Compounds that improve endometrial aging

Abstract

The present 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 obtained by screening using the following method: using D-galactose to induce endometrial organoids to form an aging endometrial model; contacting the test compound with the aging endometrial model and detecting the proportion of β-galactosidase and / or EdU-positive cells; screening for compounds that can significantly reduce β-galactosidase and / or significantly increase the proportion of EdU-positive cells as compounds that improve endometrial aging. The model constructed by the present invention can fully simulate the physiological state and pathological changes of the aging endometrium, the screening is highly reliable, the cycle is short, and ethical issues are avoided, providing a reliable method for the subsequent screening of natural products.

Description

Technical Field

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

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be regarded as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] The continued increase in global life expectancy, the widespread use of assisted reproductive technology (ART), and socioeconomic and lifestyle changes have led to a gradual postponement of the average age of childbearing. However, women's fertility declines with age, exacerbating the dual pressures of low birth rates and demographic imbalances. Therefore, identifying effective strategies to improve reproductive outcomes in women of advanced childbearing age is crucial for optimizing population structure and addressing the challenges of aging.

[0004] Traditionally, reproductive aging is primarily attributed to ovarian decline, characterized by diminished ovarian reserve, decreased oocyte quality, and insufficient hormone secretion, leading to poor or abnormal embryo quality. Studies have shown that ovarian reserve depletion accelerates with aging. Furthermore, decreased mitochondrial function, accumulated DNA damage, and increased rates of chromosomal aneuploidy contribute to decreased oocyte quality. Ovarian decline also leads to decreased estrogen and progesterone secretion, causing endometrial hypoplasia, which in turn impairs embryo implantation and pregnancy maintenance. Fertility in women of advanced reproductive age can be improved to some extent through controlled ovarian hyperstimulation, in vitro fertilization / intracytoplasmic sperm injection (IVF / ICSI), endocrine regulation, oocyte donation from younger women, and preimplantation genetic diagnosis to mitigate the risk of aneuploidy. However, even with high-quality embryos and an appropriate endometrial thickness, ART failure can still occur, a phenomenon more common in women of advanced reproductive age. A study of 11,335 embryos that underwent three generations of screening found that, although all embryos passed three generations of screening and were chromosomally normal, maternal age contributed to differences in pregnancy and live birth rates. Specifically, pregnancy and live birth rates were significantly higher in the younger group (under 35 years) than in the older group (35 years and older). This suggests that, in addition to embryo quality, maternal age is also a significant factor influencing implantation success. With aging, ovarian function declines, and endometrial function also declines. Using donor egg cycle models to eliminate the influence of oocytes, several clinical studies have compared pregnancy outcomes between older and younger women receiving the same type of donor eggs, suggesting that advanced age can affect reproductive outcomes by reducing endometrial function. A study of fresh embryo transfer involving 370 egg donor recipients found that recipient age had a significant negative impact on implantation rates. Another larger study, encompassing 1,001 donor egg cycles, found that increasing recipient age not only significantly decreased pregnancy rates but also significantly increased the incidence of hypertension and miscarriage during pregnancy. These data suggest that other factors that may affect the fertility of women of advanced childbearing age, such as endometrial function, also need to be fully valued and evaluated.

[0005] Endometrial factors in women of advanced reproductive age are not only closely associated with infertility but are also considered independent risk factors for a variety of age-related pregnancy complications and congenital defects in offspring. Epidemiological studies have shown that the spontaneous abortion rate in women of advanced reproductive age can reach 30%. Furthermore, the incidence of pregnancy complications such as gestational hypertension and gestational diabetes, as well as adverse pregnancy outcomes such as fetal growth restriction, premature birth, and stillbirth, increases significantly with age. A "young egg-old uterus" mouse model constructed through ovarian transplantation has shown a significantly increased risk of congenital heart disease in its offspring, further confirming the adverse effects of uterine factors in women of advanced reproductive age on their offspring. Therefore, the endometrium plays a vital role in female reproductive health.

[0006] Researchers have long explored effective interventions to mitigate and delay the aging process. Various approaches have been shown to delay aging, including exercise, nutritional adjustments, gene therapy, and drug interventions. Natural compounds, widely found in foods and generally considered safer than chemically synthesized compounds, have become a major source for drug discovery and development. The anti-aging effects of natural compounds involve multiple molecular mechanisms, including modulation of various signaling pathways, reduction of oxidative stress, removal of senescent cells, improved metabolism, improved mitochondrial function, and promotion of DNA repair. For example, purslane extract significantly reduced malondialdehyde (MDA) levels in the endometrium of aged mice, increased superoxide dismutase (SOD) and catalase (CAT) activities, and improved endometrial atrophy. Furthermore, vitex fruit extract effectively alleviated characteristics of both natural and Dg (D-galactose)-induced aging in mice, including endometrial atrophy, glandular swelling, elevated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, and decreased estrogen levels.

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

[0008] In response to the problems in the prior art, the present invention provides a method for screening compounds that can be used to improve endometrial aging. By establishing endometrial organoids, the endometrial changes caused by aging can be effectively simulated, and natural compounds can be screened using them as targets. The screening is highly reliable, has a short cycle, and avoids ethical issues.

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

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

[0011] A method for screening a compound that improves the aging state of the endometrium comprises the following steps:

[0012] (1) D-galactose was used to induce endometrial organoids to form an aging endometrial model;

[0013] (2) contacting the test compound with the aging endometrial model and detecting the proportion of β-galactosidase and / or EdU-positive cells;

[0014] (3) Screening compounds that can significantly reduce β-galactosidase and / or significantly increase the proportion of EdU-positive cells is a compound that improves endometrial aging.

[0015] The concentration of D-galactose is 20 mM-50 mM.

[0016] Detection of β-galactosidase includes one or more of the following: enzyme content, activity, transcription amount or expression amount.

[0017] Preferably, the detection method is high-content imaging detection.

[0018] The compound that improves endometrial aging is ganoderic acid C2 (CAS: 103773-62-2).

[0019] The above compounds can be used as effective ingredients in preparing medicines for improving the aging state of endometrium.

[0020] The above-mentioned drug can be administered orally or through the mucosa, preferably through the mucosa, and preferably in the form of a suppository or gel.

[0021] The aging state is characterized by increased endometrial inflammation, increased endometrial fibrosis, and decreased endometrial receptivity.

[0022] The present invention has the following advantages:

[0023] This invention provides a method for screening compounds that improve the aging state of the endometrium. D-galactose is used to induce endometrial organoids to form an aging endometrial model, and compounds that significantly reduce β-galactosidase and / or significantly increase the proportion of EdU-positive cells are screened. This model fully simulates the physiological state and pathological changes of the aging endometrium, provides high screening reliability, shortens the screening cycle, and avoids ethical issues, providing a reliable method for subsequent natural product screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are morphological light micrographs of endometrial organoids. The upper graph shows the morphology of primary organoids on day 3, day 6, day 9, and day 12, respectively. The lower graph shows the morphology of third-generation organoids on day 3, day 6, day 9, and day 12, respectively. Scale bar = 200 μm.

[0025] Figure 2 Immunofluorescence staining of epithelial cells (markers E-Cadherin and FOXA2) and stromal cells (marker Vimentin) in the endometrium and endometrial organoids. Scale bar = 50 μm.

[0026] Figure 3This is the validation of the D-galactose-induced endometrial organoid aging model; A: qRT-PCR was used to detect the expression of cell senescence-related genes in organoids in the NC group and different concentrations (20 mM, 40 mM and 50 mM) of D-galactose (Dg)-treated groups; B: WB was used to detect the expression levels of senescence-related β-galactosidase (SA-β-gal) and P53 in the NC group and Dg group, with GAPDH as the internal reference protein; C: Quantitative statistics of immunofluorescence and positive cell ratio of P53 in the NC group and Dg group; D: Quantitative statistics of immunofluorescence and positive cell ratio of P21 in the NC group and Dg group; Scale bar = 50 μm, and the results are shown as mean ± standard deviation. represent P <0.05, represent P <0.01, represent P <0.001;

[0027] Figure 4 The expression of inflammatory response and fibrosis-related genes during D-galactose-induced aging; A: qRT-PCR was used to detect the expression of inflammatory-related genes in organoids of the NC group and the D-galactose (Dg)-treated group; B: qRT-PCR was used to detect the expression of fibrosis-related genes in organoids of the NC group and the Dg-treated group; the results are shown as mean ± standard deviation. represent P <0.05, represent P <0.01, represent P <0.001;

[0028] Figure 5 Figure 3 is the damage of endometrial receptivity during D-galactose-induced aging; A: qRT-PCR was used to detect the expression of receptivity-related genes in organoids of the NC group and the D-galactose (Dg)-treated group; B: WB was used to detect 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, 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 shown as mean ± standard deviation. represent P <0.05, represent P <0.01;

[0029] Figure 6 The fluorescence intensity ratios of SA-β-gal in the drug group and the Dg group for nearly 200 cases in high-content imaging are shown in Figure 2. The X-axis represents the -log2 value of the fluorescence intensity ratio of SA-β-gal in the drug group compared to the D-galactose (Dg) group, and the Y-axis represents the -log2 (pvalue).

[0030] Figure 7 The results of the second round of screening based on EdU labeling are shown. A: A scatter plot showing the proportion of EdU-positive cells in 28 drug-treated cells compared to the D-galactose (Dg) group. The X-axis represents the fold change (FC) ratio of the EdU-positive cell proportions in the drug group compared to 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 five drug-treated groups: L-arginine (LA), folic acid (FA), oxaloacetic acid (OAA), chlorogenic acid (CGA), and ganoderic acid C2 (GDA). TL indicates bright field. Scale bar = 1000 μm.

[0031] Figure 8 This is a bubble chart of the ratio of the proportion of EdU-positive cells in the aged organoids treated with five drugs at different concentrations compared with the Dg group. The bubble size represents the ratio, and the bubble color represents the significance of the difference. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0033] Example 1 Construction of endometrial organoids and aging endometrial models

[0034] The study enrolled women of childbearing age with regular menstruation who had undergone hysterectomy for cervical squamous intraepithelial lesions, early cervical cancer, or uterine prolapse. Patients with medical conditions such as hypertension, diabetes, and thyroid disease, as well as those with endometrial malignancies, were excluded. All experiments involving human subjects adhered to the principles of medical ethics and the Declaration of Helsinki and were approved by the Ethics Committee (approval numbers: KYLL-202204-030, SWYX: NO. 2024-204). All participants provided written consent prior to study entry.

[0035] 1. Establishment of Endometrial Organoids

[0036] Establish endometrial organoids as follows:

[0037] Sterile, fresh endometrial tissue was collected in pre-chilled sample collection solution consisting of DMEM / F12, 10% fetal bovine serum, and 1% antibiotic-antimycotic solution. The endometrial tissue was then repeatedly and thoroughly rinsed with pre-chilled DPBS containing 1% antibiotic-antimycotic solution to remove any surface blood clots. The tissue was then cut into small pieces using fine scissors and transferred to a digestion solution containing DMEM / F12, 1% antibiotic-antimycotic solution, 0.4 mg / mL collagenase V, 1.25 U / mL proteinase II, and 10 μg / mL DNase I. This mixture was digested in a 37°C hybridization oven for approximately 20 minutes. After a brief rest, the supernatant was filtered through a 40 μm cell strainer, and the cell clumps and tissue fragments on the filter were collected.

[0038] The cell suspension obtained by removing tissue fragments and cell clusters on the filter was centrifuged at 400 g for 5 minutes, and the cell pellet was resuspended in DMEM / F12 medium and centrifuged again at 400 g for 5 minutes. 4 cells / well), calculate the volume ratio of cell suspension to Matrigel, and ensure that the ratio of cell suspension to Matrigel is 1:3. Slowly add the mixed Matrigel-cell suspension to a 24-well plate preheated to 37°C, adding 40 μL to each well. Then, place the plate in a cell culture incubator at 37°C 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) to each well. TM , 1 mM Nicotinamide, 2% B27, 1% N2, 100 ng / mL Noggin, 50 ng / mL EGF, 100 ng / mL FGF2, 200ng / 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 DMEM / F-12 of SB202190), change the fluid every other day.

[0039] Depend on Figure 1Endometrial tissue was collected under sterile conditions and digested in vitro to obtain a suspension containing small cell clusters. The cells were then embedded in a three-dimensional (3D) matrix gel and, under the guidance of specific growth factors, gradually self-organized into organoid structures. Observation of the organoid assembly process revealed that, under the guidance of the 3D matrix gel and small molecules, epithelial cells gradually self-assembled into spherical glands and expanded, with stromal cells surrounding the glands.

[0040] 2. Hormonal Treatment of Endometrial Organoids

[0041] Induce proliferative and secretory endometrial organoids as follows:

[0042] Proliferation phase: Endometrial organoids were treated with 10 nM estradiol (E2) for 2 days;

[0043] Secretory phase: Following 2 days of E2 treatment, endometrial organoids were treated with a combination of 10 nM E2, 1 μM medroxyprogesterone acetate (MPA), and 1 μM cyclic adenosine monophosphate (cAMP) for 6 days.

[0044] Immunofluorescence staining combined with multiple cell markers was used for detection. Figure 2 The results showed that, similar to in vivo endometrial tissue, endometrial organoids contained epithelial cells expressing E-Cadherin (E-Cad) and FOXA2, as well as stromal cells expressing Vimentin. Furthermore, these cells exhibited significant proliferative activity (Ki67 indicates proliferating cells). Furthermore, these endometrial organoids were able to dynamically simulate the proliferative and secretory phases of the endometrium in vitro.

[0045] 3. Inflammatory features of the aging endometrium

[0046] The volunteers were divided into two groups based on the age of 35: the "young group" aged 21-34 and the "high-childbearing age group" aged 35-46. The average ages of the young group and the high-childbearing age group were 27.9 and 39.1 years old, respectively, and their body mass index (BMI) was similar (the average BMI was 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-progestin treatment within three months before the endometrial tissue acquisition surgery.

[0047] Transcriptome analysis of the harvested endometrial tissue and organoids revealed that proinflammatory signaling pathways, such as the nuclear factor κB (NFκB) pathway, the tumor necrosis factor (TNF) pathway, T helper 17 (Th17) cell differentiation, and the interleukin-17 (IL-17) pathway, were significantly upregulated in proliferative endometrial tissue (pro-tis) from women of advanced reproductive age. These results suggest a significant increase in inflammatory responses in proliferative endometrial tissue from women of advanced reproductive age. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of upregulated differentially expressed genes (DEGs) in proliferative organoids (pro-org) revealed significant enrichment of immune and inflammatory pathways in pro-org from the advanced reproductive age group. These results further confirm that aberrant activation of inflammatory pathways in the endometrium of women of advanced reproductive age, particularly the significant upregulation of Th17 cell differentiation and the IL-17 pathway, exacerbates the endometrial inflammatory response by promoting the release of proinflammatory cytokines and the aberrant recruitment of immune cells. The above expression patterns were confirmed by qRT-PCR, and the results showed that in the high-reproductive-age group pro-tis and pro-org, inflammation-related genes ( TNF, NFKB2, CSF3, CCL2, CXCL3 ) mRNA expression levels were upregulated. Western blot (WB) and immunofluorescence (IF) experiments verified the differences in inflammatory status. Results showed that expression levels of the inflammatory activator protein AP2γ were significantly elevated in pro-tis and pro-org in the advanced reproductive age group. Furthermore, IF experiments revealed significantly enhanced expression of AP2γ in both endometrial epithelial and stromal cells, suggesting that inflammatory responses may be widespread across different cell types in the endometrium.

[0048] KEGG analysis of downregulated DEGs in the secretory phase organoids (sec-org) of the high-reproductive-age group revealed that proinflammatory signaling pathways were downregulated in the sec-org of the high-reproductive-age group, including cytokine-cytokine receptor interactions, NOD-like receptor (NLR) signaling pathway, IL-17 signaling pathway, and TNF signaling pathway. The inhibition of these pathways reflects the weakening of the inflammatory response of the endometrium in the secretory phase of women of high reproductive age. Consistent with the results of transcriptome analysis, most inflammatory genes ( TNF, NFKB2, CSF3, CCL2, CCL20, CXCL3, IL1A ) also showed significant downregulation in mRNA expression. The proteins encoded by these genes play an important role in inflammatory responses. These findings suggest that the reduced inflammatory response in the endometrium during the secretory phase in women of advanced reproductive age may be related to decreased endometrial receptivity.

[0049] In summary, the above results indicate that the inflammatory state of the endometrium in women of advanced reproductive age varies significantly during different menstrual cycles. This abnormality may play an important role in the reproductive health of women of advanced reproductive age by affecting endometrial receptivity and the embryo implantation microenvironment.

[0050] Flow cytometry analysis of organoid culture fluid from different menstrual cycles revealed elevated levels of most proinflammatory factors, including IL-23, TNF-α, IL-1β, IL-2, and IL-6, in the culture fluid of proliferative organoids (pro-org) from the advanced reproductive age group, consistent with previously reported elevated expression of proinflammatory genes and proteins in the endometrium and organoids during the advanced reproductive age period. However, in the culture fluid of secretory organoids (sec-org) from the advanced reproductive age group, we observed decreased levels of proinflammatory factors, including monocyte chemoattractant protein-1 (MCP-1), IL-23, and IL-1β. Concomitantly, increased production of the Th1 cytokines IL-2 and IFN-γ, while decreased secretion of the Th2 cytokine IL-10, suggests a shift in the Th1 / Th2 immune balance toward Th1 in the secretory endometrium of women of advanced reproductive age. Taken together, these findings confirm the presence of an inflammatory imbalance in the endometrium of women of advanced reproductive age.

[0051] 4. Fibrosis characteristics of aging endometrium

[0052] The fibrosis levels of endometrial samples in the young group and the advanced reproductive age group were compared by using multi-dimensional detection methods such as gene expression profiling, histopathology, and quantitative protein expression. qRT-PCR results showed that in the proliferative endometrial tissue (pro-tis) of the advanced reproductive age group, multiple genes related to cell adhesion ( LAMA2, LAMC3, ITGA11, ITGA5 ), collagen-related genes ( COL6A1, COL6A2, COL5A2 ) and myosin-related genes ( MYLK, MYL9 ) mRNA levels were significantly increased. This finding is consistent with the results of Masson staining, which intuitively shows a significant increase in the degree of fibrosis in endometrial samples of advanced reproductive age, and this pathological change has been shown to be closely related to impaired female fertility. In addition, Western Blot (WB) detection further confirmed this finding: in the pro-tis of the advanced reproductive age group, the protein expression levels of fibronectin and myosin light chain kinase (MYLK) were significantly upregulated. These multi-level experimental evidences together indicate that with age, the endometrium has pathological changes of aggravated fibrosis. The results of qRT-PCR detection showed that in the proliferative organoids (pro-org) of the advanced reproductive age group, multiple pro-fibrotic genes (including COL6A2、LAMC3、 MYL9、ITGA11 This finding is highly consistent with the in vivo experimental results, further supporting the conclusion that endometrial fibrosis is exacerbated in women of advanced reproductive age.

[0053] Organoids were induced to the secretory phase (sec-org) by hormone treatment. Under this experimental condition that simulates the physiological cycle, pro-fibrotic genes (such as COL6A2, MYL9, LAMC3, COL5A2, ITGA11 Transcriptional levels of endometrial fibrosis (e.g., fibronectin) remained significantly upregulated. Western blot analysis further confirmed a significant increase in fibronectin protein expression in the sec-org gene in the advanced reproductive age group. These results not only validate the phenotypic characteristics of endometrial fibrosis in women of advanced reproductive age but also reveal the persistence of this fibrotic phenomenon across different physiological phases (proliferative and secretory).

[0054] 5. Construction of Aging Endometrial Organoid Model

[0055] D-galactose (Dg) was dissolved in double-distilled water and diluted to the 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.

[0056] Endometrial organoids were treated with Dg at different concentration gradients (20 mM, 40 mM, and 50 mM), and key aging-related genes (including CDKN1A, CTNNB1, TP53, GPX1, CTGF and CDK7 ) transcriptional levels. The experimental results showed that Dg-induced organoid senescence exhibited a significant concentration-dependent effect. When the treatment concentration was 40 mM, all detected aging-related genes reached peak expression levels, among which the tumor suppressor 53 ( TP53 ) expression level was significantly increased by 1.5 times compared with the control group, and connective tissue growth factor ( CTGF ) expression increased by 1.7 times ( Figure 3 Based on the above results, 40 mM was determined to be the optimal concentration for establishing an endometrial organoid aging model.

[0057] Western blot (WB) was used to detect classic cell senescence markers. The results showed that the expression levels of senescence-associated β-galactosidase (SA-β-gal) and P53 protein were significantly increased in organoids treated with Dg ( Figure 3 B). Immunofluorescence staining (IF) results showed that the proportion of P53 and P21 positive cells in the Dg treatment group increased significantly ( Figure 3 CD). These results together confirmed that a stable and reliable endometrial aging model was successfully established.

[0058] 6. Functional Changes in Aging Endometrial Organoid Models

[0059] qRT-PCR detection revealed that after Dg successfully induced organoid senescence, inflammation-related genes ( FAS, JUN, NFKB1 and IL15 ) showed a significant upregulation in their transcription levels ( Figure 4 A). These results indicate that Dg-induced organoid senescence is accompanied by significant changes in the inflammatory microenvironment, a finding highly consistent with the characteristics of endometrial immune microenvironmental disturbances in women of advanced reproductive age.

[0060] The results of the detection of fibrosis-related marker expression levels showed that fibrosis-related genes COL1A1, COL6A1, COL6A2, ITGA1 and ITGA5 The transcription levels of Figure 4 B) Upregulated expression of COL1A1, a major collagen component, indicates extracellular matrix (ECM) deposition, while altered expression of integrin family members ITGA1 and ITGA5 may influence cell-matrix interactions and contribute to fibrosis. This finding not only confirms the significant exacerbation of Dg-induced fibrosis during aging but is also highly consistent with the phenotype of increased endometrial fibrosis in women of advanced reproductive age.

[0061] Results from uterine receptivity tests of secretory phase organoids showed that Dg treatment severely impaired the hormone 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 the receptivity-related markers progesterone receptor A / B (PRA / B) and forkhead box O1 (FOXO1) were also significantly downregulated ( Figure 5 B). In addition, immunofluorescence staining (IF) revealed that the fluorescence intensity of the receptivity markers glycodelin and insulin-like growth factor binding protein 1 (IGFBP1) was significantly reduced ( Figure 5 These data suggest that Dg can induce the aging process of endometrial organoids, promote their inflammatory response and fibrosis, and significantly reduce the receptivity of the endometrium.

[0062] Example 2 Screening of compounds that improve endometrial aging

[0063] 1. Construction of Aging Endometrium Model

[0064] The endometrial organoids constructed in Example 1 were subjected to the following procedures:

[0065] Preheat the digestion solution to 37°C in advance, aspirate the culture medium in the well plate, and wash twice with DPBS. Add 500 μL of digestion solution to each well of the 24-well plate containing endometrial organoids, blow and scrape the organoids with a pipette tip, transfer to a 15 mL centrifuge tube, and place in a 37°C molecular hybridization oven for digestion for about 20 minutes. Centrifuge the cell suspension at 400 g for 5 minutes, add DMEM / F12 medium to resuspend the cell pellet, and centrifuge again at 400 g for 5 minutes. According to the cell density requirements (2×10 4 cells / well), calculate the volume ratio of cell suspension to Matrigel, and ensure that the ratio of cell suspension to Matrigel is 1:3.

[0066] Open Corning DNA Studio software and set the print head and print bed temperatures (print head: 4°C, print bed: 37°C). Select the plate type and the number of drops to be 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 insulator onto 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 autocalibration to calibrate the print head and begin printing. Place the printed 96-well plate in a 37°C incubator and incubate 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.

[0067] 2. High-throughput drug screening

[0068] Each bioprinted 96-well plate contained 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-treated group (three replicates per drug). After seeding, the Dg and drug-treated groups were cultured in Dg-containing medium for three days. On days 4-6, the Dg group was treated with medium containing 40 mM Dg and 0.5% DMSO, while the drug-treated group was treated with medium containing 40 mM Dg and 50 μM compound.

[0069] The organoids treated above were subjected to CellEvent TMSenescence staining was performed, and the 96-well plate was scanned using the ImageXpress Micro Confocal high-content imaging system, using a 4× PhL Plan Fluor DL ​​objective and a 60 µm pinhole confocal disk. Hoechst 33342 was identified using the DAPI channel, the senescence probe β-gal was identified using the FITC channel, and organoids were identified in bright field using the TL-10 channel. Each well was scanned 200-300 µm using a 15 µm step size z-stack acquisition mode, with Best Focus selected for imaging. Image analysis was performed using Metaxpress ® The software analyzes 2D projections in a custom module workflow. Based on the FITC image, all β-gal-positive cytoplasms are identified as representing senescent cells, and the β-gal fluorescence intensity is calculated.

[0070] In the initial screening phase, the fluorescence intensity of β-galactosidase (SA-β-gal) was used as a detection indicator to evaluate the degree of cell senescence. Through systematic testing, 28 candidate drugs that can significantly reduce the level of SA-β-gal were screened out from 191 drugs ( Figure 6 ), 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, and oxaloacetic acid, indicating that these drugs have potential activity in alleviating cellular aging.

[0071] 3. High-throughput drug screening

[0072] Each bioprinted 96-well plate contained 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-treated group (three replicates per drug). After seeding, the Dg and drug-treated groups were cultured in Dg-containing medium for three days. On days 4-6, the Dg group was treated with medium containing 40 mM Dg and 0.5% DMSO, while the drug-treated group was treated with medium containing 40 mM Dg and 50 μM compound.

[0073] The organoids treated above were sequentially subjected to EdU and Beyo3D TMHoechst 33342 staining was performed and the 96-well plate was scanned using the ImageXpress Micro Confocal High-Content Imaging System, using a 4× PhL Plan Fluor DL ​​objective and a 60 µm pinhole confocal disk. Hoechst 33342 was identified using the DAPI channel, EdU was identified using the Texas Red channel, and organoids were identified using the TL-10 channel in brightfield. Each well was scanned 200–300 µm using a z-stack acquisition mode with a 15 µm step size, and imaging was performed using Best Focus. Image analysis was performed using Metaxpress. ® The software analyzes 2D projections within a custom module workflow. Based on the DAPI image, all Hoechst 33342-positive nuclei are identified to represent the total cell count, and an algorithm is used to determine the number and area of ​​organoids. Based on the Texas Red image, all EdU-positive nuclei are identified to represent proliferating cells. The percentage of EdU-positive cells in each well and the average organoid area are calculated.

[0074] Based on the initial screening of 28 potential anti-aging drug candidates, in order to further evaluate the effects of these drugs on promoting cell proliferation, the 5-ethynyl-2'-deoxyuridine (EdU) labeling method was used to detect the proportion of EdU-positive cells in the drug-treated groups. Finally, 5 drugs that could significantly increase the proportion of EdU-positive cells were screened out ( Figure 7 The five drugs are: L-arginine (LA), folic acid (FA), oxaloacetic acid (OAA), chlorogenic acid (CGA) and ganoderic acid C2 (GDA).

[0075] 4. Drug Proliferation Activity

[0076] Further drug concentration gradient tests were conducted on oxaloacetic acid, chlorogenic acid, and ganoderic acid C2. The results showed that different drugs exhibited optimal cell proliferation-promoting effects within specific concentration ranges: 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 exhibited optimal pro-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 to the cellular target.

[0077] The above research results show that the constructed aging endometrial model can be used for drug screening, and the results are highly reliable.

[0078] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. The use of ganoderic acid C2 in the preparation of a drug for improving endometrial aging, characterized in that: The aging state is characterized by increased endometrial inflammation, increased endometrial fibrosis, and decreased endometrial receptivity.

2. The use according to claim 1, characterized in that The drug is administered orally or intramucosally.

3. The use according to claim 1, characterized in that The drug dosage forms are suppositories and gels.

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