Application of GSDMD molecular inhibitor NU6300 in preparation of medicine for treating heart transplant rejection
By using the GSDMD molecular inhibitor NU6300 in heart transplantation to inhibit pyroptosis, the problems of inflammatory cytokine infiltration and shortened survival time after heart transplantation were solved, resulting in prolonged graft survival and reduced inflammatory response.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2026-03-20
AI Technical Summary
In existing technologies, GSDMD-mediated pyroptosis exists after heart transplantation, leading to inflammatory cytokine infiltration and shortened graft survival time, thus affecting quality of life.
The GSDMD molecular inhibitor NU6300 was administered via intraperitoneal injection to inhibit GSDMD-mediated pyroptosis, reduce N-GSDMD expression levels, and decrease the infiltration of inflammatory cytokines.
It prolonged the survival time of mouse heart grafts and reduced GSDMD-mediated pyroptosis levels and the infiltration of inflammatory cytokines.
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Figure CN121695142A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of medical biology and relates to the application of NU6300, a pyroptosis GSDMD molecular inhibitor, in organ transplantation. Background Technology
[0002] Organ transplantation is the most effective treatment for end-stage organ failure, improving recipient conditions, prolonging survival, and enhancing quality of life. According to literature, the overall survival rates at 1, 5, and 10 years after heart transplantation are 84.8%, 72.4%, and 53.5%, respectively, with an average survival of 14.8 years. However, due to primary end-stage heart disease, postoperative complications, and lifelong use of immunosuppressive drugs, heart transplant recipients often experience complex symptoms, with some symptoms occurring simultaneously, impacting their quality of life. Alleviating ischemia-reperfusion injury in transplanted organs and prolonging organ survival are urgent issues. Therefore, the development of novel drugs targeting these processes and the development of new treatment strategies are urgently needed.
[0003] Pyroptosis is a programmed cell death mechanism mediated by the gasdermin protein family that promotes the release of inflammatory substances. It is characterized by caspase protein cleaving gasdermin protein, separating the C-terminus from the N-terminus, forming a pore on the cell membrane at the N-terminus, causing cell swelling and membrane rupture leading to a large leakage of cytoplasmic contents, and inducing the release of further inflammatory cytokines such as IL-1β and IL-18. The gasdermin family includes several classes such as GSDMA, GSDMB, GSDMC, GSDMD, and GSDME
[13] . Among them, GSDMD is considered to be the classic molecule that mediates pyroptosis, and the classic pyroptosis pathway is the NLRP3-CASPASE1-GSDMD-IL1β pathway.
[0004] Current research on GSDMD-mediated pyroptosis mainly focuses on tumors, viral infections, and autoimmune diseases. No one has reported on it in the field of transplantation. Whether GSDMD-mediated pyroptosis occurs after heart transplantation, and research on GSDMD protein-mediated pyroptosis and its inhibitors, has great application value and development prospects in the field of organ transplantation. Summary of the Invention
[0005] The purpose of this invention is to address the shortcomings of mouse heart transplantation therapy and to provide the application of the GSDMD inhibitor NU6300 in the preparation of a drug for treating mouse heart transplantation rejection.
[0006] The specific technical solution adopted in this invention is as follows:
[0007] In a first aspect, the present invention provides the application of the GSDMD molecular inhibitor NU6300 in the preparation of a drug for treating heart transplant rejection.
[0008] It should be noted that the GSDMD molecular inhibitor NU6300 in this invention is a commercially available product, and its structural formula is as follows:
[0009]
[0010] Preferably, the drug, when administered, can improve the survival time of the mouse neck heart transplant.
[0011] Preferably, after administration of the drug, the level of GSDMD-mediated pyroptosis in mouse neck heart grafts is reduced, i.e., the level of N-GSDMD expression is reduced.
[0012] Preferably, after administration of the therapeutic drug, the infiltration of inflammatory cytokines in the mouse neck heart graft is reduced.
[0013] Preferably, the drug is an injection solution obtained by diluting the drug stock solution with phosphate buffer, wherein the drug stock solution is a dimethyl sulfoxide solution of the GSDMD molecular inhibitor NU6300.
[0014] Furthermore, the concentration of the GSDMD molecular inhibitor NU6300 administered in mouse heart transplantation was 10 mg / kg / day.
[0015] Secondly, the present invention provides a heart transplant rejection treatment drug, which is composed of the GSDMD molecular inhibitor NU6300 and pharmaceutical excipients.
[0016] It should be noted that the form of the above-mentioned heart transplant rejection treatment drugs is not limited. Theoretically, they can be made into injections, tablets, powders, etc. The specific pharmaceutical excipients need to be selected according to the form of the drug.
[0017] Secondly, the present invention provides an injectable drug for treating heart transplant rejection, which is obtained by diluting a drug stock solution with phosphate buffer, wherein the drug stock solution is a dimethyl sulfoxide solution of the GSDMD molecular inhibitor NU6300.
[0018] This invention provides a novel use for the GSDMD protein inhibitor NU6300 in a mouse neck heart transplant model. Experiments have shown that treatment with NU6300 prolonged the survival time of neck heart transplants in both the experimental and control groups, reduced GSDMD-mediated pyroptosis levels, and decreased infiltration of inflammatory cytokines in the grafts. Therefore, NU6300 can be used to prepare drugs for treating heart transplant rejection. Attached Figure Description
[0019] Figure 1This is a schematic diagram showing the mRNA levels of inflammatory factors on days 1, 3, and 5 after heart transplantation in mice according to an embodiment of the present invention.
[0020] Figure 2 This is a schematic diagram of the protein levels of GSDMD-induced pyroptosis in mice on days 1, 3, and 5 after heart transplantation in an embodiment of the present invention.
[0021] Figure 3 This is the survival curve of the heart graft in mice after treatment with NU6300 in an embodiment of the present invention.
[0022] Figure 4 This is a schematic diagram showing the mRNA levels of inflammatory factors in the heart graft on days 3 and 5 after treatment in an embodiment of the present invention.
[0023] Figure 5 This is a schematic diagram showing the levels of GSDMD-induced pyroptosis protein in the heart graft on days 3 and 5 after treatment in an embodiment of the present invention. Detailed Implementation
[0024] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0025] Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.
[0026] Example 1
[0027] In this embodiment, a mouse cervical heterotopic heart transplantation model was constructed. After grouping and administering the drug, the survival time was observed, and RT-PCR and Western blotting were performed on the graft samples. The corresponding indicators were then analyzed. The specific experimental procedure is described in detail below.
[0028] 1. Establishment of a mouse cervical heterotopic heart transplantation model and drug administration
[0029] 1.1 Experimental animals and main materials: Male purebred Balb / c mice, weighing 20-25g, were purchased from Shanghai Slico Company; Male purebred C57BL / 6 background mice, weighing 20-25g, were purchased from Shanghai Slico Company.
[0030] 1.2 Methods: Using our laboratory's mature mouse heart transplantation model, C57BL / 6 wild-type mice were used as heart donors, and BALB / c wild-type mice were used as recipients. C57BL / 6 mice were anesthetized with pentobarbital solution (0.1 g / 10 ml), shaved, locally disinfected, and heparinized systemically (100 U / ml). The donor mice's hearts were immediately removed and stored in 4℃ HTK heart preservation solution. Then, the donor's ascending aorta was anastomosed to the recipient's common carotid artery, and the donor's pulmonary artery was anastomosed to the recipient's external jugular vein using cannulas.
[0031] 1.3 Grouping and Administration: C57BL / 6 wild-type mice were used as heart donors, and BALB / c wild-type mice were used as recipients. NU6300 powder was purchased from MCE (catalog number HY-18930), and the drug was dissolved in the universal solvent dimethyl sulfoxide (DMSO) as a stock solution. The stock solution was diluted with phosphate-buffered saline (PBS) and administered to mice at a dose of 10 mg / kg / day (based on the NU6300 dosage) via intraperitoneal injection. This group served as the NU6300 experimental group. An equal volume of DMSO was used as the control group.
[0032] 2. Experimental detection methods
[0033] 2.1 Overall survival statistics of heart grafts: C57BL / 6 wild-type mice were used as transplant donors, and BALB / c wild-type mice were used as recipients. The day of transplantation was Day 0. Postoperatively, the donor heart was palpated daily in the recipient's neck. The absence of ventricular pulsation or only slight fibrillation in the donor heart was considered the endpoint of rejection.
[0034] 2.2 RT-PCR: The steps for detecting changes in inflammatory cytokines in mouse heart grafts over time, and in the control and treatment groups, are as follows: 1) Primer design; 2) Extraction of total mRNA from grafts; 3) Reverse transcription of total mRNA from grafts into cDNA; 4) Chain amplification of graft cDNA; 5) Sample statistics and calculation.
[0035] 2.3 Western blotting: Graft tissue samples from mouse hearts over time, the control group, and the drug-treated group were collected. Cells were lysed with protein lysis buffer and reacted on ice for 30 min. The supernatant was collected by centrifugation, and protein quantification was performed according to the BCA protein quantification kit instructions. Protein samples were added to 8% or 12% SDS-polyacrylamide gels and electrophoresed at 120 V for 60 min. After electrophoresis, the proteins in the gel were transferred to a PVDF membrane. Blocking buffer was applied at 4°C for 40 min. The PVDF membrane was added to primary antibody dilution buffer and incubated overnight at 4°C. After washing three times with TBST buffer, secondary antibody dilution buffer was added and incubated at 4°C for 2 h. After washing three times with TBST buffer, chromogenic buffer was added to the PVDF membrane surface, and the membrane was exposed and developed using a chemiluminescence imaging system. Finally, the exposed bands were analyzed using ImageJ software.
[0036] 3. Experimental Results
[0037] A mouse model of heterotopic heart transplantation in the neck was established. Tissue samples from the mouse heart grafts were collected on Day 1, Day 3, and Day 5, and the levels of inflammatory cytokine transcription and GSDMD-mediated pyroptosis were measured. We found that the levels of inflammatory cytokines and GSDMD-mediated pyroptosis in the grafts increased with time (results are shown in […]). Figure 1 and Figure 2 Based on this, the survival time of grafts in mice treated with NU6300 was prolonged (results are shown in...). Figure 3 We also compared changes in inflammation levels and GSDMD-mediated pyroptosis levels in the treatment group and the control group on days 3 and 5. We found that the levels of inflammatory factors in the graft were suppressed (results are available in...). Figure 4 Meanwhile, GSDMD-mediated pyroptosis levels were also suppressed in the treatment group (results are shown in...). Figure 5 Therefore, the GSDMD molecular inhibitor NU6300 can be used to prepare drugs for treating mouse heart transplants.
[0038] It should be noted that although the above embodiments use mice as the treatment subjects, the present invention is not limited to treating mouse heart transplants. It can also be used in other mammalian organs to treat heart transplant rejection and prolong graft survival time with the GSDMD molecular inhibitor NU6300.
[0039] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.
Claims
1. The application of NU6300, a GSDMD molecular inhibitor, in the preparation of drugs for treating heart transplant rejection.
2. The application according to claim 1, characterized in that, After administration of the drug, the survival time of the mouse neck heart transplant can be improved.
3. The application according to claim 2, characterized in that, After administration of the drug, the level of GSDMD-mediated pyroptosis in mouse neck heart grafts decreased, i.e., the level of N-GSDMD expression decreased.
4. The application according to claim 2, characterized in that, After administration of the therapeutic drug, the infiltration of inflammatory cytokines in the mouse neck heart graft was reduced.
5. The application according to claim 2, characterized in that, The drug is an injection solution obtained by diluting the drug stock solution with phosphate buffer, and the drug stock solution is a dimethyl sulfoxide solution of the GSDMD molecular inhibitor NU6300.
6. The application according to claim 5, characterized in that, The concentration of the GSDMD molecular inhibitor NU6300 administered to mice in heart transplantation was 10 mg / kg / day.
7. A drug for treating heart transplant rejection, characterized in that, It consists of the GSDMD molecular inhibitor NU6300 and pharmaceutical excipients.
8. A heart transplant rejection treatment injection, characterized in that, The injectable drug is obtained by diluting a drug stock solution with phosphate buffer, wherein the drug stock solution is a dimethyl sulfoxide solution of the GSDMD molecular inhibitor NU6300.