Application of clemastine fumarate in preparing medicine for treating organ transplant immune rejection

Abstract

The present invention discloses a new application of clemastine fumarate in the preparation of a drug for treating organ transplant rejection. Research in the present invention shows that clemastine fumarate can significantly reduce the occurrence of cardiac transplant rejection, specifically by significantly prolonging the survival time of mouse heart transplants, significantly reducing the degree of acute rejection graft lesions in mice, inhibiting mouse T cell allogeneic immune response, reducing the degree of rejection damage and immune infiltration of cardiac transplants, inhibiting mouse T cell proliferation, reducing the proportion of Th1 cell subsets in mouse spleen and draining lymph nodes, reducing the IFN-γ and T-bet gene expression levels in the spleen and draining lymph nodes of recipient mice and the levels of pro-inflammatory IFN-γ, TNF-α and IL-5 cytokines in the body, etc., indicating that clemastine fumarate can be used as a new immunosuppressant for treating cardiac and other organ transplant rejection, especially acute rejection after cardiac and other organ transplants.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more particularly to application of clemastine fumarate in preparing a medicament for treating organ transplant immune rejection. Background Art

[0002] Heart transplantation is the only treatment option to save patients with end-stage heart disease. However, the organs currently used for clinical organ transplantation all come from organ donations. Due to the presence of alloantigens, the recipient's immune system will spontaneously and persistently attack the transplant after organ transplantation, leading to damage to or even loss of graft function, seriously affecting the lifespan of the transplanted organ. Therefore, transplant immune rejection is a problem that urgently needs to be overcome in clinical practice.

[0003] Acute rejection is a common post-transplant rejection reaction, primarily mediated by T cells. Acute rejection caused by various factors can severely impact graft survival and even lead to graft failure. Therefore, preventing and suppressing acute rejection after surgery can help improve long-term heart graft survival.

[0004] The development of organ transplantation benefited greatly from the development and application of immunosuppressants. Early human organ transplants mostly failed until the leukemia drug azathioprine was discovered to have immunosuppressive effects. In 1963, it was first used in combination with glucocorticoids to prevent post-transplant rejection. Subsequently, organ transplantation became widespread, and the "azathioprine + glucocorticoid" maintenance regimen became the standard post-transplant treatment. The subsequent emergence and application of cyclosporine and FK506, as well as the development of immunosuppressants such as mycophenolate mofetil, mizoribine, and rapamycin, led to breakthrough clinical outcomes for various organ transplants. However, currently used immunosuppressants are not perfect and have various side effects, the most common of which is liver and kidney damage. Long-term use can lead to CNI nephropathy and liver damage. They can also cause metabolic abnormalities such as hyperlipidemia, hypertension, hyperglycemia, and hyperuricemia. Furthermore, while the widespread use of immunosuppressants has led to significant improvements in the short-term efficacy of organ transplants in recent decades, long-term outcomes remain unsatisfactory. Therefore, research and development of immunosuppressants with improved efficacy and minimal side effects is currently a key priority in organ transplantation.

[0005] Clemastine fumarate (CF) is a second-generation histamine receptor H1 antagonist. Due to its rapid onset, prolonged duration of action, and strong selectivity, it is widely used in the treatment of allergic diseases, including urticaria and allergic rhinitis, with a good safety profile. However, the effects of clemastine fumarate on immunosuppression, particularly in the immune rejection response after organ transplantation, have not been reported. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide the use of clemastine fumarate in the preparation of immunosuppressants.

[0007] The second object of the present invention is to provide a new application of clemastine fumarate in the preparation of a medicament for treating immune rejection in organ transplantation.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The present invention studies show that clemastine fumarate can significantly reduce the occurrence of immune rejection reactions in heart transplants, which is specifically manifested by significantly prolonging the survival time of mouse heart transplants and significantly reducing the degree of graft lesions in mice with acute rejection reactions. Through in vitro one-way mixed lymphocyte reaction, it was found that clemastine fumarate inhibited the allogeneic immune response of mouse T cells. Through HE and immunohistochemistry, it was shown that clemastine fumarate reduced the degree of rejection damage and immune infiltration of heart transplants. In vitro experiments showed that clemastine fumarate can inhibit mouse T cell proliferation, reduce the proportion of Th1 cell subsets in the spleen and draining lymph nodes of mice, reduce the IFN-γ and T-bet gene expression levels in the spleen and draining lymph nodes of recipient mice, and the levels of pro-inflammatory IFN-γ, TNF-α and IL-5 cytokines in the body, etc., indicating that clemastine fumarate can be used as a new immunosuppressant for treating immune rejection of organ transplants such as heart, especially acute rejection reactions after heart and other organ transplants.

[0010] Therefore, this application requests protection for the application of clemastine fumarate in the following aspects:

[0011] Application of clemastine fumarate in the preparation of immunosuppressants.

[0012] Application of clemastine fumarate in the preparation of medicines for treating organ transplant immune rejection.

[0013] Application of clemastine fumarate in the preparation of a drug for inhibiting acute rejection reaction after organ transplantation.

[0014] Use of clemastine fumarate in the preparation of a drug for reducing the function of alloreactive T cells in the blood of patients after organ transplantation.

[0015] The use of clemastine fumarate in the preparation of a drug for reducing the expression of Th1 cell-related genes in the local immune microenvironment of the body and the graft after organ transplantation and the proportion of Th1 cells in patients after organ transplantation.

[0016] Use of clemastine fumarate in the preparation of a drug for reducing the level of inflammatory cytokines in patients after organ transplantation.

[0017] Application of clemastine fumarate in the preparation of a drug for reducing the degree of immune damage and immune infiltration in organ transplantation.

[0018] Preferably, the Th1 cell-related genes are IFN-γ and T-bet.

[0019] Preferably, the inflammatory cytokines are IFN-γ, TNF-α and IL-5.

[0020] The present invention also provides a drug for the precise treatment of post-transplant immune rejection, which contains clemastine fumarate as its main active ingredient. The drug can inhibit acute rejection after heart transplantation, significantly prolong the survival of mouse heart transplants, inhibit mouse T cell allogeneic immune responses, reduce the degree of rejection damage and immune infiltration of heart transplants, inhibit mouse T cell proliferation, reduce the proportion of Th1 cell subsets in the spleen and draining lymph nodes of mice, and reduce the expression levels of IFN-γ and T-bet genes in the spleen and draining lymph nodes of recipient mice, as well as the levels of pro-inflammatory IFN-γ, TNF-α, and IL-5 cytokines in the body.

[0021] Preferably, the drug also includes a drug for synergistically treating immune rejection after heart transplantation with clemastine fumarate.

[0022] Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0023] Preferably, the dosage form of the drug includes but is not limited to oral solution, granule, capsule, tablet, granule, pill, ointment and injection.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a novel application of clemastine fumarate in the preparation of a drug for treating organ transplant rejection. Studies conducted in the present invention have shown that clemastine fumarate can significantly reduce the occurrence of acute rejection reactions in heart transplants, as demonstrated by significantly prolonging mouse heart transplant survival, significantly reducing the severity of acute rejection graft lesions in mice, inhibiting mouse T cell allogeneic immune responses, reducing the degree of rejection damage and immune infiltration in heart transplants, inhibiting mouse T cell proliferation, reducing the proportion of Th1 cell subsets in the spleen and draining lymph nodes of mice, and reducing the expression levels of IFN-γ and T-bet genes and the levels of pro-inflammatory IFN-γ, TNF-α, and IL-5 cytokines in the spleen and draining lymph nodes of recipient mice. This indicates that clemastine fumarate can be used as a new immunosuppressant for treating organ transplant rejection, particularly for inhibiting acute rejection reactions after heart and other organ transplants. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Comparison of graft survival in heart transplanted mice between the CF treatment group, the vehicle group, and the blank control group. A: Schematic diagram of the survival experimental groups; B: Graft survival curves for each group.

[0027] Figure 2 Figure 7: Appearance of the graft, spleen, and local draining lymph nodes, and HE pathological results of the grafts in the CF treatment group, vehicle group, and blank control group on day 7 after mouse heart transplantation. A: Images of the graft, spleen, and draining lymph nodes in each group on day 7 after heart transplantation; B: HE staining results of the graft on day 7 after transplantation.

[0028] Figure 3 The results of in vitro one-way mixed lymphocyte reaction were as follows. It can be found that on the 4th day of in vitro mixed culture, the CD4 + and CD8 + T cell proliferation was significantly inhibited, which was statistically significant compared with the Blank group and Vehicle group. A: Schematic diagram of one-way MLR flow cytometry results; B: CD4 + T cell proliferation; C: CD8 + T cell proliferation. (*, P<0.05; **, P<0.01)

[0029] Figure 4 These are the CD3 immunohistochemistry results of the transplanted hearts in the CF treatment group, Vehicle group, and Blank control group on the 7th day after mouse heart transplantation. It was found that T cell infiltration was obvious in the heart transplanted hearts of the Blank and Vehicle groups of mice on the 7th day after transplantation, while T cell infiltration was reduced in the CF treatment group.

[0030] Figure 5 This is the flow cytometry result of the CFSE dye dilution method to detect the effect of CF on mouse T cell proliferation.

[0031] Figure 6 Statistical results of the experiment showing the effect of CF on CD4 subset proliferation in mouse T cells. (* indicates a statistically significant difference between the CF group and the Blank group; # indicates a statistically significant difference between the CF group and the Vehicle group).

[0032] Figure 7 The statistical results of the experiment showing the effect of CF on the proliferation of CD8 subsets in mouse T cells. (* indicates a statistically significant difference between the CF group and the Blank group; # indicates a statistically significant difference between the CF group and the Vehicle group).

[0033] Figure 8Figure 7 shows the changes in Th1 subset proportions in the spleen and draining lymph nodes of mice in the CF, Vehicle, and Blank groups on day 7 after heart transplantation. A: spleen; B: draining lymph nodes.

[0034] Figure 9 qPCR results of IFN-γ, T-bet, and Lta gene expression in spleen mononuclear cells and local draining lymph nodes of mice in the CF, Vehicle, and Blank groups on day 7 after heart transplantation. A: Expression of each gene in spleen mononuclear cells; B: Expression of each gene in draining lymph nodes.

[0035] Figure 10 Figure 3 Serum cytokine levels in CF, Vehicle and Blank groups on the 7th day after mouse heart transplantation.

[0036] Figure 11 Figure 7: Liver and kidney function, weight changes, and histological structure of major organs in recipient mice in the CF, Vehicle, and Blank groups 7 days after mouse heart transplantation. A: Liver and kidney function of recipient mice 7 days after transplantation; B: Weight changes of recipient mice after transplantation; C: Histological structure of major organs in recipient mice (kidney, liver, lung, magnification 200×) DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0038] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0039] The structural formula of the clemastine fumarate of the present invention is shown below. The clemastine fumarate used for intraperitoneal injection in experimental animals of the present invention is derived from a biological reagent supplier (Aladdin, catalog number: C129211-500mg) with a purity of over 98%. The clemastine fumarate solution used in in vitro cytological experiments is derived from a biological reagent supplier (Selleck, catalog number: S1847-10mL).

[0040]

[0041] Example 1 Effect of Clemastine Fumarate on Immune Rejection after Heart Transplantation in Mice

[0042] 1. Experimental Methods

[0043] 1. Male C57BL / 6 mice and BALB / c mice were purchased from Guangdong Yaokang Laboratory Animal Technology Co., Ltd. They weighed approximately 25 g, were 6-8 weeks old, and were housed in an SPF-grade environment.

[0044] 2. Grouping of experimental animals

[0045] Before the experiment, the experimental animals were randomly divided into five groups: syngeneic group (Syngeneic group), blank control group (Blank group), vehicle group (Vehicle group), clemastine fumarate treatment group (CF group), and FK506 treatment group (FK506 group). The treatments for each group were as follows:

[0046] Table 1 Experimental groups and treatment of recipient mice in each group

[0047]

[0048] 3. Establishment of Mouse Heart Transplantation Model

[0049] The heart of the donor BALB / c mouse was removed and heterotopically transplanted into the subcutaneous area of ​​the neck of the recipient C57BL / 6 mouse. The specific operation steps are as follows: anesthetize the donor BALB / c mouse, properly fix the limbs, and disinfect the mouse chest and abdominal skin with 75% alcohol; cut the abdominal wall skin and muscle from the middle to the bilateral anterior axillary line at the level of the mouse bladder, push the mouse intestine to the right, expose the inferior vena cava, and inject 0.2mL of heparinized saline into the inferior vena cava for systemic heparinization; open the chest to expose the mouse heart, cool the heart with pre-cooled heparinized saline to stop the heart, remove the thymus, separate the aorta and pulmonary artery, clamp the distal end of the aorta with a Wenckeville clamp, and infuse heparin water into the aorta. At this time, the donor heart turns from red to khaki, cut the aorta and pulmonary artery at the same time, ligate the pulmonary vein and vena cava with silk thread, and cut out the heart for later use. The recipient C57BL / 6 mouse was then anesthetized, its limbs properly secured, the skin on the right side of the neck disinfected, and placed on a warming pad. The skin on the right side of the neck was cut open, and the external jugular vein and common carotid artery were isolated. A microvascular clamp was applied to the proximal end, and a silk suture was tied to the distal end. After cutting, the cannula was secured to the stump using a cannula and secured with silk suture. The aorta of the donor BALB / c mouse heart was cuffed over the common carotid artery, and the pulmonary artery was cuffed over the external jugular vein, secured with silk suture. The clamps were opened to restore blood flow. The transplanted heart was observed to have a rapid rhythm and bright red color. The skin was sutured, completing the heart transplant. Following successful transplantation, the heart graft was observed daily starting on the second day after surgery. When the beating became weak and undetectable, palpation was performed with the index finger and thumb. Because the transplanted heart is implanted subcutaneously, graft rejection was determined when the graft completely stopped beating. The recipient mouse was sacrificed on the seventh postoperative day, and blood was collected by enucleation of the eyeball. The collected blood was placed in EP tubes and centrifuged at 4000 rpm for 10 minutes at 4°C. The resulting serum was aliquoted into EP tubes and frozen at -80°C for Luminex analysis. The explanted heart was transversely sectioned and each half was immersed in 4% paraformaldehyde for pathological verification. The spleen and local draining lymph nodes were removed and cells were isolated for subsequent flow cytometry analysis of immune cell subsets and molecular experiments.

[0050] 4. Mouse T cell proliferation experiment in vitro

[0051] 4.1 Preparation of mouse spleen single cell suspension

[0052] 1) Execution by cervical dislocation For C57BL / 6 mice, soak the mice in 75% alcohol for 5 minutes;

[0053] 2) After removing the mouse, place it in a biosafety cabinet and use sterile ophthalmic forceps and scissors to cut the skin on the left side of the abdomen to expose the mouse spleen. Use another pair of sterile forceps to remove the spleen.

[0054] 3) Wash the spleen twice with sterile PBS. Remove the mouse spleen from the centrifuge tube and place it in a sterile 10 cm dish containing 3 mL of PBS. Use sterile forceps to place the spleen on a 200-mesh cell sieve.

[0055] 4) Take a 1 mL syringe and use the side of the plunger to gently grind the tissue until it is finely divided. Rinse the cell sieve with PBS and aspirate the cell suspension into a 15 mL centrifuge tube.

[0056] 5) Centrifuge the tube containing the splenocyte suspension at 500g for 5 minutes. Aspirate the supernatant and resuspend the cell pellet in 3 mL of pre-chilled 1× red blood cell lysis buffer. Place in a 4°C refrigerator for 3 minutes to fully lyse the red blood cells.

[0057] 6) After red blood cell lysis is complete, add 3 mL of PBS to stop the lysis, mix by inverting, and centrifuge at 500 g for 5 minutes;

[0058] 7) Aspirate the supernatant. The cell pellet will now be off-white. Add 3 mL of PBS to the cell pellet and disperse the cell clumps using a pipette. Separately, place a clean, sterile 15 mL centrifuge tube in a centrifuge tube rack. Place a 200-mesh sieve over the opening of the tube and filter the cell suspension to remove any remaining tissue debris.

[0059] 8) Once the cell suspension has been filtered into the 15 mL tube, fill the remainder of the tube with PBS and invert several times. Centrifuge at 500 g for 5 minutes.

[0060] 9) After centrifuging the spleen cells in step 8, aspirate the supernatant and resuspend the cells in 10 mL of RPMI1640 medium for cell counting.

[0061] 4.2 T cell isolation and purification

[0062] Use the Miltenyi Pan T Cell Magnetic Bead Isolation Kit (Cat. No. 130-095-130) and follow the instructions for isolation. The specific steps are as follows:

[0063] 1) Calculate the total cell volume required according to the experimental grouping requirements, pipette the required cell suspension into a sterile 15 mL centrifuge tube, centrifuge at 500 g for 5 minutes, and aspirate the supernatant;

[0064] 2) Add 40 μL AutoMACS Running Buffer for every 10^7 spleen cells (the fraction less than 10^7 spleen cells should be calculated as 10^7), resuspend the cell pellet, and pipette thoroughly to separate the cells into a single-cell suspension;

[0065] 3) Add 10 μL of Biotin-Antibody Cocktail per 10^7 spleen cells, mix thoroughly, and incubate at 4°C for 5 minutes.

[0066] 4) Add 30 μL AutoMACS Running Buffer per 10^7 spleen cells;

[0067] 5) Add 20 μL of Anti-Biotin MicroBeads per 10^7 spleen cells, mix thoroughly, and incubate at 4°C for 10 min.

[0068] 6) During the incubation period in step 5), sterilize the magnetic stand and Quadro MACS magnetic separator and place them on a clean bench. Install a new LS separation column on the magnetic separator and place a clean, sterile 15 mL centrifuge tube under the LS separation column to collect the cell suspension.

[0069] 7) Add 3 mL of AutoMACS Running Buffer to the separation column to remove any bubbles.

[0070] 8) Remove the cell suspension from step 5) (if less than 500 μL, add AutoMACS Running Buffer to 500 μL or more) and add it to the separation column. Add new cell suspension each time until the previous cell suspension is almost finished.

[0071] 9) Add 3 mL of AutoMACS Running Buffer to the separation column to fully elute the cells. The cell suspension collected in a 15 mL centrifuge tube is the enriched T cells.

[0072] 4.3CFSE staining

[0073] 1) Count the purified T cell suspension and pipette the required amount of cell suspension into a sterile 15 mL centrifuge tube according to experimental needs. Centrifuge at 500 g for 5 minutes and remove the supernatant.

[0074] 2) Resuspend the cell pellet in 0.5 mL of PBS;

[0075] 3) Add 0.5 mL of PBS to another sterile 15 mL centrifuge tube. Add 1 μL of CFSE stock solution (5 μM) in the dark and mix thoroughly. Add 0.5 mL of the cell suspension obtained in step 2) to the CFSE solution (the CFSE concentration after addition is 5 nM). Mix thoroughly and incubate in a 4°C refrigerator in the dark for 10 min.

[0076] 4) Add 10 mL of RMPI 1640 medium containing 10% FBS, pre-cooled at 4°C, mix thoroughly, neutralize at 4°C in the dark for 5 min, centrifuge at 500 g for 5 min, and aspirate the supernatant.

[0077] 5) Add 5 mL of 4°C pre-cooled RMPI 1640 medium containing 10% FBS, resuspend the cell pellet, centrifuge at 500 g for 5 minutes, and aspirate the supernatant;

[0078] 6) Repeat step 5) 2 times;

[0079] 7) Then, add an appropriate amount of complete culture medium (complete culture medium preparation: 45 mL RPMI 1640 basal medium + 5 mL heat-inactivated FBS + 0.5 mL GlutaMax + 0.5 mL penicillin / streptomycin + 50 μL β-mercaptoethanol + 50 μL Anti-mouse CD28 + 20 ng / mL mIL-2) to a cell concentration of 1×10^6 / mL.

[0080] 4.4 Laying and planting boards

[0081] 1) Plating: A single proliferation experiment was divided into a proliferation group and a non-proliferation control group. The proliferation group was coated with 1× Anti-mouse CD3 solution in a flat-bottomed 48-well plate, while the non-proliferation control group was not coated. The specific procedure was as follows: Add 10 mL of PBS to a sterile 15 mL centrifuge tube, then add 10 μL of Anti-mouse CD3 stock solution. Mix thoroughly, then add 0.3 mL to each well of the proliferation group and 0.3 mL of PBS to the non-proliferation control group. Seal the 48-well plate with sealing film and place in a 4°C refrigerator overnight.

[0082] 2) Seeding: Remove the seeded 48-well plate, aspirate the solution in the wells, wash the corresponding wells twice with PBS, and finally aspirate all the solution in the wells. Inoculate 1 mL of the cell suspension obtained in step 2.2.3.3 into each well of the 48-well plate. Add the corresponding dose of CF or DMSO to the corresponding wells according to the experimental grouping and culture in a CO2 incubator for 2-4 days.

[0083] 4.5 Flow cytometry analysis of T cell subset proliferation

[0084] 1) At a specific time point in culture, cells from each group were pipetted into flow cytometry tubes and labeled. After adding 2 mL of PBS, the tubes were centrifuged at 500 g for 5 minutes and the supernatant was discarded.

[0085] 2) Wash the cells by adding 3 mL of PBS, centrifuge at 500 g for 5 min, discard the supernatant, and leave approximately 100 μL of residual PBS at the bottom of the flow cytometry tube;

[0086] 3) Add 0.5 μL of zombie violet stock solution to each flow cytometry tube, vortex to resuspend the cells, and incubate in a 4°C refrigerator in the dark for 10 min;

[0087] 4) After incubation, add the recommended volume of flow cytometry antibodies to each tube, including: AlexaFluor700 anti-mouse CD3 (Biolegend, Inc., USA); PE / Cyanine7 anti-mouse CD4 (Biolegend, Inc., USA); and PE anti-mouse CD8 (Biolegend, Inc., USA). Mix thoroughly and incubate in a 4°C refrigerator, protected from light, for 20 minutes.

[0088] 5) Wash with 3 mL of PBS per tube, centrifuge at 500 g for 5 min, and discard the supernatant.

[0089] 6) Repeat step 5);

[0090] 7) Add 0.2 mL of PBS to each tube, resuspend the cells, and analyze on a flow cytometer.

[0091] 5. One-way mixed lymphocyte reaction (MLR)

[0092] 5.1 Preparation of stimulatory cells

[0093] adult BALB / c mouse spleen mononuclear cells were used as stimulator cells. The specific preparation steps are as follows:

[0094] 1) Preparation BALB / c mouse spleen single cell suspension: Follow step 4.1 Prepare a single-cell suspension from the spleen of BALB / c mice. Calculate the required cell amount according to experimental needs and pipette the required cell suspension into a sterile 15 mL centrifuge tube. Centrifuge at 500 g for 5 min at room temperature and discard the supernatant.

[0095] 2) Add 5 mL of culture medium (culture medium preparation: 45 mL RPMI 1640 basal medium + 5 mL heat-inactivated FBS + 0.5 mL GlutaMax + 0.5 mL penicillin / streptomycin + 50 μL β-mercaptoethanol + 20 ng / mL mIL-2) to resuspend the cells, then add 200 μL of 1 mg / mL mitomycin C solution to a final concentration of 40 μg / mL mitomycin C. Place in a CO2 incubator at 37°C for 30 min.

[0096] 3) After treatment, centrifuge at 500 g for 5 minutes at room temperature and discard the supernatant;

[0097] 4) Add 5 mL of culture medium to resuspend the cells, centrifuge at 500 g for 5 minutes at room temperature, and discard the supernatant;

[0098] 5) Repeat step 4);

[0099] 6) Add an appropriate amount of complete culture medium (complete culture medium preparation: 45 mL RPMI 1640 basal medium + 5 mL heat-inactivated FBS + 0.5 mL GlutaMax + 0.5 mL penicillin / streptomycin + 50 μL β-mercaptoethanol + 20 ng / mL mIL-2) and resuspend the cells to adjust the cell concentration to 1 × 10^6 / mL;

[0100] 5.2 Preparation of reaction cells

[0101] On the 7th day after heart transplantation, samples were collected from C57BL / 6 mice in each treatment group. C57BL / 6 mouse spleens were used as controls, and spleen single cell suspensions were prepared as reactant cells. The specific preparation steps are as follows:

[0102] 1) Prepare mouse spleen single cell suspension: Follow step 4.1 BALB / c mouse spleens were prepared into single-cell suspensions;

[0103] 2) Calculate the required cell volume according to experimental needs, pipette the required cell suspension into a sterile 15 mL centrifuge tube, centrifuge at 500 g for 5 minutes at room temperature, and discard the supernatant;

[0104] 3) Perform CFSE staining on each group of cells as in step 4.3. After staining, add an appropriate amount of complete culture medium (complete culture medium preparation: 45 mL RPMI 1640 basal medium + 5 mL heat-inactivated FBS + 0.5 mL GlutaMax + 0.5 mL penicillin / streptomycin + 50 μL β-mercaptoethanol + 20 ng / mL mIL-2) to a cell concentration of 1 × 10^6 / mL.

[0105] 5.3 types of boards

[0106] According to the experimental needs, they were divided into 5 groups:

[0107] Table 2 Grouping of one-way MLR experiment

[0108]

[0109] After mixing the stimulating cells and responding cells of each group at a ratio of 1:2, the cells were seeded into a 96-well U-shaped plate at a volume of 200 μL per well and cultured in a CO2 incubator for 2-4 days.

[0110] 5.4 Flow cytometry analysis of T cell proliferation

[0111] According to experimental step 4.5, cell surface staining was performed on cells in each group and at each time point, and the cells were detected by flow cytometry after staining.

[0112] 6. Flow cytometry detection of changes in the proportion of Th1 cell subsets

[0113] 6.1 Preparation of single cell suspensions from mouse spleen and draining lymph nodes

[0114] Prepare single-cell suspensions from the spleen or draining lymph nodes of recipient mice according to step 4.1.

[0115] 6.2 Th1 staining

[0116] 1) Calculate the total cell volume required according to the experimental grouping requirements, pipette the required cell suspension into a sterile 15 mL centrifuge tube, centrifuge at 500 g for 5 minutes, and aspirate the supernatant;

[0117] 2) Add appropriate amount of complete culture medium and stimulation blocker (culture medium preparation: 45 mL RPMI 1640 basal medium + 5 mL heat-inactivated FBS + 0.5 mL GlutaMax + 0.5 mL penicillin / streptomycin + 50 μL β-mercaptoethanol + 20 ng / mL mIL-2 + 102 μL stimulation blocker Cell Activation Cocktail (with Brefeldin A)) and resuspend the cells to adjust the cell concentration to 1 × 10^6 / mL;

[0118] 3) Inoculate 1 mL per well into a 48-well plate and incubate in a CO2 incubator for 6 h.

[0119] 4) Pipette each group of cells into a flow cytometry tube and label it. Add 2 mL of PBS, centrifuge at 500 g for 5 minutes, and discard the supernatant.

[0120] 5) Wash the cells by adding 3 mL of PBS, centrifuge at 500 g for 5 min, discard the supernatant, and leave approximately 100 μL of residual PBS at the bottom of the flow cytometry tube;

[0121] 6) Add 0.5 μL of zombie violet stock solution to each flow cytometry tube, vortex to resuspend the cells, and incubate in a dark refrigerator at 4°C for 10 min.

[0122] 7) After incubation, add the recommended volume of flow cytometry antibodies to each tube, including: AlexaFluor700 anti-mouse CD3 (Biolegend, Inc., USA); PE / Cyanine7 anti-mouse CD4 (Biolegend, Inc., USA). Mix thoroughly and incubate in a 4°C refrigerator, protected from light, for 20 minutes.

[0123] 8) Wash with 3 mL of PBS per tube, centrifuge at 500 g for 5 min, and discard the supernatant.

[0124] 9) Repeat step 8);

[0125] 10) After the final wash, discard the supernatant and vortex to dissociate cell clumps;

[0126] 11) Add 1 mL of IC Fixation Buffer (eBioscience, USA) to each tube, vortex to mix, and incubate at room temperature in the dark for 30 min.

[0127] 12) Add 2 mL of 1× Permeabilization Buffer (eBioscience, USA), centrifuge at 500 g for 5 min at room temperature, and discard the supernatant;

[0128] 13) Repeat step 12);

[0129] 14) Resuspend the pellet in the remaining volume and add the recommended volume of intracellular flow cytometry antibodies to each flow cytometry tube, including: Alexa Fluor 488 anti-mouse IFN-γ (Biolegend, Inc., USA), mix thoroughly, and incubate at room temperature in the dark for 60 minutes;

[0130] 15) After incubation, add 2 mL of 1× Permeabilization Buffer (eBioscience, USA) to each tube, centrifuge at 500 g for 5 min at room temperature, and discard the supernatant;

[0131] 16) Repeat step 15);

[0132] 17) Add 0.2 mL of PBS to each tube, resuspend the cells, and analyze on a flow cytometer.

[0133] 7. qPCR detection of IFN-γ, T-bet and Lta gene expression

[0134] 7.1 Total RNA extraction and quality control

[0135] Total RNA was extracted from mononuclear cells of the spleen and draining lymph nodes of recipient mice using the SteadyPure Universal RNA Extraction Kit II (Acori Biotech, China). RNA concentration and purity were then tested. The OD260 / OD280 of the extracted RNA solution was measured using a spectrophotometer and should be controlled between 1.8 and 2.1. If the RNA concentration was lower than 100 ng / μL, re-extraction was required.

[0136] 7.2 Reverse transcription

[0137] 1) Reverse transcription was performed using the Takara PrimeScript RT Master Mix kit. The 25 μL system was as follows:

[0138] Table 3 Each reverse transcription reaction system

[0139]

[0140] 2) Reverse transcription reaction conditions are as follows:

[0141] 37°C, 15 min (reverse transcription reaction)

[0142] 85℃, 5s (reverse transcriptase inactivation)

[0143] 4℃,∞

[0144] 7.3 Real-time fluorescence quantitative PCR reaction

[0145] The primer sequences used for qPCR were obtained from the NCBI website (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) and the PrimerBank website (https: / / pga.mgh.harvard.edu / primerbank / ). The primers were synthesized by Beijing Ruibo Xingke Biotechnology Co., Ltd. The specific primer sequences are shown in the following table:

[0146] Table 4 Primer list

[0147]

[0148] 1) Detect mRNA content using the dye method. Follow the instructions of the Takara SYBR Premix Ex TaqII dye-based fluorescence quantitative kit. The 10 μL reaction system is as follows:

[0149] Table 5 Each qPCR reaction system

[0150]

[0151] 2) Add each of the above components to a 96-well PCR plate in sequence, seal the plate with a sealing film, and centrifuge at 1000 rpm for 2 minutes to collect all components at the bottom of the tube;

[0152] 3) Analyze on a CFX-96 real-time fluorescence quantitative PCR instrument. The reaction conditions are as follows:

[0153] Cycle 1: 95°C for 30 seconds (pre-denaturation)

[0154] Cycle 2: 95°C for 5 seconds, 60°C for 30 seconds, a total of 40 cycles (PCR reaction)

[0155] Cycle 3: starting temperature 65°C, ending temperature 95°C, each temperature change is 0.5°C, and the plate is read for 5 seconds after each temperature stabilization (melting curve).

[0156] 7.4 Calculation of gene expression

[0157] After the reaction is complete, the melting curve and amplification curve are checked. Once the curve morphology is normal, the Ct values ​​of the replicate wells in the same reaction are compared. If the Ct value of one replicate well differs by more than 0.5, the well should be discarded. If the Ct values ​​of all replicate wells differ by more than 0.5, the experiment should be repeated. For qualified data, the average Ct value of the valid replicate wells will be used as the Ct value of the gene. The difference between the Ct values ​​of the target gene and the internal reference gene in the experimental group is subtracted from the difference between the Ct values ​​of the target gene and the internal reference gene in the control group to obtain ΔΔCt. This value is then converted to 2^-ΔΔCt through exponential transformation. This value represents the multiple of the target gene expression in the experimental group relative to the control group.

[0158] 8. Luminex detection of serum cytokines

[0159] 1) Add 50 μL of diluted sample or mixed standard to each well of the Luminex microplate and record the sample information corresponding to each well;

[0160] 2) Vortex to mix and dilute the microparticle cocktail, add 50 μL to each well, seal the plate with tin foil, and incubate at room temperature for 2 h on a horizontal shaker (0.12" orbit) at 800 ± 50 rpm.

[0161] 3) Place a magnetic stand against the bottom of the plate for 1 minute, discard the liquid, add 100 μL of Wash Buffer to each well, place the magnetic stand against the bottom of the plate for 1 minute, and aspirate the liquid. Repeat this wash cycle twice until all the liquid is completely aspirated.

[0162] 4) Add 50 μL of diluted Biotin-Antibody to each well, seal the plate with tin foil, and incubate at room temperature for 1 hour on a horizontal shaker (0.12" orbit) at 800 ± 50 rpm.

[0163] 5) Repeat step 4);

[0164] 6) Add 50 μL of diluted Streptavidin-PE to each well, seal the plate with tin foil, and incubate at room temperature for 30 min on a horizontal shaker (0.12" orbit) at 800 ± 50 rpm.

[0165] 7) Repeat step 4);

[0166] 8) Add 100 μL of Wash Buffer to each well to resuspend the cells. Incubate on a horizontal shaker (0.12" orbit) at 800 ± 50 rpm at room temperature for 2 min.

[0167] 9) The plate was read using a Luminex MAGPIX (R&D Systems, Inc., USA) machine.

[0168] 9. HE staining

[0169] 1) Sample Collection and Fixation: On the 7th day after heart transplantation, the eyeballs were removed and blood was collected. The mice were then sacrificed, the neck skin was cut open to expose the cardiac graft, and the graft was excised. The graft was then cut transversely and quickly placed in 4% paraformaldehyde fixative. The abdomen and chest were cut open, and the lungs, liver, kidneys, and spleen of the mice were removed and fixed in 4% paraformaldehyde fixative. The tissues were removed from the fixative and the target area was trimmed flat with a scalpel blade. The trimmed tissue and label were placed in an embedding frame.

[0170] 2) Dehydration and paraffin immersion: Place the specimen in a dehydrator and dehydrate in a gradient of alcohols: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, paraffin I melted at 65°C for 1 hour, paraffin II melted at 65°C for 1 hour, and paraffin III melted at 65°C for 1 hour.

[0171] 3) Paraffin embedding: Embed the wax-soaked tissue in an embedding machine. Pour the melted wax into the embedding frame. Before the wax solidifies, place the tissue in the embedding frame and label it accordingly. Cool the wax block at -20°C. After the wax solidifies, trim the block.

[0172] 4) Sectioning and mounting: Take out the trimmed wax block and slice it with a microtome to a thickness of 4 μm. Flatten the slices in 40°C warm water, mount them flatly on a glass slide, and bake them in a 60°C oven.

[0173] 5) Dewaxing and staining: The sections were sequentially placed in environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and rinsed with tap water. After dewaxing, the sections were immersed in hematoxylin solution and stained for 5 minutes, rinsed with tap water, differentiated with differentiation solution, rinsed with tap water, bluing solution, and rinsed with running water. The sections were immersed in 85% and 95% alcohol, dehydrated for 5 minutes each, and immersed in eosin solution and stained for 5 minutes.

[0174] 6) Dehydration and mounting: Sections were sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min until transparent. The sections were then mounted with neutral gum and observed under a microscope. Images were collected and analyzed.

[0175] 10. CD3 immunohistochemical staining of cardiac allografts

[0176] 1) Paraffin sections of the heart graft were sliced ​​to 2 μm thick, flattened in warm water, placed flat on glass slides, and baked in a 65°C oven.

[0177] 2) Dewaxing to water: 3 cylinders of xylene, 6 min each; 2 cylinders of 100% alcohol, 3 min each; 1 cylinder of 95% alcohol, 3 min;

[0178] 3) Antigen retrieval: Wash with distilled water for 3 minutes, perform high-pressure retrieval with citrate buffer for 3 minutes, and cool naturally to room temperature.

[0179] 4) Inhibit endogenous catalase with 3% hydrogen peroxide and rinse with PBS five times, 2 minutes each time;

[0180] 5) CD3 antibody was diluted 1:100, added to the specimen, and incubated at 37°C for 1 hour;

[0181] 6) After incubation, rinse with PBS five times for 2 minutes each time and incubate with goat anti-rabbit IgG antibody-HRP multimer at 37°C for 30 minutes;

[0182] 7) After incubation, rinse with PBS five times, 2 minutes each time, and develop with DAB for 2 minutes;

[0183] 8) Hematoxylin counterstaining for 2 minutes, hydrochloric acid alcohol differentiation for 5 seconds, and tap water blueing for 5 minutes;

[0184] 9) Dehydrate with gradient alcohol, transparentize with xylene, mount the slides with neutral gum, and capture images under a microscope.

[0185] 11. Data Analysis

[0186] Survival data were analyzed using the Kaplan-Meier curve method, and intergroup survival was compared using the Log-rank test. Continuous variables were expressed as mean ± standard deviation (SD), and intergroup comparisons were performed using the t-test. Significant differences were considered when p < 0.05. Data were analyzed and plotted using GraphPad Prism 8.0.1 software (GraphPad, USA). Flow cytometry data were analyzed using Kaluza 2.1 software (Beckman Coulter, USA) and FlowJo 10.5.3 software (BD Bioscience, USA). CFSE T cell proliferation assay data were fitted and analyzed using FlowJo 10.5.3 software (BD Bioscience, USA), and corresponding parameters were calculated.

[0187] 2. Experimental Results

[0188] Survival experiment group design Figure 1 As shown in A, the survival experiment was divided into CF treatment group, Vehicle group, Blank control group, FK506 treatment group and Syngeneic transplantation group (C57BL / 6 to C57BL / 6). Each group was given different treatments after heart transplantation. Figure 1 As shown in Figure B, all grafts in the Syngenenic group survived 100 days after transplantation. In an allogeneic mouse heart transplant model (BALB / c to C57BL / 6), the median survival time (MST) of the heart grafts in the Blank group was only 7 days. However, the CF group showed significantly prolonged graft survival, with the longest survival reaching 79 days and an MST of 27.5 days, significantly longer than that in the Blank group (p = 0.0004). In contrast, the Vehicle group had an MST of only 8 days, which was no different from the Blank group (p = 0.5383) and significantly shorter graft survival than the CF group (p = 0.0006). The FK506 group also showed significantly longer graft survival than the Blank group (p = 0.0004). There was no significant difference between the CF treatment group and the standard treatment FK506 group (p=0.0660), but the median survival time in the CF group was longer than that in the FK506 group (27.5 days vs. 12.5 days). These results show that clemastine fumarate can significantly prolong the survival of mouse heart grafts, with the longest survival time reaching 79 days.

[0189] On the 7th day after heart transplantation, the appearance of the graft, spleen and local draining lymph nodes in each group were as follows: Figure 2As shown in A, the heart grafts in the CF-treated group were the smallest and bright red. The grafts in the Blank and Vehicle groups were swollen, dark red, and mottled, with visible infarcts. The spleen size and local cervical lymph nodes were also the smallest in the CF-treated group. Figure 2 B, Histopathology of cardiac allografts in the Blank and Vehicle control groups revealed focal myocardial infarction, myocardial fiber necrosis, interstitial edema, and extensive inflammatory cell infiltration, demonstrating significant acute rejection pathology. CF treatment alleviated the severity of acute rejection. HE analysis of allografts showed intact myocardial fibers, interstitial edema, and minimal inflammatory cell infiltration in the CF-treated group. These results demonstrate that clemastine fumarate significantly reduces the severity of acute rejection in mice.

[0190] On the 7th day after heart transplantation, spleen cells and C57BL / 6 mouse spleen cells were labeled with CFSE and used as responder cells, and BALB / c mouse spleen cells treated with mitomycin C were used as stimulator cells. One-way MLR was performed at a ratio of stimulator cells to responder cells (1:2). Figure 3 A). The proliferation of active CD4+ and CD8+ T cells was detected on the second day of culture. The results showed that T cells in the reactive cell group did not show significant proliferation. CD4+ and CD8+ T cells in the Blank, Vehicle, and CF groups were significantly increased. + The proliferation rate of T cells increased. There was no significant difference between the two groups; the CD8+T cell proliferation ratio in the Blank group, Vehicle group and CF group was also There was no statistical difference between the two groups ( Figure 3 B and 3C). After the 4th day of culture, Active CD4 + and CD8 + After stimulation, T cells showed significant proliferation compared to the non-stimulated cell group. The proliferation of CD4+T cells in the CF group was significantly inhibited, which was significantly different from the Blank group (8.71% ± 3.95% vs. 44.67% ± 22.63%) and the Vehicle group (8.71% ± 3.95% vs. 18.40% ± 1.87%). At the same time, the proliferation of CD8+T cells in the CF group was also significantly decreased, which was statistically significant compared with the Blank group (20.90% ± 10.90% vs. 76.97% ± 18.78%). Figure 3 B and 3C). The above results indicate that CF treatment can effectively suppress allogeneic T cell responses in mice.

[0191] On the 7th day after heart transplantation, the results of CD3 immunohistochemical staining of cardiac allografts were as follows: Figure 4 As shown: T cell infiltration in the cardiac grafts of mice in the Blank and Vehicle groups was obvious, while T cell infiltration in the CF treatment group was reduced.

[0192] use In vitro T cell proliferation experiments were performed on total T cells from the spleen of C57BL / 6 mice. Different concentrations of CF solution were added to the culture medium and T cell proliferation was detected by CFSE dilution method. The results are shown in Figure 2. Figure 5 、 Figure 6 and Figure 7 As shown: Blank group proliferated significantly in this culture system, and within 2-4 days of culture, CD4 + T cells ( Figure 5 and Figure 6 ) and CD8 + T cells ( Figure 5 and Figure 7 ) There was no difference in the proliferation degree. At CF concentrations of 3μM / L and 5μM / L, CD4 + T cells and CD8 + T cell proliferation was significantly inhibited on days 2 and 3 of culture, with statistically significant differences in multiple CFSE proliferation assay indicators (including Percent Divided, Replication Index, Division Index, Expansion Index, Proliferation Index, and Std Deviation) compared to the Blank and Vehicle groups. These results suggest that clemastine fumarate can inhibit T cell proliferation in mice and reduce T cell infiltration in mouse cardiac allografts, thereby alleviating T cell-mediated rejection.

[0193] On the 7th day after mouse heart transplantation, the spleen and draining lymph nodes of each group of recipient mice were taken to extract single cell suspension to detect the changes in the proportion of Th1 cells. Figure 8 As shown in A, in the spleen of recipient mice, the proportion of Th1 cells was significantly reduced in the CF group (CF group vs. Vehicle group: 3.44% ± 1.60% vs. 6.57% ± 3.15%; CF group vs. Blank group: 3.44% ± 1.60% vs. 8.22% ± 4.08%), and there was no significant difference between the Vehicle group and the Blank group. Figure 8B shows that in the cervical draining lymph nodes, the proportion of Th1 cells in the CF group was also significantly lower than that in the Vehicle group (CF group vs. Vehicle group: 0.95% ± 0.50% vs. 2.54% ± 1.26%) and the Blank group (CF group vs. Blank group: 0.95% ± 0.50% vs. 2.58% ± 1.20%), and there was no significant difference between the Vehicle group and the Blank group. The expression levels of IFN-γ, T-bet and Lta genes in the spleen and draining lymph nodes were detected, and it was found that in the draining lymph nodes ( Figure 9 In B), the relative expression of IFN-γ and Lta in the CF group was significantly lower than that in the Blank group and the Vehicle group, and the expression of T-bet in the CF group was significantly lower than that in the Blank group. The expression of IFN-γ and T-bet in spleen cells was lower than that in the Blank group and the Vehicle group, but there was no statistical significance ( Figure 9 A). The above results show that clemastine fumarate can inhibit Th1 cell differentiation, reduce the proportion of Th1 cells, and alleviate the degree of rejection.

[0194] Effects of serum cytokines after heart transplantation on the outcome Figure 10 As shown in the figure, the serum IFN-γ, TNF-α and IL-5 levels in the CF treatment group were lower than those in the Blank group and the Vehicle group, indicating that clemastine fumarate can reduce the levels of proinflammatory cytokines after heart transplantation, reduce inflammatory response and alleviate rejection reaction.

[0195] On the 7th day after heart transplantation, the liver and kidney functions of mice in each group were measured. The serum ALT, AST, BUN and creatinine levels in the CF group did not increase significantly after treatment, and there was no significant difference in the ALT, BUN and creatinine levels among the mice in each group ( Figure 11 A). In addition, the histological examination results of the main organs (kidney, liver, and lung) of the recipient mice showed that the tissue structures of the kidney, liver, and lung of each group of recipients were clear and complete, with cells arranged neatly and the cell structure intact, indicating that CF did not cause damage to the main organs ( Figure 11 C). In addition, the weight recovery process of recipient mice in each group was similar after transplantation ( Figure 11 B) The above results indicate that clemastine fumarate has no obvious toxic side effects on recipient mice.

Claims

1. Application of clemastine fumarate in the preparation of drugs for inhibiting acute rejection after heart transplantation.

2. The use according to claim 1, wherein the drug inhibits acute rejection reaction after heart transplantation by reducing the function of alloreactive T cells in the blood of patients after heart transplantation.

3. The application according to claim 1, characterized in that The drug inhibits acute rejection reaction after heart transplantation by reducing the expression of Th1 cell-related genes in the local immune microenvironment of the body and the transplant after heart transplantation and the proportion of Th1 cells in the patient's body after heart transplantation.

4. The application according to claim 1, characterized in that The drug inhibits acute rejection reaction after heart transplantation by reducing the level of inflammatory cytokines in patients after heart transplantation.

5. The application according to claim 1, characterized in that: The drug inhibits acute rejection reaction after heart transplantation by reducing the degree of cardiac immune damage and immune infiltration after heart transplantation.

6. The application according to claim 3, characterized in that: The related genes are IFN-γ and T-bet.

7. The use according to claim 4, characterized in that The inflammatory cytokines are IFN-γ, TNF-α and IL-5.

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