Application of icariin in the preparation of drugs for preventing and treating recurrent miscarriage

Abstract

This invention discloses the application of icariin or its pharmaceutical salt in the preparation of drugs for preventing and treating recurrent miscarriage. This invention discovers that icariin can directly promote the migration, invasion, and proliferation of trophoblast cells. Through in vitro culture of human extravillous trophoblast cell lines, and using scratch healing assays, Transwell migration and invasion assays, cell cycle analysis, and immunofluorescence, it was confirmed that icariin can dose-dependently enhance the migration and invasion capabilities of trophoblast cells, promote cell cycle progression to the G2 / M phase, and upregulate the expression of PCNA and Ki-67 within the cells. This directly demonstrates that icariin has a positive promoting effect on the function of trophoblast cells, which are key cells for maintaining pregnancy.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically, it relates to the application of icariin in the preparation of drugs for preventing and treating recurrent miscarriage. Background Technology

[0002] Recurrent miscarriage (RSA) is a common and serious complication of pregnancy, generally defined as two or more consecutive pregnancies lost with the same sexual partner. This condition causes immense psychological stress and physical trauma to patients and their families, and is a pressing problem in the field of reproductive health. Currently, the clinical diagnosis and treatment of recurrent miscarriage still face many challenges. Its etiology is complex and may involve multiple aspects, including genetic factors, anatomical abnormalities, endocrine disorders, immune factors, prothrombotic states, and infections. Among these, imbalance of immune tolerance at the maternal-fetal interface and abnormal trophoblastic cell function are considered to be among the core pathological links leading to pregnancy failure. Trophoblastic cells, especially the invasive extravillous trophoblastic cells, are crucial for successful embryo implantation, placental formation, and maintaining pregnancy. Impaired function of these cells can lead to shallow placental implantation, insufficient spiral artery remodeling, and ultimately, pregnancy loss.

[0003] In current Western medicine, the treatment of recurrent miscarriage primarily employs symptomatic and supportive therapies. For example, progesterone replacement therapy is used for luteal insufficiency; low molecular weight heparin combined with low-dose aspirin is used for anticoagulation in antiphospholipid syndrome; and lymphocyte-mediated active immunotherapy or intravenous immunoglobulin may be used to address alloimmune factors. However, these treatments have certain limitations. First, many treatments lack high-level evidence-based medicine support, and their efficacy is controversial. Second, some treatments, such as immunosuppressants or biologics, may lead to increased risk of infection, liver damage, and high costs, resulting in side effects and financial burden. Furthermore, existing treatments often target a single cause, lacking a systematic, multi-target treatment strategy for patients with recurrent miscarriage of unknown etiology or multiple contributing factors. Therefore, developing a safe and effective novel drug capable of intervening in the pathological process of recurrent miscarriage at multiple stages is of significant clinical importance.

[0004] Traditional Chinese medicine (TCM) has a long history and rich experience in preventing and treating miscarriage, often based on theories such as "the kidney governs reproduction" and "the spleen is the source of qi and blood," using compound TCM formulas for treatment. However, the complex composition of traditional TCM formulas often obscures the specific material basis and mechanism of action, limiting their international recognition and promotion. With the development of modern TCM pharmacology, isolating and identifying active monomeric components with clear chemical structures from traditional TCM and elucidating their pharmacological mechanisms has become a key path for the modernization and internationalization of TCM. Epimedium, a commonly used kidney-tonifying and aphrodisiac TCM, has had its active ingredient, icariin (ICA, CAS No.: 489-32-7), extensively studied, revealing various pharmacological activities such as estrogen-like effects, antioxidant, anti-inflammatory, and improvement of bone metabolism. In the field of reproduction, some early studies suggested that icariin may have a positive impact on ovarian function and embryonic development, but these studies have mostly focused on improving egg quality or endometrial receptivity in assisted reproductive technologies, and the mechanisms have not been explored in depth.

[0005] In the context of recurrent miscarriage, current technologies do not provide clear, systematic, and in-depth answers regarding whether icariin can directly act on the key aspect of pregnancy maintenance—the functional regulation of trophoblasts—and whether it can prevent and treat recurrent miscarriage by regulating the maternal-fetal immune microenvironment. There is a lack of research utilizing standardized animal models of recurrent miscarriage (such as models constructed based on the mating of CBA / J female mice and DBA / 2 male mice) combined with trophoblast function research systems (such as the human extravillous trophoblast cell line HTR-8 / SVneo) to comprehensively explore the specific efficacy and potential molecular mechanisms of icariin in preventing and treating recurrent miscarriage at both in vivo and in vitro levels. Current technologies have not yet revealed whether icariin can play a role in maintaining pregnancy and preventing miscarriage by affecting the proliferation, migration, and invasion capabilities of trophoblasts, or by regulating related signaling pathways (such as PI3K / Akt, MAPK, etc.), or by regulating the balance of immune cells (such as natural killer cells and regulatory T cells) in the decidua. This gap at the mechanistic level hinders the development of icariin, a promising traditional Chinese medicine monomer, into a drug for preventing and treating miscarriage with a clearly defined target and mechanism. Summary of the Invention

[0006] The purpose of this invention is to provide the application of icariin in the preparation of drugs for preventing and treating recurrent miscarriage.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides the application of icariin or its medicinal salt in the preparation of drugs for preventing and treating recurrent miscarriage.

[0009] The drug for preventing recurrent miscarriage uses icariin or its medicinal salt as the sole active ingredient.

[0010] The drug for preventing recurrent miscarriage is made from icariin or its pharmaceutical salt and medically acceptable excipients.

[0011] The medications for preventing recurrent miscarriage include suspensions, granules, capsules, tablets, powders, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches, or drops.

[0012] The administration methods for the drugs for preventing recurrent miscarriage include oral administration, intravenous administration, intraperitoneal injection, intramuscular injection, subcutaneous injection, sublingual administration, transdermal administration, or rectal suppository administration.

[0013] The drug for preventing recurrent miscarriage is made from icariin or its medicinal salt and other drugs for treating recurrent miscarriage.

[0014] The drug for preventing recurrent miscarriage is a pharmaceutical composition containing icariin or its pharmaceutical salt or its derivatives.

[0015] This invention discovers that icariin can be used to reduce embryo loss rate and improve pregnancy outcomes. By constructing a classic recurrent miscarriage mouse model (CBA / J female mice mated with DBA / 2J male mice) and setting up low- and high-dose icariin intervention groups, it was confirmed that icariin can significantly reduce the embryo loss rate in the model mice, with effects comparable to known YAP pathway agonists, demonstrating that icariin has a clear efficacy in preventing and treating miscarriage in vivo.

[0016] This invention discovers that icariin can improve the pathological morphology of maternal-fetal interface tissues and promote the proliferation of decidual tissue cells. Through pathological observation and immunofluorescence detection of placental tissue in pregnant mice, this invention found that icariin intervention can reverse the pathological changes such as disordered decidual tissue arrangement and inflammatory cell infiltration caused by a recurrent miscarriage model, and significantly enhance the expression of proliferating cell nuclear antigen (PCNA) and cell proliferation marker Ki-67 in decidual tissue, indicating that icariin can promote the repair and proliferation of decidual tissue.

[0017] This invention reveals that icariin can regulate the Hippo signaling pathway to maintain pregnancy. By detecting the phosphorylation levels of key proteins in the Hippo pathway in mouse placental tissue and human chorionic villus extratrophoblast cells using Western blotting, it was found that icariin can inhibit the excessive phosphorylation of proteins such as LATS1, MST, YAP, and TAZ in this pathway, potentially activating the transcriptional activity of YAP. This provides a crucial molecular mechanism explanation for icariin's ability to promote trophoblast function and maintain pregnancy.

[0018] This invention discovers that icariin can directly promote the migration, invasion, and proliferation of trophoblast cells. Through in vitro culture of human extravillous trophoblast cell lines, and using techniques such as scratch healing assays, Transwell migration and invasion assays, cell cycle analysis, and cell immunofluorescence, it was confirmed that icariin can dose-dependently enhance the migration and invasion capabilities of trophoblast cells, promote cell cycle progression to the G2 / M phase, and upregulate the expression of PCNA and Ki-67 in cells. This directly demonstrates that icariin has a positive promoting effect on the function of trophoblast cells, which are key cells for maintaining pregnancy.

[0019] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0020] This invention addresses the lack of effective, safe, and mechanistically sound therapeutic drugs targeting the core pathological aspects of recurrent miscarriage (trophoblastic dysfunction and maternal-fetal interface immune tolerance imbalance). Current Western medical treatments are mostly symptomatic and supportive, lacking high-level evidence-based support, and have limitations such as side effects, high costs, or targeting only a single cause. Their efficacy is limited for patients with recurrent miscarriage of unknown or complex etiologies. While traditional Chinese medicine compound formulas have some efficacy, their complex components and unclear material basis and mechanisms of action limit their modernization and international acceptance.

[0021] Icariin is known to have a variety of pharmacological activities, and existing research has focused on the field of assisted reproduction. This invention uses standardized animal and cell models to systematically explore whether icariin can maintain pregnancy by regulating trophoblast function (such as proliferation, migration, and invasion) and related signaling pathways.

[0022] This invention, through systematic in vitro and in vivo experiments, reveals the specific effects and potential pathways of icariin in preventing and treating recurrent miscarriage, providing a solid scientific basis for the development of novel anti-miscarriage drugs.

[0023] In summary, the present invention aims to provide a new use for icariin, which is derived from traditional Chinese medicine, is safe and effective, and has a relatively clear mechanism of action: to prepare a drug for the prevention and treatment of recurrent miscarriage, so as to make up for the shortcomings of existing treatment methods and provide a new option for multi-target intervention in key aspects of pregnancy maintenance in clinical practice.

[0024] This invention, through in vivo experiments using a mouse model of recurrent miscarriage, demonstrates that icariin significantly reduces embryo loss rate and effectively improves pregnancy outcomes. At the tissue level, icariin reverses pathological damage to the decidua and promotes cell proliferation, as evidenced by significantly enhanced expression of PCNA and Ki-67. At the molecular level, this invention systematically reveals for the first time that icariin's prevention and treatment of recurrent miscarriage is closely related to the regulation of the Hippo signaling pathway, inhibiting the excessive phosphorylation of key proteins such as LATS1, MST, and YAP. At the cellular function level, in vitro experiments clearly demonstrate that icariin directly and dose-dependently promotes the migration and invasion of human chorionic villus extratrophoblast cells, accelerates cell cycle progression, and enhances their proliferative activity. These effects collectively indicate that icariin acts on the maternal-fetal interface through multiple targets and pathways, improving the uterine environment and directly enhancing trophoblast function, thus providing a relatively well-defined, safe, and effective candidate drug derived from traditional Chinese medicine for the prevention and treatment of recurrent miscarriage. Attached Figure Description

[0025] Figure 1 This is a schematic diagram showing the embryo loss rate of each group of mice.

[0026] Figure 2 The bar chart shows the embryo loss rate in each group of mice.

[0027] Figure 3 This is a schematic diagram showing the immunofluorescence results of Ki-67 and PCNA in the decidual tissue of mice in each group.

[0028] Figure 4 This is a schematic diagram showing the morphological characteristics of decidual tissue in each group of mice.

[0029] Figure 5 This is a schematic diagram showing the expression of Ki-67 and PCNA proteins in mouse decidual tissue.

[0030] Figure 6 This is a schematic diagram showing the expression of HIPPO pathway proteins in mouse decidual tissue.

[0031] Figure 7 This is a schematic diagram showing the expression of HIPPO pathway proteins in HTR-8 / SVneo cells of each group.

[0032] Figure 8 This is a schematic diagram showing the safe and effective dosage results for ICA.

[0033] Figure 9 This is a schematic diagram showing the cell migration results in each group under ICA intervention.

[0034] Figure 10 This is a schematic diagram showing the results of cell migration and invasion in each group under ICA intervention.

[0035] Figure 11 This is a schematic diagram showing the proportions of cells in the G0 / G1, S, and G2 / M phases for each group. Detailed Implementation

[0036] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] Icariin used to reduce embryo loss rate in a mouse model of recurrent miscarriage.

[0039] Forty SPF-grade, 7-week-old CBA / J female mice weighing 20g±5g, 16 DBA / 2J male mice of the same strain and size, and four BALB / c male mice of the same strain and size were prepared. All animals were housed in an animal experimental center that met national standards, with environmental conditions strictly controlled at room temperature (22±3)℃, relative humidity 50%~60%, 12-hour light-dark cycle, and free access to food and water. Animals underwent a one-week acclimatization period before the formal experiments to ensure their physiological stability.

[0040] Animal grouping and model establishment: Forty CBA / J female mice were randomly divided into 5 groups of 8 mice each using a random number table: normal pregnancy control group (Con group), recurrent miscarriage model group (RSA group), low-dose icariin intervention group (ICA-L group), high-dose icariin intervention group (ICA-H group), and positive control YAP pathway agonist group (XUM-MP-1 group). In the Con group, CBA / J female mice and BALB / c male mice were paired together in a 2:1 ratio every afternoon. The following morning, the presence of vaginal plugs in the female mice or the presence of sperm in vaginal smears under a microscope were used to determine successful mating. The morning of the day a vaginal plug or sperm was found was recorded as day 0.5 of pregnancy (E0.5), and the female mouse was then transferred to the Con group and housed separately. Unsuccessful mating continued to be paired with male mice. For the RSA, ICA-L, ICA-H and XUM-MP-1 groups, the classic recurrent miscarriage model was constructed by pairing CBA / J female mice with DBA / 2J male mice in a 2:1 ratio, and using the same method as above to check and confirm pregnancy. Successful pregnancies were recorded as E0.5 and included in the corresponding groups.

[0041] Drug intervention was implemented: Starting from the day of confirmed pregnancy (E0.5), mice in the ICA-L group (5 mg / kg) and ICA-H group (10 mg / kg) were administered the corresponding dose of icariin by gavage, while mice in the XUM-MP-1 group were administered the YAP agonist XUM-MP-1 (1 mg / kg, an MST1 / 2 inhibitor) by intraperitoneal injection. The Con and RSA groups were given an equal volume of DMSO (concentration not exceeding 2%). Drug intervention was administered once daily until day 10.5 of pregnancy (E10.5).

[0042] Pregnancy outcome assessment and sample collection: On day 10.5 of gestation, all pregnant mice were euthanized by cervical dislocation or other ethically acceptable methods. The abdominal cavity was quickly opened to fully expose and remove both uterine horns. The condition of each implantation site was carefully separated and counted. Embryos that were plump, rosy in color, and had obvious embryonic structures were identified as viable embryos; those that were atrophied, dark in color, had unclear structures, or had been absorbed were identified as lost embryos. The number of viable and lost embryos for each mouse was accurately recorded. The average embryo loss rate for each mouse and each group was calculated using the formula: Embryo loss rate (%) = [Number of lost embryos / (Number of viable embryos + Number of lost embryos)] × 100%. After photographing all removed placental tissues, they were quickly frozen in liquid nitrogen and then transferred to an ultra-low temperature freezer at -80°C for long-term storage for subsequent molecular biological analysis.

[0043] Data analysis was performed using professional statistical software such as GraphPad Prism 9.0 to statistically analyze the embryo loss rate data of the five groups of mice. Data are expressed as mean ± standard deviation. First, one-way ANOVA was used to test for overall differences among the groups. If significant differences were found, Tukey's multiple comparison test was used for pairwise comparisons. A statistically significant difference was defined as P < 0.05. Results are as follows: Figure 1 and Figure 2 As shown, Figure 1 Photographs illustrating the embryo loss rate in each group of mice; Figure 2 The bar chart shows the embryo loss rate in each group of mice. The results showed that compared with the Con group, the embryo loss rate in the RSA group was significantly higher (P<0.05), indicating the successful establishment of the recurrent miscarriage model. However, compared with the RSA group, the embryo loss rates in the ICA-L group, ICA-H group, and XUM-MP-1 group were significantly lower (P<0.05), and the effect of the ICA-H group was comparable to that of the positive drug XUM-MP-1 group. These results directly demonstrate that icariin intervention can effectively improve pregnancy outcomes in mice with recurrent miscarriage and significantly reduce the embryo loss rate.

[0044] This invention verifies that icariin has a clear effect in reducing embryo loss rate and preventing recurrent miscarriage in vivo by constructing a standardized animal model, setting up reasonable control and intervention groups, implementing periodic drug treatment, accurately assessing pregnancy outcomes and supplementing with statistical analysis. This provides a solid in vivo experimental basis for its preparation into a drug to improve pregnancy outcomes.

[0045] Example 2

[0046] Icariin is used to improve the pathological morphology of maternal-fetal interface tissues and promote decidual cell proliferation.

[0047] A mouse model of recurrent miscarriage was constructed according to the method described in Example 1, and the following groups were established: a normal pregnancy control group (Con group), a recurrent miscarriage model group (RSA group), a low-dose icariin intervention group (ICA-L group), a high-dose icariin intervention group (ICA-H group), and a positive control YAP pathway agonist group (XUM-MP-1 group). Samples were collected on day 10.5 of gestation, and intact placental and decidual tissues were harvested.

[0048] Pathological morphological analysis of placental decidual tissue was performed: At least two representative placental tissue samples were selected from each group of mice and immediately immersed in a sufficient amount of 4% paraformaldehyde, fixed overnight at 4°C to ensure good preservation of tissue cellular structure. After fixation, the tissues were paraffin-embedded using standard procedures including graded alcohol dehydration, xylene clearing, and paraffin embedding. The embedded blocks were serially sectioned using a paraffin microtome, with a thickness typically of 4-5 micrometers. The sections were mounted on glass slides, baked to remove wax, and then stained with hematoxylin and eosin (HE). The specific steps included: hematoxylin staining of cell nuclei, hydrochloric acid alcohol differentiation, eosin staining of cytoplasm, and finally dehydration and clearing, followed by mounting with neutral resin. The microstructure of placental decidual tissue in each group was observed under an optical microscope, focusing on the arrangement of decidual cells, cell morphological integrity, intercellular connections, presence of necrotic areas, and inflammatory cell infiltration. Representative field-of-view images were taken for comparative analysis.

[0049] The expression of cell proliferation markers in decidual tissue was detected using tissue immunofluorescence: Paraffin sections of the prepared placental tissue were dewaxed with xylene and then hydrated with a gradient of alcohols. Antigen retrieval was performed using sodium citrate buffer (pH 6.0) for heat-induced epitope retrieval. After retrieval, the sections were washed with phosphate-buffered saline (PBS). To allow antibody entry into the cell nucleus, the sections were permeabilized with 0.3% Triton X-100 at room temperature for 15 minutes. Subsequently, the sections were blocked with blocking buffer containing 5% bovine serum albumin (BSA) at room temperature for 1 hour to reduce non-specific binding. After removing the blocking buffer, diluted primary antibody working solution was added to the tissue area of ​​the section. Two primary antibodies were used: mouse anti-human / mouse Ki-67 monoclonal antibody (dilution ratio 1:400) and mouse anti-human / mouse PCNA monoclonal antibody (dilution ratio 1:400). The sections were placed in a humidified chamber and incubated overnight (approximately 16-18 hours) at 4°C.

[0050] Immunofluorescence staining and observation were completed. The next day, the slides were removed from 4°C and allowed to return to room temperature. They were then washed three times with PBS for 5 minutes each time to remove unbound primary antibody. Under light-protected conditions, a fluorescein-labeled secondary antibody (1:500 dilution) matching the primary antibody species was added and incubated at room temperature for 1 hour in the dark. After incubation, the slides were washed three more times with PBS in the dark. To visualize the cell nuclei, DAPI (4',6-diamidinyl-2-phenylindole) staining solution was added to cover the tissue and stained at room temperature in the dark for 10 minutes. After DAPI staining, the slides were washed thoroughly with PBS. Finally, an anti-fluorescence quenching mounting medium was added, and the slides were covered with coverslips. The slides were observed and images were acquired using a laser scanning confocal microscope. The signals of Ki-67, PCNA, and DAPI were acquired separately. Changes in cell proliferation activity were qualitatively assessed by comparing the fluorescence intensity of Ki-67 and PCNA in the decidual cell region among different groups.

[0051] The results are as follows Figures 3-5 As shown, Figure 3 This is a schematic diagram showing the immunofluorescence results of Ki-67 and PCNA in the decidual tissue of mice in each group. Figure 4 This is a schematic diagram showing the morphological characteristics of decidual tissue in each group of mice. Figure 5This diagram illustrates the expression of Ki-67 and PCNA proteins in mouse decidual tissue. A shows the Western blot bands of Ki-67 and PCNA proteins in mouse decidual tissue; B shows the expression of Ki-67 protein in mouse decidual tissue; and C shows the expression of PCNA protein in mouse decidual tissue. Combining the results of HE staining and immunofluorescence, it can be observed that the decidual cells in the Con group are neatly and tightly arranged, with clear structure and uniform cytoplasmic staining; the RSA group, on the other hand, shows typical pathological changes, such as disordered arrangement of decidual cells, loose connections, visible necrotic cell fragments, and extensive inflammatory cell infiltration. Figure 4 Meanwhile, the fluorescence signals of Ki-67 and PCNA were significantly weakened. In contrast, the histopathological damage in the ICA-L, ICA-H, and XUM-MP-1 groups was significantly improved, with more regular cell arrangement and reduced inflammatory infiltration. Furthermore, the fluorescence signal intensity of Ki-67 and PCNA was significantly enhanced compared to the RSA group, indicating that the proliferative activity of decidual cells was effectively promoted. Figure 3 The ICA-H group showed more significant effects, with consistent Western blotting results. Figure 5 Further analysis using Western blot revealed the expression of Ki-67 and PCNA proteins in mouse decidual tissue. Results showed a significant decrease in Ki-67 and PCNA protein expression in the RSA group, while significant increases were observed in the ICA-L, ICA-H, and XUM-MP-1 groups. This suggests a significant impairment in decidual tissue cell proliferation in the recurrent miscarriage model mouse model, and that ICA intervention can promote decidual tissue cell proliferation and repair RSA-induced decidual tissue cell proliferation damage at the maternal-fetal interface.

[0052] This invention uses histopathology (HE staining) and molecular localization techniques (immunofluorescence) to demonstrate, from both tissue morphology and cell proliferation levels, that icariin can reverse decidual tissue damage at the maternal-fetal interface caused by recurrent miscarriage and significantly promote the proliferation and repair function of decidual cells. This provides direct histological evidence for the preparation of drugs using icariin to improve the pregnancy microenvironment and promote tissue repair.

[0053] Example 3

[0054] Icariin prevents recurrent miscarriage by regulating the Hippo signaling pathway.

[0055] Protein samples for analysis were obtained from two sources: first, mouse placental tissue obtained on day 10.5 of gestation as described in Example 1; and second, cell samples from the in vitro cultured human chorionic villus exotrophic cell line HTR-8 / SVneo treated with icariin. For tissue samples, placental tissue frozen at -80°C was removed, and 10 mg of tissue was homogenized in pre-chilled tissue lysis buffer using a tissue homogenizer. For cell samples, HTR-8 / SVneo cells in good growth condition were seeded into culture dishes. After adhesion, the culture medium was replaced with complete medium containing different concentrations of icariin (0 μM, 10 μM, 20 μM, and 40 μM), and treated for 24 hours. The medium was then discarded, and the cells were gently washed twice with pre-chilled PBS.

[0056] Protein extraction and quantification: Add RIPA high-efficiency lysis buffer containing protease inhibitors and phosphatase inhibitors to tissues or cells, and lyse on ice for 30 minutes, intermittently vortexing to ensure complete lysis. Then, centrifuge at 12000-14000 rpm for 15 minutes at 4°C, carefully aspirating the supernatant, which is the total protein solution. The concentration of the extracted total protein is determined using a BCA protein quantification kit. The specific steps are as follows: Prepare BCA working solution according to the manufacturer's instructions; serially dilute protein standards and add them together with the protein samples to be tested into a 96-well plate, adding BCA working solution to each well, and incubate at 37°C for 30 minutes. Measure the absorbance at 562 nm using a microplate reader, and calculate the accurate concentration of each protein sample based on the standard curve. Adjust all samples to a uniform concentration (e.g., 2 μg / μL) with lysis buffer, add an appropriate volume of 5× protein loading buffer, mix well, and heat in a metal bath for 10 minutes to denature the protein. The protein can then be loaded immediately or stored at -80°C.

[0057] Western blotting analysis: Prepare an appropriate concentration of sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), typically using a 10% separating gel. Add equal amounts of protein (20–40 μg) sequentially to the sample wells of the gel, along with pre-stained protein molecular weight standards. Perform electrophoresis under constant pressure to separate the proteins according to their molecular weight. After electrophoresis, transfer the protein bands from the gel to a polyvinylidene fluoride (PVDF) membrane using a wet transfer method. After transfer, block the PVDF membrane with TBST buffer containing 5% skim milk at room temperature for 1 hour to block non-specific binding sites.

[0058] Antibody incubation and signal detection: After blocking, the PVDF membrane was incubated overnight at 4°C with specific primary antibodies against key proteins in the Hippo signaling pathway. The primary antibodies included: rabbit anti-phosphorylated LATS1 (p-LATS1, Ser909), rabbit anti-phosphorylated MST1 / 2 (p-MST1 / 2, Thr183 / Thr180), rabbit anti-phosphorylated YAP (p-YAP, Ser127), rabbit anti-phosphorylated TAZ (p-TAZ, Ser89), and corresponding rabbit anti-total LATS1, total MST1, total YAP, and total TAZ antibodies as internal controls. Rabbit anti-GAPDH or β-tublin antibodies were used as loading volume controls. The primary antibody dilution ratio was 1:1000. The next day, the membrane was washed three times with TBST buffer on a shaker for 10 minutes each time. Subsequently, the membrane was incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (dilution ratio 1:5000) at room temperature for 1 hour. After incubation, the membrane was washed thoroughly again with TBST. Using an enhanced chemiluminescence (ECL) colorimetric kit, equal volumes of solutions A and B were mixed and uniformly added to the membrane. Protein band signals were acquired in a dark room using a chemiluminescence imaging system.

[0059] Quantitative Data Analysis and Mechanism Elucidation: Image analysis software such as ImageJ was used to quantitatively analyze the gray values ​​of the acquired protein bands. The ratio of the gray value of the target phosphorylated protein band (e.g., p-LATS1) to the gray value of its corresponding total protein band (e.g., LATS1), or to the gray value of the internal control (e.g., GAPDH), was calculated for each sample to represent the relative phosphorylation level of the protein. The data from each group were imported into GraphPad Prism software for statistical analysis, comparing the differences in the phosphorylation levels of each protein among the Con group, RSA group, ICA-L group, ICA-H group, and XUM-MP-1 group.

[0060] The results are as follows Figure 6 As shown, Figure 6This diagram illustrates the expression of HIPPO pathway proteins in mouse decidual tissue. A shows Western blot bands of phosphorylated proteins and total proteins at key HIPPO pathway targets across groups. B shows the results of p-MST / MST, p-LATS1 / LATS1, p-YAP / YAP, and p-TAZ / TAZ in mouse placental tissue from each group. Specifically, the top left image shows p-MST / MST, the top right image shows p-LATS1 / LATS1, the bottom left image shows p-YAP / YAP, and the bottom right image shows p-TAZ / TAZ. The diagram shows that compared to the Con group, the phosphorylation levels of LATS1, MST, YAP, and TAZ in the RSA group mouse placental tissue were significantly increased, indicating overactivation of the Hippo pathway. Compared with the RSA group, the phosphorylation levels of LATS1, MST, YAP, and TAZ in the placental tissues of mice in the ICA-L, ICA-H, and XUM-MP-1 groups were significantly reduced, suggesting that ICA can inhibit the Hippo pathway.

[0061] The Hippo signaling pathway is a highly evolutionarily conserved molecular regulatory network, initially discovered in Drosophila, playing a central role in controlling cell proliferation and differentiation. In recent years, increasing research has confirmed that the Hippo signaling pathway not only plays a crucial role in tumorigenesis and development, but also, with a deeper understanding of the etiology and pathogenesis of recurrent spontaneous abortion (RSA), some literature reports that the Hippo signaling pathway is involved in regulating the invasion and growth of trophoblast cells into the endometrium. Abnormal activation of this pathway may be closely related to the occurrence of recurrent miscarriage. Its core molecular structure comprises a protein kinase chain consisting of MST1 / 2 (mammalian Sterile 20-like kinases 1 / 2), SAV1 (salvador1), Lats1 / 2 (large tumorsuppressor 1 / 2), and MOB1 (MOBKL1A / MOBKL1B), along with their substrate YAP / TAZ (Yes-associated protein / transcriptional coactivator with PDZ-binding motif).

[0062] This pathway exhibits bidirectional regulatory characteristics: when the Hippo pathway is closed, YAP is activated and enters the nucleus, where it binds to the transcription factor TEAD (TEA domain) and initiates proliferation; when the Hippo pathway is open, MST1 / 2, under the conformational stabilization of SAV1, activates the MOB1 and LATS1 / 2 kinase complex through phosphorylation. The interaction between the phosphorylated LATS1 / 2 kinase dimers is enhanced, which in turn leads to the phosphorylation and retention of YAP / TAZ in the cytoplasm and their degradation. The TEAD complex cannot be formed, and the proliferation gene is silenced.

[0063] During pregnancy, the physiological invasion of trophoblast cells is regulated by the Hippo pathway. YAP expression tends to increase during decidualization of endometrial stromal cells in early pregnancy. Abnormal activation of the Hippo pathway leads to ubiquitination and degradation of YAP in the cytoplasm, restricting EVT invasion and potentially causing placental dysfunction-related diseases such as recurrent miscarriage and preeclampsia. Results showed that abnormal activation of the Hippo pathway in RSA model mice resulted in YAP / TAZ retention and degradation in the cytoplasm, thereby inhibiting EVT proliferation and invasion. Intervention with ICA and the pathway inhibitor XUM-MP-1 reversed these changes, activating YAP nuclear translocation and restoring EVT proliferation and invasion capabilities. This suggests that excessive activation of the Hippo pathway leading to EVT dysfunction is a crucial step in the development and progression of RSA.

[0064] In cell experiments, ICA treatment (especially 40 μM) significantly altered the levels of p-LATS1, p-MST, and p-YAP in HTR-8 / SVneo cells. Results are as follows: Figure 7 As shown, Figure 7 This diagram illustrates the expression of HIPPO pathway proteins in HTR-8 / SVneo cells across different groups. A shows Western blot bands of phosphorylated proteins and total proteins at key targets of the Hippo pathway in each group. B shows the results for p-LATS1 / LATS1, p-MST / MST, p-YAP / YAP, and p-TAZ / TAZ in HTR-8 / SVneo cells across different groups. Specifically, the top left image shows the p-LATS1 / LATS1 result, the top right image shows the p-MST / MST result, the bottom left image shows the p-YAP / YAP result, and the bottom right image shows the p-TAZ / TAZ result. The diagram shows that icariin can inhibit the hyperphosphorylation of key proteins in the Hippo signaling pathway in recurrent miscarriage, potentially relieving the inhibition of downstream effector molecules YAP / TAZ and activating their proliferative and survival-promoting transcriptional activities.

[0065] The results showed a slight increase in phosphorylation levels of key proteins in the Hippo pathway in physiologically healthy HTR-8 / SVneo cells. This reflects the bidirectional regulatory role of the Hippo pathway in maintaining cellular homeostasis. In normal, unstressed trophoblast cells in vitro, ICA may act as a mild stress signal, transiently activating the Hippo pathway to maintain cellular homeostasis (preventing excessive proliferation). However, in the pathological microenvironment of RSA (involving inflammation, hypoxia, and oxidative stress), the Hippo pathway is abnormally activated, leading to excessive phosphorylation and inactivation of YAP, hindering the normal proliferation and invasion of decidual and trophoblast cells. In this situation, ICA exerts its "therapeutic effect," specifically counteracting the pathway abnormalities under pathological conditions, reversing the excessive phosphorylation of YAP, thereby relieving inhibition and restoring cell function.

[0066] This invention reveals in detail, at the molecular level, a key pathway through which icariin exerts its effect in preventing recurrent miscarriage—the Hippo signaling pathway. Using Western blotting, it was quantitatively demonstrated that icariin can regulate the phosphorylation state of the core protein in this pathway. This provides crucial molecular pharmacological evidence for its development into a drug that targets and regulates specific signaling pathways to maintain stable pregnancy.

[0067] Example 4

[0068] Icariin directly promotes the function (migration, invasion, and proliferation) of human chorionic villus extratrophoblast cells.

[0069] Cell Culture and Drug Treatment: The human chorionic villus trophoblast cell line HTR-8 / SVneo was resuscitated and routinely cultured. Cells were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated at 37°C and 5% CO2. Cells were passaged every 2-3 days according to cell density, and cells in good growth condition and in the logarithmic growth phase (passages 3-10) were used for experiments. Before the formal functional experiments, the safe concentration range of icariin (ICA) for HTR-8 / SVneo cells was determined using the CCK-8 assay. Cells were cultured at a density of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates. After adhesion, the medium was replaced with fresh medium containing different concentrations of ICA (0, 10, 20, 40, 80 μM), with 5-6 replicates for each concentration. After culturing for 24 hours, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 30 minutes. The absorbance was measured at 450 nm using a microplate reader. Relative cell viability was calculated, and concentrations (10, 20, and 40 μM ICA) that did not significantly inhibit cell viability were selected for subsequent functional experiments. Results are as follows: Figure 8 As shown, Figure 8This diagram illustrates the safe and effective dosage results of ICA. As can be seen from the figure, ICA concentrations of 10, 20, and 40 μM showed no significant inhibitory effect on cell viability after 24 hours of intervention, indicating safety for HTR-8 / SVneo cells.

[0070] Cell migration ability was assessed using a scratch healing assay: HTR-8 / SVneo cells were seeded at 6 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured until confluence reached 90% or higher. Using a sterile 10 μL pipette tip, a straight, uniformly wide "scratch" was created on the monolayer of cells, perpendicular to the pre-drawn marking lines on the back of the plate. The plates were gently washed twice with PBS to remove cell debris. The medium was then replaced with serum-free medium containing different concentrations of ICA (0, 10, 20, 40 μM). The scratched area was immediately photographed under an inverted microscope and recorded as the 0-hour image. The cells were returned to the incubator and cultured for another 24 hours, after which the same image was photographed again at the same location and recorded as the 24-hour image. The width of the scratch at 0 and 24 hours was measured using ImageJ software, and the percentage of wound healing was calculated.

[0071] [(0-hour scratch width - 24-hour scratch width) / 0-hour scratch width] × 100%

[0072] The experimental results showed that, compared with the control group, the percentage of scratch healing in HTR-8 / SVneo cells was significantly increased after 24 hours of treatment with 20 μM and 40 μM ICA (P<0.05), demonstrating that ICA can effectively promote the migration ability of trophoblast cells in a concentration-dependent manner (results are shown in Figure 1). Figure 9 As shown, Figure 9 This diagram illustrates the cell migration results under different concentrations of ICA (0, 10, 20, 40 μM). In the diagram, A shows representative results of HTR-8 / SVneo cell scratch treatment after 0h, 12h, 24h, and 48h of ICA intervention, and B shows the statistical results of cell wound healing ability after 24h of ICA intervention.

[0073] The results showed that, compared with the control group, the cell wound healing rate was significantly increased in the ICA (20, 40 μM) groups, with the highest cell wound healing rate observed at an ICA concentration of 40 μM. This suggests that ICA (20, 40 μM) can significantly enhance the migration ability of trophoblast cells.

[0074] Cell migration and invasion were assessed using Transwell assays: Migration assays used uncoated Transwell chambers (8 μm pores), while invasion assays used the upper chambers pre-coated with Matrigel (diluted to approximately 1:8 with serum-free medium) at 4°C and incubated at 37°C for 1–2 hours to solidify. HTR-8 / SVneo cells were digested and collected, resuspended in serum-free medium, and counted. Cell suspension was added to the upper Transwell chambers, and 1 × 10⁶ cells were seeded per chamber for migration assays. 4 2 × 10⁶ cells per chamber for invasion experiments 4 Cells were used. The upper chamber was filled with serum-free medium containing specified concentrations of ICA (0, 10, 20, 40 μM). The lower chamber contained 600 μL of complete medium with 20% FBS as a chemokine source. Twenty-four hours after ICA intervention, the chamber was fixed by immersing the cells in 4% paraformaldehyde at room temperature for 20 minutes. Crystal violet staining was then performed for 15 minutes, and uninvaded cells were removed with moistened cotton swabs. Cells were photographed under an inverted microscope and counted using ImageJ software. Experimental data are shown below. Figure 10 As shown, Figure 10 This diagram illustrates the cell migration and invasion results under ICA intervention in each group. A shows the Transwell migration and invasion results of HTR-8 / SVneo cells after intervention with different concentrations of ICA, and B shows the statistical results of the number of migrating and invasive cells in each group. The results show that the number of migrating and invasive cells increased after ICA intervention, with a significant increase after treatment with 40 μM ICA. This further confirms that ICA helps promote trophoblast cell migration and invasion. As shown in the figure, after intervention with 40 μM ICA, the number of cells crossing the membrane significantly increased compared to the control group (P<0.01), indicating that ICA can effectively enhance the chemotactic migration ability of trophoblast cells. Compared with the control group, treatment with 40 μM ICA significantly increased the number of cells penetrating the Matrigel barrier (P<0.01), which strongly demonstrates that ICA can enhance the invasive ability of trophoblast cells, a key function necessary for successful embryo implantation and placental formation.

[0075] Cell cycle distribution analysis (flow cytometry): HTR-8 / SVneo cells were cultured at 3 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 6-well cell culture plates. After cell attachment, they were treated with different concentrations of ICA (10 μM, 20 μM, and 40 μM) for 24 hours, with a control group included. After treatment, cells were digested with EDTA-free trypsin and gently washed twice with pre-chilled phosphate-buffered saline (PBS) to remove drug residues. Subsequently, the cell cycle assay kit (Shanghai Beyotime Biotechnology Co., Ltd.) was followed as follows: first, cells were fixed with pre-chilled 70% ethanol at 4°C for at least 2 hours; after fixation, the ethanol was removed by centrifugation, and the cells were resuspended and washed with PBS; then, a staining working solution containing propidium iodide (PI) staining solution and RNase A was added, and the cells were incubated at 37°C in the dark for 30 minutes to allow the PI dye to specifically intercalate into the nuclear DNA double strand. Finally, flow cytometry was used to analyze the stained single-cell suspensions. By analyzing the distribution of cellular DNA content, the cell population was divided into G0 / G1 phase (pre-DNA synthesis), S phase (DNA synthesis phase), and G2 / M phase (post-DNA synthesis / mitosis phase). The results were processed and plotted using flow cytometry data analysis software (FlowJo).

[0076] Experimental data such as Figure 11 As shown, Figure 11 This diagram illustrates the proportions of cells in the G0 / G1, S, and G2 / M phases for each group. The left diagram shows the flow cytometry results for each group's cell cycle, while the right diagram shows the statistical results of the proportions of HTR-8 / SVneo cells in the G0 / G1, S, and G2 / M phases for each group. The diagram shows that, compared to the control group, the proportion of cells in the G2 / M phase was significantly increased in the ICA-treated cell population (P<0.05), indicating that ICA can promote more cells to enter the mitotic phase, thereby accelerating the cell proliferation cycle. The results show that the proportion of cells in the G2 / M phase significantly increased after intervention with different concentrations of ICA. This indicates that ICA can promote DNA synthesis and division in trophoblast cells, thereby enhancing the proliferative capacity of trophoblast cells at the maternal-fetal interface.

[0077] Icariin regulates the maternal-fetal interface through multiple targets, promotes trophoblast function and tissue repair, and prevents recurrent miscarriage.

[0078] This invention reveals the mechanism of action of icariin in preventing recurrent miscarriage through in vitro and in vivo experimental systems, mainly manifested in the following four synergistic effects:

[0079] 1. Significantly reduces embryo loss rate and improves pregnancy outcomes (Example 1)

[0080] In the classic CBA / J×DBA / 2J recurrent miscarriage mouse model, icariin intervention (low-dose and high-dose intervention groups) significantly reduced embryo loss rate, with effects comparable to the YAP pathway agonist XUM-MP-1. This indicates that icariin has a clear protective effect against pregnancy in vivo and can effectively improve pregnancy outcomes in the recurrent miscarriage model.

[0081] 2. Repairing pathological damage to maternal-fetal interface tissues and promoting decidual cell proliferation (Example 2)

[0082] Histopathological examination (HE staining) showed that icariin could reverse the pathological changes in decidual tissue, such as disordered arrangement, cell necrosis, and inflammatory cell infiltration, caused by recurrent miscarriage. Immunofluorescence results further indicated that icariin significantly upregulated the expression of proliferation markers Ki-67 and PCNA in decidual tissue, suggesting that it can promote decidual cell proliferation and tissue repair, and improve the microenvironment at the maternal-fetal interface.

[0083] 3. Regulating the Hippo signaling pathway to inhibit the excessive phosphorylation of key proteins (Example 3)

[0084] Western blot results showed that in recurrent miscarriage, the phosphorylation levels of key Hippo pathway proteins (p-LATS1, p-MST, p-YAP, p-TAZ) were significantly increased in mouse placental tissue and HTR-8 / SVneo cells, indicating abnormal activation of this pathway. After intervention with icariin, the phosphorylation levels of these proteins significantly decreased, returning to near-normal levels. This indicates that icariin promotes cell proliferation, migration, and survival by inhibiting the overactivation of the Hippo pathway and relieving the inhibition of downstream YAP / TAZ transcriptional activity.

[0085] 4. Enhances the migration, invasion, and proliferation capabilities of human extravillous trophoblasts (Example 4)

[0086] In vitro functional experiments further confirmed that, within a safe dosage range (10–40 μM), scratch healing assays and Transwell assays showed that ICA significantly enhanced the migration and invasion abilities of HTR-8 / SVneo cells in a concentration-dependent manner. Flow cytometry showed that ICA promoted cell cycle progression to the G2 / M phase, indicating that it accelerates cell division and proliferation. Immunofluorescence assays showed that ICA upregulated the expression of PCNA and Ki-67, further verifying its promoting effect on trophoblast proliferation.

[0087] In summary, icariin prevents and treats recurrent miscarriage through a multi-target, multi-stage regulatory mechanism. At the tissue level, it repairs pathological damage to the decidual tissue at the maternal-fetal interface and promotes cell proliferation; at the cellular level, it enhances the migration, invasion, and proliferation capabilities of trophoblasts; at the molecular level, it inhibits abnormal activation of the Hippo signaling pathway, restores YAP / TAZ transcriptional activity, and promotes cell survival and function. These effects collectively improve the microenvironment for embryo implantation and placental formation, reducing embryo loss rates and thus effectively preventing and treating recurrent miscarriage. This invention provides solid experimental evidence and mechanistic explanation for icariin as a novel anti-miscarriage drug, possessing significant clinical application value and development prospects.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of icariin or its medicinal salt in the preparation of drugs for preventing and treating recurrent miscarriage.

2. The application of icariin or its medicinal salt according to claim 1 in the preparation of drugs for preventing and treating recurrent miscarriage, characterized in that, The drug for preventing recurrent miscarriage uses icariin or its medicinal salt as the sole active ingredient.

3. The application of icariin or its medicinal salt according to claim 1 in the preparation of drugs for preventing and treating recurrent miscarriage, characterized in that, The drug for preventing recurrent miscarriage is made from icariin or its pharmaceutical salt and medically acceptable excipients.

4. The application of icariin or its medicinal salt according to claim 1 in the preparation of drugs for preventing and treating recurrent miscarriage, characterized in that, The medications for preventing recurrent miscarriage include suspensions, granules, capsules, tablets, powders, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches, or drops.

5. The application of icariin or its medicinal salt according to claim 1 in the preparation of drugs for preventing and treating recurrent miscarriage, characterized in that, The administration methods for the drugs for preventing recurrent miscarriage include oral administration, intravenous administration, intraperitoneal injection, intramuscular injection, subcutaneous injection, sublingual administration, transdermal administration, or rectal suppository administration.

6. The application of icariin or its medicinal salt according to claim 1 in the preparation of drugs for preventing and treating recurrent miscarriage, characterized in that, The drug for preventing recurrent miscarriage is made from icariin or its medicinal salt and other drugs for treating recurrent miscarriage.

7. The application of icariin or its medicinal salt according to claim 1 in the preparation of drugs for preventing and treating recurrent miscarriage, characterized in that, The drug for preventing recurrent miscarriage is a pharmaceutical composition containing icariin or its pharmaceutical salt or derivatives.

Images