Application of soluble epoxide hydrolase inhibitor in transplantation field

Abstract

The invention relates to an application of a soluble epoxide hydrolase inhibitor in the field of transplantation, the soluble epoxide hydrolase inhibitor is TPPU, and the soluble epoxide hydrolase inhibitor is applied in the fields of organ transplantation and tissue transplantation. The TPPU is used for relieving allogeneic mouse heart and skin transplantation immunological rejection, reducing immune cell infiltration in transplantation operation, and inhibiting CD4 + T cell proliferation, CXCR3 chemokine receptor positive and Th1 cell subset proportion. The TPPU inhibits proliferation of primary CD4 + T cells of normal mice and differentiation of Th1 cell subgroups. In organ transplantation and tissue transplantation, the invention provides a single soluble epoxide hydrolase inhibitor TPPU, which can relieve allogeneic mouse heart and skin transplantation immunological rejection, inhibit normal mouse primary CD4 + T cell proliferation and differentiation, and reduce toxic and side effects caused by the use of an immunosuppressant.

Description

Technical Field

[0001] This invention relates to the medical field, and more particularly to the application of soluble epoxide hydrolase inhibitors in the field of organ transplantation. Background Technology

[0002] Soluble epoxide hydrolase (sEH) is an enzyme widely found in mammalian cells. It belongs to the epoxide hydrolase family and plays a key role in physiological and pathological processes, especially in cardiovascular, neurological, renal, and inflammation-related diseases.

[0003] TPPU (1-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea), chemically named N-[1-(1-oxypropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl]urea, is a small molecule compound belonging to the class of soluble epoxide hydrolase (sEH) inhibitors. It exerts anti-inflammatory and neuroprotective biological activities by regulating the epoxide fatty acid metabolic pathway. It is primarily used to study the function of sEH enzymes and their roles in cardiovascular diseases, inflammation, metabolic disorders, and neuroprotection.

[0004] The applications of soluble epoxide hydrolases (sEHs) are primarily focused on drug development and disease treatment. Their core logic is to inhibit sEH activity, reducing the hydrolysis of epoxide fatty acids (such as EETs), thereby enhancing the physiological effects of these active substances. They are mainly used for vasodilation, anti-inflammation, and neuroprotection, providing treatment strategies for various chronic diseases. The application potential of soluble epoxide hydrolases covers multiple fields, including cardiovascular, neurological, metabolic, and inflammatory diseases. Their core value lies in providing new therapeutic targets for chronic diseases by regulating epoxide fatty acid metabolism.

[0005] Post-organ transplant immune rejection is the most common and serious complication, often requiring patients to take oral immunosuppressive drugs. Immunosuppressive drugs frequently have various adverse reactions, making the development of novel immunosuppressive drugs with fewer side effects and multiple protective effects urgently needed. Soluble epoxide hydrolase inhibitors are investigational drugs not yet used clinically; current research focuses primarily on their application in treating inflammation, hypertension, and cardiovascular diseases, with no relevant studies published in the field of transplant immunology. Summary of the Invention

[0006] In view of the above technical problems, the present invention provides a new use for soluble epoxide hydrolase inhibitors, namely, the application of soluble epoxide hydrolase inhibitors in the field of transplantation.

[0007] The soluble epoxide hydrolase inhibitor is TPPU, which is used in organ transplantation and tissue transplantation.

[0008] TPPU alleviates immune rejection reactions after transplantation of allogeneic liver, heart, kidney, lung, and pancreas in mice.

[0009] TPPU alleviates immune rejection reactions after allogeneic mouse cornea, skin, bone marrow, and blood vessel transplantation.

[0010] The dosage of TPPU used in organ transplantation is 3 mg / kg / day, administered daily.

[0011] The dosage of TPPU used in tissue transplantation is 3 mg / kg / day, administered daily.

[0012] TPPU can reduce immune cell infiltration during transplantation surgery.

[0013] TPPU inhibited CD4 in the spleen of an allogeneic mouse skin transplant model. + T cell proliferation, proportion of CXCR3 chemokine receptor-positive cell subsets, and proportion of Th1 cell subsets.

[0014] TPPU inhibits primary CD4 in normal mice + T cell proliferation and Th1 cell subset differentiation.

[0015] The dosage of TPPU used was 50 μM, and TPPU was added to the culture medium.

[0016] The beneficial effects of the present invention are:

[0017] The invention proposes the use of soluble epoxide hydrolase inhibitor TPPU in organ and tissue transplantation. The use of soluble epoxide hydrolase inhibitor TPPU alone can alleviate the immune rejection reaction of allogeneic mouse heart and skin transplants, reduce the side effects of immunosuppressant use, and significantly reduce toxic side effects, which can make a significant contribution to improving the postoperative quality of life of transplant patients.

[0018] The soluble epoxide hydrolase inhibitor TPPU significantly prolonged the survival curve of allogeneic mouse heart and skin transplant models and alleviated the immune rejection response in these models. TPPU also reduced immune cell infiltration during transplantation and inhibited CD4+. + T cell proliferation, CXCR3 chemokine receptor positivity, and the proportion of Th1 cell subsets. The soluble epoxide hydrolase inhibitor TPPU significantly inhibited primary CD4+ in normal mice. + T cell proliferation and Th1 cell subset differentiation. Attached Figure Description

[0019] As shown in the figure

[0020] Figure 1A -D is the experimental result diagram of TPPU alleviating immune rejection in the allogeneic mouse heart transplantation model in Example 1 of the present invention;

[0021] Figure 1A This is an image of the donor-recipient vascular anastomosis after allogeneic mouse heart transplantation in Example 1 of the present invention;

[0022] Figure 1B Survival curves of the control group and TPPU group allogeneic mouse heart transplantation models in Example 1 of this invention;

[0023] Figure 1C HE staining results of transplanted hearts from allogeneic mice after heart transplantation in the control group and TPPU group in Example 1 of this invention;

[0024] Figure 1D The results are CD4 immunohistochemical staining of the transplanted hearts from allogeneic mice after heart transplantation in the control group and TPPU group in Example 1 of this invention.

[0025] Figure 2A -E is the experimental result diagram of TPPU alleviating immune rejection in the allogeneic mouse skin transplantation model in Example 2 of the present invention;

[0026] Figure 2A This is a photograph of the transplanted skin from the control group allogeneic mice after skin transplantation in Example 2 of the present invention;

[0027] Figure 2B This is a photograph of the transplanted skin from the TPPU group of allogeneic mice after skin transplantation in Example 2 of this invention;

[0028] Figure 2C The survival curves of the control group and TPPU group allogeneic mouse skin transplantation models in Example 2 of this invention are shown.

[0029] Figure 2D HE staining results of transplanted skin from allogeneic mice after skin transplantation in the control group and TPPU group in Example 2 of this invention;

[0030] Figure 2E The results are CD4 immunohistochemical staining of the transplanted skin from the control group and TPPU group after allogeneic mouse skin transplantation in Example 2 of this invention.

[0031] Figure 3A -C is a flow cytometry result of TPPU inhibiting spleen immune cells after allogeneic mouse skin transplantation in Example 3 of the present invention;

[0032] Figure 3A The CD4+ cells in the spleen immune cells of the control group and TPPU group after allogeneic mouse skin transplantation in Example 3 of this invention are... + Flow cytometry and statistical plot of CXCR3 in T cell subsets;

[0033] Figure 3B The CD4+ cells in the spleen immune cells of the control group and TPPU group after allogeneic mouse skin transplantation in Example 3 of this invention are... + Flow cytometry and statistical plot of IFNγ in T cell subsets;

[0034] Figure 3C The CD4+ cells in the spleen immune cells of the control group and TPPU group after allogeneic mouse skin transplantation in Example 3 of this invention are... + Ki67 flow cytometry and statistical plot in T cell subsets.

[0035] Figure 4A -C represents the inhibition of primary CD4 in normal mice by TPPU in Example 4 of this invention. + Figure showing experimental results of T cells;

[0036] Figure 4A The control group and groups with different concentrations of TPPU in Example 4 of this invention are used to evaluate the effects of TPPU on primary CD4 in normal mice. + Microscopic images of cultured T cells and a statistical graph of CCK8 absorbance values;

[0037] Figure 4B The control group and groups with different concentrations of TPPU in Example 4 of this invention are used to evaluate the effects of TPPU on primary CD4 in normal mice. + Flow cytometry and statistical graphs of CFSE in T cell culture;

[0038] Figure 4C The control group and TPPU group in Example 4 of this invention are examples of the effects of primary CD4 in normal mice. + Flow cytometry and statistical graph of IFNγ in T cell culture. Detailed Implementation

[0039] Example 1

[0040] The applicant uses the most classic and representative animal model in the field of immunology—the inbred mouse heterotopic heart transplantation model—to conduct experimental research. This model has a strong degree of immune rejection and the experimental results are easy to observe.

[0041] The soluble epoxide hydrolase inhibitor used in the experiment was TPPU. The following is an experiment on the use of TPPU to alleviate the immune rejection response in an allogeneic mouse heart transplantation model:

[0042] A mouse model of ectopic heart transplantation was established by transplanting the hearts of Balb / c mice into the abdomen of C57 mice. Figure 1A Images of the donor-recipient vascular anastomosis after transplantation. Multiple transplant surgeries were performed consecutively by a skilled allogeneic mouse heart transplant surgeon. Patients were randomly assigned to either a control group or a TPPU group. The control group received physiological saline solution, while the TPPU group received TPPU solution (3 mg / kg, daily). Post-transplantation, the pulsation of the transplanted heart in the abdominal cavity was palpated daily, and the duration of cardiac arrest was recorded to plot survival curves. On day 10 post-transplantation, the transplanted heart was harvested for HE and immunohistochemical staining to assess transplant rejection.

[0043] I. Allogeneic mouse heart transplantation model:

[0044] 1. Laboratory animals and equipment

[0045] Donor mice were Balb / c mice (8-10 weeks old, male, weighing 20-25g), and recipient mice were C57 mice (8-10 weeks old, male, weighing 22-25g). Both donor and recipient mice were fasted for 12 hours before surgery but had free access to water.

[0046] Instruments: Micro forceps, micro scissors, micro needle holder, vascular clamp, 10-0 needle-supported sutures, sterile gauze, cotton swabs, iodine solution, heparinized saline, ice box (for preserving donor hearts), electrosurgical unit, warming pad, microscope, anesthesia machine, surgical board.

[0047] 2. Donor Heart Acquisition

[0048] Anesthesia: Inhalation of isoflurane (2%–3%) for anesthesia;

[0049] Fixation: The patient is placed in a supine position and fixed to the surgical board. The limbs are secured with tape. The chest and abdomen are shaved and disinfected with iodine.

[0050] Incision: Make an incision along the center of the sternum through the skin and muscles, pull back the chest cavity, and expose the heart;

[0051] Cardiac perfusion: Inject approximately 1–2 ml of heparinized saline along the inferior vena cava proximal to the heart, while simultaneously cutting the thoracic segment of the thoracic aorta;

[0052] Heart dissection: Cut the mesentery around the heart, cover the heart with gauze, ligate the inferior vena cava and superior vena cava in sequence, and dissect the ascending aorta and pulmonary artery to the bifurcation and cut them;

[0053] Heart harvesting: The fibrous connective tissue in the posterior mass of the heart is completely removed, then the pulmonary veins are ligated and cut, and the heart is removed and placed in heparinized saline in a pre-prepared ice box for preservation.

[0054] 3. Recipient heart transplantation

[0055] Anesthesia: Inhalation of isoflurane (2%–3%) for anesthesia;

[0056] Fixation: The patient is placed in a supine position and fixed to the surgical board. The limbs are secured with tape. The abdomen is shaved and disinfected with iodine.

[0057] Incision: The skin and muscles are incised along the midline of the abdomen to retract the abdominal cavity and expose the inferior vena cava and abdominal aorta;

[0058] Vascular dissection: Bluntly dissect the abdominal aorta and inferior vena cava, use an electrocautery to burn off small branch vessels on the surface, ligate the small posterior branches with 11-0 needle and suture, and gently dissect the vessels to an appropriate length to avoid bleeding or dissecting them too high or low.

[0059] Vascular occlusion: The abdominal aorta and inferior vena cava are simultaneously occluded at the upper and lower ends of the vascular clamp, so that sufficient space is left in the middle of the occlusion to facilitate suturing;

[0060] Vascular trimming: The abdominal aorta and inferior vena cava are clamped and then cut open vertically at the middle to trim the openings so that the donor and recipient vessels can be anastomosed.

[0061] Vascular anastomosis: The donor abdominal aorta and recipient abdominal aorta, and the donor pulmonary artery and recipient inferior vena cava are continuously anastomosed end-to-end. The upper and lower ends of the donor and recipient vessels are fixed to fully expose the posterior wall. The anterior wall is sutured in a circular motion, maintaining uniform stitch spacing and consistent edge distance to avoid stenosis or gaps.

[0062] Opening of blood vessels: First, loosen the distal end of the vascular clamp, apply pressure to the vascular anastomosis with a cotton ball to stop bleeding, and then loosen the proximal end of the vascular clamp and continue to apply pressure to stop bleeding for 2-3 minutes.

[0063] Heart rewarming and re-beating: The surface of the transplanted heart is irrigated with warm saline solution to rapidly rewarm it, allowing the transplanted heart to gradually resume beating within the recipient's abdominal cavity.

[0064] 4. Postoperative management of mouse heart transplantation

[0065] Examine the surgical field: observe that the transplanted heart beats evenly and strongly, and check that the recipient mouse's respiratory and heart rates gradually return to stability and that there is no active bleeding in the abdominal cavity;

[0066] Muscle and skin suturing: The abdominal wall muscles and skin are sutured layer by layer using 5-0 sutures;

[0067] Postoperative warming: After placing the recipient mice back into the incubator (37℃) for 2-4 hours, they were then transferred to a normal culture environment for individual rearing.

[0068] Postoperative observation: The condition of the transplanted heart and the recipient will be observed daily after the operation, and intraperitoneal medication will be administered daily.

[0069] II. HE staining experimental method:

[0070] The transplanted heart tissue was fixed with formalin and other fixatives to preserve its structure. It was then dehydrated using a gradient of alcohols and cleared with xylene to allow paraffin penetration. The tissue was subsequently impregnated in molten paraffin and embedded into paraffin blocks. The paraffin blocks were sectioned, flattened in a water bath, mounted on glass slides, and baked. After dewaxing with xylene and hydration with a gradient of alcohols, the sections were stained with hematoxylin for the nuclei (requiring differentiation and blue reversion) and eosin for the cytoplasm. Finally, the sections were dehydrated and cleared again, and then mounted with resin for preservation.

[0071] III. Immunohistochemical staining methods:

[0072] The transplanted heart tissue underwent a series of pretreatments, including fixation, dehydration, clearing, paraffin embedding, sectioning, baking, and dewaxing (same as HE staining). Then, antigen retrieval was performed using heat retrieval buffer or enzyme digestion to expose the antigen. Subsequently, endogenous enzyme activity was blocked with hydrogen peroxide, and serum blocking was used to reduce non-specific binding. A specific primary antibody was added to bind to the target antigen, and then a labeled secondary antibody was used to bind the primary antibody for signal amplification. DAB chromogenic solution was added, and the chromogenic time was controlled under a microscope to display a positive signal; chromogenic development was terminated by running water. Finally, the cell nuclei were lightly counterstained with hematoxylin, and the tissue was dehydrated, cleared, and mounted.

[0073] IV. Experimental Results:

[0074] 1. The pulsation of the transplanted heart was palpated daily after heart transplantation in mice. The cessation of heart pulsation was defined as the survival endpoint. The time of cessation of heart pulsation was recorded, and a heart graft survival curve was plotted. The median survival of the heart graft in the control group was 8 days, and the median survival of the heart graft in the TPPU group was 13 days. Figure 1B ).

[0075] 2. Ten days after heart transplantation, the recipient mice were euthanized, and the transplanted hearts were harvested for HE and CD4 immunohistochemical staining.

[0076] HE staining in the control group showed fragmented inner cardiomyocyte structure, complete lysis and necrosis of the outermost cardiomyocytes, and diffuse inflammatory cell infiltration. HE staining in the TPPU group showed neatly arranged cardiomyocytes with intact nuclei and structures, but localized inflammatory cell infiltration was observed. Figure 1C ).

[0077] CD4 immunohistochemical staining in the control group showed diffuse CD4 immune cell infiltration in the myocardium, while CD4 immunohistochemical staining in the TPPU group showed a small amount of CD4 immune cell infiltration in the myocardium. Figure 1D ).

[0078] In summary, TPPU significantly prolonged the survival curve of the allogeneic mouse heart transplantation model and alleviated the immune rejection response in the allogeneic mouse heart transplantation model.

[0079] Example 2

[0080] The soluble epoxide hydrolase inhibitor used in the experiment was TPPU. The following is an experiment on the immune rejection response of TPPU in a Balb / c-C57 allogeneic mouse skin transplantation model:

[0081] Tail skin from Balb / c mice was transplanted onto the backs of C57 mice to establish an allogeneic mouse skin graft model. Multiple transplant surgeries were performed consecutively by a skilled allogeneic mouse skin graft surgeon, and patients were randomly assigned to either a control group or a TPPU group. The control group received physiological saline solution, while the TPPU group received TPPU solution (3 mg / kg, daily). The transplanted skin was observed and photographed daily post-transplantation. A rejection rate greater than 90% was defined as the endpoint of skin graft rejection, and survival curves were plotted based on the rejection endpoint. On day 10 post-transplantation, skin samples were harvested for HE and immunohistochemical staining to assess skin graft rejection.

[0082] I. Allogeneic mouse skin transplantation model:

[0083] 1. Laboratory animals and equipment

[0084] Donor mice were Balb / c mice (8-10 weeks old, male, weighing 20-25g), and recipient mice were C57 mice (8-10 weeks old, male, weighing 22-25g). Both donor and recipient mice were fasted for 12 hours before surgery but had free access to water.

[0085] Instruments: Micro forceps, micro scissors, micro needle holder, vascular clamp, 6-0 suture with needle, sterile gauze, cotton swabs, iodine, heparinized saline, ice box (for preserving donor heart), electrosurgical unit, warming pad, microscope, anesthesia machine, surgical board.

[0086] 2. Donor skin acquisition

[0087] Anesthesia: Inhalation of isoflurane (2%–3%) for anesthesia;

[0088] Fixation: The patient is placed in a supine position and fixed to the surgical board. The limbs are secured with tape, and the tail is disinfected with iodine.

[0089] Incision: Use a blade to make a circular cut along the end of the tail to expose the tailbone, then make a small longitudinal cut;

[0090] Skin harvesting: Use hemostatic forceps to tear off the entire skin from the tail along the small incision, remove it and place it in heparinized saline in a pre-prepared ice box for preservation, and cut it into square-sized skin pieces.

[0091] 3. Recipient heart transplantation

[0092] Anesthesia: Inhalation of isoflurane (2%–3%) for anesthesia;

[0093] Fixation: The patient is placed in a supine position and fixed to the surgical board. The limbs are secured with tape. The back is shaved and disinfected with iodine.

[0094] Incision: Along the symmetrical area on both sides of the spine on the back, lift the edge of the skin with micro forceps, and remove the full-thickness skin of a square area with micro-scissors. The square area is similar to the area of ​​the donor skin.

[0095] Skin grafting: The donor skin is laid flat in the recipient incision, with the edges aligned. Intermittent sutures are made along the four corners and midpoints of the four sides of the square using 6-0 needle sutures, for a total of 8 sutures. After suturing, the donor and recipient skin are pulled to ensure there are no wrinkles or tension.

[0096] Skin fixation: Wrap the skin graft site on the abdomen and back with adhesive bandages and bandages in a circular motion. Cut about 1 cm in the middle of the abdomen to prevent breathing pressure.

[0097] 4. Postoperative management of mouse skin grafts

[0098] Examination of recipients: Observe that the transplanted skin and bandages are properly fixed, and check that the respiratory and heart rates of the recipient mice gradually return to a stable state;

[0099] Postoperative warming: After placing the recipient mice back into the incubator (37℃) for 2-4 hours, they were then transferred to a normal culture environment for individual rearing.

[0100] Postoperative observation: The bandage was observed daily after surgery to prevent it from falling off and the transplanted skin grafts from being chewed off by the mice. Administered medication intraperitoneally daily.

[0101] II. HE and Immunohistochemical Staining Methods:

[0102] The procedure was performed using the same HE and immunohistochemical staining protocols as in Example 1.

[0103] III. Experimental Results:

[0104] 1. After skin grafting in mice, the bandage was observed daily to prevent it from falling off. The bandage was removed on the 7th day after the grafting. The condition of the grafted skin was observed and photographed daily. Figure 2A , Figure 2B The rejection rate was defined as greater than 90% as the survival endpoint for the transplanted skin. The rejection time was recorded, and skin graft survival curves were plotted. The median survival of the skin grafts in the control group was 8 days, and the median survival of the skin grafts in the TPPU group was 10 days. Figure 2C ).

[0105] 2. Ten days after the mouse skin transplant, the recipient mice were euthanized, and the transplanted skin was harvested for HE and CD4 immunohistochemical staining.

[0106] HE staining in the control group showed extensive tissue necrosis and congestion in the skin. HE staining in the TPPU group showed that the skin tissue was neatly arranged, with no obvious congestion. Figure 2D ).

[0107] In the control group, CD4 immunohistochemical staining showed diffuse CD4 immune cell infiltration in the skin. In the TPPU group, CD4 immunohistochemical staining showed a small amount of CD4 immune cell infiltration in the skin. Figure 2E ).

[0108] In summary, TPPU significantly prolonged the survival curve of the Balb / c-C57 allogeneic mouse skin transplantation model and alleviated the immune rejection response in the Balb / c-C57 allogeneic mouse skin transplantation model.

[0109] Example 3

[0110] The soluble epoxide hydrolase inhibitor used in the experiment was TPPU. The following is a flow cytometry experiment on the effect of TPPU on spleen immune cells in a Balb / c-C57 allogeneic mouse skin transplantation model:

[0111] Tail skin from Balb / c mice was transplanted to the backs of C57 mice to establish an allogeneic mouse skin transplantation model. Six pairs of transplant surgeries were performed consecutively by a skilled allogeneic mouse skin transplant surgeon. Patients were randomly assigned to either a control group or a TPPU group. The control group received a solvent injection, while the TPPU group received TPPU solution (3 mg / kg, daily). On day 10 post-surgery, spleen tissue was harvested from the recipient mice in both the control and TPPU groups. The spleen was ground, split, and used to prepare a single-cell suspension for flow cytometry analysis of CD4+. + T cells contain chemokines, cytokines, and transcription factors.

[0112] I. Allogeneic mouse skin transplantation model:

[0113] The mouse skin transplantation model was treated using the same experimental method as in Example 2.

[0114] II. Flow cytometry experimental methods:

[0115] After euthanasia of mouse skin graft recipients in the control and TPPU groups, spleens were harvested, ground and filtered, erythrocytes were lysed, and cells were washed to prepare spleen single-cell suspensions. The cell suspension concentration was adjusted to 1×10⁻⁶. 7 / mL, 100μL of cell suspension was stained with corresponding indicator antibodies and analyzed using FlowJo software.

[0116] The flow cytometry gate logic consists of FSC and SSC gates with main cell population in the middle, Zombie gate with live cell population in the middle, and CD4 gate with CD4 in the middle. +T cell populations, then CXCR3 and CCR6 phyla were circled one by one. + Cell population, CD4 and IFNγ phylum, IFNγ in the middle circle + Cell population, CD4 and Ki67 phylum, middle circle Ki67 + Cell population.

[0117] III. Experimental Results:

[0118] CD4 in the control group and TPPU group + Flow cytometry analysis of T cell subsets CXCR3 and CCR6 showed that TPPU administration significantly inhibited CXCR3. + Cell ratio ( Figure 3A ).

[0119] CD4 in the control group and TPPU group + Flow cytometry analysis of IFNγ in T cell subsets showed that TPPU administration significantly inhibited IFNγ. + Cell ratio ( Figure 3B ).

[0120] CD4 in the control group and TPPU group + In flow cytometry analysis of Ki67 in T cell subsets, TPPU administration significantly inhibited Ki67. + Cell ratio ( Figure 3C ).

[0121] In conclusion, TPPU significantly inhibited CD4 in the spleen of an allogeneic mouse skin transplant model. + T cell proliferation, proportion of CXCR3 chemokine receptor-positive cell subsets, and proportion of Th1 cell subsets.

[0122] Example 4

[0123] The soluble epoxide hydrolase inhibitor used in the experiment was TPPU. Below is the effect of TPPU on primary CD4+ isolated from and cultured spleens of normal C57 mice. + Experiments with T cells:

[0124] After euthanizing C57 mice, the spleen was obtained, ground, filtered, and washed to prepare a single-cell suspension of the spleen. Primary CD4 cells were extracted from the mice using a kit. + T cell culture. Cells were randomly assigned to a control group and a TPPU group. The control group was cultured with 0.5% DMSO solution, while the TPPU group was cultured with different concentrations of TPPU solution. After 72 hours of culture, cell number, cell proliferation, and the proportion of Th1 cell subsets were measured.

[0125] I. Original CD4 + T cell isolation and culture:

[0126] After euthanizing C57 mice, the spleen was obtained, ground, filtered, and washed to prepare a single-cell suspension of the spleen. Primary CD4 cells were extracted from the mice using a kit. + T cells were cultured in CD3 antibody-coated plates in complete medium containing CD28 antibody and IL-2 cytokine. Cells were randomly assigned to a control group and a TPPU group. The control group was cultured in 0.5% DMSO, while the TPPU group was cultured in different concentrations of TPPU solution for 72 hours. II. Experimental methods for detecting cell number, cell proliferation, and Th1 cell subset ratio:

[0127] Cell count was determined by measuring the absorbance of the culture medium after adding CCK8 reagent; a lower absorbance value indicated a lower cell count. Cell proliferation was assessed by flow cytometry of the proportion of cells cultured with CFSE reagent for 72 hours; a lower CFSE proportion indicated slower cell proliferation. The proportion of Th1 cell subsets was determined by flow cytometry of the proportion of IFNγ-positive cells; a lower IFNγ-positive proportion indicated a lower Th1 cell subset.

[0128] III. Experimental Results:

[0129] Primary CD4 cells were cultured by adding TPPU solutions of different concentrations to the culture medium. + After adding T cells, the absorbance of the culture medium was measured using CCK8 reagent. Higher TPPU solution concentration resulted in lower absorbance values ​​and fewer cells. Figure 4A ).

[0130] Primary CD4 cells were cultured by adding TPPU solutions of different concentrations to the culture medium. + After T cells were cultured, CFSE reagent was added to detect the CFSE ratio after 72 hours of cell culture. The higher the TPPU solution concentration, the lower the CFSE ratio and the slower the cell proliferation. Figure 4B ).

[0131] Original CD4 + T cells were cultured in a medium that induces Th1 cell subset differentiation and were divided into a control group and a 50 μM TPPU group. Flow cytometry analysis showed that the proportion of IFNγ-positive cells was significantly decreased in cells cultured in the 50 μM TPPU solution. Figure 4C ).

[0132] In conclusion, TPPU significantly inhibited primary CD4+ isolated from mouse spleen culture. + The number of T cells, cell proliferation, and the proportion of Th1 cell subsets.

[0133] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. The various components mentioned in this invention are common technologies in the existing field. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. The application of soluble epoxide hydrolase inhibitors in the field of transplantation, characterized in that, The soluble epoxide hydrolase inhibitor is TPPU, which is used in organ transplantation and tissue transplantation.

2. The application of the soluble epoxide hydrolase inhibitor according to claim 1 in the field of transplantation, characterized in that, The TPPU alleviates the immune rejection response to allogeneic mouse liver, heart, kidney, lung, and pancreas transplantation.

3. The application of the soluble epoxide hydrolase inhibitor according to claim 1 in the field of transplantation, characterized in that, The TPPU alleviates the immune rejection response of allogeneic mouse cornea, skin, bone marrow, and blood vessel transplantation.

4. The application of the soluble epoxide hydrolase inhibitor according to claim 2 in the field of transplantation, characterized in that, The TPPU used in organ transplantation is at a dose of 3 mg / kg / day, administered daily.

5. The application of the soluble epoxide hydrolase inhibitor according to claim 3 in the field of transplantation, characterized in that, The TPPU used in tissue transplantation is at a dose of 3 mg / kg / day, injected daily.

6. The application of the soluble epoxide hydrolase inhibitor according to claim 1 in the field of transplantation, characterized in that, The TPPU can reduce immune cell infiltration during transplantation surgery.

7. The application of the soluble epoxide hydrolase inhibitor according to claim 6 in the field of transplantation, characterized in that, The TPPU inhibited CD4 in the spleen of an allogeneic mouse skin transplant model. + T cell proliferation, proportion of CXCR3 chemokine receptor-positive cell subsets, and proportion of Th1 cell subsets.

8. The application of the soluble epoxide hydrolase inhibitor according to claim 1 in the field of transplantation, characterized in that, The TPPU inhibits primary CD4 in normal mice. + T cell proliferation and Th1 cell subset differentiation.

9. The application of the soluble epoxide hydrolase inhibitor according to claim 8 in the field of transplantation, characterized in that, The TPPU dosage was 50 μM, and the TPPU was added to the culture medium.

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