Use of beta cell protein in the preparation of a medicine for preventing miscarriage

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of beta cell protein in preparation of medicines for preventing and treating abortion. The application finds and proves for the first time that the application of beta cell protein (BTC) promotes invasion and tube formation of the extravillous trophoblast cell line-HTR8 cells, BTC has obvious rescue effect on embryo resorption in a mouse model of early-mid pregnancy abortion, and BTC increases placental weight and improves placental development when injected into the mouse model of early-mid pregnancy abortion, thereby indicating that BTC can have certain therapeutic effect on abortion, and thus has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of β-cell protein in the preparation of drugs for preventing and treating miscarriage. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Miscarriage refers to the termination of pregnancy before 28 weeks of gestation and with a fetus weighing less than 1000g. Termination before 12 weeks of gestation is considered early miscarriage; termination between 12 and 28 weeks is considered late miscarriage. The causes are complex, with common causes including chromosomal factors, maternal factors (including abnormalities in reproductive anatomy, hormonal imbalances due to endocrine disorders, autoimmune diseases, and coagulation abnormalities), and environmental exposure, unhealthy lifestyle habits, medications, and infections can all be contributing factors. After excluding all the above causes, 10%–20% of miscarriages are of unknown cause, which may be related to abnormal embryo implantation microenvironment and epigenetic changes. The core issue of miscarriage is "poor embryo quality" or "the mother's inability to accommodate the embryo." Trophoblastic cells are the core cells for "embryo implantation and placental formation," and their normal function is a prerequisite for maintaining pregnancy. Any factor (chromosomal, immune, endocrine, infection, etc.) that leads to abnormal proliferation, invasion, differentiation, and function of trophoblastic cells directly affects the stability of embryo implantation and the quality of placental formation, resulting in embryo implantation failure or miscarriage. Therefore, trophoblastic cell function is an important direction for the investigation of the causes of miscarriage and for treatment intervention.

[0004] Although miscarriage is currently believed to be related to the function of trophoblast cells during placental development, its specific pathogenesis remains unclear. Clinically, aside from some diagnostic methods and treatments targeting the underlying cause, there is a lack of effective therapeutic drugs targeting trophoblast cell function. Therefore, to better facilitate clinical intervention, it is necessary to thoroughly elucidate the pathogenesis of miscarriage and screen and identify effective therapeutic targets. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, the inventors, through long-term technical and practical exploration, have provided the application of β-cell protein in the preparation of drugs for preventing and treating miscarriage. This invention, through research, has for the first time discovered and confirmed that the application of β-cell protein (BTC) promotes the invasion and tube formation of the chorionic villus extratrophoblastic cell line HTR8. BTC has a significant rescue effect on embryo resorption in mouse models of early to mid-pregnancy miscarriage. Intraperitoneal injection of BTC into mouse models of miscarriage in early to mid-pregnancy increases placental weight and improves placental development, thus indicating that BTC can have a certain therapeutic effect on miscarriage. Based on the above research results, this invention has been completed.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the invention provides the use of β-cell protein in the preparation of a medicament for preventing and treating miscarriage. Therefore, β-cell protein is effective in preventing and / or treating miscarriage-related conditions.

[0008] The prevention and treatment of miscarriage includes one or more of the following functions: (a) Promotes trophoblast cell invasion; (b) Promotes the acquisition of endothelial properties in trophoblast cells; (c) Improve pregnancy outcomes mediated by miscarriage.

[0009] The product can be a drug or a general experimental reagent for non-medical purposes. The general experimental reagent is used for basic research and can be used to construct relevant cell or animal models.

[0010] Specifically, in (c), improving miscarriage-mediated pregnancy outcomes is manifested in reducing embryo resorption rate, increasing placental weight, rescuing miscarriage phenotype, and improving maternal-fetal outcomes.

[0011] A second aspect of the present invention provides a pharmaceutical composition comprising β-cell protein and at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutical active ingredient.

[0012] The other active pharmaceutical ingredients include substances that prevent miscarriage.

[0013] The non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients, and / or diluents.

[0014] A third aspect of the invention provides a method for preventing and / or treating miscarriage-related diseases, the method comprising administering the above-described β-cell protein or the above-described pharmaceutical composition to a subject.

[0015] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects: Experiments have shown that the above-mentioned technical solution can increase the invasiveness and endothelial property acquisition of human trophoblast cells, improve insufficient trophoblast cell invasion and poor vascular remodeling, salvage the miscarriage phenotype, and improve maternal and fetal outcomes. It has important value and application prospects for the prevention and treatment of miscarriage, and therefore has important clinical significance and social benefits. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 The images show the results of promoting trophoblast cell invasion using recombinant human BTC in Example 1 of this invention; A shows the cell invasion analysis of HTR8 / SVneo cells treated with blank vector or BTC (50 ng / ml) for 40 hours; B shows the cell invasion analysis of primary human EVT cells treated with blank vector or BTC (50 ng / ml) for 40 hours; the left image shows representative images, and the right image shows quantitative results; scale bar, 200 μm; Figure 2 This is an analysis of the formation of endothelial tubules in human trophoblast cells treated with blank carrier or BTC (50 ng / ml) for 10 hours in Example 2 of the present invention; the left image shows a representative image, and the right image shows the quantitative results; scale bar, 200 μm.

[0018] Figure 3 The results of the animal experiment in Example 3 of this invention, which supplemented a miscarriage mouse model with recombinant BTC, are shown in Figure A. The animal model treatment procedure is as follows: male and female mice were placed in the same cage at a ratio of 1:2 for mating. Vaginal plugs were detected the following day, confirming pregnancy, recorded as gestational day (G) 0.5. Pregnant mice were randomly divided into four groups: control group (PBS group), miscarriage group (LPS group), rescue group (LPS+BTC group), and BTC group. PBS or lipopolysaccharide (LPS) or recombinant BTC was injected intraperitoneally into the mice at gestational day (G) 7.5. Recombinant BTC or PBS was supplemented intraperitoneally on G9.5 and G11.5 days. Mice were sacrificed at G12.5 for tissue collection. Figure B shows representative images of embryonic absorption in each group (N=6). Figure C shows representative images of placental development in each group. Quantitative results are expressed as mean ± standard deviation. p<0.05, p<0.01, p<0.001, statistical method was ANOVA, ns, no significant difference. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0021] As mentioned earlier, although it is currently believed that the occurrence of miscarriage is related to the function of trophoblast cells during placental development, its specific pathogenesis is still unclear. In addition to some diagnostic methods and treatments targeting the cause, there is a lack of effective therapeutic drugs targeting the function of trophoblast cells.

[0022] Through research, the inventors discovered that during the early development of the human placenta, chorionic trophoblast (CTB) progenitor cells follow one of two differentiation pathways: (1) they differentiate into multinucleated syncytiotrophoblasts (STBs) through cell membrane fusion; and (2) they differentiate into extravillous trophoblast cells (EVTs) by acquiring invasive and vascular remodeling functions through directed activation, phenotypic transformation, migration and invasion, and subtype specialization. Among these, EVTs are crucial for normal placental function and participate in numerous processes, including forming stable attachments by invading the endometrium; modifying maternal spiral arteries to establish a "mother-placenta-fetus" blood supply circulation; and secreting blocking antibodies and anti-inflammatory factors to inhibit maternal immune attacks on the embryo. Abnormal differentiation and function of EVTs can directly lead to early pregnancy failure and mid-to-late pregnancy complications, and are closely related to miscarriage.

[0023] Betacellulin (BTC) is a secreted protein belonging to the epidermal growth factor (EGF) family. Some members of this family have been shown to participate in numerous physiological processes and are key regulators of cell proliferation, differentiation, division, and survival. However, current research on BTC is very limited. Regarding ovarian function, BTC has been shown to promote cumulus cell proliferation, hyaluronic acid synthesis, and phosphorylation of gap junction proteins, thereby maintaining communication between cumulus cells. In human luteinized granulosa cells, recombinant BTC can affect corpus luteum formation by regulating gap junctions. Regarding endometrial function, BTC promotes endometrial epithelial cell proliferation and the expression of adhesion molecules, which is beneficial for embryo adhesion and invasion. Currently, no studies have elucidated the effects of BTC on EVT differentiation and function, nor has any research shown that its application helps treat miscarriage phenotypes. Through experiments, this invention has found that the application of BTC promotes the invasion and tube formation process of the chorionic villus trophoblast cell line HTR8. BTC has a significant rescue effect on embryo resorption in mice with early to mid-pregnancy miscarriage models. Intraperitoneal injection of BTC into mice with miscarriage models in early to mid-pregnancy increases placental weight and improves placental development, thus indicating that BTC can have a certain therapeutic effect on miscarriage.

[0024] In view of this, in a typical embodiment of the present invention, the use of β-cell protein in the preparation of a medicament for preventing and treating miscarriage is provided. Therefore, β-cell protein is effective in preventing and / or treating miscarriage-related diseases.

[0025] The prevention and treatment of miscarriage includes one or more of the following functions: (a) Promotes trophoblast cell invasion; (b) Promotes the acquisition of endothelial properties in trophoblast cells; (c) Improve pregnancy outcomes mediated by miscarriage.

[0026] The trophoblast cells can be HTR8 cells or human primary EVT cells.

[0027] Specifically, in (c), improving miscarriage-mediated pregnancy outcomes manifests as reducing embryo resorption rate, increasing placental weight, rescuing the miscarriage phenotype, and improving maternal-fetal outcomes.

[0028] In another specific embodiment of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising β-cell protein and at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutical active ingredient.

[0029] The other active pharmaceutical ingredients include substances that prevent miscarriage.

[0030] The non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients, and / or diluents.

[0031] In another specific embodiment of the present invention, the non-pharmaceutical active ingredient includes: Pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and nonionic surfactants or lipids; The ingredients include pharmacologically harmless salts (preferably sodium chloride), flavoring agents, vitamins (preferably vitamin A or vitamin E, tocopherol or provitamins), antioxidants (preferably ascorbic acid), and stabilizers and / or preservatives.

[0032] In another specific embodiment of the present invention, a method for preventing and / or treating miscarriage-related diseases is provided, the method comprising administering the above-mentioned β-cell protein or the above-mentioned pharmaceutical composition to a subject.

[0033] The subjects can be humans or non-human mammals, such as mice, rats, pigs, cattle, sheep, orangutans, etc. In some embodiments, the subjects are mice, and more specifically, a miscarriage mouse model constructed with lipopolysaccharide.

[0034] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Any simple modifications, equivalent variations, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the present invention.

[0035] Example 1 Human recombinant BTC promotes trophoblast cell invasion (1) Human trophoblast cells were treated with recombinant human BTC protein (50 ng / ml) (R&D, #261-CE-010) or blank vector (control group, denoted as Ctrl) for later use.

[0036] (2) Pre-treat with blank vector or BTC (50 ng / ml) for 24 hours, then digest and resuspend in DMEM medium containing 0.1% (vol / vol) fetal bovine serum. Take 250 μl of DMEM medium containing 8 x 10⁻⁶ ng / ml of BTC. 4Cell suspensions of 100 cells were seeded into Transwell chambers (the Transwell chambers were pre-coated with 40 µl of 1 mg / ml matrix gel; the pore size of the Transwell chambers was 8 μm). 750 μl of DMEM medium containing 10% (vol / vol) fetal bovine serum was added to the lower layer of the Transwell chamber. The seeded cells were incubated at 37°C for 40 h. After incubation, the cells in both the upper and lower portions of the chamber membrane were fixed with tissue fixation solution for 30 min. After fixation, crystal violet staining was performed for 30 min. After staining, the chambers were washed with PBS, and uninvaded cells on the upper part of the membrane were wiped away. Imaging was performed using an inverted Olympus microscope, and the number of invading cells was analyzed using Image-J software. Figure 1 As shown in Figure A, BTC culture enhanced the invasiveness of the villous trophoblast cell line HTR8; as Figure 1 As shown in Figure B, BTC culture enhanced the invasiveness of primary human EVT cells.

[0037] Example 2 Human recombinant BTC promotes the acquisition of trophoblast cell endothelial properties (1) Human trophoblast cells were treated with recombinant human BTC protein (50 ng / ml) (R&D, #261-CE-010) or blank vector (control group, denoted as Ctrl) for later use.

[0038] (2) Dilute 10 mg / mL of Matrix gel with DMEM medium containing 0.1% (vol / vol) fetal bovine serum 1:1 (vol / vol), add 50 μL of the diluted Matrix gel to each well of a 96-well plate, and incubate at 37°C for 2 hours to solidify. Digest cells that have been pretreated with blank vector or BTC (50 ng / ml) for 24 hours, and then resuspend them in DMEM medium containing 0.1% fetal bovine serum 3×10⁻⁶ mg / mL of blank vector or BTC. 4 Cell suspensions of [number] cells were seeded in Matrigel and incubated at 37°C for 10 hours. Tube formation was observed using an Olympus inverted microscope, and measurements and analysis were performed using ImageJ software. Figure 2 As shown, BTC treatment can promote the formation of endothelial-like tubules in trophoblast cells.

[0039] Studies have found that BTC treatment significantly increases the invasiveness and endothelial property acquisition of human trophoblast cells compared to the control group. In conclusion, BTC treatment may improve trophoblast cell invasion insufficiency and poor vascular remodeling by promoting the trophoblast cell invasion process, thereby improving adverse pregnancy outcomes such as miscarriage.

[0040] Example 3 Recombinant BTC can improve pregnancy outcomes in a mouse model of miscarriage. (1) Establishment of mouse model The mice used in this experiment were wild-type C57BL / 6J mice, purchased from Beijing Vital River Laboratory Animal Co., Ltd. All animals were cared for in accordance with international animal care guidelines and received ethical approval from the Animal Care and Research Committee of Shandong University.

[0041] To investigate whether BTC can maintain normal pregnancy, a miscarriage model mouse was constructed using lipopolysaccharide LPS (R&D, L4391). The mouse embryo absorption rate was observed by intraperitoneal injection of exogenous recombinant BTC protein (R&D, #1025-CE-025 / CF) to confirm whether BTC helps maintain pregnancy and rescue the miscarriage phenotype.

[0042] Wild-type female mice aged 6-8 weeks (18-20g) were mated with male mice of similar age and weight. Vaginal plugs were detected in the female mice on the morning of the second day after mating and marked as day 0.5 of pregnancy (G0.5).

[0043] Pregnant mice were randomly divided into four groups: control group (PBS group) (n=6), abortion group (LPS group) (n=6), rescue group (LPS+BTC group) (n=6), and BTC group (n=6). PBS or LPS (0.20 mg / kg) or recombinant BTC (0.01 mg / kg) was administered intraperitoneally to mice on day 7.5 of gestation (G7.5). Recombinant BTC or PBS supplementation was administered intraperitoneally on days G9.5 and G11.5 as a control. Mice were sacrificed on day G12.5 and their tissues were collected.

[0044] (2) Observation of mouse embryonic development Mice were anesthetized with isoflurane on day 12.5 and removed while unconscious. The neck of the pregnant mouse was cut open with tissue scissors and placed in a beaker of tap water. The mouse was squeezed to expel blood, ensuring the uterus was as clean as possible. The mouse was then euthanized by cervical dislocation. The limbs were fixed to a board. After disinfection of the abdomen, the skin was longitudinally cut open with ophthalmic scissors to expose the uterus. The uterine tissue was separated from the mesentery using ophthalmic scissors and removed. Surrounding fat and mesentery tissue were trimmed before photographing. Embryos that were shrunken or severely congested were considered resorbed embryos. Embryo resorption rate (%) = number of resorbed embryos / total number of embryos × 100%. After photographing, the placenta, decidua, and fetal mouse were carefully separated using ophthalmic curved forceps, and the placental weight was measured.

[0045] Figure 3 A in the diagram represents the flowchart for constructing a miscarriage mouse model and its rescue. Figure 3 In Figure B, embryo resorption was observed in each group after mice were sacrificed on day G12.5 following intraperitoneal injection of the corresponding drugs. Figure 3The middle section (C) shows the embryonic development and placental weight observed after the removal of the placenta, decidua, and fetal mice. The study found that the LPS group mice exhibited significantly increased embryo resorption and decreased placental weight, indicating the successful establishment of a mouse model of miscarriage using LPS. In the LPS+rmBTC group mice, intraperitoneal injection of recombinant mouse BTC protein at G7.5, G9.5, and G11.5 significantly reduced embryo resorption and increased placental weight, rescuing the miscarriage phenotype and improving maternal and fetal outcomes. In contrast, pregnant mice supplemented solely with recombinant mouse BTC protein showed no significant difference in embryo resorption and placental weight compared to the control group, indicating that recombinant BTC protein supplementation alone had no adverse effect on pregnancy outcomes.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of β-cell protein in the preparation of drugs for the prevention and treatment of miscarriage in the first and second trimesters.

2. The application as described in claim 1, characterized in that, Preventing miscarriage includes promoting the invasion of trophoblast cells, which are HTR8 cells or human primary EVT cells.

3. The application as described in claim 1, characterized in that, Preventing miscarriage includes promoting the acquisition of trophoblast cell endothelial properties.

4. The application as described in claim 1, characterized in that, Preventing miscarriage includes reducing embryo resorption and increasing placental weight.

5. The application as described in claim 3, characterized in that, The trophoblast cells are HTR8 cells or human primary EVT cells.

6. The application as described in claim 1, characterized in that, The drug is administered by injection.

7. The application as described in claim 6, characterized in that, The injection was administered intraperitoneally.

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