Application of HIF-1alpha agonist / p53 inhibitor in treatment of URSA

Abstract

The invention relates to an application of an HIF-1alpha agonist / p53 inhibitor in treatment of URSA, in particular to an application in preparation of a medicine for treating URSA, especially SIRT1 gene-mediated unexplained recurrent abortion.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of HIF-1α agonists / p53 inhibitors in the treatment of URSA. Background Technology

[0002] Recurrent spontaneous abortion (RSA) refers to two or more consecutive spontaneous abortions before 28 weeks of gestation with the same sexual partner, with an incidence rate of approximately 2%–5%, and this rate is increasing annually. The etiology of RSA is complex, involving known factors such as genetic defects, immune dysfunction, infection, endocrine disorders, and thrombotic tendency. However, the cause remains unclear in about 50% of cases, termed unexplained recurrent spontaneous abortion (URSA). Given that the pathological mechanisms of URSA have not been systematically elucidated, modern medicine has significant limitations in its prevention and treatment, and its safety is controversial. It is difficult to effectively address the problem of repeated pregnancy loss, potentially negatively impacting patients' physical and mental health, family relationships, and even social stability. Therefore, in-depth elucidation of the molecular mechanisms of URSA and identification of its key pathological links will provide important scientific support for optimizing precise prevention and treatment strategies for URSA.

[0003] Currently, clinical treatment strategies for URSA are mainly based on interventions targeting the underlying causes, but these have significant limitations: Immunotherapy, such as active lymphocyte immunotherapy or intravenous immunoglobulin, has controversial efficacy, lacks high-level evidence-based medicine support, and carries potential safety risks and high costs; Anticoagulation therapy, such as low molecular weight heparin and aspirin, is the standard treatment for patients in prethrombotic states like antiphospholipid syndrome, but it is ineffective for nonthrombotic URSA patients; Lutein support, such as progesterone supplementation, is a common method, but it is mainly suitable for patients with endocrine insufficiency and has limited efficacy for most URSA patients; Empirical treatments, such as glucocorticoids and immunosuppressants, lack specificity, have significant side effects, and uncertain efficacy.

[0004] Current research indicates that SIRT1 deficiency leads to embryonic developmental disorders and consequently pathological pregnancies. Animal experiments have confirmed that activating SIRT1 expression in vivo can improve abnormal embryonic development. Furthermore, SIRT1 expression is decreased in the decidual tissue during the implantation window of patients with recurrent implantation failure, and oxidative stress homeostasis is disrupted, ultimately affecting the decidualization process of ESCs. Therefore, elucidating the specific function of SIRT1 in the female reproductive environment and developing novel targeted therapies to reverse URSA caused by SIRT1 deficiency or insufficiency has become a key technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0005] The problem the invention aims to solve This invention aims to address at least one of the technical problems existing in the prior art. This invention intends to explore the inhibitory effect of HIF-1α agonists / p53 inhibitors on URSA and the related matrix, providing new insights for the clinical treatment of URSA.

[0006] Solution for solving the problem On the one hand, the present invention provides the use of HIF-1α agonists in the preparation of medicaments for the treatment and / or prevention of unexplained recurrent miscarriage.

[0007] Preferably, the drug can promote decidual angiogenesis.

[0008] Preferably, the unexplained recurrent miscarriage is SIRT1 gene-mediated unexplained recurrent miscarriage.

[0009] Preferably, the HIF-1α agonist is DMOG.

[0010] On the one hand, the present invention provides the use of p53 inhibitors in the preparation of medicaments for the treatment and / or prevention of unexplained recurrent miscarriage.

[0011] Preferably, the drug can regulate the autophagy / apoptosis balance of uterine cells.

[0012] Preferably, the unexplained recurrent miscarriage is SIRT1 gene-mediated unexplained recurrent miscarriage.

[0013] Preferably, the p53 inhibitor is Pifithrin-α.

[0014] On one hand, the present invention provides the application of a traditional Chinese medicine composition in the preparation of a drug for treating and / or preventing unexplained recurrent miscarriage, the traditional Chinese medicine composition comprising the following components and the weight parts of each component as follows: 5-15 parts of Salvia miltiorrhiza, 10-30 parts of Cuscuta chinensis, 10-20 parts of Taxillus chinensis, 10-20 parts of Astragalus membranaceus, 5-15 parts of donkey-hide gelatin, and 10-20 parts of Dipsacus asper.

[0015] Preferably, the traditional Chinese medicine composition comprises the following components and the weight parts of each component are as follows: 10 parts of Salvia miltiorrhiza, 20 parts of Cuscuta chinensis, 15 parts of Taxillus chinensis, 15 parts of Astragalus membranaceus, 10 parts of donkey-hide gelatin, and 15 parts of Dipsacus asper.

[0016] Preferably, the drug can regulate the autophagy / apoptosis balance of uterine cells and / or promote decidual angiogenesis.

[0017] Preferably, the unexplained recurrent miscarriage is SIRT1 gene-mediated unexplained recurrent miscarriage.

[0018] The effects of the invention This invention provides a method for constructing a uterine tissue-specific SIRT1 conditional gene knockout (CKO) mouse model, thereby systematically and multidimensionally revealing the specific molecular mechanisms by which the SIRT1 gene affects decidual angiogenesis and influences the autophagy / apoptosis balance and decidualization through the p53 pathway.

[0019] Furthermore, this invention has found that HIF-1α agonists, p53 inhibitors, and the aforementioned traditional Chinese medicine compositions can improve or treat URSA. Therefore, HIF-1α agonists, p53 inhibitors, and the aforementioned traditional Chinese medicine compositions show promise as novel therapeutic agents for URSA. Attached Figure Description

[0020] Figure 1 This invention provides a strategy for constructing CKO heterozygous and homozygous mice. Figure 2 SIRT1 was obtained through breeding for this invention. flox / - Cre + / － The image shows the results of mouse PCR gene identification, where M represents the marker and the red box represents SIRT1. flox / - Cre + / － The yellow box indicates that Cre was not identified. + / － Amplified band, only SIRT1flo x / - The green box represents WT mice, and the blue box represents samples loaded with water and TE (used to detect contamination). Figure 3 SIRT1 was obtained through breeding for this invention. flox / flox Cre + / － The image shows the results of mouse PCR gene identification, where M represents the marker and the red box represents SIRT1. flox / flox Cre + / － The green box represents WT mice, and the blue box represents samples loaded with water and TE (used to detect contamination). Figure 4 Image of the mouse vaginal plug from Example 2; Figure 5 This is a schematic diagram showing the results of Western Blot (WB) detection of SIRT1 protein expression in decidual tissues of mice in the ZC and CM groups in Example 2. Figure 6 This is a schematic diagram showing the expression and distribution of SIRT1 in the decidual tissues of mice in the ZC and CM groups as detected by immunohistochemistry in Example 2. Figure 7 This is a schematic diagram showing the number of blastocysts in groups ZC, CM, and U in Example 2; Figure 8 For example, calculate the number of implantation sites and embryo loss rate for groups ZC, CM, and U in Example 2; Figure 9 The image shows the decidual tissue morphology of mice in groups ZC, CM, and U on day 8 of pregnancy, as observed by HE staining in Example 2. Figure 10 This is a schematic diagram showing the results of WB detection of PRL and IGFBP-1 protein expression in decidual tissues of mice in groups ZC, CM, and U on day 8 of pregnancy in Example 2. Figure 11 This is a schematic diagram showing the results of ELISA determination of the expression level of IGFBP-1, a decidualization marker, in the peripheral blood of three groups of mice on days 4, 6, and 8 of pregnancy in Example 3. Figure 12 This is a schematic diagram showing the results of real-time fluorescence PCR determination of the mRNA expression levels of decidualization markers PRL and IGFBP-1 in the decidual tissues of three groups of mice on days 4, 6, and 8 of pregnancy in Example 3. Figure 13 This is a schematic diagram showing the results of WB assay in detecting the protein expression of PRL and IGFBP-1 in the decidual tissues of three groups of mice at gestational days 4, 6, and 8 in Example 3. Figure 14 This is a bar chart showing the percentage of PRL-positive area in the decidual tissue of three groups on day 8 of pregnancy, as determined by immunohistochemical detection in Example 3. Figure 15 This is a schematic diagram showing the results of ELISA measurements of the expression levels of HIF-1α, VEGFA, VEGFR2, and sFLT-1 in the peripheral blood of three groups of mice on days 4, 6, and 8 of gestation in Example 3. Figure 16 This is a schematic diagram showing the results of real-time fluorescence PCR determination of the mRNA expression levels of HIF-1α, VEGFA, VEGFR2, sFlt-1, and PLGF in the decidual tissues of three groups of mice on days 4, 6, and 8 of pregnancy in Example 3. Figure 17 This is a schematic diagram showing the results of Western blot analysis (WB) of the expression levels of angiogenesis-related proteins HIF-1α, VEGFA, VEGFR2, sFLT-1, and PLGF in the decidual tissue of three groups of mice on days 4, 6, and 8 of pregnancy in Example 3. Figure 18 The bar chart shows the expression of HIF-1α, VEGFA, and VEGFR2 in the decidual tissues of three groups of mice in Example 3, as well as the percentage of positive area. Figure 19 This is a schematic diagram showing the results of immunofluorescence detection of VEGFA expression in decidual tissue of three groups on day 6 of pregnancy in Example 3; Figure 20 This is a schematic diagram showing the results of immunofluorescence detection of VEGFA expression in decidual tissue of three groups on day 8 of pregnancy in Example 3; Figure 21Schematic diagram of the uterine embryonic status of pregnant mice in each group on days 6 and 8 of gestation; Figure 22 This is a schematic diagram showing the results of real-time fluorescence PCR detection of the expression levels of SIRT1, p53, BAX, BCL-2, PRL, and IGFBP-1 mRNA in the uterine tissues of three groups of mice on days 4, 6, and 8 of pregnancy in Example 3. Figure 23 This is a schematic diagram showing the results of Western blot analysis (WB) in Example 3 for detecting the protein levels of SIRT1, acetylated p53 (A-p53), p53, BAX, BCL-2, mTOR, p62, Beclin-1, LC3Ⅱ / Ⅰ, PRL, and IGFBP-1 in the uterine tissues of three groups of mice on days 4, 6, and 8 of gestation. * indicates P < 0.05 compared to the CKO model group; ns indicates P ≥ 0.05 compared to the CKO model group. Figure 24 This is a schematic diagram showing the results of ELISA measurements of p53, BAX, BAD, p62, mTOR, Beclin-1, and IGFBP-1 levels in the peripheral blood of three groups of mice on days 4, 6, and 8 of gestation in Example 3. Figure 25 This is a graph showing the results of HE staining assessment of uterine decidualization in three groups of mice on days 4, 6, and 8 of pregnancy in Example 3. Figure 26 This is a schematic diagram showing the immunohistochemical detection results of LC3B, p62, Beclin-1, BAX, BCL-2, and PRL expression in the uterine tissues of three groups of mice on days 4, 6, and 8 of pregnancy in Example 3. * indicates P < 0.05 compared to the CKO model group; ns indicates P ≥ 0.05 compared to the CKO model group. Figure 27 This is a schematic diagram showing the results of immunofluorescence detection of LC3B and Beclin-1 levels in uterine tissues of three groups of mice on days 4, 6, and 8 of pregnancy in Example 3. Figure 28 This is a schematic diagram illustrating the TUNEL staining detection of cell apoptosis in the uterine tissue of three groups of mice on days 4, 6, and 8 of pregnancy in Example 3. Figure 29 This is a schematic diagram of autophagy in the uterine tissue of three groups of mice observed by transmission electron microscopy on days 4, 6, and 8 of pregnancy in Example 3. Figure 30 Images of uterine tissue and embryonic morphology in the three groups of mice on day 8 of pregnancy in Example 4; Figure 31 The bar chart shows the abortion rates of the three groups of mice in Example 4; Figure 32 The images show the decidual tissue and vascular morphology of three groups of pregnant women on days 4-8 of pregnancy, as observed by HE staining in Example 4. Figure 33 This is a schematic diagram showing the results of ELISA determination of the expression level of IGFBP-1, a marker of decidualization in peripheral blood, in three groups in Example 4; Figure 34 This is a schematic diagram showing the results of real-time fluorescence PCR determination of the mRNA expression levels of decidualization markers PRL and IGFBP-1 in the decidual tissues of three groups of mice on days 4, 6, and 8 of pregnancy in Example 4. Figure 35 This is a schematic diagram showing the protein expression of PRL and IGFBP-1 in the decidual tissues of three groups at days 4, 6, and 8 of pregnancy detected by Western blot in Example 4. Figure 36 This is a bar chart showing the percentage of PRL-positive area in the decidual tissue of the three groups on day 8 of pregnancy, as determined by immunohistochemical staining in Example 4. Figure 37 This is a schematic diagram showing the results of ELISA measurements of the expression levels of HIF-1α, VEGFA, VEGFR2, and sFLT-1 in the peripheral blood of three groups of mice on days 4, 6, and 8 of gestation in Example 4. Figure 38 This is a schematic diagram of the ultrastructure of decidual tissue and vascular endothelial cells of three groups of pregnant mice on days 4, 6, and 8 of gestation in Example 4. Figure 39 This is a schematic diagram showing the results of real-time fluorescence PCR determination of the mRNA expression levels of HIF-1α, VEGFA, VEGFR2, sFLT-1, and PLGF in the decidual tissues of three groups of mice on days 4, 6, and 8 of pregnancy in Example 4. Figure 40 This is a schematic diagram showing the results of Western blot analysis of the expression levels of angiogenesis-related proteins VEGFA, VEGFR2, sFLT-1, and PLGF in three groups of mice on days 4, 6, and 8 of gestation in Example 4. Figure 41 The bar chart shows the expression of HIF-1α, VEGFA, and VEGFR2 in the decidual tissue of three groups of mice on day 8 of pregnancy, as well as the percentage of positive area, as detected by immunohistochemistry in Example 4. Figure 42 This is a schematic diagram of the co-localization results of vWF and VEGFR2 fluorescence in decidual tissue on day 8 of pregnancy in Example 4; Figure 43 This is a schematic diagram showing the immunofluorescence staining results of decidual tissues from three groups of mice on days 4, 6, and 8 of pregnancy, labeled with CD31 monoclonal antibody, in Example 4. Figure 44 This is a schematic diagram showing the results of Western blot analysis of the expression levels of HIF-1α and ac-HIF-1α proteins in the decidual tissue of three groups of mice on days 4, 6, and 8 of pregnancy in Example 4. Figure 45This is a schematic diagram showing the results of ELISA measurements of the expression levels of MDA and SOD, oxidative stress-related indicators, in the peripheral blood of mice in four groups (ZC, CM, H, and TM) on days 4, 6, and 8 of gestation in Example 4. Figure 46 This is a schematic diagram of the uterine embryonic status of pregnant mice in each group on days 6 and 8 of gestation in Example 4; Figure 47 This is a schematic diagram showing the results of real-time fluorescence PCR detection of the expression levels of p53, BAX, BCL-2, PRL, and IGFBP-1 mRNA in the uterine tissues of three groups of mice on days 4, 6, and 8 of pregnancy in Example 4. Figure 48 This is a schematic diagram showing the results of Western blotting (WB) analysis of protein levels of p53, BAX, BCL-2, mTOR, Beclin-1, p62, LC3Ⅱ / Ⅰ, IGFBP-1, and PRL in uterine tissues of three groups of mice on days 4, 6, and 8 of gestation. Figure 49 This is a schematic diagram showing the results of ELISA measurements of p53, BAX, BAD, mTOR, Beclin-1, p62, and IGFBP-1 levels in the peripheral blood of three groups of mice on days 4, 6, and 8 of gestation in Example 4. Figure 50 This is a schematic diagram of the uterine decidualization status of three groups of mice on days 4, 6, and 8 of pregnancy, as assessed by HE staining in Example 4. Figure 51 This is a schematic diagram showing the results of immunohistochemical detection of the expression of BAX, BCL-2, Beclin-1, LC3B, p62, and PRL in the uterine tissues of three groups of mice on days 4, 6, and 8 of pregnancy in Example 4. Figure 52 Schematic diagram showing the results of immunofluorescence detection of LC3B and Beclin-1 levels in uterine tissues of three groups of mice on days 4, 6, and 8 of gestation. Figure 53 This is a schematic diagram showing the results of TUNEL staining in the uterine tissue of three groups of mice on days 4, 6, and 8 of pregnancy, as described in Example 4. Figure 54 This is a schematic diagram of autophagy in the uterine tissue of three groups of mice observed by transmission electron microscopy on days 4, 6, and 8 of pregnancy in Example 4. In this application, * in the figures represents P<0.05 compared to the CKO model group; ns represents P≥0.05 compared to the CKO model group. Detailed Implementation

[0021] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0022] The inventors of this invention, through extensive and in-depth research, used C57BL / 6-SIRT1-em1 (flox) Cya mice (genotype SIRT1) flox / flox ) and PR-Cre (genotype Cre) + / － A conditional knockout mouse model of the SIRT1 gene in uterine stromal cells was successfully constructed using tool mice hybridization. This model provides a highly accurate animal model for studying the biological function of SIRT1 in the endometrial stroma, directly focusing on the target gene under controllable conditions, and allowing for a clearer and more intuitive observation of the target gene's role in the disease process. This is used to study the effects of promoting angiogenesis, autophagy, apoptosis balance, and decidualization in URSA, and to screen potential drugs to regulate these mechanisms and improve pregnancy outcomes. Furthermore, the inventors discovered that HIF-1α agonists, p53 inhibitors, and the aforementioned traditional Chinese medicine combinations can improve or treat URSA. Based on this, this invention was completed.

[0023] This invention provides a method for constructing a SIRT1 conditional gene knockout mouse model, the method comprising: using a mouse model with the SIRT1 genotype... flox / flox Mice were bred with Cre tool mice, and the offspring were then compared with the SIRT1 genotype. flox / flox Mice were bred to obtain the SIRT1 conditional gene knockout mouse model. The strategy for constructing the SIRT1 conditional gene knockout mouse model of this invention is described below. Figure 1 .

[0024] In this invention, the genotype is SIRT1. flox / flox The mice and Cre tool mice were purchased from commercial sources or constructed using known methods. Preferably, the genotype used in this application is SIRT1. flox / flox Mice and Cre tool mice were purchased from Cyagen Biosciences.

[0025] This invention also provides the application of the SIRT1 conditional gene knockout mouse model constructed by the above-described method in screening drugs for treating diseases related to SIRT1 gene defects.

[0026] In some embodiments, the disease associated with SIRT1 gene deficiency is unexplained recurrent miscarriage.

[0027] On the one hand, the present invention provides the use of HIF-1α agonists in the preparation of medicaments for the treatment and / or prevention of unexplained recurrent miscarriage.

[0028] In some embodiments, the drug can promote decidual angiogenesis.

[0029] In some embodiments, the unexplained recurrent miscarriage is SIRT1 gene-mediated unexplained recurrent miscarriage.

[0030] In some implementations, the HIF-1α agonist is DMOG.

[0031] On the one hand, the present invention provides the use of p53 inhibitors in the preparation of medicaments for the treatment and / or prevention of unexplained recurrent miscarriage.

[0032] In some embodiments, the drug can regulate the autophagy / apoptosis balance of uterine cells.

[0033] In some embodiments, the unexplained recurrent miscarriage is SIRT1 gene-mediated unexplained recurrent miscarriage.

[0034] In some implementations, the p53 inhibitor is Pifithrin-α.

[0035] On one hand, the present invention provides the application of a traditional Chinese medicine composition in the preparation of a drug for treating and / or preventing unexplained recurrent miscarriage, the traditional Chinese medicine composition comprising the following components and the weight parts of each component as follows: 5-15 parts of Salvia miltiorrhiza, 10-30 parts of Cuscuta chinensis, 10-20 parts of Taxillus chinensis, 10-20 parts of Astragalus membranaceus, 5-15 parts of donkey-hide gelatin, and 10-20 parts of Dipsacus asper.

[0036] In some embodiments, the weight parts of Danshen in the traditional Chinese medicine composition are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 parts.

[0037] In some embodiments, the weight parts of Cuscuta chinensis in the traditional Chinese medicine composition are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 parts.

[0038] In some embodiments, the weight parts of mulberry mistletoe in the traditional Chinese medicine composition are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 parts.

[0039] In some embodiments, the weight parts of Astragalus membranaceus in the traditional Chinese medicine composition are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 parts.

[0040] In some embodiments, the weight parts of donkey-hide gelatin in the traditional Chinese medicine composition are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 parts.

[0041] In some embodiments, the weight parts of Dipsacus asperoides in the traditional Chinese medicine composition are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 parts.

[0042] In some embodiments, the traditional Chinese medicine composition includes the following components and the weight parts of each component as follows: 8-12 parts of Salvia miltiorrhiza, 18-22 parts of Cuscuta chinensis, 12-18 parts of Taxillus chinensis, 12-18 parts of Astragalus membranaceus, 8-12 parts of donkey-hide gelatin, and 12-18 parts of Dipsacus asper.

[0043] In some embodiments, the traditional Chinese medicine composition includes the following components and the weight parts of each component as follows: 10 parts of Salvia miltiorrhiza, 20 parts of Cuscuta chinensis, 15 parts of Taxillus chinensis, 15 parts of Astragalus membranaceus, 10 parts of donkey-hide gelatin, and 15 parts of Dipsacus asper.

[0044] In some embodiments, the drug can regulate the autophagy / apoptosis balance of uterine cells and / or promote decidual angiogenesis.

[0045] In some embodiments, the unexplained recurrent miscarriage is SIRT1 gene-mediated unexplained recurrent miscarriage.

[0046] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sam brook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor, Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0047] In this invention, SIRT1 is used to establish a SIRT1 conditional gene knockout mouse model. flox / floxMice and Cre tool mice were purchased from Cyagen Biosciences Co., Ltd., and were of the C57BL / 6J strain. All laboratory animals were SPF (Specific Pathogen Free) grade, and all animal experiments were conducted in accordance with the "Regulations on the Management of Laboratory Animals" promulgated by the state.

[0048] Example 1

[0049] The method for constructing the SIRT1 conditional gene knockout mouse model of the present invention includes: (1) Male Cre obtained after sperm resuscitation + / － Mice and female Cre + / － Mice mating in the same cage or male WT mice mating with female Cre + / － Mice were mated together in the same cage with a male-to-female ratio of 1:2 to obtain a sufficient number of PR-Cre mice; (2) Male fluxed mice obtained after sperm resuscitation were mated with female fluxed mice in the same cage, or male WT mice were mated with female fluxed mice in the same cage, with a male-to-female ratio of 1:2 to expand the fluxed population, and SIRT1 was screened. flox / flox Homozygous mice.

[0050] (3) Male SIRT1 flox / flox Genotype mice and female Cre + / － Genotype mice mated in the same cage or male Cre + / － Genotype mice and female SIRT1 flox / flox Genotype mice were mated together in the same cage with a male-to-female ratio of 1:2 to screen for SIRT1. flox / － Cre + / － Genotype of mouse.

[0051] (4) Male SIRT1 flox / － Cre + / － Genotype mice and female SIRT1 flox / flox Mice were mated in the same cage with a male-to-female ratio of 1:2 to screen for female SIRT1 mice. flox / flox Cre + / － Genotype mice are the SIRT1 conditional gene knockout mouse models that need to be constructed.

[0052] All mice were housed in an SPF-grade environment with strictly controlled temperature, humidity, noise, and ventilation, and were provided with nutritional supplements. Offspring mice were separated, ear-tagged, and toe-clipping samples were taken at 21 days of age. Genomic DNA was extracted using a kit method, and PCR amplification was performed using specific primers. Genotypes were identified by agarose gel electrophoresis. The flox and Cre primer sequences verified by PCR are shown in Table 1. The SIRT1 mice bred in this example... flox / -Cre + / － Mouse PCR gene identification results are shown in Figure 2 SIRT1 bred in this embodiment flox / flox Cre + / － Mouse PCR gene identification results are shown in Figure 3 .

[0053] Table 1

[0054] Example 2 The method for studying abortion and decidualization in the SIRT1 conditional gene knockout mouse model includes the following steps: S1, Genotype identified as SIRT1 flox / flox Cre + / － Mice were used as the experimental model group (CM group), with the genotype SIRT1. flox / flox Cre － / － Mice were used as the control group (ZC group). CBA / J female mice were mated with DBA / 2 male mice in a 1:2 male-to-female ratio to establish an URSA pregnancy model (U group). In the CM and ZC groups, the estrous cycle of female mice was observed via vaginal smears. During proestrus, female mice were mated with WT male mice to establish a pregnancy model. Mice in all groups were mated at 6 PM with a male-to-female ratio of 1:2. The vaginal plugs were examined at 8 AM the following day. After moistening the plugs with saline solution, vaginal smears were prepared. The vaginal plugs (…) were found to be… Figure 4 If the fetus contains sperm, it is recorded as the first day of pregnancy (D1).

[0055] Five mice from each of the S2, ZC and CM groups were collected on days 4, 6 and 8 of gestation, and five mice from the U group were collected on day 8 of gestation. Fundus venous blood was collected from the mice, and uterine, decidual tissue, liver, spleen and kidney organs were collected.

[0056] S3. Western blotting (WB) was used to detect the expression of SIRT1 protein in the decidual tissue of mice in the ZC and CM groups.

[0057] S4. Immunohistochemical staining was used to detect the expression and distribution of SIRT1 in the decidual tissues of mice in the ZC and CM groups.

[0058] S5. Visually observe the number of blastocysts in the ZC, CM, and U groups, calculate the number of implantation sites and the embryo loss rate, and observe the loss of mouse embryos after SIRT1 gene knockout.

[0059] S6 and HE staining were used to observe the morphology of decidual tissue in mice on day 8 of pregnancy in groups ZC, CM and U.

[0060] The protein expression of PRL and IGFBP-1 in the decidual tissue of mice in the ZC, CM and U groups on day 8 of pregnancy was detected by S7 and WB methods.

[0061] The expression results of SIRT1 protein in the decidual tissue of mice in each group are shown in the figure. Figures 5 to 6 The number of blastocysts in the three groups of mice is shown in the figure. Figure 7 Implantation site number and embryo loss rate are shown in [reference]. Figure 8 Morphological images of decidual tissue in three groups of mice at 8 days of gestation are shown below. Figure 9 The protein expression levels of PRL and IGFBP-1 in the decidual tissue of the three groups of mice on day 8 of gestation are shown in the figure. Figure 10 .

[0062] The results above indicate that SIRT1 protein expression was not observed in the CM group. Immunohistochemistry showed that SIRT1 expression was successfully knocked out in the uterine stromal cells of the CM group mice, with no obvious positive signal detected. Phenotypic analysis on day 8 of gestation (D8) showed that the embryo loss rate in the CM group was significantly higher than that in the ZC group, and the phenotype was similar to that of the U group. HE staining showed no obvious embryos in the uterine tissue of the CM group, but significant proliferation of uterine stromal cells was observed, although only a small number of uterine stromal cells decidualized into decidual cells. No obvious placental structure was observed, and only a small number of microvessels were visible in the endometrium. Western blot analysis further showed that the protein expression levels of the key decidualization markers PRL and IGFBP-1 were significantly downregulated in the CM group. These results collectively confirm that the SIRT1 conditional gene knockout mouse model successfully constructed in this invention is similar to the core pathological phenotype of recurrent miscarriage, providing a reliable tool for subsequent mechanism research.

[0063] Example 3 To elucidate the specific mechanism of action of SIRT1 in recurrent miscarriage, this invention utilizes the CKO model constructed in Example 1 for systematic analysis. The experiment established a normal control group (ZC group) and a CKO model group (CM group), and interventions were initiated with the HIF-1α agonist DMOG (40 mg / kg / d intraperitoneal injection) and the p53 inhibitor Pifithrin-α (2.2 mg / kg / d intraperitoneal injection), respectively.

[0064] The research method for studying the role of the SIRT1 gene in decidual angiogenesis includes the following steps: S1. Based on the above method for constructing the SIRT1 conditional knockout mouse model, construct ① a conditional knockout (CKO) model group (CM group): genotype SIRT1. flox / flox Cre + / － ① Mice with SIRT1 genotype; ② Normal control group (ZC group): genotype SIRT1 flox / flox Cre － / －③ HIF-1α agonist group (H group): CKO model mice were administered HIF-1α agonist DMOG (40 mg / kg / d) via intraperitoneal injection.

[0065] S2. From the first day of pregnancy confirmed by the discovery of vaginal plugs, designated as D1, mice were given intraperitoneal injections of physiological saline (40 mg / kg / d) and DMOG (40 mg / kg / d) in the ZC and CM groups at 9:00 AM every day, once daily until D8. Fundus venous blood and decidual tissue were collected from mice on D4, D6, and D8.

[0066] The decidualization level of the three groups of mice was detected.

[0067] S3. The expression levels of IGFBP-1, a decidualization marker, in the peripheral blood of three groups of mice on days 4, 6, and 8 of gestation were determined by ELISA. The results are shown in [Figure 3]. Figure 11 .

[0068] S4. The mRNA expression levels of decidualization markers PRL and IGFBP-1 in the decidual tissues of three groups of mice on days 4, 6, and 8 of gestation were determined by real-time fluorescence PCR. The results are shown in the table below. Figure 12 .

[0069] The protein expression of PRL and IGFBP-1 in the decidual tissues of three groups of mice at days 4, 6, and 8 of pregnancy was detected by S5 and Western blotting. The results are shown in the figure. Figure 13 .

[0070] S6. Immunohistochemical detection of the percentage of PRL-positive area in decidual tissue of the three groups on day 8 of pregnancy. Results are shown in […]. Figure 14 .

[0071] Furthermore, the decidual angiogenesis status of the three groups of mice was examined.

[0072] S7. The expression levels of HIF-1α, VEGFA, VEGFR2, and sFLT-1 in peripheral blood of three groups of mice on days 4, 6, and 8 of gestation were determined by ELISA. The results are shown in [Figure 7]. Figure 15 .

[0073] S8. The mRNA expression levels of HIF-1α, VEGFA, VEGFR2, sFlt-1, and PLGF in the decidual tissue of three groups of mice on days 4, 6, and 8 of pregnancy were determined by real-time fluorescence PCR. The results are shown in [Figure 8]. Figure 16 .

[0074] The expression levels of angiogenesis-related proteins HIF-1α, VEGFA, VEGFR2, sFLT-1, and PLGF in decidual tissues of three groups of mice on days 4, 6, and 8 of gestation were detected by S9 and Western blotting. Results are shown in [Figure 1]. Figure 17 .

[0075] S10. Immunohistochemical staining was performed on the expression of HIF-1α, VEGFA, and VEGFR2 in the decidual tissues of the three groups of mice, and the percentage of positive areas was compared (see results). Figure 18 The observation was conducted during the period when decidualization and angiogenesis were most pronounced, specifically on day 8 of gestation.

[0076] S11. Combining immunohistochemical analysis, immunofluorescence was used to observe the expression of VEGFA in decidual tissues of the three groups on days 6 and 8 of pregnancy. The expression level of VEGFA in decidual tissues of the three groups on day 6 of pregnancy observed by immunofluorescence is shown in [the table below]. Figure 19 Immunofluorescence observation of VEGFA expression levels in decidual tissue of three groups on day 8 of pregnancy is shown in the figure. Figure 20 .

[0077] The research method for studying the mechanism by which the SIRT1 gene affects autophagy / apoptosis balance and decidualization through the p53 pathway includes the following steps: S1. Based on the above method for constructing the SIRT1 conditional gene knockout mouse model, construct ① the CKO model group (CM group): genotype SIRT1. flox / flox Cre + / － ① Mice with SIRT1 genotype; ② Normal control group (ZC group): genotype SIRT1 flox / flox Cre － / － Mice; ③ P53 inhibitor group, half of each of the three groups were randomly selected as the autophagy detection group, and the other half as the apoptosis and decidualization detection group.

[0078] S2. From the first day of pregnancy confirmation upon discovery of vaginal plugs to the day of tissue collection, the p53 inhibitor group received an intraperitoneal injection of 2.2 mg / kg / day of Pifithrin-α, while the normal group and CKO model group received an equal volume of saline intraperitoneal injection. Four hours before tissue collection, the autophagy detection group (detecting LC3, p62, Beclin-1, and mTOR) received an intraperitoneal injection of leucopeptide 20 mg / kg, while the apoptosis and decidualization detection group received an equal volume of saline intraperitoneal injection. Fundus venous blood and decidual tissue were collected from mice in all groups on days 4, 6, and 8.

[0079] Furthermore, the effects of the SIRT1 gene on autophagy / apoptosis balance and decidualization via the p53 pathway were verified.

[0080] S3. Observe the condition of the uterine embryos in each group of pregnant mice on days 6 and 8 of gestation. The results are as follows: Figure 21 As shown.

[0081] S4. The expression levels of SIRT1, p53, BAX, BCL-2, PRL, and IGFBP-1 mRNA in uterine tissues of three groups of mice on days 4, 6, and 8 of pregnancy were detected by real-time fluorescence PCR. The results are as follows: Figure 22 As shown.

[0082] The protein levels of SIRT1, p53, acetylated p53 (A-p53), BAX, BCL-2, mTOR, p62, Beclin-1, PRL, IGFBP-1, and LC3Ⅱ / Ⅰ in the uterine tissues of three groups of mice on days 4, 6, and 8 of gestation were detected by Western blotting (S5) and Western blotting (WB). The results are as follows: Figure 23 As shown.

[0083] S6. The levels of p53, BAX, BAD, p62, mTOR, Beclin-1, and IGFBP-1 in the peripheral blood of three groups of mice on days 4, 6, and 8 of gestation were measured by ELISA. The results are as follows: Figure 24 As shown.

[0084] S7 and HE staining were used to assess the decidualization of the endometrium in the three groups of mice on days 4, 6, and 8 of gestation. The results are as follows: Figure 25 As shown.

[0085] S8. Immunohistochemical detection of LC3B, Beclin-1, and BCL-2 in uterine tissues of three groups of mice on days 4, 6, and 8 of gestation. The expressions for PRL, p62, and BAX are as follows: Figure 26 As shown.

[0086] S9. Immunofluorescence assay was performed on the levels of LC3B and Beclin-1 in the uterine tissues of the three groups of mice on days 4, 6, and 8 of gestation. The results are as follows: Figure 27 As shown.

[0087] S10 and TUNEL staining were used to detect cell apoptosis in the uterine tissues of mice on days 4, 6, and 8 of gestation in the three groups of mice. The results are as follows: Figure 28 As shown.

[0088] S11. Transmission electron microscopy was used to observe autophagy in the uterine tissue of three groups of mice on days 4, 6, and 8 of gestation. The results are as follows: Figure 29 As shown.

[0089] ELISA, real-time fluorescence PCR, Western blotting, and immunohistochemistry / fluorescence assays revealed that, compared to the ZC group, the CM group showed significantly downregulated expression of decidualization markers (PRL, IGFBP-1), a slow increase in decidualization levels, and impaired decidual angiogenesis. HIF-1α and key angiogenesis factors (VEGFA, VEGFR2) expression were also impaired. Simultaneously, the CM group exhibited abnormally elevated levels of p53 and A-p53, increased apoptosis, and decreased autophagy and decidualization. After administration of an HIF-1α agonist, both angiogenesis and decidualization levels showed some recovery. Intervention with a p53 inhibitor effectively reversed excessive activation of the p53 pathway, restored the autophagy / apoptosis balance, and improved decidualization. These results indicate that SIRT1 deficiency may impair decidual angiogenesis through the HIF-1α / VEGF pathway and disrupt the homeostasis of autophagy and apoptosis through the p53 pathway, jointly leading to pregnancy failure, thus fully revealing the core regulatory network of SIRT1 in maintaining pregnancy.

[0090] Example 4 This study investigates the effects of a kidney-tonifying and blood-activating formula on decidual angiogenesis in SIRT1 conditional gene knockout mice, as well as its role in regulating autophagy / apoptosis balance and improving decidualization.

[0091] The kidney-tonifying and blood-activating formula comprises the following components: 10g of Salvia miltiorrhiza, 20g of Cuscuta chinensis, 15g of Taxillus chinensis, 15g of Astragalus membranaceus, 10g of donkey-hide gelatin, and 15g of Dipsacus asper. Preparation method and administration: Prepare the raw materials according to the above weight proportions. Mix the raw materials and soak them in a clay pot for 30 minutes. Then add water to cover the herbs and simmer over medium heat until the liquid reaches approximately 200 ml. Pour out the liquid, add water again to cover the herbs, and simmer until approximately 200 ml of liquid remains. Pour out the liquid and mix it with the first decoction. This is the kidney-tonifying and blood-activating formula.

[0092] The study on the effect of a kidney-tonifying and blood-activating formula on decidual angiogenesis in SIRT1 conditional gene knockout mice included the following steps: S1. Based on the above method for constructing the SIRT1 conditional gene knockout mouse model, construct ① the aforementioned SIRT1 conditional gene knockout mice (CKO model group, CM group): genotype SIRT1 flox / flox Cre + / － ① Mice with SIRT1 genotype; ② Normal control group (ZC group): genotype SIRT1 flox / flox Cre － / － ③ Traditional Chinese Medicine Group (TM Group): CKO model mice were treated with a traditional Chinese medicine formula for tonifying the kidney and promoting blood circulation by gavage.

[0093] S2. From the first day of pregnancy confirmed by the discovery of vaginal sac, at 9:00 AM every day, the ZC and CM groups were given saline by gavage, and the TM group was given a kidney-tonifying and blood-activating formula by gavage (0.15ml / 10g), once a day until the day of collection. The dosage of saline and traditional Chinese medicine groups was the same.

[0094] Furthermore, the effects of the kidney-activating and blood-activating formula on the morphology of the uterus and embryo, abortion rate, decidual tissue, and blood vessel morphology in the CKO mouse model were investigated.

[0095] S3. Compare the uterine tissue and embryonic morphology of the three groups of mice on day 8 of gestation. The results are as follows: Figure 30 .

[0096] S4. Count the number of mouse embryos in the three groups and calculate their miscarriage rate. The results are as follows: Figure 31 .

[0097] S5 and HE staining were used to observe the effects on decidual tissue and vascular morphology in the three groups during days 4-8 of pregnancy. The results are as follows: Figure 32 .

[0098] Furthermore, the effects of the kidney-tonifying and blood-activating formula on the decidualization level of mice at different gestational ages were investigated.

[0099] S6. The expression levels of IGFBP-1, a marker of decidualization in peripheral blood, were measured in three groups using ELISA. The results are as follows: Figure 33 .

[0100] S7. The mRNA expression levels of decidualization markers PRL and IGFBP-1 in the decidual tissue of three groups of mice on days 4, 6, and 8 of gestation were determined by real-time fluorescence PCR. The results are as follows: Figure 34 .

[0101] The protein expression of PRL and IGFBP-1 in decidual tissues of three groups at days 4, 6, and 8 of gestation was detected by S8 and Western blotting methods. The results are as follows: Figure 35 .

[0102] S9. Immunohistochemical detection of the percentage of PRL-positive area in the decidual tissue of the three groups on day 8 of pregnancy. Results are as follows: Figure 36 .

[0103] Furthermore, we investigated the effect of the kidney-tonifying and blood-activating formula on the angiogenesis status during decidualization.

[0104] The expression levels of HIF-1α, VEGFA, VEGFR2, and sFLT-1 in peripheral blood of three groups of mice on days 4, 6, and 8 of gestation were determined by S10 and ELISA. The effects of the kidney-tonifying and blood-activating formula on the levels of HIF-1α, VEGFA, VEGFR2, and sFLT-1 in peripheral blood were investigated. The results are as follows: Figure 37 .

[0105] S11. Transmission electron microscopy was used to observe the ultrastructure of vascular endothelial cells. The ultrastructure of decidual tissue and vascular endothelial cells in three groups of pregnant mice at days 4, 6, and 8 of gestation were observed under an electron microscope. The results are as follows: Figure 38 .

[0106] S12. Real-time fluorescence PCR was used to determine the mRNA expression levels of HIF-1α, VEGFA, VEGFR2, sFLT-1, and PLGF in the decidual tissue of three groups of mice on days 4, 6, and 8 of gestation. The effects of the kidney-tonifying and blood-activating formula on the levels of angiogenesis-related genes in mice at different gestational days were investigated. The results are as follows: Figure 39 .

[0107] S13. Western blotting was used to detect the expression levels of angiogenesis-related proteins VEGFA, VEGFR2, sFLT-1, and PLGF in three groups of mice on days 4, 6, and 8 of gestation. The results are as follows: Figure 40 S14. Immunohistochemical staining was performed on the expression of HIF-1α, VEGFA, and VEGFR2 in the decidual tissue of the three groups of mice on day 8 of pregnancy. The percentage of positive area was calculated. The results are as follows: Figure 41 S15, co-localization of vWF and VEGFR2 fluorescence in decidual tissue on day 8 of pregnancy, results as follows: Figure 42 .

[0108] S16. Decidual tissues from three groups of mice on days 4, 6, and 8 of gestation were labeled with CD31 monoclonal antibody and subjected to immunofluorescence staining. Microvessel density (MVD) was calculated based on CD31 expression levels. The results are as follows: Figure 43 .

[0109] Furthermore, the effects of the kidney-tonifying and blood-activating formula on the expression of HIF-1α and its acetylated ac-HIF-1α were investigated.

[0110] S17 and WB were used to detect the expression levels of HIF-1α and ac-HIF-1α proteins in the decidual tissue of three groups of mice on days 4, 6, and 8 of gestation. The results are as follows: Figure 44 .

[0111] S18. The expression levels of oxidative stress-related markers MDA and SOD in the peripheral blood of mice in the ZC, CM, H (same as the HIF-1α agonist group in Example 3), and TM groups on days 4, 6, and 8 of gestation were measured by ELISA. The results are as follows: Figure 45 .

[0112] Furthermore, the research methods for regulating the autophagy / apoptosis balance and improving decidualization using the kidney-tonifying and blood-activating formula include the following steps: S1. Based on the above method for constructing the SIRT1 conditional gene knockout mouse model, construct ① the CKO model group (CM group): genotype SIRT1. flox / flox Cre+ / － ① Mice with SIRT1 genotype; ② Normal control group (ZC group): genotype SIRT1 flox / flox Cre － / － ③ The traditional Chinese medicine group; half of each of the three groups was randomly selected as the autophagy detection group and the other half as the apoptosis and decidualization detection group.

[0113] S2. From the first day of pregnancy confirmation by vaginal plug discovery to the day of tissue collection, the normal group and CKO model group were given an equal volume of saline intraperitoneally, while the traditional Chinese medicine group was given 0.15 mL / 10 g of kidney-tonifying and blood-activating formula by gavage according to the mouse's body weight. Four hours before tissue collection, the autophagy detection group (detecting LC3, p62, Beclin-1, and mTOR) was given 20 mg / kg of leucopeptide intraperitoneally, while the apoptosis and decidualization detection group was given an equal volume of saline intraperitoneally. Fundus venous blood and decidual tissue were collected from mice in each group on days 4, 6, and 8.

[0114] Further, this study validated the kidney-tonifying and blood-activating formula's ability to regulate autophagy and apoptosis and improve decidualization based on the SIRT1-p53 pathway.

[0115] S3. Observe the condition of the uterine embryos in each group of pregnant mice on days 6 and 8 of gestation. The results are as follows: Figure 46 .

[0116] S4. The expression levels of p53, BAX, BCL-2, PRL, and IGFBP-1 mRNA in uterine tissues of three groups of mice on days 4, 6, and 8 of pregnancy were detected by real-time fluorescence PCR. The results are as follows: Figure 47 .

[0117] The protein levels of p53, BAX, BCL-2, LC3Ⅱ / Ⅰ, p62, mTOR, Beclin-1, PRL, and IGFBP-1 in uterine tissues of three groups of mice on days 4, 6, and 8 of gestation were detected by Western blotting (S5) and Western blotting (WB). The results are as follows: Figure 48 .

[0118] S6. The levels of p53, BAX, BAD, p62, mTOR, Beclin-1, and IGFBP-1 in the peripheral blood of three groups of mice on days 4, 6, and 8 of gestation were measured by ELISA. The results are as follows: Figure 49 .

[0119] S7 and HE staining were used to assess the decidualization of the uterus in the three groups of mice on days 4, 6, and 8 of gestation. The results are as follows: Figure 50 .

[0120] S8. Immunohistochemical detection of LC3B, Beclin-1, and BCL-2 in uterine tissues of three groups of mice on days 4, 6, and 8 of gestation. The expressions for PRL, p62, and BAX are as follows: Figure 51 .

[0121] S9. Immunofluorescence assay was performed on the levels of LC3B and Beclin-1 in the uterine tissues of the three groups of mice on days 4, 6, and 8 of gestation. The results are as follows: Figure 52 .

[0122] S10 and TUNEL staining were used to detect cell apoptosis in the uterine tissues of mice on days 4, 6, and 8 of gestation in the three groups of mice. The results are as follows: Figure 53 .

[0123] S11. Transmission electron microscopy was used to observe autophagy in the uterine tissue of three groups of mice on days 4, 6, and 8 of gestation. The results are as follows: Figure 54 .

[0124] The results showed that the Kidney-Nourishing and Blood-Activating Formula significantly improved pregnancy outcomes in CKO mice. Compared with the CM group, the miscarriage rate was significantly reduced, PRL and IGFBP-1 expression was increased, and decidual tissue morphology was improved. At the molecular mechanism level, the Kidney-Nourishing and Blood-Activating Formula reduced ac-HIF-1α protein levels, enhanced HIF-1α stability, upregulated the expression levels of HIF-1α, VEGFA, VEGFR2, and PLGF, downregulated the expression levels of anti-angiogenic factors, increased decidual tissue microvessel density, and improved vascular endothelial cell ultrastructure. Regarding cell homeostasis, the Kidney-Nourishing and Blood-Activating Formula reduced p53 and apoptosis levels, and increased low levels of autophagy and decidualization. This example demonstrates that the Kidney-Nourishing and Blood-Activating Formula can effectively improve decidualization defects, angiogenesis disorders, and autophagy / apoptosis imbalance in CKO model mice by regulating the p53 and HIF-1α pathways downstream of SIRT1, fully validating the effectiveness and application value of the research method established in this invention in screening and evaluating drugs for the treatment of recurrent miscarriage.

[0125] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. The use of HIF-1α agonists in the preparation of drugs for the treatment and / or prevention of unexplained recurrent miscarriage.

2. The application according to claim 1, characterized in that, The drug can promote decidual angiogenesis.

3. The application according to claim 1, characterized in that, The unexplained recurrent miscarriage refers to SIRT1 gene-mediated unexplained recurrent miscarriage. Preferably, the HIF-1α agonist is DMOG.

4. The use of p53 inhibitors in the preparation of drugs for the treatment and / or prevention of unexplained recurrent miscarriage.

5. The application according to claim 4, characterized in that, The drug can regulate the autophagy / apoptosis balance of uterine cells.

6. The application according to claim 4, characterized in that, The unexplained recurrent miscarriage refers to SIRT1 gene-mediated unexplained recurrent miscarriage. Preferably, the p53 inhibitor is Pifithrin-α.

7. The use of a traditional Chinese medicine composition in the preparation of a medicament for treating and / or preventing unexplained recurrent miscarriage, characterized in that, The traditional Chinese medicine composition includes the following components and their weight parts: 5-15 parts of Salvia miltiorrhiza, 10-30 parts of Cuscuta chinensis, 10-20 parts of Taxillus chinensis, 10-20 parts of Astragalus membranaceus, 5-15 parts of donkey-hide gelatin, and 10-20 parts of Dipsacus asper.

8. The application according to claim 7, characterized in that, The traditional Chinese medicine composition includes the following components and their weight proportions: 10 parts of Salvia miltiorrhiza, 20 parts of Cuscuta chinensis, 15 parts of Taxillus chinensis, 15 parts of Astragalus membranaceus, 10 parts of donkey-hide gelatin, and 15 parts of Dipsacus asper.

9. The application according to claim 7, characterized in that, The drug can regulate the autophagy / apoptosis balance of uterine cells and / or promote decidual angiogenesis.

10. The application according to claim 7, characterized in that, The unexplained recurrent miscarriage refers to SIRT1 gene-mediated unexplained recurrent miscarriage.

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