge at 1500rpm for 5 minutes; then, discard the supernatant, add 100μl of fixed medium A (invitrogen, product number: GAS004) in the FIX&PERM ^TM cell permeabilization kit, mix well, and keep in the dark at room temperature Incubate for 15min.

(4) Add 2ml of PBS buffer and mix well, centrifuge at 1500rpm for 5 minutes; then, discard the supernatant, add 100μl of permeabilization medium B (invitrogen, product number: GAS004) in the FIX&PERM ^TM cell permeabilization kit, mix well, add 1μl Intracellular flow cytometry antibody NF-κB p65 (Cell Signaling Technology, Danvers, MA, USA), incubated at room temperature for 15 minutes in the dark.

(5) Add 2ml of PBS buffer and mix well, and centrifuge at 1500rpm for 5 minutes; then, discard the supernatant, add 100μl of the permeabilization medium B in the FIX&PERM ^TM cell permeabilization kit, mix well, and add rabbit anti-mouse secondary antibody (Cell Signaling Technology , Danvers, MA, USA), and incubated at room temperature for 15 min in the dark.

(6) Add 2ml of PBS buffer and mix well, centrifuge at 1500rpm for 5 minutes; then, discard the supernatant, add 200μl of PBS buffer to resuspend the cells, and test on the machine within 4 hours. Flow image analysis uses Diva 7.0 software (BD, USA).

The results are shown in Figure 3H.

4. Calculation of CI value

Test cells: CD3 ⁺ T cells prepared in Step 1.

Set two drug combinations: MP and 3PO combination, MP and MTX combination.

A variety of proportioning concentrations are set for each drug combination.

Refer to the group processing method in step 2 for processing.

The CI values corresponding to the inhibition rates of 25%, 50%, 75%, 90%, and 95% for the synthesis of IFNγ by CD3 ⁺ T cells were calculated by Com-puSyn software. The CI values corresponding to the inhibition rates of CD3 ⁺ T cell proliferation of 25%, 50%, 75%, 90%, and 95% were calculated by Com-puSyn software.

The results are shown in Figure 3J. The average CI value of MP and 3PO for T cell IFNγ synthesis was 0.364; the average CI value of MP and MTX for T cell IFNγ synthesis was 0.554. The average CI value of MP and 3PO for T cell proliferation was 0.475; the average CI value of MP and MTX for T cell proliferation was 0.406.

Combined application of glucocorticoid (MP) and glycolysis regulator (3PO or MTX) to peripheral T cells of patients with aGVHD in vitro, and found that combined application of glucocorticoid and glycolysis regulator can synergistically inhibit glycolysis, and then synergistically inhibit aGVHD Activation and proliferation of peripheral T cells in patients.

Through the dose-response effect, it was found that MP (1 μM) and MTX (0.5 μm) had the best inhibitory effect on T cells in patients with aGvHD, but had no significant effect on T cell survival. In vitro application of MP and MTX significantly reduced the rate of glucose consumption and lactate production. MP reduces the glycolytic activity of T cells by downregulating the expression of GLUT1 and PFKFB3. MTX reduces the glycolytic activity of T cells by downregulating the expression of GLUT1. In addition, MP and MTX reduced the proportion of pro-inflammatory cells, including Th1 and Tc1 cells, in the T cells of patients with aGVHD. And MP and MTX decreased the proliferation of T cells in aGvHD patients, but did not significantly affect the apoptosis of T cells. These results suggest that MP inhibits T cell activation and proliferation by inhibiting GLUT1- and PFKFB3-stimulated glycolysis, and MTX inhibits T-cell activation and proliferation by inhibiting GLUT1-stimulated glycolysis.

The combined use of glucocorticoids and glycolytic regulators has a synergistic effect on the improvement of T cell activity in patients with aGvHD, and the mechanism may be achieved by reducing glycolytic activity. Compared with cells treated with single drugs, the combination of glucocorticoids and glycolysis regulators has a synergistic inhibitory effect on T cell differentiation to pro-inflammatory Th1, Tc1 and T cell proliferation. In addition, the combined use of glucocorticoids and glycolytic modulators was superior to glucocorticoids alone in downregulating glycolytic activity in vitro.

It is worth noting that the combined regimen has a strong synergistic effect on T cells, and the average CI value of MP and 3PO for T cell IFNγ synthesis is 0.364. In addition, the average CI value of MP and MTX for T cell IFNγ synthesis was 0.554. The average CI value of MP and 3PO for T cell proliferation was 0.475. In addition, the average CI value of MP and MTX for T cell proliferation was 0.406.

Example 4. Cooperative treatment of aGVHD with glucocorticoids and glycolysis regulators

The synergistic therapeutic effect of glucocorticoids and glycolytic modulators was verified using aGVHD mouse model.

NPG mice (NOD.Cg-Prkdcscid Il2rgtm1Vst/Vst mice): Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

Luciferase luciferase ⁺ THP-1 leukemia cells (THP-1-luc cells), described in the following literature: Azacytidine prevents experimental xenogeneic graft-versus-host disease without abrogating graft-versus-leukemia effects, ONCOIMMUNOLOGY, 2017, VOL.6, NO .5, e1314425.

1. Suspend human peripheral blood stem cells with 1× hemolysin, suspend and shake for 5 seconds, then place on ice in the dark for 15 minutes, then centrifuge at 1500rpm for 5 minutes, discard the supernatant; then suspend with PBS buffer, and then centrifuge at 1500rpm for 5 minutes Minutes, discard the supernatant; then suspend with PBS buffer to obtain cell suspension. 1 volume part of 10× hemolysin (BD Biosciences) was mixed with 9 volume parts of sterile water for injection to obtain 1× hemolysin.

2. NPG mice began to drink antibiotic water one week before cell injection to prevent infection, and put them into air laminar flow cabinets for aseptic feeding.

3. The whole body of NPG mice was irradiated with ⁶⁰ Coγ-rays at 1.5Gy (semi-lethal dose) one day before cell injection.

4. Each mouse was injected with the cell suspension prepared in Step 1 (5×10 ⁶ cells/mouse) and luciferase ⁺ THP-1 leukemia cells (1×10 ⁶ cells/mouse) through the tail vein. Start counting days after you finish your injection.

5. The mice that completed step 4 were randomly divided into six groups (PBS group, MP group, 3PO group, MTX group, MP combined with 3PO group, MP combined with MTX group), with 11 mice in each group. The mice in the PBS group were intraperitoneally injected with PBS buffer once a day, with an injection volume of 200 μl/mouse. Mice in the MP group were intraperitoneally injected with MP solution once a day, the injection volume was 200 μl/mouse, and the dose of MP was 2 mg/kg body weight/time. The mice in the 3PO group were intraperitoneally injected with 3PO solution once a day, the injection volume was 200 μl/mouse, and the dose of 3PO was 25 mg/kg body weight/time. The mice in the MTX group were intraperitoneally injected with MTX solution once a day, the injection volume was 200 μl/mouse, and the dose of MTX was 1 mg/kg body weight/time. Mice in MP combined with 3PO group were intraperitoneally injected with MP-3PO solution once a day, the injection volume was 200 μl/mouse, the dose of MP was 2 mg/kg body weight/time, and the dose of 3PO was 25 mg/kg body weight/time. Mice in MP combined with MTX group were intraperitoneally injected with MP-MTX solution once a day, the injection volume was 200 μl/mouse, the dose of MP was 2 mg/kg body weight/time, and the dose of MTX was 1 mg/kg body weight/time. Continuous administration for 30 days. The PBS group is also called the CTL group.

MP solution is obtained by dissolving MP in sterile water. 3PO solution is obtained by dissolving 3PO in sterile water. MTX solution is obtained by dissolving MTX in sterile water. MP-3PO solution is obtained by dissolving MP and 3PO in sterile water. MP-MTX solution is obtained by dissolving MP and MTX in sterile water.

The survival rate was continuously counted, and the results are shown in Figure 4.

On day 28, acute GVHD scoring was performed. The acute GVHD clinical scoring system is based on six parameters: weight loss, posture, activity, fur texture, skin integrity, and diarrhea. The results are shown in Figure 5.

On the 30th day, the mice were sacrificed, the liver and spleen were collected, and mononuclear cells were collected, and the proportion of CD4 ⁺ cells, CD8 ⁺ cells, Th1 cells, Tc1 cells and the proliferation ability of T cells were detected by flow cytometry. The results are shown in Figure 6.

On the 28th day, the peripheral blood of the mice was collected, mononuclear cells were collected, and the proportion of CD4 ⁺ cells and the proportion of CD8 ⁺ cells were detected by flow cytometry. The results are shown in Figure 7.

On the 28th day, the mice were sacrificed, and tissues of acute GVHD target organs (liver, spleen, skin, intestinal tract, lung) were taken, fixed with 4% paraformaldehyde, and paraffin sections were prepared and stained with HE. Acute GVHD pathological scoring in mice was performed according to the scoring system. The results are shown in Figure 8.

On day 21, clearance of leukemic cells after transplantation in NPG mice was assessed by in vivo bioluminescent imaging. The results are shown in Figure 9. Finding a way to effectively treat GVHD while maintaining the GVL effect is crucial to improving the efficacy of hematopoietic stem cell transplantation and reducing transplant-related complications. In order to further explore whether the combined treatment of MP with 3PO or MTX affects the GVL effect, luciferase ⁺ THP-1 leukemia cells were reinfused, and the clearance of leukemia cells after transplantation in NPG mice was evaluated by in vivo bioluminescence imaging. Compared with MP alone, combined treatment of MP with 3PO or MTX had no significant effect on GVL. These results suggest that MP in combination with 3PO or MTX can synergistically reduce T cell alloreactivity and improve aGvHD without loss of GVL effect by downregulating glycolytic activity in a humanized mouse model.

Combined administration of glucocorticoids (MP) and glycolysis modulators (3PO or MTX) reduced aGvHD clinical scores and lethality in mice compared with monotherapy. Pathological evidence showed that the GVHD target organ pathology score of mice combined with MP and 3PO or MTX was significantly reduced. The results showed that combined application of MP and 3PO or combined application of MP and MTX could synergistically treat aGVHD.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

Claims (2)

1. The application of the combination of glucocorticoid and glycolysis regulator in the preparation of medicines for acute graft-versus-host disease; The glycolysis regulator is 3PO; The glucocorticoid is methylprednisolone; In the medicine, the molar ratio of the methylprednisolone and the 3PO is 1:10. 2. A medicine whose active ingredient is a glucocorticoid and a glycolysis regulator; the medicine is a medicine for acute graft-versus-host disease; The glycolysis regulator is 3PO; The glucocorticoid is methylprednisolone; In the medicine, the molar ratio of the methylprednisolone and the 3PO is 1:10.

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