Use of organic acid compounds in improving endometrial aging

Abstract

The application belongs to the technical field of natural products and medicine industry, and provides application of an organic acid compound in improving endometrial aging. The compound is screened by the following method: a D-galactose-induced endometrial organoid is used to form an aging endometrial model; the compound to be tested is contacted with the aging endometrial model, and the proportion of beta-galactosidase and / or EdU positive cells is detected; the compound that can significantly reduce the beta-galactosidase and / or significantly increase the proportion of EdU positive cells is the compound for improving endometrial aging. The model constructed by the application can fully simulate the physiological state and pathological changes of the aging endometrium, has high reliability, short cycle and avoids ethical problems, and provides a reliable method for subsequent screening of natural products.

Description

Technical Field

[0001] This invention belongs to the field of natural products and pharmaceutical technology, and relates to a compound that slows down the aging of the reproductive system. Background Technology

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

[0003] The continuous increase in global life expectancy, the widespread use of assisted reproductive technology (ART), and changes in socioeconomic and lifestyle factors have led to a gradual postponement of the average age of childbirth for women (Kontis V, Bennett JE, Mathers CD, et al. Future life expectancy in 35 industrialized countries: projections with a Bayesian model ensemble[J]. Lancet, 2017, 389(10076):1323-1335. Lakatta EG, Sollott S J. Perspectives onmammalian cardiovascular aging: humans to molecules[J]. Comp Biochem PhysiolA Mol Integr Physiol, 2002, 132(4):699-721. Mills M, Rindfuss RR, McDonald P, et al. Why do people postpone parenthood? Reasons and social policy incentives[J]. Hum Reprod Update, 2011, 17(6):848-860.). However, with increasing age, women's fertility gradually declines (Crawford NM, Steiner A Z. Age-related infertility[J].Obstet Gynecol Clin North Am, 2015, 42(1):15-25.), a phenomenon that exacerbates the dual pressures of low birth rate and population imbalance. Therefore, finding effective strategies to improve reproductive outcomes for women of advanced reproductive age is of great significance for optimizing population structure and addressing the challenges of aging.

[0004] Traditionally, reproductive aging is primarily attributed to ovarian dysfunction, characterized by reduced ovarian reserve, decreased oocyte quality, and insufficient hormone secretion. These factors lead to decreased or abnormal embryo quality (Cimadomo D, Fabozzi G, Vaiarelli A, et al. Impact of Maternal Age on Oocyte and EmbryoCompetence[J]. Front Endocrinol (Lausanne), 2018,9:327. Li Q, Geng X, ZhengW, et al. Current understanding of ovarian aging[J]. Sci China Life Sci,2012,55(8):659-669.). Studies have shown that ovarian reserve depletion accelerates with age (Faddy MJ, Gosden RG, Gougeon A, et al. Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause[J]. Hum Reprod, 1992,7(10):1342-1346.). Meanwhile, decreased mitochondrial function, accumulated DNA damage, and increased chromosomal aneuploidy rate in oocytes also lead to a decline in oocyte quality (Broekmans FJ, Soules MR, Fauser B C. Ovarianaging: mechanisms and clinical consequences[J]. Endocr Rev, 2009,30(5):465-493. Kasapoğlu I, Seli E. Mitochondrial Dysfunction and Ovarian Aging[J].Endocrinology, 2020,161(2):bqaa1.).Ovarian dysfunction can also lead to a decrease in the secretion of estrogen and progesterone, causing poor endometrial development, which in turn affects embryo implantation and pregnancy maintenance (Pathare A, Loid M, Saare M, et al. Endometrial receptivity in women of advanced age: an underrated factor in infertility[J]. Hum Reprod Update, 2023,29(6):773-793. Colella M,Cuomo D, Peluso T, et al. Ovarian Aging: Role of Pituitary-Ovarian AxisHormones and ncRNAs in Regulating Ovarian Mitochondrial Activity[J]. FrontEndocrinol (Lausanne), 2021,12:791071.). Methods such as controlled ovarian hyperstimulation, in vitro fertilization / intracytoplasmic sperm injection (IVF / ICSI), endocrine regulation, egg donation from young women, and preimplantation genetic diagnosis to eliminate the risk of aneuploidy can improve the fertility of women of advanced maternal age to some extent (Seshadri S, Morris G, Serhal P, et al. Assisted conception in women of advanced maternal age[J]. Best Pract Res Clin Obstet Gynaecol, 2021,70:10-20. Qiao J, Wang ZB, Feng HL, et al. The root of reduced fertility in aged women and possible therapeutic options: current status and future perspects[J]. Mol AspectsMed, 2014,38:54-85.).However, even with high-quality embryos and adequate endometrial thickness, ART can still fail, a situation more common in women of advanced reproductive age (Reig A, Franasiak J, Scott RJ, et al. The impact of age beyond ploidy: outcome data from 8175 euploid single embryotransfers[J]. J Assist Reprod Genet, 2020,37(3):595-602. Vitagliano A,Paffoni A, Vigano P. Does maternal age affect assisted reproduction technology success rates after euploid embryo transfer? A systematic review and meta-analysis[J]. Fertil Steril, 2023,120(2):251-265.). A study including 11,335 embryos that underwent three-generation screening found that, although all embryos passed three-generation screening and had normal chromosomes, differences in maternal age led to differences in pregnancy and live birth rates. Specifically, the pregnancy and live birth rates in the younger group (under 35 years of age) were significantly higher than those in the older group (35 years and above) (Vitagliano A, Paffoni A, Vigano P. Does maternal age affect assisted reproduction technology success rates after euploid embryo transfer? A systematic review and meta-analysis[J]. FertilSteril, 2023,120(2):251-265.). This indicates that, in addition to embryo quality, maternal age is also an important factor affecting the success rate of embryo transfer. With increasing age, ovarian function declines, and the function of the endometrium also decreases.After excluding oocyte-related factors through oocyte donation cycle models, several clinical studies compared pregnancy outcomes in older and younger women receiving the same type of donated eggs. These studies suggest that advanced age can affect reproductive outcomes by reducing endometrial function (Moomjy M, Cholst I, Mangieri R, et al. Oocyte donation: insights into implantation[J]. FertilSteril, 1999,71(1):15-21. Yaron Y, Ochshorn Y, Amit A, et al. Oocyte donation in Israel: a study of 1001 initiated treatment cycles[J]. Hum Reprod, 1998,13(7):1819-1824. Sterzik K, Dallenbach C, Schneider V, et al. In vitrofertilization: the degree of endometrial insufficiency varies with the type of ovarian stimulation[J]. Fertil Steril, 1988,50(3):457-462.). A fresh embryo transfer study involving 370 egg donor recipients found that recipient age had a significant negative impact on implantation rates (Moomjy M, Cholst I, Mangieri R, et al. Oocyte donation: insights into implantation[J]. Fertil Steril, 1999,71(1):15-21.). Another larger study covering 1001 egg donation cycles found that as recipient age increased, not only did pregnancy rates decline significantly, but the incidence of gestational hypertension and miscarriage also increased significantly (Yaron Y, Ochshorn Y, Amit A, et al. Oocyte donation in Israel: a study of 1001 initiated treatment cycles[J]. Hum Reprod, 1998,13(7):1819-1824.). These data suggest that other factors that may affect fertility in older women, such as endometrial function, also need to be given sufficient attention and evaluation.

[0005] Endometrial factors in women of advanced maternal age are not only closely associated with infertility, but are also considered an independent risk factor for various age-related pregnancy complications and congenital defects in offspring (Laopaiboon M, Lumbiganon P, Intarut N, et al. Advanced maternal age and pregnancy outcomes: a multicountryassessment[J]. BJOG, 2014,121 (Suppl 1):49-56. Woods L, Perez-Garcia V, Kieckbusch J, et al. Decidualisation and placentation defects are a major cause of age-related reproductive decline[J]. Nat Commun, 2017,8(1):352.). Epidemiological studies show that the spontaneous abortion rate of women of advanced maternal age can reach 30%. At the same time, 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, also increase significantly with age (De la Rochebrochard E, Thonneau P. Paternal age and maternal age are risk factors for miscarriage; results of a multicentre European study[J]. HumReprod, 2002,17(6):1649-1656. Khalil A, Syngelaki A, Maiz N, et al. Maternalage and adverse pregnancy outcome: a cohort study[J]. Ultrasound ObstetGynecol, 2013,42(6):634-643.).A mouse model of "young oocytes-aged uterus" constructed through ovarian transplantation showed a significantly increased risk of congenital heart disease in its offspring (Jay PY, Akhirome E, Magnan RA, et al. Transgenerational cardiology: One way to a baby's heart is through themother[J]. Mol Cell Endocrinol, 2016,435:94-102.), further confirming the adverse effects of uterine factors in older women on their offspring. Therefore, the endometrium plays a crucial role in female reproductive health.

[0006] Researchers have long been exploring effective interventions to mitigate and slow the aging process. Several methods have been shown to delay aging, such as exercise, nutritional adjustments, gene therapy, and drug intervention. Natural compounds, widely found in food 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 regulating various signaling pathways, reducing oxidative stress, removing senescent cells, improving metabolism, improving mitochondrial function, and promoting DNA repair (Muzammil A, Waqas M, Umar A, et al. Anti-aging Natural Compounds and their Role in the Regulation of Metabolic Pathways Leading to Longevity[J]. Mini Rev Med Chem, 2021,21(18):2630-2656. Gherardi G, Corbioli G, Ruzza F, et al. CoQ(10) and ResveratrolEffects to Ameliorate Aged-Related Mitochondrial Dysfunctions[J]. Nutrients,2022,14(20):4326.). For example, purslane extract can significantly reduce malondialdehyde (MDA) content in the endometrium of aging mice, increase the activity of superoxide dismutase (SOD) and catalase (CAT), and improve endometrial atrophy (Ahangarpour A, Lamoochi Z, Fathi MH, et al. Effects of Portulaca oleraceaethanolic extract on reproductive system of aging female mice[J]. Int JReprod Biomed, 2016,14(3):205-212.).Vitex agnus-castus fruit extract can effectively alleviate the characteristics of natural aging and Dg (D-galactose)-induced aging in mice, such as endometrial atrophy, glandular swelling, elevated levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and decreased estrogen levels (Ahangarpour A, Najimi SA, Farbood Y. Effects of Vitex agnus-castus fruit on sex hormones and antioxidant indices in a d-galactose-induced aging female mouse model[J]. J Chin Med Assoc, 2016,79(11):589-596.).

[0007] Although some 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 a deep understanding of the specific mechanisms of action of many compounds is still lacking. Therefore, it is necessary to develop more efficient and systematic screening methods to discover potential anti-aging natural compounds. Summary of the Invention

[0008] To address the problems in existing technologies, this invention provides a method for screening compounds that can improve endometrial aging. By establishing endometrial organoids, the changes in the endometrium caused by aging can be effectively simulated. Natural compounds can be screened using these organoids as targets. The screening method is highly reliable, has a short cycle, and avoids ethical issues.

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

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

[0011] A method for screening compounds that improve endometrial aging includes the following steps:

[0012] (1) D-galactose-induced endometrial organoids to form a model of aging endometrium;

[0013] (2) The test compound was brought into contact with an aging endometrial model, and the proportion of β-galactosidase and / or EdU-positive cells was detected;

[0014] (3) Compounds that can significantly reduce β-galactosidase and / or significantly increase the proportion of EdU-positive cells are compounds that improve 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 level, or expression level.

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

[0018] The compounds that improve endometrial aging are selected from: D-alanine, nicardipine, vorminoxetine, loxapine, guanethidine, triamcinolone A, metformin, melatonin, sulfamethoxazole, acarbose, thioridazine, nicotinamide adenine dinucleotide, quercetin, diallyl trisulfide, kaempferol, vitexin, sesamin, L-arginine, piperidin, doxycycline hydrochloride, quercetin (dihydrate), chicoric acid, folic acid, chlorogenic acid, and oxaloacetic acid. The L-arginine is selected from L-arginine, L-arginine hydrochloride, or L-glutamate of L-arginine.

[0019] Preferably, the above-mentioned compound is selected from L-arginine (CAS: 74-79-3), folic acid (CAS: 59-30-3), oxaloacetic acid (CAS: 328-42-7), or chlorogenic acid (CAS: 327-97-9). More preferably, the above-mentioned compound is selected from oxaloacetic acid or chlorogenic acid.

[0020] The above compounds can be used as active ingredients in the preparation of drugs that improve the aging state of the endometrium.

[0021] The above-mentioned drugs can be administered orally or via mucosal administration, preferably via mucosal administration. The preferred dosage forms are suppositories or gels.

[0022] The aging state is characterized by increased endometrial inflammation, enhanced endometrial fibrosis, and decreased endometrial receptivity.

[0023] The present invention has the following advantages:

[0024] This invention provides a method for screening compounds that significantly reduce β-galactosidase and / or significantly increase the proportion of EdU-positive cells using an aging endometrial model. This screening method is highly reliable, has a short cycle time, and avoids ethical issues, providing a reliable method for subsequent screening of natural products. The two organic acid compounds provided in this invention are widely available, low in cost, and highly safe, possessing clinical application value. Attached Figure Description

[0025] Figure 1 These are light micrographs of the morphology of endometrial organoids. The top image shows the morphology of the primary (passage 0) organoids on day 3, day 6, day 9, and day 12, while the bottom image shows the morphology of the third-generation (passage 3) organoids on day 3, day 6, day 9, and day 12. Scale bar = 200 μm.

[0026] Figure 2 Immunofluorescence staining of endometrial and endometrial organoid epithelial cells (markers E-Cadherin and FOXA2) and stromal cells (marker Vimentin), scale bar = 50 μm;

[0027] Figure 3 This study validated a D-galactose-induced aging model of endometrial organoids. A: qRT-PCR was used to detect the expression of cellular aging-related genes in organoids treated with different concentrations (20 mM, 40 mM, and 50 mM) of D-galactose (Dg). B: Western blotting was used to detect the expression levels of aging-related β-galactosidase (SA-β-gal) and P53 in the NC and Dg groups, with GAPDH as an internal reference protein. C: Quantitative statistics of P53 immunofluorescence and the proportion of positive cells in the NC and Dg groups. D: Quantitative statistics of P21 immunofluorescence and the proportion of positive cells in the NC and Dg groups. Scale bar = 50 μm. All results are presented as mean ± standard deviation. represent P <0.05, represent P <0.01, represent P <0.001;

[0028] Figure 4 This study investigated the expression of genes related to inflammation and fibrosis during D-galactose-induced aging. Specifically, A: qRT-PCR was used to detect the expression of inflammation-related genes in organoids from the NC and D-galactose (Dg) treatment groups; B: qRT-PCR was used to detect the expression of fibrosis-related genes in organoids from the NC and Dg treatment groups. All results are presented as mean ± standard deviation. represent P <0.05, represent P <0.01, represent P <0.001;

[0029] Figure 5This study examines the impairment of endometrial receptivity during D-galactose-induced aging. A: Expression of receptivity-related genes in organoids from the NC and D-galactose (Dg) treatment groups was detected by qRT-PCR. B: Expression levels of receptivity-related markers progesterone receptor A / B (PRA / B) and forkhead box O1 (FOXO1) were detected by Western blotting in the NC and Dg groups, with GAPDH as an internal reference protein. C: Immunofluorescence and quantitative comparison of the receptivity-related marker glycodelin were performed between the NC and Dg groups. D: Immunofluorescence and quantitative comparison of the receptivity-related marker insulin-like growth factor binding protein 1 (IGFBP1) were performed between the NC and Dg groups. Scale bar = 50 μm. All results are presented as mean ± standard deviation. represent P <0.05, represent P <0.01;

[0030] Figure 6 The ratio of SA-β-gal fluorescence intensity of nearly 200 drugs compared to the Dg group in high-content imaging; the X-axis represents the -log2 value of the ratio of SA-β-gal fluorescence intensity of the drug group compared to the D-galactose (Dg) group, and the Y-axis represents the -log2 (pvalue) value;

[0031] Figure 7 This is the result of the second round of screening based on EdU labeling; A: A scatter plot showing the proportion of EdU-positive cells in the 28 drug groups compared to the D-galactose (Dg) group, with the X-axis representing the Fold change (FC) ratio of EdU-positive cells in the drug group compared to the Dg group, and the Y-axis representing the P-value; B: Representative images of EdU staining in organoid high-content cell imaging of the Dg group and the five drug treatment groups, namely 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;

[0032] Figure 8 This is a bubble chart showing the ratio of the proportion of EdU-positive cells in the Dg group after treatment of aging organoids with five drugs at different concentrations. The size of the bubbles represents the ratio, and the color of the bubbles represents the significance of the difference. Detailed Implementation

[0033] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0034] Example 1: Construction of endometrial organoids and aging endometrial models

[0035] Women of childbearing age with regular menstrual cycles who had undergone hysterectomy for cervical squamous intraepithelial lesion, early-stage cervical cancer, or uterine prolapse were included in the study. Patients with hypertension, diabetes, thyroid disease, or other internal medical conditions, as well as those with endometrial malignancies, were excluded. All human experiments were conducted in accordance with medical ethics principles and the Declaration of Helsinki, and were approved by the ethics committee (approval numbers: KYLL-202204-030, SWYX: NO.2024-204). All participants signed written consent forms before joining the study.

[0036] 1. Establishment of endometrial organoids

[0037] Endometrial organoids were constructed using the following method:

[0038] Sterile, fresh endometrial tissue was collected in a pre-chilled collection solution consisting of DMEM / F12, 10% fetal bovine serum, and 1% antibiotic-antifungal solution. The endometrial tissue was then thoroughly rinsed repeatedly with pre-chilled DPBS containing 1% antibiotic-antifungal solution to remove 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-antifungal solution, 0.4 mg / mL collagenase V, 1.25 U / mL proteinase II, and 10 μg / mL DNase I. The mixture was digested in a molecular hybridization oven at 37°C for approximately 20 minutes. After a brief settling period, the supernatant cell suspension was filtered through a 40 μm cell filter, and cell clusters and tissue debris were collected from the filter.

[0039] The cell suspension obtained from the tissue debris and cell clusters on the filter screen was centrifuged at 400 g for 5 minutes. The cell pellet was resuspended in DMEM / F12 medium and centrifuged again at 400 g for 5 minutes. The cells were then separated according to the required cell density (2 × 10⁻⁶). 4 Calculate the volume ratio of cell suspension to Matrigel (cells / well), ensuring a 1:3 ratio. Slowly add 40 μL of the mixed Matrigel-cell suspension to a preheated 37°C 24-well plate. Then, incubate the plate at 37°C for 30 minutes until the Matrigel solidifies. Finally, add 500 μL of ExM endometrial organoid growth medium (containing 1% Antibiotic-Antimycotic (100×), 1% ITS, and 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.

[0040] Depend on Figure 1 As can be seen, endometrial tissue collected under sterile conditions and processed in vitro yielded a suspension containing small cell clusters. The cells were then embedded in three-dimensional (3D) matrix gel and, under the induction of specific growth factors, gradually self-organized to form organoid structures. Observation of the organoid assembly process revealed that epithelial cells, under the induction of the 3D matrix gel structure and small molecules, gradually self-assembled into spherical glands and expanded, with stromal cells arranged around the glands.

[0041] 2. Hormone treatment of endometrial organoids

[0042] Inducing proliferative and secretory endometrial organoids using the following methods:

[0043] Proliferative phase: Endometrial organoids were treated with 10 nM estradiol (E2) for 2 days;

[0044] Secretory phase: Following E2 treatment for 2 days, endometrial organoids were treated for 6 days with a combination of 10 nM E2, 1 μM medroxyprogesterone acetate (MPA), and 1 μM cyclic adenosine monophosphate (cAMP).

[0045] Detection was performed using immunofluorescence staining combined with multiple cell markers. Figure 2 The results showed that, similar to in vivo endometrial tissue, the endometrial organoids also 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 represents proliferating cells). In addition, this endometrial organoid was able to dynamically simulate the proliferative and secretory phases of the endometrium in vitro.

[0046] 3. Inflammatory characteristics of aging endometrium

[0047] Volunteers were divided into two groups based on age 35: the "younger group" (21-34 years old) and the "older reproductive age group" (35-46 years old). The average age of the younger and older reproductive age groups was 27.9 years and 39.1 years, respectively, with similar body mass indexes (BMI) (average BMI of 20.8 kg / m²). 2 and 22.1 kg / m 2 She had no internal medical complications or infertility, had regular menstrual cycles, and had not received any estrogen or progesterone treatment within three months prior to the endometrial tissue retrieval procedure.

[0048] Transcriptomic analysis of the obtained endometrial tissue and organoids revealed that pro-inflammatory signaling pathways, such as nuclear factor-κB (NFκB) signaling, tumor necrosis factor (TNF) signaling, Th17 helper cell differentiation, and interleukin-17 (IL-17) signaling, were significantly upregulated in the proliferative endometrial tissue (pro-tis) of women of advanced reproductive age. These results indicate a significantly enhanced inflammatory response in the proliferative endometrial tissue of women of advanced reproductive age. KEGG enrichment analysis of upregulated differentially expressed genes (DEGs) in proliferative organoids (pro-org) revealed a significant enrichment of immune and inflammation-related pathways in the pro-org of women of advanced reproductive age. These results further confirm the abnormal activation of inflammatory pathways in the endometrium of women of advanced reproductive age, especially the significant upregulation of Th17 cell differentiation and IL-17 signaling, which can exacerbate the inflammatory response of the endometrium by promoting the release of pro-inflammatory factors and the abnormal recruitment of immune cells. The expression patterns described above were confirmed by qRT-PCR, and the results showed that in the older reproductive age group, inflammation-related genes ( ) were expressed in pro-tis and pro-org. TNF, NFKB2, CSF3, CCL2, CXCL3 The mRNA expression level of AP2γ was upregulated. Differences in inflammatory status were verified by Western blot (WB) and immunofluorescence (IF) experiments. Results showed that the expression level of the inflammatory activating protein AP2γ was significantly increased in the advanced reproductive age groups pro-tis and pro-org. Furthermore, immunofluorescence experiments also showed significantly enhanced expression of AP2γ in both endometrial epithelial cells and stromal cells, suggesting that the inflammatory response may be widespread in different cell types of the endometrium.

[0049] KEGG analysis of downregulated DEGs in secretory organoids (sec-org) from women of advanced reproductive age revealed that pro-inflammatory signaling pathways were downregulated in these organoids, including cytokine-cytokine receptor interactions, the NOD-like receptor (NLR) signaling pathway, the IL-17 signaling pathway, and the TNF signaling pathway. This inhibition of these pathways reflects a reduced inflammatory response in the endometrium during the secretory phase in women of advanced reproductive age. Consistent with transcriptomic analysis, most inflammatory genes ( ) were downregulated in the secretory organoids of women of advanced reproductive age. TNF NFKB2, CSF3, CCL2, CCL20, CXCL3, IL1A The mRNA expression levels of these genes were also significantly downregulated. The proteins encoded by these genes play important roles in the inflammatory response. These findings suggest that the reduced inflammatory response of the endometrium during the secretory phase in older women of reproductive age may be related to decreased endometrial receptivity.

[0050] In summary, the above results indicate that the inflammatory status of the endometrium in women of advanced reproductive age varies significantly across 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.

[0051] Flow cytometry was used to analyze organoid cultures from different menstrual cycles. The results showed that in the proliferative phase organoid (pro-org) culture medium of women of advanced reproductive age, the concentrations of most pro-inflammatory factors, such as IL-23, TNF-α, IL-1β, IL-2, and IL-6, were elevated, consistent with the previously observed elevated expression levels of pro-inflammatory genes and proteins in the proliferative endometrium and organoids of women of advanced reproductive age. However, in the secretory phase organoid (sec-org) culture medium of women of advanced reproductive age, we found decreased concentrations of pro-inflammatory factors such as monocyte chemoattractant protein-1 (MCP-1), IL-23, and IL-1β. Simultaneously, the production of Th1 cytokines IL-2 and IFN-γ increased, while the secretion of Th2 cytokine IL-10 decreased. This result indicates a shift in the Th1 / Th2 immune balance towards Th1 in the secretory phase endometrium of women of advanced reproductive age. In conclusion, these results confirm the presence of an inflammatory imbalance in the endometrium of women of advanced reproductive age.

[0052] 4. Fibrous characteristics of aging endometrium

[0053] This study compared the differences in fibrosis levels between endometrial samples from younger and older reproductive-age groups using multidimensional detection methods including gene expression profiling, histopathology, and quantitative protein expression. qRT-PCR results showed that in the proliferative endometrial tissue (pro-tis) of the older reproductive-age group, multiple genes related to cell adhesion were present. LAMA2, LAMC3, ITGA11, ITGA5 ), collagen-related genes ( COL6A1, COL6A2, COL5A2 ) and myosin-related genes ( MYLK, MYL9The mRNA levels of fibronectin and myosin light chain kinase (MYLK) were significantly elevated. This finding corroborated the Masson staining results, which visually showed a significant increase in the degree of fibrosis in endometrial samples from women of advanced reproductive age, a pathological change that has been proven to be closely related to impaired female fertility. Furthermore, Western blot (WB) analysis further confirmed this finding: in pro-tis samples from women of advanced reproductive age, the protein expression levels of fibronectin and MYLK were significantly upregulated. These multi-level experimental evidences collectively indicate that endometrial fibrosis worsens with age. qRT-PCR results showed that in proliferative organoids (pro-org) from women of advanced reproductive age, multiple pro-fibrosis genes (including...) were... COL6A2, LAMC3 MYL9, ITGA11 The transcriptional levels of [various substances] were significantly elevated. This finding is highly consistent with in vivo experimental results and further supports the conclusion that endometrial fibrosis is exacerbated in women of advanced reproductive age.

[0054] Organoids were induced to the secretory phase (sec-org) through hormone treatment. Under these experimental conditions that mimic the physiological cycle, pro-fibrotic genes (such as...) were observed in the sec-org of older reproductive-age groups. COL6A2, MYL9, LAMC3, COL5A2, ITGA11 The transcriptional level of fibronectin (etc.) remained significantly upregulated. Furthermore, Western blotting further confirmed that fibronectin protein expression was significantly elevated in the sec-org of women of advanced reproductive age. These results not only validated the phenotypic characteristics of endometrial fibrosis in women of advanced reproductive age but also revealed the persistence of this fibrosis phenomenon across different physiological stages (proliferative and secretory phases).

[0055] 5. Construction of an organoid model of aging endometrium

[0056] 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.

[0057] Endometrial organoids were treated with different concentration gradients (20 mM, 40 mM, and 50 mM) of Dg, and key aging-related genes (including Dg) were quantitatively detected by qRT-PCR. CDKN1A, CTNNB1, TP53, GPX1, CTGF and CDK7 The study investigated changes in the transcriptional levels of Dg-induced organoid senescence. Results showed that Dg-induced organoid senescence exhibited a significant concentration-dependent effect. At a treatment concentration of 40 mM, all detected senescence-related genes reached peak expression levels, including tumor suppressor 53 (…). TP53 The expression level of connective tissue growth factor (FT4) was significantly increased by 1.5 times compared with the control group. CTGF The expression level of ) increased by 1.7 times ( Figure 3A). Based on the above results, 40 mM was determined to be the optimal concentration for establishing an endometrial organoid aging model.

[0058] Western blot (WB) was used to detect classic markers of cellular senescence. 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 was significantly increased in the Dg treatment group ( Figure 3 These results collectively confirm the successful construction of a stable and reliable model of endometrial aging.

[0059] 6. Functional changes in aging endometrial organoid models

[0060] qRT-PCR analysis revealed that after Dg successfully induced organoid aging, inflammation-related genes ( FAS, JUN, NFKB1 and IL15 The transcriptional level of ) was significantly upregulated ( Figure 4 A). These results indicate that Dg-induced organoid aging is accompanied by significant changes in the inflammatory microenvironment, a finding that is highly consistent with the characteristics of endometrial immune microenvironment dysregulation in women of advanced reproductive age.

[0061] The results of detecting the expression levels of fibrosis-related biomarkers showed that fibrosis-related genes... COL1A1、 COL6A1, COL6A2, ITGA1 and ITGA5 The transcription levels were significantly increased ( Figure 4 (B) Among them, the upregulation of COL1A1, a major collagen component, suggests extracellular matrix (ECM) deposition, while altered expression of integrin family members ITGA1 and ITGA5 may affect cell-matrix interactions, jointly promoting the fibrotic process. This finding not only confirms the significant exacerbation of fibrosis during Dg-induced aging, but also closely matches the phenotype of increased endometrial fibrosis in women of advanced reproductive age.

[0062] Results of uterine receptivity assays on secretory 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 exhibited 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 5B). Furthermore, immunofluorescence staining (IF) revealed a significant decrease in the fluorescence intensity of the receptivity markers glycodelin and insulin-like growth factor binding protein 1 (IGFBP1). Figure 5 The above data collectively indicate that Dg can induce the aging process of endometrial organoids, promote their inflammatory response and fibrosis, and significantly reduce endometrial receptivity.

[0063] Example 2: Screening of compounds that improve endometrial aging

[0064] 1. Construction of an aging endometrial model

[0065] The endometrial organoids constructed in Example 1 were operated on as follows:

[0066] Preheat the digestion solution to 37°C, discard the culture medium in the well plate, and wash twice with DPBS. Add 500 μL of digestion solution to each well of a 24-well plate containing endometrial organoids, pipette and scrape off the organoids, transfer them to 15 mL centrifuge tubes, and incubate at 37°C for approximately 20 minutes. Centrifuge the cell suspension at 400 g for 5 minutes, resuspend the cell pellet in DMEM / F12 medium, and centrifuge again at 400 g for 5 minutes. According to the cell density requirement (2 × 10⁻⁶ cells / well), [the cell density is determined by the cell density requirement (2 × 10⁻⁶ cells / well)]. 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.

[0067] Open Corning DNA Studio software and set the printhead and print bed temperatures (printhead 4°C, print bed 37°C). Select the plate type and the number of drops per well (96 wells, 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 heatsink on the printhead, add the mixed Matrigel-cell suspension to the syringe, connect the syringe plunger holder, and adjust the position of the plunger holder. Use automatic calibration to calibrate the printhead, and then start printing. Incubate 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, changing the medium every other day.

[0068] 2. High-throughput initial screening of drugs

[0069] In each 96-well plate of the bioprinted material, a control group (6 wells, standard growth medium, with the same medium change frequency as the other two groups), a Dg group (6 wells, standard growth medium containing Dg), and a drug-treated group (3 replicates for each drug) were set up. After seeding, the Dg group and the drug-treated group were cultured in medium containing Dg for 3 days. From day 4 to 6, the Dg group was treated with medium containing 40 mM Dg and 0.5% DMSO, and the drug-treated group was treated with medium containing 40 mM Dg and 50 μM of the compound.

[0070] The organoids processed above are subjected to CellEvent. TM Senescence staining was performed, followed by scanning of 96-well plates using the ImageXpress Micro Confocal high-content imaging system with 4×PhL Plan Fluor DL ​​objectives and a 60 µm pinhole confocal rotary disk. Hoechst 33342 was identified using the DAPI channel, the senescence probe β-gal using the FITC channel, and organoids under bright-field conditions using the TL-10 channel. Each well was scanned 200–300 µm using a z-stack acquisition mode with a 15 μm step, and imaging was performed using Best Focus. Image analysis was performed using Metaxpress. ® The software, within its custom module workflow, analyzes 2D projections. Based on FITC images, it identifies all β-gal-positive cytoplasm, representing senescent cells, and calculates the fluorescence intensity of β-gal.

[0071] In the initial screening stage, the fluorescence intensity of β-galactosidase (SA-β-gal) was used as a detection indicator to assess the degree of cellular senescence. Through systematic detection, 28 candidate drugs that could significantly reduce SA-β-gal levels were screened from 191 drugs. Figure 6 The 28 drugs identified were: D-alanine, nicardipine, vorminopeptide, loxapine, guanethidine, triamcinolone A, metformin, melatonin, sulfamethoxazole, acarbose, thioridazine, nicotinamide adenine dinucleotide, quercetin, diallyl trisulfide, kaempferol, vitexin, sesamin, L-arginine, L-arginine (hydrochloride), piperidin, doxycycline hydrochloride, quercetin (dihydrate), L-arginine (L-glutamate), chicoric acid, folic acid, chlorogenic acid, ganoderic acid C2, and oxaloacetic acid. This suggests that these drugs have potential activity in alleviating cellular aging.

[0072] 3. High-throughput drug rescreening

[0073] In each 96-well plate of the bioprinted material, a control group (6 wells, standard growth medium, with the same medium change frequency as the other two groups), a Dg group (6 wells, standard growth medium containing Dg), and a drug-treated group (3 replicates for each drug) were set up. After seeding, the Dg group and the drug-treated group were cultured in medium containing Dg for 3 days. From day 4 to 6, the Dg group was treated with medium containing 40 mM Dg and 0.5% DMSO, and the drug-treated group was treated with medium containing 40 mM Dg and 50 μM of the compound.

[0074] The processed organoids were then subjected to EdU and Beyo3D experiments sequentially. TM Hoechst 33342 staining was performed, followed by scanning of 96-well plates using the ImageXpress Micro Confocal high-content imaging system with 4×PhL Plan Fluor DL ​​objectives and a 60 µm pinhole confocal rotary disk. Hoechst 33342 was identified using the DAPI channel, EdU using the Texas Red channel, and organoids under bright-field conditions using the TL-10 channel. Each well was scanned 200–300 µm using a z-stack acquisition mode with a 15 μm step, and Best Focus was selected for imaging. Image analysis was performed using Metaxpress. ® The software analyzes 2D projections within its custom module workflow. Based on DAPI images, it identifies all Hoechst 33342-positive cell nuclei, representing the total cell count, and uses an algorithm to identify the number and area of ​​organoids. Based on Texas Red images, it identifies all EdU-positive cell nuclei, representing proliferating cells. It then calculates the percentage of EdU-positive cells per well relative to the total cell count and the average area of ​​the organoids.

[0075] Based on the initial screening of 28 potential anti-aging drug candidates, to further evaluate the promoting effect of these drugs on cell proliferation, the proportion of EdU-positive cells in the drug-treated groups was detected using the 5-ethynyl-2'-deoxyuridine (EdU) labeling method. Ultimately, five drugs that could significantly increase the proportion of EdU-positive cells were screened. Figure 7 The five drugs are: L-Arginine (LA), folic acid (FA), oxaloacetic acid (OAA), chlorogenic acid (CGA), and ganoderic acid C2 (GDA).

[0076] 4. Drug-induced proliferation activity

[0077] Further drug concentration gradient tests were conducted on oxaloacetic acid, chlorogenic acid, and ganoderic acid C2. The results showed that different drugs exhibited the best cell proliferation-promoting effects at specific concentration ranges: LA, FA, OAA, and CGA showed 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 mechanism of action and its affinity for cellular targets.

[0078] The above findings demonstrate that the constructed aging endometrial model can be used for drug screening, and the results are highly reliable.

[0079] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. The application of organic acid compounds in the preparation of drugs to improve endometrial aging, characterized in that, The organic acid compound is selected from oxaloacetic acid or chlorogenic acid.

2. The application according to claim 1, characterized in that, The aging state is characterized by increased endometrial inflammation, enhanced endometrial fibrosis, and decreased endometrial receptivity.

3. The application according to claim 1, characterized in that, The drug is administered orally or via mucosal membrane.

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

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