Use of bavachin in the preparation of a drug for alleviating and / or preventing senescence of uterine decidual stromal cells

Abstract

This invention discloses the application of bavachin in the preparation of drugs for alleviating and / or preventing senescence of uterine decidual stromal cells, belonging to the field of biomedical technology. Through drug library screening and experimental verification, this invention found that bavachin can significantly alleviate the senescent phenotype of human uterine decidual stromal cells, reduce the expression of senescence-related markers, and alleviate decidual stromal cell senescence by regulating intracellular copper ion metabolism and transport to maintain intracellular and extracellular copper ion homeostasis. This invention provides a novel drug target and intervention strategy for clinically improving endometrial decidual function and treating related reproductive health problems. This invention also provides the application of bavachin in the preparation of drugs for alleviating senescence of uterine decidual stromal cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of Bavachin in the preparation of drugs for alleviating and / or preventing senescence of uterine decidual stromal cells. Background Technology

[0002] Decidualization is the differentiation and transformation of the endometrial stroma, which is crucial for embryo implantation, placental development, and the establishment of the maternal-fetal interface. Decidual stromal cell senescence is closely associated with adverse pregnancy outcomes, including implantation failure, early miscarriage, late-pregnancy preeclampsia, and fetal growth restriction.

[0003] Cellular senescence has become an important research direction in the fields of reproductive medicine and perinatal medicine in recent years, and it is closely related to adverse pregnancy outcomes. It is mainly manifested in the following aspects: (1) Premature placental cell senescence is closely related to pregnancy complications (such as preeclampsia and premature rupture of membranes), manifested as telomere shortening, ROS accumulation and release of inflammatory factors (IL-6, IL-8); (2) In the reproductive system, oocyte senescence is associated with chromosomal abnormalities and increased risk of miscarriage, the mechanism of which involves cumulus cells secreting senescence-promoting factors and telomere damage caused by free radical exposure. (3) Senescence of immune cells at the maternal-fetal interface can lead to recurrent miscarriage by disrupting immune tolerance. Among them, decidual stromal cells are an important component of the maternal-fetal interface. Premature senescence of DSCs is an important cause of pregnancy complications such as preeclampsia and premature birth, and its pathological mechanism involves replicative senescence caused by telomere shortening and abnormal accumulation of mitochondrial ROS. Based on this, the senolytic strategy of targeting and clearing senescent cells has become a cutting-edge direction in reproductive senescence research.

[0004] Therefore, finding anti-aging drugs that meet the requirements of long-term safety and efficacy has always been an important strategy in the field of aging intervention. Flavonoids are a variety of natural phenolic compounds. In addition to their well-known antioxidant activities, flavonoids also possess anti-inflammatory, vasodilatory, anticoagulant, cardioprotective, antidiabetic, chemoprotective, neuroprotective, and anti-obesity activities. Previous studies have revealed that, in an ovarian granulosa cell aging model, quercetin and baicalin can significantly inhibit oxidative stress and restore cell proliferation capacity through the synergistic regulation of the Nrf2 / ARE-PI3K / Akt dual pathway. In endometrial diseases, the progesterone compound acetin improves the pathological microenvironment of the endometrium by blocking the NF-κB signaling cascade. These findings provide direct evidence for the molecular mechanisms by which flavonoids regulate cellular aging and improve the aging of the female reproductive system.

[0005] Intracellular copper ions play a crucial role in maintaining normal cellular physiological functions as essential cofactors for many key enzymes, such as antioxidant and energy metabolism enzymes. However, precise balance of copper levels is critical; imbalance can have a profound and complex impact on the cellular senescence process. Conversely, cellular senescence itself disrupts copper homeostasis. Senescent cells often exhibit decreased metabolic reprogramming and autophagy, potentially leading to abnormal accumulation of copper ions intracellularly, thereby amplifying their toxic effects. Recent studies have also revealed the potential for bidirectional regulation of this relationship. For example, inducing "copper death"—a newly discovered copper-dependent cell death mechanism—through specific methods (such as copper ion carriers) can selectively eliminate stubborn senescent cells, providing a promising new strategy for developing anti-aging and treatment strategies for age-related diseases such as cancer and neurodegenerative diseases.

[0006] Psoralen, an important member of the natural flavonoid family, has been shown to have anti-inflammatory effects, including inhibiting the SASP pathway, reducing neuroinflammation through antioxidation, inhibiting the mitochondrial electron transport chain, oxidative phosphorylation, and inducing apoptosis. However, whether bavachin alleviates decidual stromal cell senescence by regulating intracellular and extracellular copper ion metabolism and transport, and its specific molecular mechanism, remains unclear. Further investigation into the regulatory role of bavachin on decidual stromal cell senescence and copper ion metabolism could provide new targets and theoretical basis for the prevention and treatment of pregnancy-related diseases. Summary of the Invention

[0007] The purpose of this invention is to solve the technical problem that the key factors and their specific regulatory mechanisms for alleviating the senescence of uterine decidual stromal cells are still unclear in the prior art.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] Use of Bavachin in the preparation of drugs for alleviating and / or preventing senescence of uterine decidual stromal cells.

[0010] In some embodiments, the drug is used to treat or prevent pregnancy-related disorders caused by senescence of uterine decidual stromal cells, the disorders being selected from at least one of implantation failure, recurrent implantation failure, early spontaneous abortion, preeclampsia, fetal growth restriction, and premature rupture of membranes.

[0011] In some embodiments, the Bavachin exerts its effects through at least one of the following mechanisms: reducing aging-related β-galactosidase activity, downregulating the expression of cyclin-dependent kinase inhibitor P21 and / or tumor suppressor protein P53, inhibiting the expression of aging-related secretory phenotype factors IL-1β and / or CXCL2, reducing apoptosis, and reducing lactate dehydrogenase release.

[0012] In some embodiments, the Bavachin alleviates cellular senescence by regulating copper ion homeostasis within uterine decidual stromal cells.

[0013] In some embodiments, the Bavachin regulates intracellular copper homeostasis by upregulating the expression of the copper ion transporter ATP7B.

[0014] A pharmaceutical composition for alleviating and / or preventing senescence of uterine decidual stromal cells, comprising Bavachin as the active ingredient and a pharmaceutically acceptable carrier or excipient.

[0015] In some embodiments, the concentration of Bavachin in the pharmaceutical composition is from 1 μM to 50 μM.

[0016] A method for screening compounds with the potential to alleviate the senescence of uterine decidual stromal cells includes the following steps:

[0017] (a) Provide uterine decidual stromal cells or an in vitro aging model thereof;

[0018] (b) Treat the cells with the candidate compound;

[0019] (c) Detect changes in the intracellular copper ion concentration or the expression level of the copper ion transporter ATP7B;

[0020] Among them, compounds that can increase intracellular copper ion concentration or upregulate ATP7B expression were identified as potentially effective compounds.

[0021] One application of Bavachin in constructing an in vitro model for studying the aging mechanism of uterine decidual stromal cells or screening anti-aging drugs is described, in which Bavachin is used as a positive control compound or tool drug to regulate intracellular copper ion homeostasis.

[0022] Compared with the prior art, the beneficial effects of this application are as follows:

[0023] This application screened the target compound Bavachin from a library of natural flavonoids, constructed a decidualization model to verify the low toxicity and anti-apoptotic effects of Bavachin on decidual stromal cells, its ability to alleviate cell damage, inhibit SASP secretion in senescent cells, and reduce the recruitment of inflammatory cells. It elucidates that Bavachin regulates intracellular copper ion metabolism and transport, maintains intracellular and extracellular copper ion homeostasis, and alleviates decidual stromal cell senescence. This could provide a new drug candidate for the clinical treatment of infertility, recurrent implantation failure, and early miscarriage caused by decidual senescence, and also provide a theoretical basis for exploring the relationship between cellular senescence and intracellular copper ion metabolism. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the construction and verification of the decidualization model in Embodiment 1 of the present invention:

[0025] In A and B, human endometrial stromal cells (HESCs) were treated with 8-Br-cAMP and MPA. Cell morphology was observed after 72 hours, showing that the cells changed from elongated spindle-shaped to elliptical, with the decidualization morphology being most pronounced at 72 hours. In C, the expression of decidual marker molecules PRL and IGFBP1 was significantly increased. In D, the senescence value gradually increased over time (SA-β-gal activity increased). These findings validated the successful establishment of a senescence model for decidualized stromal cells in the experiment. P < 0.05;

[0026] Figure 2 This is a schematic diagram of screening the target compound Bavachin in Example 2 of the present invention;

[0027] Among them, the positive criterion is: SA-β-gal inhibition rate > 50%;

[0028] Figure 3 The figure shows the experimental results of Bavachin in Example 3 of this invention, which verified the anti-aging effects of Bavachin on decidual cell proliferation, viability, apoptosis, LDH, ATP, and aging phenotype.

[0029] in, Figure 3 -A: The CCK8 assay was used to detect cell proliferation and cytotoxicity between the two groups. P < 0.05; Figure 3 -B: Apoptosis assay kit showed reduced apoptosis in the control group cells. P < 0.05;

[0030] Figure 3 -C: LDH and ATP kits were used to detect differences between the two groups of cells. P < 0.05;

[0031] Figure 3 -D: Cell β-galactosidase staining assay was used to detect the difference in senescence between the two groups of cells. Blue represents senescent cells. The scale bar in the figure is 100μm.

[0032] Figure 3 -E: Statistical analysis of SA-β-gal staining area in two groups. P < 0.05;

[0033] Figure 3 -F, G, H, I: SASP level qPCR validation, P < 0.05;

[0034] Figure 4-1 This is a schematic diagram of proteomics sequencing in Example 4 of the present invention;

[0035] In this diagram, A: The differential volcano plot shows the levels of 351 proteins that were significantly downregulated and 378 proteins that were significantly upregulated in decidual stromal cells after Bavachin treatment; B: The differential scatter plot shows the changes in the levels of proteins related to copper ion transport.

[0036] Figure 4-2 This is a graph showing the qPCR validation results of differentially expressed proteins related to copper ion transport. P < 0.05;

[0037] Figure 5 This refers to the level of intracellular copper ion concentration detected by Bavachin in Example 5 of the present invention.

[0038] Figure 6 To verify the effects of different concentrations of copper ions on decidual cell proliferation, viability, apoptosis, LDH, ATP, and aging phenotype in Example 6 of this invention on day 4 and day 7;

[0039] in, Figure 6 -A: CCK8 assay was used to detect the effects of adding different concentrations of copper ions on cell proliferation and toxicity. P < 0.05; Figure 6 -B: Apoptosis kit to detect the apoptotic effect of adding different concentrations of copper ions on cells. P < 0.05; Figure 6 -C: LDH and ATP kits were used to detect differences in the effects of adding different concentrations of copper ions on cells. P < 0.05; Figure 6 -D: Validation of cell senescence phenotypes using appropriate concentrations of copper ions. P < 0.05. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0041] The above content will be explained in conjunction with specific verification experiments:

[0042] Example 1: Constructing a decidualization model and verifying its successful construction

[0043] Specifically, it includes the following steps:

[0044] 1. Endometrial stromal cells were revived and cultured in a 37°C, 5% CO2 incubator in DMEM / F12 medium supplemented with 10% fetal bovine serotonin (FBS) (Gibco), 100 IU / ml penicillin, and 100 μg / ml streptomycin (HyColne). In this application, cells passaged 2 to 4 times were used.

[0045] 2. Induction of decidualization: A decidual stromal cell (DSC) model was induced in vitro by adding 8-bromocyclic adenosine monophosphate (8-Br-cAMP) (0.5 mM) and medroxyprogesterone acetate (MPA) (1 μM) to human endometrial stromal cells, and the culture medium was replaced with 2.5% fetal bovine serum. Cell morphology was observed after 72 hours, and the cells changed from elongated spindle-shaped to elliptical.

[0046] 3. RNA Extraction and qPCR Analysis from Decidual Stromal Cells: Total RNA was extracted from decidual stromal cells according to the Invitrogen RNA Isolation Kit instructions. The absorbance (A260 / 280 nm) was measured using a nucleic acid protein analyzer, and the RNA concentration and purity were calculated. PRL and IGFBP1 primers were ordered from Shanghai Sangon Biotech Co., Ltd. (see Table 1 below). Reverse transcription and qPCR reactions were performed on the total RNA using Takara's reverse transcription kit and Takara's SYBR Green PCR kit, respectively. A 10 μl reaction mixture was prepared, and the reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 40 cycles. β-actin was used as an internal control, and the reaction was performed on a Roche real-time quantitative PCR instrument. The results were calculated and statistically analyzed, with β-actin used as an internal reference to correct the copy number of the PCR template and eliminate inter-group loading errors.

[0047] Table 1 Primers used for decidual-related qPCR

[0048]

[0049] The results are as follows Figure 1As shown, this application uses 8-Br-cAMP and MPA to induce decidualization. Cell morphology was observed at 72 hours, showing that cells changed from elongated spindle-shaped to elliptical, indicating a decidualization phenotype. Quantitative PCR analysis showed that, compared with the untreated control group (human endometrial stromal cells), the experimental group (decidual stromal cells) after the addition of 8-Br-cAMP and MPA showed significantly increased expression of decidual marker molecules PRL and IGFBP1, and the senescence values ​​gradually increased over time (increased SA-β-gal activity). Figure 1 CD). The above experimental results indicate that the in vitro induced decidualization cell model was successfully constructed. P<0.05, P<0.01, P<0.001.

[0050] Example 2: Screening for the target compound Bavachin

[0051] Compounds that intervene in decidual stromal cell senescence were detected by high-throughput SA-β-gal in the Progesterone Compound Library (n=260).

[0052] Specifically, it includes the following steps:

[0053] Endometrial stromal cells were revived, passaged, and their stability ensured. According to the experimental design, cells were cultured at approximately 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in white ELISA-labeled 96-well plates and cultured until adherent. At approximately 75% confluence, 8-Br-cAMP (0.5 mM) and MPA (1 μM) were added to induce in vitro decidual stromal cells. After 72 hours, 260 compounds from a progesterone compound library (final concentration 10 μM) were added to each well, with a blank control group (no compound added). Each compound was applied in triplicate. The medium was changed after 48 hours, and the 96-well plates were removed after another 48 hours. Following the Promega aging kit instructions, 100 μL of working solution + 100 μL of culture medium were added to each well. The plates were incubated at room temperature with shaking for 30-60 minutes. Chemiluminescence logarithmic analysis was performed using an ELISA reader to calculate the relative aging level.

[0054] The results are as follows Figure 2 As shown, Bavachin can significantly reduce SA-β-gal activity in decidual stromal cells. P<0.05, P<0.01, P < 0.001. The results show that Bavachin is the target compound with anti-aging activity.

[0055] Example 3: Verifying the cytotoxic and anti-aging effects of Bavachin

[0056] Specifically, it includes the following steps:

[0057] 1. CCK8 detection

[0058] The in vitro decidualization treatment in clear 96-well plates was the same as above. The original culture medium was discarded, and 0.1 μL Lavachin (10 mM) + 100 μL 2.5% complete culture medium was added to each well. A blank control group (culture medium only, no cells) and a negative control group (cells + drug-free culture medium) were also set up, with three replicates for each group. After adding the drug, the medium was changed after 48 hours, and the 96-well plate was removed after another 48 hours. According to the instructions for the CCK8 kit, 100 μL of CCK8 working solution was prepared in each well at a 1:10 ratio of the assay reagent to basal culture medium. The 96-well plate was gently shaken to ensure thorough mixing of the reagent and culture medium, avoiding air bubbles. The 96-well plate was placed back in a 37°C, 5% CO2 incubator and incubated in the dark for 60 minutes. After incubation, the 96-well plate was removed, and the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Data calculation: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0059] 2. Apoptosis, LDH, and ATP detection

[0060] The in vitro decidualization treatment in white ELISA-labeled 96-well plates was the same as above. The original culture medium was discarded, and 0.1 μL Lavachin (10 μM) + 100 μL 2.5% complete culture medium was added, with 3 replicates. After adding the drug, the medium was changed after 48 hours, and the 96-well plate was removed after another 48 hours. According to the Promega apoptosis kit instructions, the complete culture medium was preheated. Annexin V-LgBiT and Annexin V-SmBiT were added to 10% complete culture medium and shaken. CaCl2 and Annexin V NanoBiT® Substrate were added and mixed by inverting. The original culture medium was discarded, and 100 μL of apoptosis detection drug + 100 μL of 10% complete culture medium (200 μL total) were added. The 96-well plate was returned to a 37°C, 5% CO2 incubator and incubated for 6-8 hours. After incubation, the 96-well plate was removed, and the chemiluminescence value (LUM value) of each well was measured using a microplate reader. Using the Promega ATP assay kit, the assay reagent was mixed 1:1 with cell supernatant and added to each well. After incubation for 10 min, the chemiluminescence (LUM) value was measured using a microplate reader. Using the Promega LDH assay kit, the cell supernatant was removed, diluted 50-fold with LDH buffer, mixed 1:1 with LDH working solution, and then placed back into a cell-free white microplate. After incubation at room temperature for 60 min, the chemiluminescence value was measured.

[0061] 3. Cell smear SA-β-gal staining

[0062] The in vitro decidualization treatment is the same as above. Seed the cells to be tested in 24-well cell slides and culture until the cell density reaches 80%. After completing the treatment as described above, aspirate the old culture medium from the 24-well plate and gently wash the cells twice with pre-cooled PBS, incubating for 3-5 minutes each time to remove residual culture medium. Aspirate the PBS, add 4% paraformaldehyde fixative to cover the cell surface, and fix at room temperature for 15-20 minutes. After fixation, aspirate the fixative and wash the cells three times with PBS, 3-5 minutes each time, to thoroughly remove residual fixative. Prepare the staining working solution according to the kit instructions. The preparation process should be carried out at room temperature, ensuring thorough mixing to avoid precipitation. Add the working solution to the 24-well plate until it covers the slide, ensuring complete cell coverage. Seal the culture dish with parafilm and incubate at 37°C in a CO2-free incubator in the dark for 24-48 hours, until the blue signal is clear and the background is low. After incubation, aspirate the staining working solution and wash the cells twice with PBS to remove unbound stain. Add a small amount of PBS to keep the cells moist and observe them under a light microscope: blue-stained cells are SA-β-gal positive cells (senescent cells), and colorless cells are negative cells (non-senescent cells). Randomly select 5 fields of view and use ImageJ software to calculate the positive cell coverage area: positive cell coverage area / total cell coverage area × 100%.

[0063] 4. Total RNA extraction and qPCR analysis

[0064] Total RNA was extracted from the control group (naturally senescent decidual stromal cells) and the experimental group (decidual stromal cells treated with Bavachin) according to the Invitrogen RNA Isolation Kit instructions. The absorbance (A260 / 280 nm) was measured using a nucleic acid protein analyzer, and the RNA concentration and purity were calculated. Primers for P21, P53, IL-1β, and CXCL2 were ordered from Shanghai Sangon Biotech and Qingke Biotechnology (see Table 2 below). Total RNA was reverse transcribed and qPCR was performed using Takara's reverse transcription kit and SYBR Green PCR kit, respectively. A 10 μl reaction volume was prepared, and the reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 40 cycles. β-actin was used as an internal control, and the reaction was performed on a Roche real-time quantitative PCR instrument. The results were calculated and statistically analyzed, with β-actin used as an internal reference to correct the copy number of the PCR template and eliminate inter-group loading errors.

[0065] Table 2 Primers used in aging-related qPCR

[0066]

[0067] The results are as follows Figure 3 , 4-1 As shown in Figure 4-2, the CCK8 experiment showed no significant difference between the control group and the experimental group. Figure 3 -A). Apoptosis experiments showed that the apoptosis rate in the 10 μM Bavachin treatment group was significantly lower than that in the control group. Figure 3 -B). LDH and ATP tests showed no significant difference between the two groups ( Figure 3 -C), further reflecting that the drug did not cause significant damage to the cells. SA-β-gal staining showed a decrease in the number of senescent cells in the experimental group ( Figure 3 -D, E). P53 and P21 are classic pathway molecules mediating cellular senescence, and their expression levels directly reflect the core regulatory state of cellular senescence. After cellular senescence, a large number of soluble molecules such as inflammatory factors and chemokines are secreted, forming a senescence-associated secretory phenotype (SASP). IL-1β and CXCL2 are representative core molecules in SASP, and their expression levels directly reflect the functional phenotypic effects of cellular senescence. Therefore, detecting their mRNA expression levels can indicate the degree of cellular senescence. In qPCR experiments, the mRNA expression levels of P21, P53, IL-1β, and CXCL2 were significantly lower than those of the control group ( Figure 3 -F, G, H, I) P<0.05, P<0.01, P<0.001. This application found that Bavachin did not alter the proliferative capacity of decidual stromal cells, nor did it cause a decrease in cell viability due to toxicity. 10 μM Bavachin significantly inhibited apoptosis without causing cell damage or affecting ATP production, thus maintaining cell viability. These experiments simultaneously verified, from two key dimensions—the mechanism of aging and the phenotypic effect of aging—that Bavachin treatment alleviated cell cycle arrest, inhibited SASP secretion in senescent cells, and reduced the recruitment of inflammatory cells, thereby comprehensively demonstrating that Bavachin has a role in alleviating cellular senescence.

[0068] Example 4: Proteomics Sequencing

[0069] Deciduous cells from the 10 μM Bavachin treatment group and the control group were collected, and total protein was extracted. Proteomics sequencing was performed using LC-MS / MS to screen for differentially expressed proteins. GO functional enrichment analysis and KEGG analysis were conducted on the differentially expressed proteins. Candidate differentially expressed proteins with significant differences were validated by qPCR (primers are shown in Table 3 below) to understand the mechanisms related to the copper ion transport pathway.

[0070] Table 3 Primers used in copper ion transport-related qPCR

[0071]

[0072] The results are shown in Figure 4. A volcano plot of differentially expressed genes was drawn, and a total of 729 differentially expressed proteins were screened, of which 378 were upregulated and 351 were downregulated. Figure 4-1 A). KEGG enrichment suggests a close correlation with fatty acid degradation, TCA cycle, oxidative phosphorylation, 2-oxocarboxylic acid metabolism, and degradation of valine, leucine, and isoleucine. Figure 4-1 B), the scatter plot showed changes in the level of ATP7B, a protein related to copper ion transport. qPCR was used to verify its mRNA expression, and the results showed that ATP7B mRNA expression was upregulated and statistically significant. P<0.05, P<0.01, P<0.001, Figure 4-2 ).

[0073] Example 5: Validation of the copper ion transport pathway

[0074] Specifically, it includes the following steps:

[0075] 1. Intracellular copper ion measurement

[0076] Cells were arranged at a ratio of 2 × 10 6 Cells were seeded in 6-well plates and subjected to in vitro decidualization induction as described above. On days 0, 4, and 7 post-treatment, collected cells were added to approximately 0.2 mL of reagent tetralysis buffer per 2 × 10⁶ cells. After mixing, the cells were lysed on ice for 10 min, followed by centrifugation at 12000 × g for 10 min at 4°C. The supernatant was collected for analysis, and a portion of the supernatant was retained for protein concentration determination. The protein concentration was determined according to Elabscience® Cell Copper (Cu) assay. 2+ The intracellular copper ion content in Bavachin and control cells was detected using a colorimetric assay kit, and the protein concentration in each well was measured according to the Beyotime BCA kit. The calculation formulas are: Standard curve fitting: y = ax + b; Cellular copper ion concentration calculation formula: Copper ion content Notes: y: OD value of standard wells - OD value of blank wells (OD value when the concentration of standard is 0) x: Concentration of standard a: Slope of standard curve b: Intercept of standard curve ΔA580: OD value of sample determination - OD value of blank wells (OD value when the concentration of standard is zero) f: Dilution factor of sample before addition to the detection system Cpr: Protein concentration of sample before addition to the detection system (gprot / L) The results indicate that the copper ion content in the drug-treated group is significantly higher than that in the control group, and the intracellular copper ion content shows a decreasing trend with the increase of days. Figure 5 ).

[0077] Example 6: Validation of Copper Ion Homeostasis in Alleviating Cellular Senescence

[0078] According to the experimental design, cells were spaced at approximately 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in white ELISA-labeled 96-well plates and cultured until adherent. At approximately 75% confluence, 8-Br-cAMP (0.5 mM) and MPA (1 μM) were added to induce in vitro decidual stromal cells. CCK8, LDH, ATP, apoptosis, and senescence were measured on days 4 and 7 using 0.2 μmol, 0.4 μmol, 0.6 μmol CuCl2 (Sigma), and 40 nmol Elesclomol (Sellck), respectively, following the same procedures as described above. Results are as follows: Figure 6 As shown, the simultaneous addition of 0.2 μmol and 0.4 μmol CuCl2 and 40 nmol Elesclomol can alleviate the senescence of uterine decidual stromal cells without affecting cell viability, proliferation, apoptosis, or ATP production. According to the experimental results, the intracellular copper ion concentration in the untreated group cells gradually decreased over time (i.e., during the senescence process). It is further speculated that ATP7B retains copper ions in the cells to maintain the homeostasis of intracellular copper ions, thereby maintaining normal cell function and improving the senescent state of uterine decidual stromal cells.

[0079] Based on the construction of a decidualization model and the screening of the target compound Bavachin from a library of natural flavonoids, this application found that Bavachin effectively alleviates the senescence of decidual stromal cells and has no cytotoxic effects through senescence assays, CCK8 assays, apoptosis assays, and detection of the expression of classic senescence markers P21, P53, IL-1β, and CXCL2.

[0080] Furthermore, this application discovered a potential close relationship between Bavachin and copper ion transport through proteomics sequencing. By analyzing the mRNA expression level of the copper ion transport-related differentially expressed protein ATP7B, intracellular copper ion content, and senescence assays after copper ion addition, it was found that Bavachin significantly alleviates decidual stromal cell senescence by regulating intracellular copper ion transport to maintain intracellular copper ion homeostasis, which has significant theoretical and clinical value.

[0081] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Use of Bavachin in the preparation of drugs for alleviating and / or preventing senescence of uterine decidual stromal cells.

2. The application according to claim 1, characterized in that, The drug is used to treat or prevent pregnancy-related diseases caused by aging of uterine decidual stromal cells, the diseases being selected from at least one of implantation failure, recurrent implantation failure, early spontaneous abortion, preeclampsia, fetal growth restriction, and premature rupture of membranes.

3. The application according to claim 1 or 2, characterized in that, The Bavachin exerts its effects through at least one of the following mechanisms: reducing aging-related β-galactosidase activity, downregulating the expression of cyclin-dependent kinase inhibitor P21 and / or tumor suppressor protein P53, inhibiting the expression of aging-related secretory phenotype factors IL-1β and / or CXCL2, reducing apoptosis, and decreasing lactate dehydrogenase release.

4. The application according to claim 1 or 2, characterized in that, The Bavachin alleviates cellular senescence by regulating copper ion homeostasis within uterine decidual stromal cells.

5. The application according to claim 4, characterized in that, The Bavachin regulates intracellular copper homeostasis by upregulating the expression of the copper ion transporter ATP7B.

6. A pharmaceutical composition for alleviating and / or preventing senescence of uterine decidual stromal cells, characterized in that, Its active ingredient is Bavachin, along with a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, characterized in that, The concentration of Bavachin in the pharmaceutical composition is from 1 μM to 50 μM.

8. A method for screening compounds with the potential to alleviate the senescence of uterine decidual stromal cells, characterized in that, Includes the following steps: (a) Provide uterine decidual stromal cells or an in vitro aging model thereof; (b) Treat the cells with the candidate compound; (c) Detect changes in the intracellular copper ion concentration or the expression level of the copper ion transporter ATP7B; Among them, compounds that can increase intracellular copper ion concentration or upregulate ATP7B expression were identified as potentially effective compounds.

9. An application of Bavachin in constructing an in vitro model for studying the senescence mechanism of uterine decidual stromal cells or screening anti-aging drugs, characterized in that, Bavachin was used as a positive control compound or tool drug to regulate intracellular copper ion homeostasis.

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