Compound for improving endometrial senescence

Abstract

The invention belongs to the technical field of natural products and pharmaceutical industry, and provides a compound for improving endometrial aging. The compound is ganoderic acid C2 and is screened by the following method: inducing an endometrial organ to form an aging endometrial model by using D-galactose; contacting a compound to be detected with the senescence endometrial model, and detecting the proportion of beta-galactosidase and / or EdU positive cells; and screening a compound capable of remarkably reducing beta-galactosidase and / or remarkably improving the proportion of EdU positive cells, namely the compound for improving endometrial aging. The model constructed by the invention can fully simulate the physiological status and pathological change of the senescent endometrium, the screening reliability is high, the period is short, the ethical problem is avoided, and a reliable method is provided for subsequent screening of natural products.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural products and the pharmaceutical industry, and relates to a compound for slowing down the aging of the reproductive system. Background Art

[0002] Disclosing the information of this background art is intended to enhance the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] The continuous increase in the global average life expectancy, the wide application of assisted reproductive technology (ART), and the changes in social economy and lifestyle have led to a gradual postponement of the average childbearing age of women. However, with the increase in age, the fertility of women gradually declines, and this phenomenon exacerbates the dual pressures of low birth rate and population structure imbalance. Therefore, finding effective strategies to improve the reproductive outcomes of high-fertility-age women is of great significance for optimizing the population structure and coping with the challenges of aging.

[0004] In traditional views, reproductive aging is mainly attributed to ovarian function decline, which is characterized by reduced ovarian reserve, decreased oocyte quality, insufficient hormone secretion, etc. These factors lead to reduced or abnormal embryo quality. Research has shown that with age, the depletion of ovarian reserve accelerates. At the same time, the decline in mitochondrial function, the accumulation of DNA damage, and the increase in chromosomal aneuploidy rate of oocytes also result in decreased oocyte quality. Ovarian function decline also leads to reduced secretion of estrogen and progesterone, causing poor endometrial development, which in turn affects embryo implantation and pregnancy maintenance. By methods such as controlled ovarian hyperstimulation, in vitro fertilization / intracytoplasmic sperm injection (IVF / ICSI), endocrine regulation, egg donation from young women, preimplantation genetic diagnosis to eliminate the risk of aneuploidy, etc., the fertility of high-fertility-age women can be improved to a certain extent. However, even with high-quality embryos and appropriate endometrial thickness, ART may still fail, and this situation is more common in high-fertility-age women. A study involving 11,335 embryos screened by the third generation found that although all embryos passed the third-generation screening and had normal chromosomes, differences in pregnancy rates and live birth rates were caused by different maternal ages. Specifically, the pregnancy rate and live birth rate of the young group under 35 years old were significantly higher than those of the high-fertility-age group of 35 years old and above. This shows that in addition to embryo quality, maternal age is also an important factor affecting the success rate of transplantation. With age, while ovarian function declines, the function of the endometrium also declines. After excluding the interference of egg factors through the egg donation cycle model, multiple clinical studies compared the pregnancy outcomes of older and non-older women who received the same type of egg donation, and believed that advanced age would affect reproductive outcomes by reducing endometrial function. A fresh embryo transfer study involving 370 egg donation recipients found that the age of the recipients had a significant negative impact on the implantation rate. Another larger-scale study covering 1001 egg donation cycles found that as the age of the recipients increased, not only did the pregnancy rate decrease significantly, but the incidence of pregnancy-induced hypertension and the abortion rate also increased significantly. These data all suggest that other factors that may affect the fertility of high-fertility-age women, such as endometrial function, also need to be fully emphasized and evaluated.

[0005] The endometrial factors of high-fertility-age women are not only closely related to infertility but also considered an independent risk factor for various age-related pregnancy complications and congenital defects in offspring. Epidemiological studies show that the natural abortion rate of high-fertility-age women can reach 30%, and at the same time, the incidence of pregnancy complications such as pregnancy-induced hypertension and gestational diabetes, as well as adverse pregnancy outcomes such as fetal growth restriction, preterm birth, and stillbirth, also increase significantly with age. The "young egg - old uterus" mouse model constructed by ovarian transplantation shows that the risk of congenital heart disease in its offspring increases significantly, further confirming the adverse effects of uterine factors in high-fertility-age women on offspring. Therefore, the endometrium plays a crucial role in female reproductive health.

[0006] All along, researchers have been exploring effective intervention measures to alleviate and delay the aging process. A variety of methods have been proven to delay aging, such as exercise, nutritional adjustment, gene therapy, and drug intervention. Natural compounds are widely present in foods and are generally considered safer than chemically synthesized compounds, making them one of the main sources for drug discovery and development. The anti-aging effects of natural compounds involve multiple molecular mechanisms, including regulating various signaling pathways, reducing oxidative stress, removing senescent cells, improving metabolism, improving mitochondrial function, promoting DNA repair, etc. For example, the extract of Portulaca oleracea can significantly reduce the content of malondialdehyde (MDA) in the endometrium of aging mice, increase the activities of superoxide dismutase (SOD) and catalase (CAT), and improve endometrial atrophy. The extract of Vitex negundo var. cannabifolia can effectively alleviate the characteristics of naturally aging and D-galactose (D-gal)-induced aging mice, such as endometrial atrophy, gland swelling, elevated levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and decreased estrogen.

[0007] Although certain progress has been made in the treatment of uterine aging with natural compounds, the functional screening of natural compounds remains a slow and complex process, and there is still a lack of in-depth understanding of the specific mechanisms of action of many compounds. Therefore, more efficient and systematic screening methods need to be developed to discover potential anti-aging natural compounds. Summary of the Invention

[0008] Aiming at the problems in the prior art, the present invention provides a method for screening compounds that can improve endometrial aging. By establishing endometrial organoids, the endometrial changes caused by aging can be effectively simulated, and it is used as a target for screening natural compounds, with high reliability, short cycle, and avoidance of ethical issues.

[0009] Another object of the present invention is to provide a compound for improving endometrial aging.

[0010] To achieve the above object, the present invention adopts the following technical solutions.

[0011] A method for screening compounds that can improve the aging state of the endometrium, comprising the following steps: (1) Inducing the formation of an aging endometrial model with D-galactose in endometrial organoids; (2) Contacting the test compound with the aging endometrial model and detecting the proportion of β-galactosidase and / or EdU-positive cells; (3) Screening the compounds that can significantly reduce β-galactosidase and / or significantly increase the proportion of EdU-positive cells as the compounds for improving endometrial aging.

[0012] The concentration of D-galactose is 20 mM - 50 mM.

[0013] Detecting β-galactosidase includes one or more of the content, activity, transcription amount, or expression amount of the enzyme.

[0014] Preferably, the detection method is high-content imaging detection.

[0015] The compound for improving endometrial senescence is ganoderic acid C2 (CAS: 103773-62-2).

[0016] The above compound can be used as an active ingredient for preparing a drug for improving the senescent state of the endometrium.

[0017] The above drug can be administered orally or by mucosal administration, preferably by mucosal administration. Preferred dosage forms are suppositories and gels.

[0018] The senescent state is enhanced endometrial inflammation, increased endometrial fibrosis, and decreased endometrial receptivity.

[0019] The present invention has the following advantages: The present invention provides a method for screening compounds for improving the senescent state of the endometrium. A senescent endometrial model is formed by inducing endometrial organoids with D-galactose, and compounds that can significantly reduce β-galactosidase and / or significantly increase the proportion of EdU-positive cells are screened. The model constructed by the present invention can fully simulate the physiological state and pathological changes of the senescent endometrium, and the screening is highly reliable, has a short cycle, and avoids ethical issues, providing a reliable method for the subsequent screening of natural products. Description of the Drawings

[0020] Figure 1 It is a morphological light microscope image of endometrial organoids. The upper figure shows the morphology of primary organoids on day 3, day 6, day 9, and day 12 respectively, and the lower figure shows the morphology of the third-generation organoids on day 3, day 6, day 9, and day 12 respectively. Scale bar = 200 μm; Figure 2 It is an immunofluorescence staining of endometrium and endometrial organoid epithelial cells (markers E-Cadherin and FOXA2) and stromal cells (marker Vimentin). Scale bar = 50 μm; Figure 3It is the verification of the D-galactose-induced senescence model of endometrial organoids. Among them, A: The expression of cell senescence-related genes in organoids of the NC group and the D-galactose (Dg) treatment groups at different concentrations (20 mM, 40 mM, and 50 mM) was detected by qRT-PCR; B: The expression levels of senescence-related β-galactosidase (SA-β-gal) and P53 in the NC group and the Dg group were detected by WB, with GAPDH as the internal reference protein; C: Quantitative statistics of the immunofluorescence and positive cell ratio of P53 in the NC group and the Dg group; D: Quantitative statistics of the immunofluorescence and positive cell ratio of P21 in the NC group and the Dg group; Scale bar = 50 μm, and the results are all shown as mean ± standard deviation. represents P <0.05, represents P <0.01, represents P <0.001; Figure 4 It is the expression of inflammation-related and fibrosis-related genes during the D-galactose-induced senescence process. Among them, A: The expression of inflammation-related genes in organoids of the NC group and the D-galactose (Dg) treatment group was detected by qRT-PCR; B: The expression of fibrosis-related genes in organoids of the NC group and the Dg treatment group was detected by qRT-PCR; The results are all shown as mean ± standard deviation. represents P <0.05, represents P <0.01, represents P <0.001; Figure 5 It is the impairment of endometrial receptivity during the D-galactose-induced senescence process. Among them, A: The expression of receptivity-related genes in organoids of the NC group and the D-galactose (Dg) treatment group was detected by qRT-PCR; B: The expression levels of receptivity-related markers progesterone receptor A / B (PRA / B) and forkhead box O1 (FOXO1) in the NC group and the Dg group were detected by WB, with GAPDH as the internal reference protein; C: Immunofluorescence and quantitative comparison of the receptivity-related marker glycodelin in the NC group and the Dg group; D: Immunofluorescence and quantitative comparison of the receptivity-related marker insulin-like growth factor binding protein 1 (IGFBP1) in the NC group and the Dg group; Scale bar = 50 μm, and the results are all shown as mean ± standard deviation. represents P <0.05, represents P <0.01; Figure 6It is the fluorescence intensity ratio of SA-β-gal of nearly 200 drugs in high-content imaging compared with the Dg group; the X-axis represents the -log2 value of the fluorescence intensity ratio of SA-β-gal in the drug group compared with the D-galactose (Dg) group, and the Y-axis represents the -log2 (pvalue) value; Figure 7 It is the result of the second-round screening based on EdU labeling; among them, A: a scatter plot showing the proportion of EdU-positive cells of 28 drugs compared with the D-galactose (Dg) group. The X-axis represents the ratio (Fold change, FC) of the proportion of EdU-positive cells in the drug group compared with the Dg group, and the Y-axis represents the P value; B: Representative images of EdU staining in high-content cell imaging of organoids in the Dg group and 5 drug-treated groups. The five drugs are L-arginine (LA), folic acid (FA), oxaloacetic acid (OAA), chlorogenic acid (CGA), and ganoderic acid C2 (GDA). TL represents bright field, scale bar = 1000 μm; Figure 8 It is a bubble chart of the ratio of the proportion of EdU-positive cells in senescent organoids treated with 5 drugs at different concentrations compared with the Dg group. The size of the bubble represents the ratio, and the color of the bubble represents the significance of the difference. Detailed implementation methods

[0021] The present invention will be further described below in conjunction with examples and drawings, but the present invention is not limited by the following examples.

[0022] Example 1 Construction of endometrial organoids and senescent endometrial models The women of childbearing age included in the study had regular menstruation and underwent hysterectomy due to cervical squamous intraepithelial lesion, early cervical cancer, or uterine prolapse; patients with medical diseases such as hypertension, diabetes, and thyroid diseases, as well as patients with endometrial malignant lesions, were excluded. All experiments involving humans followed medical ethical principles and the Declaration of Helsinki and were approved by the ethics committee (approval number: KYLL-202204-030, SWYX: NO.2024-204). All participants signed a written consent form before joining the study.

[0023] 1. Establishment of endometrial organoids Endometrial organoids were established according to the following method: Collect sterile fresh endometrial tissue into pre-cooled sample collection fluid, which consists of DMEM / F12, 10% fetal bovine serum, and 1% antibiotic-antimycotic solution. Then, use pre-cooled DPBS containing 1% antibiotic-antimycotic solution to thoroughly rinse the endometrial tissue repeatedly to remove the blood clots on the surface. Next, use fine scissors to cut the tissue into small pieces and transfer them to the digestion solution (the digestion solution contains DMEM / F12, 1% antibiotic-antimycotic solution, 0.4 mg / mL collagenase V, 1.25 U / mL protease II, and 10 μg / mL DNase I). Place the mixture in a 37°C hybridization oven and digest for about 20 minutes. After a short static settlement, filter the upper cell suspension through a 40 μm cell strainer, and collect the cell clusters and tissue fragments on the strainer; Centrifuge the cell suspension obtained from the tissue fragments and cell clusters on the strainer at 400 g for 5 minutes. After resuspending the cell pellet with DMEM / F12 medium, centrifuge again at 400 g for 5 minutes. According to the cell density requirement (2×10 4 cells / well), calculate the volume ratio of the cell suspension to Matrigel to ensure that the ratio of the cell suspension to Matrigel is 1:3. Slowly add the mixed Matrigel-cell suspension to a pre-warmed 24-well plate at 37°C, adding 40 μL to each well. Then, place the plate in a 37°C cell culture incubator and incubate for 30 minutes until Matrigel solidifies. Finally, add 500 μL of endometrial organoid growth medium ExM (containing 1% Antibiotic-Antimycotic (100×), 1% ITS, 2 mM GlutaMAX TM , 1 mM Nicotinamide, 2% B27, 1% N2, 100 ng / mL Noggin, 50 ng / mL EGF, 100 ng / mL FGF2, 200 ng / mL WNT-3A, 200 ng / mL R-Spondin-1, 0.5 μM A83-01, 1.25 mM N-acetyl-L-cysteine, 10 μM p38 inhibitor SB202190 in DMEM / F-12) to each well and change the medium every other day.

[0024] It can be seen that Figure 1 under sterile conditions, endometrial tissue is collected, and after in vitro digestion treatment, a suspension containing small cell clusters is obtained. Subsequently, the cells are embedded in a three-dimensional (3D) matrix gel, and under the induction of specific growth factors, they gradually self-organize to form an organoid structure. Observing the assembly process of the organoids, it is found that epithelial cells gradually self-assemble into spherical glands and expand under the induction of the 3D structure of the matrix gel and small molecules, and stromal cells are arranged around the glands.

[0025] 2. Hormonal treatment of endometrial organoids Induce proliferative and secretory endometrial organoids according to the following method: Proliferative phase: Treat endometrial organoids with 10 nM estradiol (E2) for 2 days; Secretory phase: After 2 days of E2 treatment, treat endometrial organoids with a combination of 10 nM E2, 1 μM medroxyprogesterone acetate (MPA), and 1 μM cyclic adenosine monophosphate (cAMP) for 6 days.

[0026] Detect by immunofluorescence staining combined with multiple cell markers Figure 2 The results showed that, similar to in vivo endometrial tissue, there were also epithelial cells expressing E-Cadherin (E-Cad) and FOXA2 and stromal cells expressing Vimentin in endometrial organoids. In addition, these cells showed significant proliferative activity (Ki67 represents proliferating cells). In addition, these endometrial organoids could dynamically simulate the endometrial state in the proliferative and secretory phases in vitro.

[0027] 3. Inflammatory characteristics of senescent endometrium Taking 35 years old as the boundary, the volunteers were divided into two groups. Those aged 21 - 34 were the "young group", and those aged 35 - 46 were the "high reproductive age group". The average ages of the young group and the high reproductive age group were 27.9 years old and 39.1 years old respectively, and the body mass index (BMI) was similar (average BMIs were 20.8 kg / m 2 and 22.1 kg / m 2 ), without any medical complications and infertility, with regular menstrual cycles, and without receiving any estrogen and progesterone treatments within three months before the operation to obtain endometrial tissue.

[0028] Transcriptome analysis was performed on the obtained endometrial tissues and organoids. The results showed that in the proliferative-phase endometrial tissues (pro-tis) of the high-fertility-age group, pro-inflammatory signaling pathways such as the nuclear factor κB (NFκB) signaling pathway, tumor necrosis factor (TNF) signaling pathway, T helper 17 (Th17) cell differentiation, and interleukin-17 (IL-17) signaling pathway were all significantly upregulated. These results indicate a significant enhancement of the inflammatory response in the proliferative-phase endometrial tissues of high-fertility-age women. Through KEGG enrichment analysis of the upregulated differentially expressed genes (DEGs) in the proliferative-phase organoids (pro-org), it was found that immune and inflammation-related pathways were also significantly enriched in the pro-org of the high-fertility-age group. These results further confirm the abnormal activation of the inflammatory pathway in the endometrium of high-fertility-age women, especially the significant upregulation of Th17 cell differentiation and IL-17 signaling pathway, which can exacerbate the inflammatory response of the endometrium by promoting the release of pro-inflammatory factors and abnormal recruitment of immune cells. The above expression patterns were confirmed by qRT-PCR, and the results showed that the mRNA expression levels of inflammation-related genes ( TNF, NFKB2, CSF3, CCL2, CXCL3 ) were upregulated in the pro-tis and pro-org of the high-fertility-age group. The differences in the inflammatory status were verified by Western blot (WB) and immunofluorescence (IF) experiments. The results showed that the expression levels of the inflammatory activation protein AP2γ were significantly increased in the pro-tis and pro-org of the high-fertility-age group. In addition, the immunofluorescence experiment also showed that the expression of AP2γ was significantly enhanced in both endometrial epithelial cells and stromal cells, suggesting that the inflammatory response may be widely present in different cell types of the endometrium.

[0029] Through KEGG analysis of the downregulated DEGs in the secretory-phase organoids (sec-org) of the high-fertility-age group, it was found that the pro-inflammatory signaling pathways were downregulated in the sec-org of the high-fertility-age group, including cytokine-cytokine receptor interaction, NOD-like receptor (NLR) signaling pathway, IL-17 signaling pathway, and TNF signaling pathway. The inhibition of these pathways reflects the attenuation of the inflammatory response in the endometrium of high-fertility-age women during the secretory phase. Consistent with the transcriptome analysis results, the mRNA expression levels of most inflammation-related genes ( TNF, NFKB2, CSF3, CCL2, CCL20, CXCL3, IL1A ) were also significantly downregulated in the sec-org of the high-fertility-age group. The proteins encoded by these genes play important roles in the inflammatory response. The above research results suggest that the attenuation of the inflammatory response in the endometrium of high-fertility-age women during the secretory phase may be related to the decline in endometrial receptivity.

[0030] In summary, the above results indicate that there are significant changes in the inflammatory status of the endometrium of high-fertility-age women in different menstrual cycles, and this abnormality may play an important role in the reproductive health of high-fertility-age women by affecting endometrial receptivity and the embryo implantation microenvironment.

[0031] Flow cytometry multi-factor detection was performed on the organoid culture media of different menstrual cycles. The results showed that in the culture media of proliferative-phase organoids (pro-org) in the high-fertility-age group, the concentrations of most pro-inflammatory factors such as IL-23, TNF-α, IL-1β, IL-2, and IL-6 were increased, which was consistent with the increased expression levels of pro-inflammatory genes and proteins in the endometrium and organoids during the proliferative phase of the high-fertility-age group mentioned above. However, in the culture media of secretory-phase organoids (sec-org) in the high-fertility-age group, we found that the concentrations of pro-inflammatory factors such as Monocyte Chemoattractant Protein-1 (MCP-1), IL-23, and IL-1β were decreased, while the production of Th1-type cytokines IL-2 and IFN-γ was increased, and the secretion of Th2-type cytokine IL-10 was decreased. This result indicates that the Th1 / Th2 immune balance in the secretory-phase endometrium of high-fertility-age women shifts towards Th1. Taken together, these results confirm the existence of inflammatory imbalance in the endometrium of high-fertility-age women.

[0032] 4. Fibrotic characteristics of the senescent endometrium The fibrotic levels of endometrial samples in the young group and the high-fertility-age group were compared by multi-dimensional detection methods such as gene expression profiling, histopathology, and quantitative protein expression. The qRT-PCR results showed that in the proliferative-phase endometrial tissue (pro-tis) of the high-fertility-age group, the mRNA levels of multiple genes related to cell adhesion ( LAMA2, LAMC3, ITGA11, ITGA5 ), collagen-related genes ( COL6A1, COL6A2, COL5A2 ), and myosin-related genes ( MYLK, MYL9 ), were all significantly increased. This finding was corroborated by the results of Masson staining, which visually showed a significant increase in the degree of fibrosis in the high-fertility-age endometrial samples, and this pathological change has been confirmed to be closely related to impaired female fertility. In addition, Western Blot (WB) detection further confirmed this finding: in the pro-tis of the high-fertility-age group, the protein expression levels of fibronectin and myosin light chain kinase (MYLK) were both significantly upregulated. These multi-level experimental evidences together indicate that with the increase of age, there is a pathological change of aggravated endometrial fibrosis. The qRT-PCR detection results showed that in the proliferative-phase organoids (pro-org) of the high-fertility-age group, the transcriptional levels of multiple profibrotic genes (including COL6A2, LAMC3, MYL9, ITGA11 etc.) were all significantly increased. This finding was highly consistent with the in vivo experimental results, further supporting the conclusion of aggravated endometrial fibrosis in high-fertility-age women.

[0033] The organoids were induced into the secretory phase (sec-org) by hormonal treatment. Under such experimental conditions that mimic the physiological cycle, the transcriptional levels of pro-fibrotic genes (such as COL6A2, MYL9, LAMC3, COL5A2, ITGA11 etc.) in sec-org of the high reproductive age group were still significantly upregulated. Meanwhile, WB detection further confirmed that the protein expression of fibronectin in sec-org of the high reproductive age group was significantly increased. The experimental results not only verified the phenotypic characteristics of endometrial fibrosis in high reproductive age women, but also revealed the persistence of this fibrosis phenomenon at different physiological stages (proliferative phase and secretory phase).

[0034] 5. Construction of senescent endometrial organoid model D-galactose (Dg) was dissolved in double-distilled water and diluted to an appropriate concentration before use. To test the effect of Dg on endometrial organoids, endometrial organoids were treated with different doses of Dg (20, 40 or 50 mM) for 3 days.

[0035] Endometrial organoids were treated with different concentration gradients (20 mM, 40 mM and 50 mM) of Dg, and the transcriptional level changes of key senescence-related genes (including CDKN1A, CTNNB1, TP53, GPX1, CTGF and CDK7 ) were quantitatively detected by qRT-PCR. The experimental results showed that Dg-induced organoid senescence exhibited a significant concentration-dependent effect. When the treatment concentration was 40 mM, the expression levels of all detected senescence-related genes reached the peak levels. Among them, the expression level of tumor suppressor 53 ( TP53 ) was significantly increased by 1.5-fold compared with the control group, and the expression level of connective tissue growth factor ( CTGF ) was increased by 1.7-fold ( Figure 3 A). Based on the above results, 40 mM was determined as the optimal concentration for establishing the senescent endometrial organoid model.

[0036] Classic cellular senescence markers were detected by Western blot (WB). The results showed that the expression levels of senescence-associated β-galactosidase (SA-β-gal) and P53 protein in the Dg-treated organoids were both significantly increased ( Figure 3 B). Immunofluorescence staining (IF) results showed that the proportions of P53- and P21-positive cells in the Dg treatment group were significantly increased ( Figure 3 C-D). These results together confirmed the successful construction of a stable and reliable endometrial senescence model.

[0037] 6. Functional changes of the senescent endometrial organoid model It was detected by qRT-PCR that after Dg successfully induced organoid senescence, inflammation-related genes ( FAS, JUN, NFKB1 and IL15)showed significant upregulation at the transcriptional level ( Figure 4 A). These results indicate that Dg-induced organoid senescence is accompanied by significant changes in the inflammatory microenvironment, a finding that highly coincides with the characteristics of the disrupted immune microenvironment in the endometrium of high-fertility-age women.

[0038] The detection results of the expression levels of fibrosis-related markers showed that the fibrosis-related genes COL1A1, COL6A1, COL6A2, ITGA1 and ITGA5 both showed significantly increased transcriptional levels ( Figure 4 B). Among them, COL1A1, as the main collagen component, its upregulation indicates the deposition of the extracellular matrix (ECM), while the altered expressions of integrin family members ITGA1 and ITGA5 may affect cell-matrix interaction and jointly promote the fibrosis process. This finding not only confirms the significant exacerbation of fibrosis during Dg-induced senescence but also highly coincides with the phenotype of increased endometrial fibrosis in high-fertility-age women.

[0039] The results of the detection of uterine receptivity-related in secretory organoids showed that Dg treatment severely impaired the hormonal responsiveness and receptivity of endometrial organoids. At the gene expression level, key receptivity regulators (including PGR, ESR2, GPX3, and IHH) all showed significant transcriptional repression ( Figure 5 A). At the protein expression level, the expression levels of receptivity-related markers progesterone receptor A / B (PRA / B) and forkhead box O1 (FOXO1) were also significantly downregulated ( Figure 5 B). In addition, through immunofluorescence staining (IF) observation, it was found that the fluorescence intensities of receptivity markers glycodelin and insulin-like growth factor-binding protein 1 (IGFBP1) were significantly weakened ( Figure 5 C-D). The above data comprehensively indicate that Dg can induce the senescence process of endometrial organoids, promote the occurrence of their inflammatory responses and fibrosis, and significantly reduce the receptivity of the endometrium.

[0040] Example 2 Screening of Compounds for Improving the Senescent State of the Endometrium 1. Construction of the senescent endometrial model The endometrial organoids constructed in Example 1 were operated as follows: Preheat the digestive fluid to 37°C in advance. Aspirate the culture medium in the well plate and wash it twice with DPBS. Add 500 μL of digestive fluid to each well of the 24-well plate containing endometrial organoids, pipette and scrape off the organoids, transfer them to a 15 mL centrifuge tube, and place it in a 37°C hybridization oven for digestion for about 20 minutes. Centrifuge the cell suspension at 400 g for 5 minutes. After resuspending the cell pellet with DMEM / F12 medium, centrifuge it again at 400 g for 5 minutes. According to the cell density requirement (2×10 4 cells / well), calculate the volume ratio of the cell suspension to Matrigel to ensure that the ratio of the cell suspension to Matrigel is 1:3.

[0041] Open the Corning DNA Studio software, set the print head and print bed temperatures (print head at 4°C, print bed at 37°C), select the well plate type and the number of drops printed per well (96-well, 1 drop per well). Set parameters such as extrusion rate, extrusion volume, retract volume, droplet volume (10 μL), z-offset, and extra preflow volume. Install the nozzle-type insulator on the print head, add the mixed Matrigel-cell suspension to the syringe, connect the syringe to the syringe plunger holder, and adjust the position of the syringe plunger holder. Use automatic calibration to calibrate the print head, and then start printing. Place the printed 96-well plate in a 37°C incubator for 30 minutes until the Matrigel solidifies. Finally, add 200 μL of endometrial organoid growth medium or medium containing 40 mM Dg to each well and change the medium every other day.

[0042] 2. High-throughput primary screening of drugs In each 96-well plate for bioprinting, set up a control group (6 wells, normal growth medium, with the same medium change frequency as the other two groups), a Dg group (6 wells, normal growth medium containing Dg), and a drug addition group (3 replicates for each drug). After seeding the plates, the Dg group and the drug addition group are cultured with medium containing Dg for 3 days. On days 4 - 6, the Dg group is treated with medium containing 40 mM Dg and 0.5% DMSO, and the drug addition group is treated with medium containing 40 mM Dg and 50 μM compound.

[0043] Perform CellEvent on the above-treated organoids TMSenescence staining was performed, and then the 96-well plate was scanned using the ImageXpressMicro Confocal high-content imaging system with a 4×PhL Plan Fluor DL objective and a 60 µm pinhole confocal spinning disk. Hoechst 33342 was identified using the DAPI channel, the senescence probe β-gal was identified using the FITC channel, and the organoids in bright field were identified using the TL-10 channel. Each well was scanned with a z-stack acquisition mode with a 15 μm step size for 200 - 300 µm, and Best Focus was selected for imaging. Image analysis was performed using Metaxpress ® software. In the custom module workflow of the software, 2D Projection was analyzed. All β-gal-positive cytoplasm was identified based on the FITC image, representing senescent cells, and the fluorescence intensity of β-gal was calculated.

[0044] In the primary screening stage, the fluorescence intensity of β-galactosidase (SA-β-gal) was used as the detection index to evaluate the degree of cell senescence. Through systematic detection, 28 candidate drugs that could significantly reduce the SA-β-gal level were screened out from 191 drugs ( Figure 6 ), and the 28 drugs were: D-alanine, nicardipine, wortmannin, loxapine, guanabenz, trichostatin A, metformin, melatonin, sulfamethoxazole, acarbose, thioridazine, nicotinamide adenine dinucleotide, quercetin, diallyl trisulfide, kaempferol, vitexin, sesamin, L-arginine, L-arginine (hydrochloride), taxifolin, doxycycline hydrochloride, quercetin (dihydrate), L-arginine (L-glutamate), chicoric acid, folic acid, chlorogenic acid, ganoderic acid C2, oxaloacetic acid, indicating that these drugs have potential activity in alleviating cell senescence.

[0045] 3. High-throughput re-screening of drugs In each 96-well plate for bioprinting, a control group (6 wells, normal growth medium, with the same medium change frequency as the other two groups), a Dg group (6 wells, normal growth medium containing Dg), and a drug addition group (3 replicates for each drug) were set up. After seeding the plates, the Dg group and the drug addition group were cultured with medium containing Dg for 3 days. On days 4 - 6, the Dg group was treated with medium containing 40 mM Dg and 0.5% DMSO, and the drug addition group was treated with medium containing 40 mM Dg and 50 μM compound.

[0046] The above-treated organoids were successively subjected to EdU and Beyo3D TMStain with Hoechst 33342, then scan the 96-well plate using an ImageXpress Micro Confocal high-content imaging system, with a 4×PhL Plan Fluor DL objective and a 60 µm pinhole confocal spinning disk. Identify Hoechst 33342 using the DAPI channel, EdU using the Texas Red channel, and the organoids in bright field using the TL-10 channel. Scan each well with a z-stack acquisition mode with a 15 μm step size for 200 - 300 µm, and select Best Focus for imaging. Image analysis was performed using Metaxpress ® software. In the custom module workflow of the software, analyze the 2D Projection. Based on the DAPI image, identify all Hoechst 33342-positive cell nuclei, representing the total number of cells, and use an algorithm to identify the number and area of the organoids; based on the Texas Red image, identify all EdU-positive cell nuclei, representing the proliferating cells; calculate the percentage of EdU-positive cells in each well relative to the total number of cells and the average area of the organoids.

[0047] On the basis of initially screening 28 potential anti-aging candidate drugs, to further evaluate the promoting effect of these drugs on cell proliferation ability, the 5-Ethynyl-2’-deoxyuridine (EdU) labeling method was used to detect the proportion of EdU-positive cells in the drug treatment groups, and finally 5 drugs that could significantly increase the proportion of EdU-positive cells were screened out ( Figure 7 ). These 5 drugs are: L-Arginine (LA), Folic acid (FA), Oxaloacetic acid (OAA), Chlorogenic acid (CGA), and Ganoderic acid C2 (GDA).

[0048] 4. Drug proliferative activity Further drug concentration gradient tests were carried out on oxaloacetic acid, chlorogenic acid, and ganoderic acid C2. The results showed that different drugs exhibited the best cell proliferation promoting effect within a specific concentration range: LA, FA, OAA, and CGA had the most significant effect on restoring the proportion of EdU-positive cells in senescent endometrial organoids at a concentration of 50 μM, while GDA showed the best proliferative activity at a concentration of 100 μM ( Figure 8 ). This concentration-dependent difference may be related to the drug's own mechanism of action and its affinity for cell targets.

[0049] The above research results show that the constructed senescent endometrial model can be used for drug screening, and the results are highly reliable.

[0050] The above are only examples of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. Use of ganoderic acid C2 in the preparation of a medicament for improving endometrial senescence.

2. The application according to claim 1, wherein The senescent state is enhanced endometrial inflammation, increased endometrial fibrosis, and decreased endometrial receptivity.

3. The application according to claim 1, wherein The medicament is administered orally or by mucosal administration.

4. The application according to claim 1, wherein The pharmaceutical dosage form is suppository or gel.

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