Application method of romidepsin in drug for pre-sensitization of rejection reaction

Abstract

The invention belongs to the technical field of application of pre-sensitization rejection drugs, and particularly relates to an application method of romidepsin in a pre-sensitization rejection drug. According to the application, by controlling the use mode of the romidepsin, the effects of inhibiting HDAC1 and HDAC2 by using the romidepsin are realized, the acetylation levels of different sites corresponding to histone H2A and H3 are up-regulated, the expression of BLIMP-1, AID and the like is reduced, the activity of an endoplasmic reticulum IRE1alpha-XBP1 pathway is inhibited, the transcriptional levels of Pridm1 and Xbp1 are further reduced, the generation of plasma cells is inhibited, the generation of donor specific antibodies is reduced, and the application of the romidepsin in the preparation of the medicine for treating the endoplasmic reticulum IRE1alpha-XBP1 is promoted. The synthetic secretion of antibodies is inhibited, thereby improving the survival of the graft.

Description

Technical Field

[0001] The present invention belongs to the technical field of the application of drugs for presensitized rejection reaction, and particularly relates to an application method of romidepsin in drugs for presensitized rejection reaction. Background Art

[0002] Organ transplantation has saved millions of lives globally and is the only hope for patients with end-stage organ dysfunction to obtain a new life. The severe shortage of organs and the huge risk of rejection seriously restrict the development of organ transplantation. China has become the second largest country in organ transplantation in the world, but still cannot meet the waiting needs of more than 300,000 people every year. While urgently seeking more transplant organ resources, it is particularly important to improve the survival rate of transplanted organs.

[0003] Based on the nature of the underlying immunopathological mechanisms, acute rejection has been further divided into two major categories, namely cellular rejection and humoral rejection. Performing sensitive lymphocytotoxic crossmatch testing before transplantation can avoid the occurrence of hyperacute rejection against the graft. In acute and chronic rejection reactions, the mediation of T cells and antibodies both play important roles. In the past few decades, with the continuous in-depth study of the adaptive immune response in solid organ transplantation, many immunosuppressive drugs targeting key alloreactive T cell mechanisms have been developed, and the probability of acute rejection has been significantly reduced. Compared with the significantly improved short-term survival rate, the long-term survival rate of organ transplantation has not been significantly improved. Antibody-mediated graft rejection is one of the main reasons for graft failure and even graft loss, and is also the core element interfering with long-term survival. The production of antibodies is crucial for humoral immunity. B cells are stimulated by antigen receptor-dependent signals and costimulated by follicular helper T cells to form germinal centers (GCs). GCs are the extensive selective proliferation of antigen receptors modified based on somatic hypermutation (SHM). GCs ultimately produce long-lived plasma cells capable of maintaining a high level of high-affinity antibody secretion, and at the same time produce memory cells. To achieve this secretory capacity, plasma cells need to greatly expand the endoplasmic reticulum (ER) and Golgi complex networks. This huge expansion and the ultra-high rate of antibody synthesis in plasma cells both require efficient regulatory measures to ensure that production matches the supply of biosynthesis, immunoglobulin folding, and the secretory capacity of the cell.

[0004] Histone deacetylase inhibitors (HDAC inhibitors, HDACI) are compounds that can balance histone acetylation by inhibiting the activity of HDACs and interfering with their functions. HDACI can also affect the activities of many non-histone proteins because they can also be targeted for lysine acetylation or deacetylation. This leads to an increase in the acetylation rate of many gene clusters, resulting in increased transcriptional activity and, in turn, upregulation of specific genes. If these upregulated genes are transcription factors, they may greatly alter the expression profiles of many other responsive genes, thus potentially changing certain cell phenotypes. HDACI have broad inhibitory activities by targeting different types of HDACs, and many unexpected results are produced by modifying the chemical and biological mechanisms of intracellular interactions.

[0005] Romidepsin (FK228) is a selective class I HDAC inhibitor, a bicyclic dipeptide antibiotic with antitumor activity extracted from Corynebacterium violaceum. After entering cells, FK228 inhibits HDAC1 and HDAC2, thereby altering the expression of related genes, inducing cell cycle arrest, cell differentiation, and apoptosis. Romidepsin was approved by the US Food and Drug Administration in 2009 for the treatment of certain T-cell lymphomas. Currently, there are multiple clinical trial studies on FK228 for other diseases underway, and it has broad clinical application prospects. Summary of the Invention

[0006] The object of the present invention is to provide a method for the application of romidepsin in drugs for pre-sensitized rejection reactions, which is used to improve the survival rate of grafts.

[0007] To achieve the above object, the present invention adopts the following technical solutions.

[0008] A method for the application of romidepsin in drugs for pre-sensitized rejection reactions, which is used to inhibit the expression of HDAC1 and HDAC2, including reducing the expression levels of DSA-IgG and DSA-IgM.

[0009] A further improvement or preferred embodiment of the above method for the application of romidepsin in drugs for pre-sensitized rejection reactions, which is used to upregulate the acetylation levels of histone H2A and H3 at different sites.

[0010] A further improvement or preferred embodiment of the above method for the application of romidepsin in drugs for pre-sensitized rejection reactions, which is used to intervene and downregulate the expression of BLIMP-1 and AID.

[0011] A further improvement or preferred embodiment of the above method for the application of romidepsin in drugs for pre-sensitized rejection reactions, which is used to downregulate the proportion of Tfh cells and inhibit plasma cell differentiation.

[0012] A further improvement or preferred embodiment of the aforementioned method for using romidepsin in drugs for pre-sensitized rejection reactions, which is used to inhibit the relative mRNA expression levels of Hdac1 and Hdac2.

[0013] A further improvement or preferred embodiment of the aforementioned method for using romidepsin in drugs for pre-sensitized rejection reactions, which is used to inhibit the transcription of Pridm-1 and Xbp1, and to transform the transcription of transplanted B cells into plasma cell transcription; inhibit the transcription of Aicda, and inhibit somatic hypermutation and antibody class switching.

[0014] A further improvement or preferred embodiment of the aforementioned method for using romidepsin in drugs for pre-sensitized rejection reactions, which is used to inhibit the activity of endoplasmic reticulum IRE1α-XBP1, inhibit BLIMP-1, and reduce antibody synthesis and modification.

[0015] A further improvement or preferred embodiment of the aforementioned method for using romidepsin in drugs for pre-sensitized rejection reactions, wherein the graft in the pre-sensitized rejection reaction is skin.

[0016] A further improvement or preferred embodiment of the aforementioned method for using romidepsin in drugs for pre-sensitized rejection reactions, and the administration method is intraperitoneal injection.

[0017] A further improvement or preferred embodiment of the aforementioned method for using romidepsin in drugs for pre-sensitized rejection reactions, and the dosage of romidepsin is 500 μg / kg, and it is administered once a day.

[0018] Its beneficial effects are as follows:

[0019] The present invention relates to a method for using romidepsin in drugs for pre-sensitized rejection reactions. By reasonably controlling the usage mode of romidepsin, the effects of using romidepsin to inhibit HDAC1 and HDAC2 are realized, the acetylation levels of corresponding different sites of histone H2A and H3 are up-regulated, the expressions of BLIMP-1, AID, etc. are reduced, the activity of the endoplasmic reticulum IRE1α-XBP1 pathway is inhibited, the transcription levels of Pridm1 and Xbp1 are further reduced, thereby inhibiting the generation of plasma cells, reducing the production of donor-specific antibodies, inhibiting the synthesis and secretion of antibodies, and thus improving the survival of the graft. Description of the Drawings

[0020] Figure 1 It is a graph of DSA-IgM levels in healthy mice and mice after primary transplantation;

[0021] Figure 2 It is a graph of DSA-IgG levels in healthy mice and mice after primary transplantation;

[0022] Figure 3It is a graph of the survival time of the grouped transplanted skin under different intervention treatments;

[0023] Figure 4 It is a graph of the average fluorescence intensity of DSA-IgG in the sera of the recipients at D0, D7, and D14 after surgery;

[0024] Figure 5 It is a graph of the average fluorescence intensity of DSA-IgM in the sera of the recipients at D0, D7, and D14 after surgery;

[0025] Figure 6 It is a graph of the relative expression levels of the Hdac1 gene amplified by reverse transcription of the total RNA from the spleens of mice in each group;

[0026] Figure 7 It is a graph of the relative expression levels of the Hdac2 gene amplified by reverse transcription of the total RNA from the spleens of mice in each group;

[0027] Figure 8 It is a graph of the expression levels of acetylated histone Lys4 in the spleens of mice in each group;

[0028] Figure 9 It is a graph of the expression levels of acetylated histone Lys5 in the spleens of mice in each group;

[0029] Figure 10 It is a graph of the expression levels of acetylated histone Lys9 in the spleens of mice in each group;

[0030] Figure 11 It is a graph of the expression levels of acetylated histone Lys27 in the spleens of mice in each group;

[0031] Figure 12 It is a graph of the expression levels of acetylated histone Lys56 in the spleens of mice in each group;

[0032] Figure 13 It is a graph of the proportion of PD-1 in the splenocytes of the secondarily skin-transplanted mice in each group;

[0033] Figure 14 It is a graph of the proportion of Tfh in the splenocytes of the secondarily skin-transplanted mice in each group;

[0034] Figure 15 It is a graph of the proportion of CD138 in the splenocytes of the secondarily skin-transplanted mice in each group;

[0035] Figure 16 It is a graph of the proportion of plasma cells in the splenocytes of the secondarily skin-transplanted mice in each group;

[0036] Figure 17 It is a graph of the levels of the IL-2 cytokine on the 10th day after surgery in the secondarily skin-transplanted mice in each group;

[0037] Figure 18It is the graph of the IL-4 cytokine level in mice with secondary skin transplantation on the 10th day after surgery for each group;

[0038] Figure 19 It is the graph of the IL-10 cytokine level in mice with secondary skin transplantation on the 10th day after surgery for each group;

[0039] Figure 20 It is the graph of the IFN-γ cytokine level in mice with secondary skin transplantation on the 10th day after surgery for each group;

[0040] Figure 21 It is the graph of the IL-21 cytokine level in mice with secondary skin transplantation on the 10th day after surgery for each group;

[0041] Figure 22 It is the graph of the relative expression level of the spleen B cell gene Pax5 in mice;

[0042] Figure 23 It is the graph of the relative expression level of the spleen B cell gene Bcl-6 in mice;

[0043] Figure 24 It is the graph of the relative expression level of the spleen B cell gene Bach2 in mice;

[0044] Figure 25 It is the graph of the relative expression level of the spleen B cell gene Prdm-1 in mice;

[0045] Figure 26 It is the graph of the relative expression level of the spleen B cell gene Irf4 in mice;

[0046] Figure 27 It is the graph of the relative expression level of the spleen B cell gene Xbp1 in mice;

[0047] Figure 28 It is the graph of the relative expression level of the total spleen protein HDAC1 in mice;

[0048] Figure 29 It is the graph of the relative expression level of the total spleen protein HDAC2 in mice;

[0049] Figure 30 It is the graph of the Prdm-1 expression level;

[0050] Figure 31 It is the graph of the relative expression level of Xbp1;

[0051] Figure 32 It is the graph of the relative expression level of AID;

[0052] Figure 33 It is the graph of the phosphorylation level of endoplasmic reticulum IRE. Detailed implementation methods

[0053] The present invention is described in detail below in conjunction with specific embodiments.

[0054] Romidepsin (FK-228) is a cyclopentapeptide compound isolated from the culture medium of Gram-negative Bacillus violaceus by Fujisawa Pharmaceutical Co., Ltd. of Japan. It mainly forms the basic structural unit of bicyclic tetrapeptide disulfide through disulfide bonds and ester bonds. In the same year, it was preliminarily confirmed that it has potential anti-cancer activity.

[0055] The skin is one of the largest "organs" and is highly immunogenic. In the allogeneic skin transplant model, transplant immune rejection will eventually lead to graft loss. The rejection reaction caused by transplantation can be divided into three stages, namely the induction stage in which the host is sensitized by the graft antigen, the proliferation and differentiation stage in which the host T cells are activated, and the effector stage in which the graft is attacked and destroyed by the host immune system. The entire transplant immune response process is a T cell-dependent immune response, and the central link is the activation and proliferation of T cells. In clinical practice, immunosuppressants are usually used for anti-rejection treatment of rejection reactions caused by organ transplantation. The most common ones include cyclosporine A (CsA) and the like.

[0056] At present, most skin transplant models use back skin transplantation and tail skin transplantation. Back skin transplantation and tail skin transplantation have high surgical requirements for donor skin sampling, poor uniformity of skin taken from different animals, and are prone to infection.

[0057] In order to verify the effectiveness of the application method of romidepsin (FK228) in the presensitization rejection reaction drug of the present invention, a skin transplantation experiment was established by transplanting the tail of the donor BALB / c mouse to the back of the recipient C57BL / 6 mouse to verify the application effect of the present invention.

[0058] In this experiment, the skin transplantation model was established by transplanting ear skin to the back of mice, which has the advantages of simple and quick operation, good uniformity between different animals, low infection, good growth state of transplanted skin, and easy survival.

[0059] 1. Experimental design:

[0060] A. Source of experimental subjects

[0061] Male Balb / c (H-2d) and C57BL / 6 (H-2b) strains of SPF mice, weighing about 19-24 g and 6-8 weeks old, were purchased from Beijing Weitonglihua Biological Co., Ltd. All experimental mice were kept in SPF animal rooms.

[0062] B. Experimental Equipment

[0063] Pure water system, incubator, laminar flow hood, ordinary optical microscope, automatic enzyme labeler, electronic balance, ordinary refrigerator, -80°C low-temperature refrigerator, room temperature centrifuge, constant temperature shaker, flow cytometer, refrigerated centrifuge, transfer tank, stirrer, oscillator, cell counter, ice maker, micropipette, etc.

[0064] C. Transplantation and sample collection steps:

[0065] (1) Donor surgery: Anesthetize Balb / c mice, fix them on the operating table, disinfect the tail with iodophor, cut off the mouse tail, place it in physiological saline, peel off the skin from the incision, and remove the subcutaneous appendages, trim it into a 1.5 cm × 1.5 cm skin, and store the graft on a gauze soaked with sterile phosphate buffered saline (PBS) for later use, and place it in a petri dish on ice for later use.

[0066] (2) Recipient surgery: Anesthetize C57BL / 6 mice, fix them on the operating table, scrape off the hair on the side of the back of the animal where the graft will be implanted with a scalpel, disinfect with iodophor, and cut off a 1.5 cm × 1.5 cm square of skin with ophthalmic scissors as shallow as possible, and the defect size is slightly larger than the graft.

[0067] Place the graft on the edge of the incision in the direction reverse to the recipient's hair, avoiding folding along the edge. Place 4 sutures at the four corners and the middle of each edge. For each suture, pass the needle through the graft and then through the tissue under the surrounding recipient skin and suture and tie a knot. Let the animal partially recover from anesthesia, and then use a bandage. Cover the surface of the graft with a vaseline gauze, and wrap the recipient mouse with a bandage. Observe the mouse closely during the recovery process to ensure that the bandage does not restrict the offset of the chest and breathing. If the respiratory rate decreases or the animal starts to pant or breathe shallowly, remove the bandage.

[0068] After the operation, the mice are raised in an SPF-class animal room, and the activities of the mice are observed and recorded every day. The mice in the FK228 group are given FK228 every day. Anesthetize the mice on the seventh day after the operation, remove the back bandage of the mice, and then observe the growth and change of the skin in the transplantation area of the mice every day.

[0069] Observe the changes such as the curling of the edge and the purple and black color of the transplanted skin. If more than 90% of the transplanted skin area is involved, it is considered complete rejection, and record the time when the graft is completely rejected.

[0070] (3) Specimen collection: The recipient mice were anesthetized with sodium pentobarbital at different time points after the second operation (day 0, day 7, day 14). Peripheral blood of the recipient mice was collected by intracanthal venous blood sampling, 200 μl each time, and the supernatant was obtained by centrifugation. In addition, on the 10th day after the second operation, the mice were anesthetized with sodium pentobarbital and fixed. First, the grafts were cut off, and then peripheral blood and spleen specimens were obtained. The skin specimens were placed in 4% paraformaldehyde, and the spleen specimens were divided into two parts and stored at -80 °C for later use.

[0071] 2. Experimental verification process:

[0072] A model was established by using the method of allogeneic mouse tail-back heterotopic skin transplantation.

[0073] On day -14, the first presensitized skin transplantation surgery from BALB / C to C57BL / 6 mice was performed. On day 0, the second skin transplantation was performed on the same recipient mice.

[0074] The recipient mice were divided into three groups: C57BL / 6 - C57BL / 6 syngeneic group, BALB / c - C57BL / 6 allogeneic group, BALB / c - C57BL / 6 + FK228 treatment group;

[0075] FK228 was intraperitoneally injected at a dose of 0.5 mg / kg / d starting from day 0, with n = 6 in each group; the survival of the skin grafts was monitored daily after the surgery, and the survival time of the transplanted skin of the recipient mice was recorded. A necrotic area of the transplanted skin > 90% was defined as the loss of the skin graft.

[0076] On day 0, day 7, and day 14, peripheral blood specimens of the recipient mice were obtained, and the levels of donor-specific antibody (DSA)-IgG and IgM in the peripheral circulation of the mice were detected by flow cytometry-antibody binding assay.

[0077] On day 10, peripheral blood, spleen, and transplanted skin specimens of the recipient mice in each group were collected, and the transplanted skin tissues were stained with HE to evaluate their pathological changes.

[0078] The following methods were used to analyze the sample status before and after transplantation. Specifically:

[0079] ELISA technology was used to detect the cytokine levels in the serum;

[0080] Flow cytometry was used to detect the proportions of CD4+PD-1+CXCR5+Tfh and B220lowCD138+ plasma cells in the spleen tissue;

[0081] Total RNA was extracted from the spleen, and the mRNA levels of B cell-related Pax5, Bcl-6, and Bach2, the mRNA levels of plasma cell generation-related Blimp-1, Irf4, and Xbp1, as well as the transcriptional levels of Hdac1 and Hdac2 were detected using RT-PCR technology;

[0082] Total protein was extracted from the spleen, and the levels of acetylation of HDAC1, HDAC2, histone H2A, and H3 were detected using Western blot technology, as well as the protein expression levels of the IRE1α-XBP1 pathway in the endoplasmic reticulum stress UPR related to antibody synthesis modification and the expression levels of BLIMP-1 related to antibody secretion and AID related to CSR.

[0083] 3. Analysis of experimental results

[0084] Through analysis, the healing time of the graft and the recipient skin was 6-7 days after surgery, and the graft rejection process often occurred 10-14 days after surgery. After the first transplantation, specific antibodies against the donor mice were produced in the recipient mice. The DSA levels in healthy C57BL / 6 mice and recipient mice on the 14th day after the first transplantation were detected, and the results were as Figure 1 、 Figure 2 shown. The DSA level in healthy C57BL / 6 mice was not zero, and there were no antibodies against BALB / c in healthy mice, which we evaluated as being caused by non-specific binding of the flow antibody detection method. However, after pre-sensitized skin transplantation stimulation, the levels of DSA-IgM and DSA-IgG were significantly increased (P<0.001), indicating successful pre-sensitization.

[0085] A secondary skin transplantation model was established by pre-sensitizing with the first skin transplantation. Groups were divided under different intervention treatments and the survival period of the transplanted skin was observed. The results were as Figure 3 shown. After the first transplantation, the median survival time (MST) of the skin graft was 12 days. After the second skin transplantation, the transplanted skin of mice in the same genome grew well within 28 days after surgery, and no phenomena such as curling, turning purple or black occurred; the MSTs of the transplanted skin of mice in the heterologous genome and FK228 group were 11 and 25 days respectively, and the difference between groups was significant (P<0.0001), proving that intraperitoneal injection of 0.5 mg / kg / d of FK228 after surgery improved the survival of the graft.

[0086] In the allogeneic mouse skin transplantation model, antibody-mediated rejection plays an important role. To study the effect of FK228 on the production of DSA, peripheral blood was collected on postoperative days D0, D7, and D14 in this part of the experiment, and flow cytometry was used to detect the median fluorescent intensity (MFI) of DSA-IgM and DSA-IgG in the recipient serum. The experimental results are as Figure 4 , Figure 5 shown. After the second skin transplantation, DSA-IgG and IgM in the circulation of mice in each group reached a certain level (29900±2700 MFI). After the second transplantation, the level of DSA-IgG in mice of the same gene group increased, and the level of IgM did not change significantly, while the level of DSA-IgG in mice of the different gene group increased significantly. With the use of FK228, the levels of DSA-IgG in the FK228 group on days 7 and 14 were lower than those in the different gene group (P<0.01, P<0.01), and the levels of DSA-IgM in the FK228 group on days 7 and 14 were also lower than those in the different gene group (P<0.01, P<0.01). Therefore, the above experimental results indicate that FK228 can significantly reduce the level of DSA in pre-sensitized secondary skin transplantation mice.

[0087] In view of the above experimental results that FK228 can significantly reduce the production of DSA in the pre-sensitized allogeneic skin transplantation model, we further detected the effect of FK228 on HDAC enzymes. By obtaining the spleens of recipient mice in each group, extracting total RNA for reverse transcription amplification, and then detecting the relative expression differences of Hdac1 and Hdac2 genes in each group by Real-Time PCR. The results of the RT-PCR experiment are as Figures 6 - 7 shown. Compared with the same gene group, the mRNA transcription levels of Hdac1 and Hdac2 in the spleen of the different gene group were significantly increased (P<0.05), which was statistically significant; however, after the intervention of FK228, the relative expression levels of Hdac1 and Hdac2 mRNA were significantly decreased compared with the different gene group (P<0.05), and the difference was statistically significant.

[0088] To explore and verify the specific histone acetylation sites regulated by FK228 in the transplantation model, we extracted nuclear proteins from the spleens of recipient mice and detected the levels of acetylated histones H2A and H3 by western-blot technology. The results are as Figures 8 - 9 and Figures 10 - 12 shown. The expression levels of acetylated histones H2A (Lys4 and Lys5) and H3 (Lys9, Lys27, and Lys56) in the FK228 group were significantly higher than those in the different gene group (P<0.05), indicating that FK228 up-regulated the acetylation levels of histones H2A and H3.

[0089] The spleen is a key lymphoid organ for the differentiation and induction of humoral immunity mediated by B lymphocytes. To explore the effects of FK228 on mouse immune cells, we first isolated splenocytes from mice with secondary skin transplantation, and then performed flow cytometry. The experimental results are shown in Figures 13 - 16 Figure Figures 13 - 16 . Compared with the allogeneic group, the proportions of CD4+PD-1+CXCR5+Tfh and B220lowCD138+ plasma cells in the FK228 group were significantly decreased (P<0.05), indicating that FK228 downregulated the proportion of Tfh cells and inhibited the differentiation of plasma cells.

[0090] To study the levels of Th1, Th2 and Tfh-related cytokines in the serum of pre-sensitized skin transplantation mice treated with FK228, the serum of mice with secondary skin transplantation on the 10th day after surgery was collected, and the cytokine levels were detected by ELISA. The experimental results are as follows Figures 17 - 21 Figure Figures 17 - 21 . There were no significant differences in the levels of IL-2, IFN-r, IL-4 and IL-10 between the FK228 group and the allogeneic group (P>0.05), indicating that FK228 may not affect the Th1 and Th2-related differentiation regulation during the rejection reaction. Notably, compared with the allogeneic group, the level of IL-21 in the FK228 group was significantly decreased (P<0.05), which indirectly confirmed the inhibitory effect of FK228 on Tfh cells and plasma cells mentioned above.

[0091] Furthermore, total RNA was extracted from the spleens of recipient mice, and then the relative expression levels of the key genes for maintaining B cells, Pax5, Bcl-6, Bach2, and the key genes for plasma cell generation, Pridm-1, Irf4, Xbp1, were detected by reverse transcription amplification and Real-Time PCR. As shown in Figures 22 - 27 Figure Figures 22 - 27 .

[0092] The results of RT-PCR showed that there were no significant differences in the transcriptional levels of Pax5, Bcl-6 and Bach2 between the allogeneic group and the FK228 group (P>0.05). FK228 downregulated the mRNA expression levels of Pridm-1 and Xbp1 (P<0.05), indicating that FK228 inhibited the transcription of Pridm-1 and Xbp1 and hindered the regulation of the transition from B cell transcription to plasma cell transcription. In addition, FK228 inhibited the transcription of Aicda, which also inhibited somatic hypermutation and antibody class switch.

[0093] Analyze the hypertrophy of the endoplasmic reticulum (ER) and Golgi apparatus during the antibody production process to meet the requirements of efficient protein synthesis and secretion in antibody-secreting cells (ASCs). The main change in ASC metabolism lies in adapting to the stress of the unfolded protein response (UPR) pathway to play a greater physiological role. The IRE1α-XBP1 branch plays a key role in the ER hypertrophy of plasma cells. To further verify the inhibitory effect of FK228 on antibody synthesis and modification at the protein level, total proteins from the spleen were extracted, and Western blot was used to detect the effects of FK228 on the expression levels of activation-induced cytidine deaminase (AID) and B lymphocyte-induced maturation protein-1 (BLIMP-1), as well as the activation status of the IRE1α-XBP1 pathway. The experimental results are as Figures 28 - 29 and Figures 30 - 33 shown. The figure shows results consistent with the above PCR experiments. Compared with the same genome, the expression of histone deacetylase 1 (HDAC1) and histone deacetylase 2 (HDAC2) was significantly increased in the heterogeneous genome. It can be seen that FK228 significantly inhibited the expression of HDAC1 and HDAC2 (P < 0.05). The expression levels of AID and BLIMP-1 were significantly increased in the heterogeneous genome, indicating that FK228 can intervene and downregulate their expression.

[0094] In the IRE1α-XBP1 axis, the expression level of XBP1 and the phosphorylation level of IRE1α were significantly increased in the heterogeneous genome (P < 0.05). KF228 reduced the expression of XBP1 and the phosphorylation level of IRE1α to a certain extent (P < 0.05).

[0095] Based on the above process, it can be seen that FK228 inhibited the differentiation of B cells into plasma cells in pre-sensitized mice, reduced the level of donor-specific antibodies (DSAs) in mice, and significantly improved the survival of grafts in pre-sensitized skin transplantation mice. FK228 directly inhibited the activities of HDAC1 and HDAC2, upregulated the levels of acetylated histone H2A and H3 at corresponding sites, and downregulated the expression of key transcription factors related to plasma cells, Prdm-1 and Xbp1. In addition, FK228 decreased the proportion of follicular helper T (Tfh) cells in the spleen, inhibited the production of interleukin-21 (IL-21), and weakened the role of assisting antibody production. FK228 reduced antibody synthesis and modification by inhibiting the activity of IRE1α-XBP1 in the endoplasmic reticulum, simultaneously reduced antibody production by inhibiting BLIMP-1, and intervened in the expression of AID, thus affecting the subsequent class switch recombination (CSR) process of antibodies. Similarly, during the in vitro culture of B cells, FK228 inhibited the differentiation of B cells into plasma cells and reduced the production of IgM and IgG1.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for the application of romidepsin in drugs for pre-sensitized rejection reaction, characterized in that, For inhibiting the expression of HDAC1 and HDAC2, including reducing the expression levels of DSA-IgG and DSA-IgM.

2. The application method of romidepsin in the drug for presensitized rejection reaction according to claim 1, wherein For upregulating the acetylation levels of histone H2A and H3 at different sites.

3. The application method of romidepsin in the drug for pre-sensitized rejection reaction according to claim 1, characterized in that, For intervening in and downregulating the expression of BLIMP-1 and AID.

4. The application method of romidepsin in the drug for pre-sensitized rejection reaction according to claim 1, wherein For downregulating the proportion of Tfh cells and inhibiting the differentiation of plasma cells.

5. The method for the use of romidepsin in a drug for pre-sensitized rejection reaction according to claim 1, characterized in that, For inhibiting the relative mRNA expression levels of Hdac1 and Hdac2.

6. The application method of romidepsin in drugs for pre-sensitized rejection reaction according to claim 1, wherein For inhibiting the transcription of Pridm-1 and Xbp1, transplanting the transcriptional transformation of B cells to plasma cells; inhibiting the transcription of Aicda, and inhibiting somatic hypermutation and antibody class switch.

7. The method for the use of romidepsin in a drug for pre-sensitized rejection reaction according to claim 6, characterized in that, For inhibiting the activity of endoplasmic reticulum IRE1α-XBP1, inhibiting BLIMP-1, and reducing antibody synthesis and modification.

8. The application method of romidepsin in the drug for pre-sensitized rejection reaction according to any one of claims 1 to 6, characterized in that, The graft in the pre-sensitized rejection is skin.

9. The method for the use of romidepsin in a drug for pre-sensitized rejection reaction according to any one of claims 1 to 6, characterized in that, The administration method is intraperitoneal injection.

10. The application method of romidepsin in the drug for pre-sensitized rejection reaction according to any one of claims 1 to 6, characterized in that, The dosage of romidepsin is 500 μg / kg, administered once a day.

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