Application of palmitoylation inhibitor 2-BP in preparation of drugs for spontaneous abortion

Abstract

The invention relates to application of a palmitoylation inhibitor in preparation of a medicine for treating spontaneous abortion. The palmitoylation inhibitor is 2-BP (2-Butyl Phosphate). The spontaneous abortion is recurrent abortion caused by unknown reasons. The 2-BP is used for promoting the dCD4 < + > T cells to secrete Th2 and Treg type cell factors. The 2-BP reduces embryo loss and promotes the tolerance phenotype of dCD4 + T cells, facilitating maternal-fetal immune tolerance. The invention provides an application of 2-BP in a medicine for treating spontaneous abortion. In particular, in-vitro experiments and animal experiments prove that 2-BP promotes Th2-type bias and Treg amplification of dCD4 + T cells, reduces embryo absorption and improves bad pregnancy outcome.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to the medicinal use of palmitoylation inhibitor 2-bromopalmitic acid (2-BP) (targeting DHHC protein palmitoyltransferase), specifically to the use of 2-BP for preparing a drug for treating spontaneous abortion. Background Art

[0002] The prior art discloses that recurrent spontaneous abortion (RSA) or recurrent pregnancy loss (RPL) refers to the pregnancy loss that occurs 2 or more times before 28 weeks of gestation, including biochemical pregnancy. The incidence of early pregnancy failure is as high as 9-50%, and its recurrence risk increases with the increase in the number of abortions. RSA not only brings a heavy economic burden to patients and their families, but also seriously damages the physical and mental health of patients. Due to the complexity of the causes of RSA and the high individual heterogeneity among patients, it is particularly urgent to find a diagnosis and treatment method for RSA in the current situation of low fertility and increasing aging.

[0003] Unexplained recurrent spontaneous abortion (URSA) refers to the pregnancy loss that occurs 2 or more times before 28 weeks of gestation in women, and genetic, endocrine, autoimmune factors, thrombophilia, anatomical abnormalities of the reproductive tract, acute infections and other factors have been excluded.

[0004] Normal physiological pregnancy is actually a kind of allogeneic semi-allograft. The embryo, as an allograft, is not rejected by the mother, which is the only exception to the immunological principle, reflecting the maternal immune tolerance to embryonic antigens; while pregnancy failure is mostly due to the maternal immune rejection of embryonic antigens. Decidual immune cells (DICs) are the basis of maternal-fetal immune tolerance. In the maternal-fetal immune tolerance mechanism, the function of CD4 helper T cells (Th) is crucial. The helper T cell type 2 (Th2) bias and regulatory T cell (Treg) expansion of dCD4 + T cells are the key to pregnancy maintenance. Palmitic acid is modified to the sulfhydryl group of protein cysteine through a thioester bond, which is called palmitoylation, and its process is regulated by palmitoyltransferases (ZDHHC1-9, 11-24). Palmitoylation modification is the most common and the only reversible post-translational modification in lipid modification, which can affect protein trafficking, stability and protein-protein interactions, etc., and then regulate cell signal transduction, apoptosis, metabolism, differentiation, etc., and plays an important role in the occurrence and development of tumors, inflammation, autoimmune diseases, etc. However, there are few reports on the impact of palmitoylation modification on early pregnancy outcomes. Whether palmitoylation modification regulates the function of decidual T cells and thus affects pregnancy outcomes has not been studied yet.

[0005] However, there are no reports on the use of palmitoylation inhibitor 2-BP in the preparation of drugs for the treatment of spontaneous abortion. Summary of the invention

[0006] The purpose of the present invention is to provide an application of a palmitoylation inhibitor 2-BP in the preparation of a drug for treating spontaneous abortion in view of the deficiencies in the prior art.

[0007] In a first aspect, the present invention provides the use of a palmitoylation inhibitor in the preparation of a drug for treating spontaneous abortion.

[0008] As a preferred example, the palmitoylation inhibitor is 2-BP.

[0009] As another preferred embodiment, the spontaneous abortion is unexplained recurrent spontaneous abortion.

[0010] As another preferred example, the 2-BP promotes dCD4 + T cells secrete Th2 and Treg type cytokines.

[0011] As another preferred example, the 2-BP reduces embryonic loss and promotes dCD4 + The tolerant phenotype of T cells is conducive to maternal-fetal immune tolerance.

[0012] In this study, we found through palmitoylation sequencing that the palmitoylation level of DICs in URSA patients was significantly higher than that in normal early pregnant women; and palmitoylated protein testing suggested that dCD4 + The palmitoylation protein level of T cells was also significantly higher than that of the normal pregnancy group. The palmitoylation agonist ML348 (an inhibitor of acyl protein thioesterase 1 (APT1)) reduced the expression of human dCD4 + The secretion of Th2 and Treg cytokines by T cells; 2-BP can reverse the effect of ML348, and its use alone promotes the secretion of dCD4 + T cell tolerance phenotype. In vivo experiments also confirmed that 2-BP can improve the adverse pregnancy outcomes of SA mice.

[0013] The inventors of the present application evaluated the potential value of the palmitoylation inhibitor 2-BP in the treatment of spontaneous abortion through experimental studies, provided the use of 2-BP in the preparation of drugs for the treatment of spontaneous abortion, and provided new methods and new ideas for the clinical treatment of URSA.

[0014] The present invention is achieved by the following technical scheme: DICs of normal early pregnant women and URSA patients were collected for palmitoylation group sequencing. The results showed that compared with normal early pregnant women, 3262 proteins in DICs of URSA patients had increased palmitoylation levels and 1577 proteins had decreased palmitoylation levels (such asFigure 1 As shown in Figure A. Detection of palmitoylated proteins indicated that the level of palmitoylated proteins in dCD4 + T cells of URSA patients was significantly higher than that of women with normal pregnancy (as Figure 1 shown in Figure B). Treatment with ML348 inhibited the expression of Th2-type cytokines (IL-4, IL-13) and Treg-type cytokines (IL-10, TGF-β1) in dCD4 + T cells; 2-BP could reverse the effect of ML348, and its treatment alone could significantly promote the expression of Th2 and Treg-type cytokines in dCD4 + T cells (as Figure 2 shown). Detection of palmitoylated proteins indicated that the level of palmitoylated proteins in dCD4 + T cells of SA mice was significantly higher than that of normal pregnancy mice (normal pregnancy mouse model, NP) (as Figure 3 shown in Figure A). Further, the present invention constructed a SA mouse model treated with 2-BP, that is, SA mice were intraperitoneally injected with 2-BP three times a week. The results showed that the embryo resorption rate of the mice in the 2-BP treatment group was significantly decreased, and at the same time, dCD4 + T cells produced more Th2 and Treg-type cytokines beneficial to pregnancy (as Figure 3 shown in Figures B and 3C).

[0015] The advantages of the present invention are as follows: The present invention provides the use of 2-BP as a drug for treating spontaneous abortion; in particular, it is confirmed by in vitro experiments and animal experiments that 2-BP promotes Th2 bias and Treg expansion of dCD4 + T cells, reduces embryo resorption, and improves adverse pregnancy outcomes. Brief Description of the Drawings

[0016] Figure 1 Palmitoylation modification enhancement is closely related to early pregnancy failure

[0017] Among them, HNP: human normal pregnancy, normal early pregnancy group,

[0018] URSA: unexplained recurrent spontaneous abortion, URSA, unexplained recurrent spontaneous abortion group.

[0019] ****p<0.0001.

[0020] Figure 2 The palmitoylation inhibitor 2-BP can promote the tolerogenic phenotype of dCD4 + T cells

[0021] Among them, Ctrl: control, control group,

[0022] ML348: palmitoylation agonist,

[0023] 2-BP: palmitoylation inhibitor,

[0024] *p < 0.05, **p < 0.01, ****p < 0.0001, compared with the control group;

[0025] ### p < 0.001, #### p < 0.0001, compared with the ML348 group.

[0026] Figure 3 2-BP improves the pregnancy outcome of SA mice

[0027] Among them, NP: normal pregnancy mouse model, normal pregnancy model group

[0028] SA: spontaneous-abortion-prone mouse model, spontaneous abortion model group

[0029] SA + 2BP: spontaneous abortion model group treated with palmitoylation inhibitor 2-BP.

[0030] Embryo absorption rate: embryo absorption rate, calculated as follows: number of surviving embryos / (number of surviving embryos + bleeding absorption points) × 100%.

[0031] *p < 0.05, **p < 0.01, ***p < 0.001. Specific implementation manners

[0032] The present invention will be further described below in conjunction with specific implementation manners. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0033] Example 1 Palmitoylation modification is closely related to early pregnancy failure

[0034] Materials

[0035] 1. Human decidual tissue and cells:

[0036] All clinical samples collected in this study were approved by the Ethics Committee of Obstetrics and Gynecology Hospital of Fudan University and informed consent was obtained from the patients themselves. The decidual tissue specimens of HNP were obtained from pregnant women with normal early pregnancy (7 ～ 9 weeks) who underwent induced abortion in the day surgery unit of Obstetrics and Gynecology Hospital of Fudan University from 2023 to 2024. Inclusion criteria: The age of the pregnant women was 20 ～ 40 years old, terminated pregnancy for non-medical reasons, had no history of adverse pregnancy outcomes such as spontaneous abortion, and had no vaginal bleeding, abdominal pain or other discomfort during this pregnancy. B-ultrasound showed intrauterine pregnancy, with germ, primitive cardiovascular pulsation (+), no intrauterine fluid accumulation, and no infection in preoperative examinations. The decidual tissue specimens of URSA patients were obtained from patients who underwent curettage due to fetal arrest or poor fetal development in early pregnancy. Inclusion criteria: The gestational age was calculated according to the last menstrual period as 7 ～ 12 weeks, and no primitive cardiac tube pulsation was seen in two vaginal B-ultrasound examinations with an interval of one week. Abortions caused by endocrine factors, genital malformations, genetic abnormalities, autoimmune diseases, infections and traumas were excluded. Neither of the above two groups had a history of allergies or a family history of allergies. The tissues were stored in ice-precooled DMEM / F12 serum-free medium, and primary cell isolation and extraction were performed as soon as possible after sampling.

[0037] 2. Reagents for isolation, purification and culture of human primary cells:

[0038] DMEM / F12 medium, RPMI 1640 medium (Hyclone), fetal bovine serum (Viva Cell), penicillin-streptomycin-amphotericin B (100×, hereinafter referred to as triple antibiotics) (Xinsaimi), Antimicrobial agent for primary cells (Invivogen), phosphate buffer solution (PBS) (1×, 10×) (Saiweier), Percoll density gradient centrifugation medium (Cytiva), type IV collagenase (Gibco), universal nuclease (Yeasen).

[0039] 3. Palmitoylated Protein Assay Kit (Red) (abcam)

[0041] 4. Flow cytometry antibody: Brilliant Violet 510-conjugated anti-human CD4 (Biolegend).

[0043] 5. Main consumables and equipment:

[0044] 15 mL / 50 mL centrifuge tubes, T25 / T75 cell culture flasks, 96-well cell culture plates (Saiweier), 3 mL extended Pasteur pipettes (Shanghai Suhai, biosharp), 5 mL Pasteur pipettes (biosharp), 1 mL / 200 μL / 10 μL pipette tips (Axygen), 1.5 mL / 2 mL microcentrifuge tubes (EP tubes) (Axygen), 1000 μL, 200 μL, 100 μL, 20 μL, 10 μL, 2.5 μL micropipettes (Eppendorf), ophthalmic scissors, ophthalmic forceps, 100-mesh / 200-mesh / 400-mesh stainless steel cell sieves (Nanjing Dongmai Technology), glass test tubes, small glass bottles. Carbon dioxide cell incubator (HealForce), laminar flow hood (Shanghai Jinghua Instrument and Equipment Factory), constant temperature shaker (Shanghai Zhicheng), low-temperature centrifuge (Eppendorf), low-speed large-capacity centrifuge (Xiangyi), flow cytometer (Beckman / CytoFLEX), ice maker (Sanyo), tray balance (Shanghai Precision Instrument and Meter Co., Ltd.), autoclave (SANYO), -20 °C / 4 °C refrigerator (Haier), -80 °C refrigerator (Thermo Fisher Scientific)

[0045] Method

[0046] 1. Isolation, purification and culture of human DICs

[0047] 1) Human decidual tissues obtained clinically were washed thoroughly with PBS. After removing visible blood clots, they were transferred to small glass bottles. The tissues were minced thoroughly with ophthalmic scissors and transferred to 50 mL centrifuge tubes using a 3 mL Pasteur pipette;

[0048] 2) Each tube was labeled with the tissue amount, and the total digestion system was estimated accordingly. The volume of type IV collagenase added to each tube was 20% of the total system, and the remaining volume was made up with PBS. Incubated in a constant temperature shaker at 37 °C at 140 rpm for about 35 ～ 45 minutes;

[0049] 3) Add PBS to terminate digestion. The liquid in the tube and the remaining tissues were passed through 100-mesh, 200-mesh and 400-mesh stainless steel cell sieves in sequence. The filtered cell suspension was transferred to a new 50 mL centrifuge tube, centrifuged at 1500 rpm for 7 minutes, the supernatant was discarded, and the cell pellet was resuspended thoroughly with an appropriate volume of DMEM / F12 serum-free medium according to the cell pellet amount;

[0050] 4) Use a 3 mL extended Pasteur pipette to aspirate the cell suspension and slowly add it dropwise along the wall of the glass tube pre-coated with Percoll, so that about 2 mL of cell suspension is laid on the upper layer of the pre-coated Percoll solution in each glass tube (laying Percoll: First, prepare a Percoll working solution (100%) by mixing the Percoll stock solution and 10×PBS at a volume ratio of 9:1. Then, dilute the Percoll working solution with 1×PBS to 20%, 40%, and 60% Percoll working solutions. Slowly lay 2 mL of 60%, 40%, and 20% Percoll working solutions in the glass tube in sequence. After standing, layering of each density of Percoll working solution can be seen);

[0051] 5) Centrifuge at 2500 rpm for 30 minutes at room temperature using a low-speed large-capacity centrifuge. After centrifugation, cell layering can be seen. The cell layer between the 40% and 60% Percoll working solutions is mainly DICs. Aspirate the cells in this layer into a 50 mL centrifuge tube, add sufficient PBS and shake, then centrifuge at 1500 rpm for 7 minutes, and discard the supernatant;

[0052] 6) Resuspend the cell pellet with RPMI 1640 complete medium containing 10% fetal bovine serum and triple antibodies, seed into a flask, and culture in an incubator. After the stromal cells and others adhere to the wall, take the supernatant, which is DICs.

[0053] 2. Palmitoylation omics sequencing

[0054] 1) After trypsin digestion of the DICs samples from HNP and URSA patients, desalt the peptides using a C18 spin column (Thermo Scientific). After drying, resuspend the peptides in 200 μL of loading buffer and mix with 100 μL of high-capacity streptavidin beads;

[0055] 2) Bind the eluted peptides with 50 mM iodoacetamide to block the reduced cysteine residues (which are potential palmitoylation sites). Subsequently, further desalt the peptides using C18 Stage Tips and perform LC-MS / MS analysis.

[0056] 3. Palmitoylated protein assay

[0057] 1) Seed the DICs of HNP and URSA patients in a 96-well cell culture plate, with a system of 100 μL of RPMI 1640 complete medium containing 1×Palmitic Acid Label in each well. At the same time, set up negative control cells (cells not exposed to Palmitic Acid Label and Fluorescent Azide I / Fluorescent Azide) and background control cells (cells not exposed to Palmitic Acid Label and only exposed to Fluorescent Azide I / Fluorescent Azide), and culture them at 37°C in 5% CO2 for 24 hours.

[0058] 2) Transfer the DICs to a 1.5 mL EP tube, centrifuge at 500×g for 5 minutes, discard the supernatant, and then wash with PBS 1 ～ 2 times.

[0059] 3) Fixation and permeabilization: Resuspend the cells in 100 μL of Fixative Solution I / Fixative Solution and incubate in the dark at room temperature for 15 minutes, then centrifuge at 500×g for 5 minutes and discard the supernatant. Wash the cells once with 100 μL of 1×wash Buffer IV / wash Buffer, centrifuge at 500×g for 5 minutes and discard the supernatant. Resuspend the cells in 100 μL of 1×Permeabilization Buffer and incubate at room temperature for 10 minutes, centrifuge at 500×g for 5 minutes and discard the supernatant, then add 20 μL of Permeabilization Buffer.

[0060] 4) Palmitic Acid reaction: Operate in the dark at room temperature. Within 15 minutes, sequentially add the following reagents to each sample:

[0061]

[0062] Add 100 μL of PBS to the negative control, and add 100 μL of 1×Reaction Cocktail (prepared as shown in the above table) to each sample of background control cells and positive control cells, and incubate in the dark at room temperature for 30 minutes.

[0063] 5) Discard the Reaction Cocktail and wash the cells 3 times with 100 μL of 1×wash Buffer IV / wash Buffer.

[0064] 6) Resuspend the DICs in approximately 100 μL of PBS, add anti-human CD4 flow antibody, stain in the dark at room temperature for 30 minutes, then wash with PBS, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant.

[0065] 7) Detect and analyze dCD4 + The palmitoylated protein level of T cells by flow cytometry.

[0066] Results

[0067] 1. The results of palmitoylation proteomics showed that there were significant differences in the palmitoylated protein levels of DICs between normal early pregnant women and URSA patients, and the palmitoylated protein level of DICs in URSA patients was higher (as Figure 1 shown in A).

[0068] 2. Detection of palmitoylated protein and flow cytometry indicated that the palmitoylated protein level of dCD4 + T cells in URSA patients was significantly higher than that in normal early pregnant women (as Figure 1 shown in B).

[0069] Example 2

[0070] Explore the functional regulation of palmitoylation agonist ML348 and inhibitor 2-BP on dCD4 + T cells

[0071] Materials

[0072] 1. Human decidual tissues and cells (same as Example 1).

[0073] 2. Reagents for isolation, purification and culture of human primary cells (same as Example 1): additionally add Cell Activation Cocktail (with Brefeldin A) (Biolegend).

[0074] 3. Palmitoylation regulators: 2-BP (21604, Sigma-Aldrich), ML348 (MCE, HY-100736), dimethyl sulfoxide (DMSO) (Sigma).

[0075] 4. Flow cytometry reagents: PE / CY7-conjugated anti-human IL-10, TGF-β1; PerCP / Cy5.5-conjugated anti-human IL-4; Alexa Fluor 647-conjugated anti-human IL-13; Brilliant Violet 421-conjugated anti-human IL-4; Brilliant Violet 510-conjugated anti-human CD4; Fix / Perm kit (Biolegend).

[0076] 5. Main consumables and instruments (same as in Example 1).

[0077] Method

[0078] 1. Treatment with DICs drugs:

[0079] Collect DICs from normal early pregnant women (n = 5) and divide them into 4 groups, which are treated with DMSO, 10 μM ML348, 50 μM 2-BP, and ML348 + 2-BP respectively for 48 hours. Among them, each group is activated with anti-CD3 and anti-CD28 antibodies, and Cell Activation Cocktail (with Brefeldin A) is added 4 hours before harvesting the cells.

[0080] 2. Detection of dCD4 + Cytokine levels of T cells

[0081] 1) Collect the treated DICs in 1.5 mL EP tubes, centrifuge at 1500 rpm for 5 minutes to discard the culture medium, and then wash with PBS 1 ～ 2 times. Make necessary groupings according to the fluorescence channels corresponding to the stained indicators (CD4, IL-4, IL-13, IL-10, TGF-β1), and leave single-label tubes for each fluorescence channel for compensation adjustment and blank tubes for voltage adjustment;

[0082] 2) Resuspend the washed DICs in about 100 μL of PBS, add surface antibodies according to the flow antibody instruction manual, stain at room temperature in the dark for 30 minutes, then wash with PBS, and centrifuge at 1500 rpm for 5 minutes to discard the supernatant;

[0083] 3) Add 100 μL of Fix Perm buffer to each tube, fix and permeabilize at room temperature in the dark for 30 minutes; then add 1 mL of 1×Perm Wash buffer to each tube, centrifuge at 350×g for 5 minutes to discard the supernatant;

[0084] 4) Resuspend the DICs in approximately 100 μL of 1× Perm Wash buffer, add antibodies according to the flow cytometry antibody usage instructions, stain in the dark at room temperature for 30 minutes, then wash with 1× Perm Wash buffer, centrifuge at 350×g for 5 minutes, discard the supernatant, resuspend the DICs in PBS, and detect by flow cytometry.

[0085] Results

[0086] Compared with the control group (DMSO), ML348 treatment inhibited the expression of Th2-type (IL-4, IL-13) and Treg-type cytokines (IL-10, TGF-β1) in dCD4 + T cells; the combined treatment with 2-BP could reverse the effect of ML348, and its single treatment could significantly promote the expression of the above cytokines in dCD4 + T cells (as Figure 2 shown).

[0087] Animal experiment 3 2-BP improves the poor pregnancy outcome in SA mice

[0088] Materials

[0089] 1. Experimental animals:

[0090] CBA / J female mice (SPF grade, 8 weeks old) were purchased from Beijing Huafukang Bioscience Co., Ltd., DBA / 2 male mice (SPF grade, 8 weeks old) and BALB / c male mice (SPF grade, 8 weeks old) were purchased from Shanghai Slack Experimental Animal Co., Ltd. All the above mice were housed in the Experimental Animal Center of Obstetrics and Gynecology Hospital of Fudan University.

[0091] 2. Mouse primary experimental reagents:

[0092] RPMI 1640 medium, fetal bovine serum, triple antibody, PBS, type IV collagenase, universal nuclease, 2-BP, DMSO, PEG300 (153757, TargetMol), Tween-80 (162666, TargetMol), physiological saline, Ultra-LEAF TM Purified anti-mouse CD3ε Antibody (100359, Biolegend, USA), Ultra-LEAF TM Purified anti-mouse CD28 Antibody (102116, Biolegend, USA), Cell Activation Cocktail (with Brefeldin A).

[0093] 3. Palmitoylated Protein Detection Kit (same as Example 1).

[0094] 4. Main Consumables and Equipment (same as Example 1): Additionally, a 1 mL syringe with a needle, a 5 mL syringe, a cell sieve (Corning), and sterile gauze are added.

[0095] Method

[0096] 1. Construction of pregnant mouse models:

[0097] CBA / J female mice at 8 weeks of age and BALB / c male mice at 8 weeks of age were caged together at a ratio of 2:1 - 3:1 to construct the NP model; CBA / J female mice at 8 weeks of age and DBA / 2 male mice at 8 weeks of age were caged together at a ratio of 2:1 - 3:1 to construct the SA model.

[0098] 2. Construction of 2 - BP - treated SA mouse models:

[0099] Prepare the 2 - BP drug solution for intraperitoneal injection: Weigh 0.032 g of 2 - BP powder and dissolve it in 2 mL of DMSO. Then, add 8 mL of PEG30, 1 mL of Tween - 80, and 9 mL of normal saline in sequence. After mixing, a clear and transparent liquid is obtained. Each mouse is injected at a dose of 0.2 mL / 10 g. Additionally, prepare a control drug solution: Take 2 mL of DMSO, add 8 mL of PEG30, 1 mL of Tween - 80, and 9 mL of normal saline in sequence, and store it in a 4°C refrigerator. Ensure that the solution is clear and transparent. The experimental group is injected with 2 - BP intraperitoneally three times a week, and the control group is injected with the control drug solution intraperitoneally three times a week.

[0100] 3. Primary experiments on mice and isolation, purification, and culture of mouse DICs:

[0101] 1) All the above - constructed pregnant mice were deeply anesthetized at 13.5 days of pregnancy and then sacrificed by cervical dislocation. The mice were dissected in a laminar flow hood, and their uteri were separated and placed in PBS.

[0102] 2) Remove the adipose and other connective tissues attached to the mouse uterus, retain the bilateral ovaries, place the uterus on a sterile gauze pad, and take a photo beside a scale for recording.

[0103] 3) Dissect out the embryo - placenta unit and place it in a 24 - well plate pre - filled with PBS. In particular, try to separate the decidual tissue on the placental attachment surface as much as possible. Transfer the mouse decidual tissue to a 2 mL EP tube, add a few drops of PBS, and finely cut the tissue with ophthalmic scissors. Then, add 1 mL of type IV collagenase and make up to 2 mL with PBS. Digest in a thermostatic shaker at 37°C and 140 rpm for 40 ～ 45 minutes;

[0104] 4) The digested mouse decidual tissue was passed through a cell sieve, and the tissue was ground with the plunger of a 5 mL syringe to accelerate sieving. The decidual cell suspension was collected in a 50 mL centrifuge tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 complete medium, seeded into a flask or plate, and anti-mouse CD3 (5 μg / mL) and anti-mouse CD28 (1 μg / mL) were added according to the experimental needs. The cells were cultured in a 37 °C 5% CO2 cell incubator to allow the stromal cells to adhere fully. Four hours before harvesting the cells for flow cytometry staining, Cell Activation Cocktail (with Brefeldin A) was added to the culture medium;

[0105] 5) Carefully separate the embryo and placenta, arrange them neatly according to the scale and take a photo for record.

[0106] 4. Detection of palmitoylated proteins (same as Example 1)

[0107] Results

[0108] 1. Detection of palmitoylated proteins and flow cytometry results showed that the level of palmitoylated proteins in dCD4 + T cells of SA mice was significantly higher than that of NP mice (as Figure 3 shown in A).

[0109] 2. After intraperitoneal injection of 2-BP to SA model mice, the embryo resorption rate decreased, and dCD4 + T cells secreted more Th2 and Treg-type cytokines (as Figure 3 shown in B and 3C).

[0110] The in vitro and in vivo experimental results of the present invention confirmed that 2-BP can promote the tolerance phenotype of dCD4 + T cells and significantly improve the pregnancy outcome of SA mice.

[0111] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Application of palmitoylation inhibitors in the preparation of drugs for the treatment of spontaneous abortion.

2. The use according to claim 1, characterized in that: The palmitoylation inhibitor is 2-BP.

3. The use according to claim 1, characterized in that: The spontaneous abortion is unexplained recurrent spontaneous abortion.

4. The use according to claim 2, characterized in that: The 2-BP promotes dCD4 + T cells secrete Th2 and Treg type cytokines.

5. The use according to claim 2, characterized in that: The 2-BP reduces embryonic loss and promotes dCD4 + The tolerant phenotype of T cells is conducive to maternal-fetal immune tolerance.

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