Use of biochanin a in the preparation of a medicament for inhibiting or ameliorating decidual stromal cell senescence

Abstract

The application discloses application of Biochanin A in preparation of a medicine for inhibiting or improving uterine decidua stromal cell senescence, and belongs to the field of biomedical technology. Through drug chemical library screening and experimental verification, it is found that Biochanin A can significantly alleviate the senescence phenotype of human uterine decidua stromal cells, reduce the expression of senescence-related markers, and alleviate decidua stromal cell senescence through regulation of mitochondrial energy metabolism. The application provides a brand-new drug target and intervention strategy for improving endometrial decidua function and treating related reproductive health problems in clinic, and provides application of Biochanin A in preparation of a medicine for alleviating uterine decidua stromal cell senescence.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Biochanin A in the preparation of drugs for inhibiting or improving the senescence of uterine decidual stromal cells. Background Technology

[0002] Endometrial decidualization is a crucial step in the establishment and maintenance of pregnancy, closely related to the formation of endometrial receptivity, trophoblastic cell invasion, and spiral artery remodeling. It provides a suitable microenvironment for embryo implantation and development, and has direct or indirect effects on extracellular matrix remodeling, immune response regulation, and anti-oxidative stress, forming the biological basis for successful pregnancy establishment and maintenance. When decidual stromal cells age, it can lead to pregnancy-related complications such as implantation failure and miscarriage. Mitochondria, as the core organelle of cellular energy metabolism, are closely related to cellular senescence due to functional abnormalities. Impaired mitochondrial respiratory chain function, reduced ATP production, and accumulation of oxidative stress products can all accelerate cellular senescence. Biochanin A (BCA) is a natural isoflavone compound. Previous studies have suggested that BCA regulates steroid production in ovarian granulosa cells by activating TAS2Rs. In polycystic ovary syndrome (PCOS), BCA can alleviate DHEA-induced PCOS by upregulating GDF9 and MBP15 signaling in vivo. However, whether BCA alleviates decidual stromal cell senescence by regulating mitochondrial energy metabolism pathways, and its specific molecular mechanisms, remain unclear. Further investigation into the regulatory role of BCA in decidual stromal cell senescence and its mitochondrial-related mechanisms could provide new targets and theoretical basis for the prevention and treatment of pregnancy-related diseases. Summary of the Invention

[0003] 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.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] Application of Biochanin A in the preparation of drugs for inhibiting or improving the senescence of uterine decidual stromal cells.

[0006] In some embodiments, the drug is used to prevent or treat pregnancy-related diseases caused by decidual stromal cell aging.

[0007] In some embodiments, the pregnancy-related conditions include implantation failure, recurrent implantation failure, miscarriage, or unexplained infertility.

[0008] In some embodiments, Biochanin A inhibits the senescence process of decidual stromal cells by downregulating the expression of cell cycle arrest proteins P21 and / or P53.

[0009] In some embodiments, Biochanin A exerts its anti-aging effect by inhibiting the expression of aging-related secretory phenotypes; preferably, the aging-related secretory phenotypes include at least one of IL-1α, IL-1β, and CXCL2.

[0010] In some embodiments, Biochanin A alleviates decidual stromal cell senescence by improving mitochondrial energy metabolism function.

[0011] In some embodiments, the improvement of mitochondrial energy metabolism function includes: increasing at least one of mitochondrial maximum respiratory capacity, residual respiratory capacity, and ATP production efficiency.

[0012] In some embodiments, Biochanin A improves mitochondrial energy metabolism by upregulating the expression of mitochondrial function-related proteins; preferably, the mitochondrial function-related proteins are selected from at least one of synaptic proteins, heat shock protein family members, NADH dehydrogenase complex subunits, and cytochrome C oxidase subunits.

[0013] In some embodiments, the mitochondrial function-related protein is COX2.

[0014] A pharmaceutical composition comprising a therapeutically effective amount of Biochanin A as the active ingredient, and a pharmaceutically acceptable carrier or excipient, for inhibiting or improving senescence of uterine decidual stromal cells.

[0015] A method for screening candidate compounds with inhibitory activity against decidual cell senescence, the method comprising: evaluating the effect of the candidate compounds on improving mitochondrial energy metabolism function in uterine decidual stromal cells; wherein the effect on improving mitochondrial energy metabolism function includes enhancing mitochondrial respiration capacity and / or upregulating the expression of mitochondrial function-related proteins.

[0016] In some embodiments, the method assesses the enhancing effect of candidate compounds on mitochondrial respiratory capacity by detecting the effects of candidate compounds on mitochondrial maximum respiratory capacity, residual respiratory capacity, or ATP production efficiency.

[0017] In some embodiments, the mitochondrial function-related protein is COX2; the method assesses the function of candidate compounds in improving mitochondrial energy metabolism by detecting the upregulation effect of candidate compounds on the expression level of COX2 protein or its encoding gene.

[0018] Compared with the prior art, the beneficial effects of this application are as follows:

[0019] This application screened the target compound Biochanin A from a library of natural flavonoids, constructed a decidualization model to verify the low toxicity and anti-apoptotic effects of Biochanin A on decidual stromal cells, alleviated cell cycle arrest, inhibited SASP secretion in senescent cells, and reduced the recruitment of inflammatory cells. It elucidates that Biochanin A enhances mitochondrial electron transport efficiency or increases reserve respiration capacity, thereby protecting cells from oxidative stress damage. It emphasizes that Biochanin A exerts its biological effects through a dual mechanism of "regulating mitochondrial energy metabolism + downregulating aging-related pathways." This application could provide a new drug candidate for the clinical treatment of infertility, recurrent implantation failure, and early miscarriage caused by decidual aging, while also providing a theoretical basis for exploring the link between cellular senescence and mitochondrial energy metabolism. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the construction and verification of the decidualization model in Embodiment 1 of the present invention:

[0021] In the following experiments, A) human endometrial stromal cells (HESCs) were treated with 8-Br-cAMP and MPA. Cell morphology was observed at 24h, 48h, and 96h. The cells changed from elongated spindle-shaped to elliptical, with the most obvious decidualization morphology at 96h. The scale bars in the figures are all 100μm. B) The expression of decidual marker molecules PRL and IGFBP1 was significantly increased. P < 0.05;

[0022] Figure 2 This is a schematic diagram of screening the target compound Biochanin A in Example 2 of the present invention;

[0023] Among them, the positive criterion is: SA-β-gal inhibition rate > 50%;

[0024] Figure 3 This is a schematic diagram illustrating the effects of Biochanin A on decidual cell proliferation, viability, apoptosis, and senescence phenotypes at the cellular level in Example 3 of the present invention.

[0025] in, Figure 3 -A. CCK8 assay was used to detect cell proliferation and cytotoxicity between the two groups. P < 0.05; Figure 3 -B. Apoptosis kit detection showed reduced cell apoptosis in the control group. P < 0.05; Figure 3-C. Cell smear β-galactosidase staining experiment was used to detect the difference in the degree of cell senescence between the two groups of cells. Blue represents senescent cells. The scale bars in the figure are all 100μm. Figure 3 -D. Statistical analysis of SA-β-gal staining area in the two groups. P < 0.05;

[0026] Figure 4 This is a schematic diagram illustrating the effects of Biochanin A on decidual cell proliferation, viability, apoptosis, and senescence phenotypes at the protein level, as shown in Example 3 of this invention; wherein, Figure 4 -A, B, protein-level qPCR validation P < 0.05; Figure 4 -C, D, E, and SASP level qPCR validation P < 0.05;

[0027] Figure 5-1 The images show the volcano plot and KEGG enrichment analysis of proteomics sequencing in Example 4 of this invention. A) The volcano plot shows 425 genes significantly downregulated and 499 genes significantly upregulated in decidual stromal cells treated with Biochanin A. B) KEGG enrichment analysis suggests that fatty acid degradation, TCA cycle, oxidative phosphorylation, 2-oxocarboxylic acid metabolism, and valine, leucine, and isoleucine degradation are closely related to mitochondrial energy metabolism.

[0028] Figure 5-2 This is a diagram showing the GO functional analysis and qPCR validation of proteomics sequencing in Example 4 of this invention; wherein, C, GO functional analysis suggests that cellular respiration, respiratory electron transport chain, aerobic respiration, oxidoreductase activity, mitochondrial membrane, etc., are closely related to mitochondrial energy metabolism; D, E, qPCR validation of mitochondrial-related differentially expressed proteins. P < 0.05;

[0029] Figure 6 This is a schematic diagram of the detection of mitochondrial energy metabolism related by Biochanin A in Example 5 of the present invention;

[0030] Among them, the oxygen consumption rate between groups A and Sea Horse XF mitochondrial stress test was detected. P < 0.05; B. Comparison of baseline respiratory rate (BR), maximum respiratory rate (MR), and residual respiratory capacity (SRC) between the groups. P < 0.05; C, comparison between the two groups regarding proton leakage (PL) and ATP production. P < 0.05; D. Mitochondrial morphology observed under transmission electron microscopy in both groups. Detailed Implementation

[0031] 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.

[0032] The present invention will be further described in detail below with reference to specific embodiments.

[0033] This application provides the use of Biochanin A in the preparation of drugs to alleviate the aging of uterine decidual stromal cells;

[0034] This application also provides a method for verifying the effect of Biochanin A in alleviating the senescence of decidual stromal cells, including the following steps:

[0035] S1: Construction and validation of the decidualization model;

[0036] S2: Screening for target compound Biochanin A from a library of natural flavonoids that has an intervention effect on decidual stromal cell senescence;

[0037] S3: The anti-aging effect of Biochanin A was verified by detecting cell proliferation, viability, apoptosis and aging phenotype.

[0038] S4: Screening key pathways and proteins involved in the action of Biochanin A through proteomics sequencing;

[0039] S5: Detect the effect of Biochanin A on mitochondrial energy metabolism.

[0040] The above content will be explained in conjunction with specific verification experiments:

[0041] Example 1: Constructing a decidualization model and verifying its successful construction

[0042] Specifically, it includes the following steps:

[0043] 1. Collect primary human endometrial stromal cells.

[0044] 2. Induction of decidualization: A decidual stromal cell (DSC) model was induced in vitro by adding 8-bromocyclic adenosine monophosphate (8-Br-cAMP) (1 mM) and medroxyprogesterone acetate (MPA) (2 μM) to human endometrial stromal cells. Cell morphology was observed at 24 h, 48 h, and 96 h, showing that the cells changed from elongated spindle-shaped to elliptical.

[0045] 3. RNA Extraction and qPCR Analysis from Decidual Stromal Cells: Total RNA was extracted from decidual stromal cells according to the Ambion 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 the Thermo-K1622 reverse transcription kit and the Thermo-F-415XL SYBR Green PCR kit, respectively. A 20 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 an ABI-7500 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.

[0046] Table 1 Primers used for decidual-related qPCR

[0047]

[0048] The results are as follows Figure 1 As shown, 8-Br-cAMP and MPA were used to induce decidualization in this application. Cell morphology was observed at 24h, 48h, and 96h. The cells changed from elongated spindle-shaped to elliptical, with the most pronounced decidualization morphology at 96h. Figure 1 A), indicating that the cell morphology conformed to the decidualization phenotype. Quantitative PCR analysis showed that, after the addition of 8-Br-cAMP and MPA, the expression of decidual marker molecules PRL and IGFBP1 was significantly increased in the experimental group (decidual stromal cells) compared with the untreated control group (human endometrial stromal cells). Figure 1 B). The results of this experiment indicate that the in vitro induced decidualization cell model was successfully constructed. P<0.05, P<0.01, P<0.001.

[0049] Example 2: Screening for the target compound Biochanin A

[0050] Compounds that intervene in decidual stromal cell senescence were detected by high-throughput SA-β-gal in the Progesterone Compound Library (n=260).

[0051] Specifically, it includes the following steps:

[0052] Human endometrial stromal cells were resuscitated and passaged to ensure stable cell condition. Following the experimental design, cells were seeded at a density of approximately 1-5 × 10³ cells per well into white ELISA-labeled 96-well plates and cultured until adherent. At approximately 80-90% confluence, 8-Br-cAMP (1 mM) and MPA (2 μM) were added to induce in vitro cell induction of decidual stromal cells. After 96 hours, 260 compounds from a progesterone compound library (final concentration 10 μM) were added, 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, 40 μL of working solution + 40 μL of culture medium were added to each well, and the plates were incubated at room temperature with shaking for 45 minutes. Blue-positive cells were counted under an inverted microscope, and the positivity rate was calculated.

[0053] The results are as follows Figure 2 As shown, Biochanin A can significantly reduce SA-β-gal activity in decidual stromal cells. P<0.05, P<0.01, P < 0.001. The results show that Biochanin A is the target compound with anti-aging activity.

[0054] Example 3: Verification of the cytotoxicity and anti-aging effects of Biochanin A

[0055] Specifically, it includes the following steps:

[0056] 1. CCK8 detection

[0057] The in vitro decidualization induction process in clear 96-well plates was performed as described above. The original culture medium was discarded, and 0.1 μL Biochanin A (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 Beyotime CCK8 kit instructions, 10 μL of CCK8 reagent + 90 μL of basal culture medium was added to each well, for a total of 100 μL per well. The 96-well plate was gently shaken to ensure thorough mixing of the reagent and culture medium, avoiding the formation of air bubbles. The 96-well plate was returned to a 37°C, 5% CO2 incubator and incubated in the dark for 45 minutes. After incubation, the 96-well plate was removed, and the absorbance (OD value) of each well was measured using a microplate reader at a wavelength of 450 nm. 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%.

[0058] 2. Apoptosis detection

[0059] 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 of L Biochanin A (10 μM) + 100 μL of 2.5% complete culture medium was added, with 3 replicates. After adding the drugs, the medium was changed after 48 hours, and the 96-well plate was removed after another 48 hours. Following the Promega apoptosis kit instructions, the plate was preheated. Drugs 1 and 2 were added to 10% complete culture medium and then shaken. Drugs 3 and 4 were added and then mixed by inverting. The original culture medium was discarded, and 32 μL of apoptosis detection drug + 48 μL of 10% complete culture medium (80 μL total) were added. The 96-well plate was returned to a 37°C, 5% CO2 incubator and incubated for 12 hours. After incubation, the 96-well plate was removed, and the absorbance (OD) of each well was measured at 450 nm using an ELISA reader. 450 value).

[0060] 3. Cell smear SA-β-gal staining

[0061] 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%.

[0062] 4. Total RNA extraction and qPCR analysis

[0063] Total RNA was extracted from the control group (naturally senescent decidual stromal cells) and the experimental group (decidual stromal cells treated with Biochanin A) according to the Ambion 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-1α, 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 the Thermo-K1622 reverse transcription kit and the Thermo-F-415XL SYBR Green PCR kit, respectively. A 20 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 an ABI-7500 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.

[0064] Table 2 Primers used in aging-related qPCR

[0065]

[0066] The results are as follows Figure 3 , 4 As shown in the CCK8 experiment, there was 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 Biochanin A treatment group was significantly lower than that in the control group, while the apoptosis rate in the 50 μM group was slightly higher than that in the control group. Therefore, 10 μM was determined to be the optimal concentration. Figure 3 -B). SA-β-gal staining showed a decrease in the number of senescent cells in the experimental group ( Figure 3 -C, D). Extensive research has demonstrated that 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 the senescence-associated secretory phenotype (SASP). IL-1α, 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-1α, IL-1β, and CXCL2 were significantly lower than those in the control group (…). Figure 4 ) P<0.05, P<0.01, P<0.001. This application found that Biochanin A did not alter the proliferative capacity of decidual stromal cells, nor did it cause a decrease in cell viability due to toxicity. Low concentrations (10 μM) of Biochanin A could inhibit apoptosis and maintain cell viability. Furthermore, from two key dimensions—senescence mechanisms and senescence phenotype effects—it was verified that cell cycle arrest was alleviated, SASP secretion function in senescent cells was inhibited, and the recruitment of inflammatory cells was reduced, thus comprehensively demonstrating that Biochanin A has a role in alleviating cellular senescence.

[0067] Example 4: Proteomics Sequencing

[0068] Deciduous cells from the 10 μM Biochanin A treatment group and the control group were collected, and total protein was extracted. Proteomics sequencing was performed using LC-MS / MS to screen differentially expressed proteins. GO functional enrichment analysis and KEGG analysis were performed on the differentially expressed proteins. Candidate differentially expressed proteins with significant differences were validated by qPCR (primers are shown in Table 3 below) to explore possible mitochondrial energy metabolism pathways.

[0069] Table 3 Primers used for mitochondrial-related qPCR

[0070]

[0071] The results are as follows Figure 5-1 and Figure 5-2 As shown, a volcano plot of differentially expressed genes was drawn, and a total of 924 differentially expressed proteins were screened, of which 499 were upregulated and 425 were downregulated. Figure 5-1 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 5-1 B), GO functional analysis suggests a close relationship with cellular respiration, the respiratory electron transport chain, aerobic respiration, oxidoreductase activity, and mitochondrial membrane. Figure 5-2 C). The KEGG pathway is largely a key link in mitochondrial energy metabolism, while GO function directly relates to mitochondrial structure, core energy metabolism processes, and molecular functions. Both corroborate each other from the perspectives of "metabolic pathway" and "functional characteristics," indicating that GO is primarily enriched in core mitochondrial energy metabolism processes. Differentially expressed proteins related to mitochondrial energy metabolism (SNPH, DNAJC6, NDUFA5, COX2) were selected, and their mRNA expression was verified by qPCR. The results showed that the mRNA expression of all four differentially expressed proteins was upregulated and statistically significant, with COX2 upregulated to 10-fold compared to the control group, suggesting that COX2 may be a key target for Biochanin A in regulating mitochondrial energy metabolism. P<0.05, P<0.01, P<0.001, Figure 5-2 D, E).

[0072] Example 5: Validation of the mitochondrial energy metabolism pathway

[0073] Specifically, it includes the following steps:

[0074] 1. Seahorse XFe24 Mitochondrial Stress Measurement

[0075] The oxygen consumption rate (OCR) of mitochondria in decidual stromal cells was measured using the Agilent Seahorse XF Cell Mitochondrial Stress Assay Kit. The decidual stromal cells were subjected to 10K...

[0076] Cells were seeded at a density of 100 μL / well in XF24 cell culture plates. 100 μL of cell growth medium was added to the background calibration wells, but no cells were added. After successful cell adhesion, 150 μL of growth medium was added to all wells of the cell culture plate to bring the total volume to 250 μL. When the cells reached 70-80% confluence, Biochanin A (10 μM) was added, and the medium was changed after 48 hours. The probe plate was hydrated the day before the experiment by adding 1 mL of XF calibration solution (XFCalibrant) to each well and incubating overnight at 37°C in a CO2-free cell culture incubator. On the day of the experiment, Seahorse assay solution was prepared (supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose (pH = 7.4)). The cultured adherent cells were removed from the CO2 cell culture incubator and observed under a microscope. Cells were washed, incubated with the detection solution at 37°C, and the growth medium was replaced with the detection solution. The cells were washed three times, with a final volume of 500 μL. Cells were incubated in a CO2-free cell culture incubator at 37°C for 60 minutes, and then analyzed on an XF probe plate. Oligomycin (2.5 μmol / L), FCCP (2.0 μmol / L), and Rotenon & antimycin A (0.5 μmol / L) were prepared and added to the XF probe plate. OCR was measured using a Seahorse XFe24 analyzer (Agilent Technologies, USA).

[0077] 2. Transmission electron microscopy

[0078] The in vitro decidualization treatment was the same as above. Cells to be tested were seeded into 60mm culture dishes containing complete culture medium. When the cells reached 70%–80% confluence, Biochanin A (10μM) was added. After 48 hours, the medium was changed. After another 48 hours, the cells were digested with trypsin, the trypsin was discarded, and digestion was terminated after complete culture. Cells were collected and centrifuged at 1000 rpm for 5 min in a 1.5ml centrifuge tube. The supernatant was discarded, and 1ml of fixative was added, taking care not to break up the cell clumps. The tube was then placed at 4°C for 2 hours. The fixative was gently aspirated, and a suitable amount of 1% agarose solution at approximately 40°C was gently added dropwise near the wall of the tube, stirring constantly, not exceeding 100µl. After the cell clumps solidified with the agar, the clumps were gently removed, and the cell-free gel around the edges was removed before placing them back into the fixative. Cells were fixed for 2 hours with 1% osmium tetroxide at room temperature in the dark, using 0.1mol / L phosphate buffer PB (pH = 7.4).

[0079] Rinse three times, 15 minutes each time. Dehydrate using a gradient of alcohols (30%, 50%, 70%, 80%, 95%, 100%, 100%) at room temperature, 20 minutes each time. After dehydration, soak twice in 100% acetone, 15 minutes each time. Infiltration embedding (acetone: 812 embedding agent = 1:1, 37°C for 2-4 hours; acetone: 812 embedding agent = 1:2, 37°C overnight; pure 812 embedding agent, 37°C, 5-8 hours). Add 812 embedding agent to the embedding plate, place the sample in and incubate overnight at 37°C, then polymerize in a 60°C oven for 46 hours. Remove the resin block for later use. Slice the resin block (70 nm). Under light-protected conditions, stain the slices with 2% uranium acetate saturated alcohol solution for 8 minutes, wash three times with 70% alcohol, and wash three times with ultrapure water. Under CO2-free conditions, the sections were stained with 2.6% lead citrate solution for 8 minutes, washed three times with ultrapure water, and dried overnight at room temperature. The sections were then observed and photographed using a transmission electron microscope.

[0080] The results are as follows Figure 6 As shown, the OCR value of the control group increased and reached its peak after FCCP treatment, while the OCR value decreased after the addition of Rotenoon & antimycin A, consistent with the normal mitochondrial oxygen consumption curve. The experimental group's basal respiration was close to that of the control group, and the OCR was significantly improved after FCCP treatment, exceeding that of the control group. Figure 6 -A). There was no significant difference in basal respiration and proton leakage, but maximal respiration, residual respiratory capacity, and ATP production were significantly improved. Figure 6(BR: Basal Respiration, MR: Maximum Respiration, SRC: Spare Respiration Capacity, PL: Proton Leak). In the control group, some mitochondrial cristae were club-shaped and enlarged, electron density increased, mitochondria became smaller and rounder, showing condensation, mitochondrial mass decreased significantly, and atypical mitochondria and other characteristics of mitochondrial damage appeared. The basic morphology of mitochondria in the experimental group was relatively normal. Compared with the control group, the experimental group alleviated the damage to mitochondrial morphology, specifically manifested in a fuller central matrix and an increase in tubular structures. Figure 6 -D) P<0.05, P<0.01, P<0.001.

[0081] The above results suggest that Biochanin A enhances mitochondrial electron transport efficiency or increases reserve respiratory capacity, without significantly altering the basal oxygen consumption of mitochondria to maintain basic cellular life activities, nor causing an increase in ineffective proton leakage due to abnormal mitochondrial membrane potential. This indicates that the drug has no negative impact on mitochondrial basal structure and homeostasis. Simultaneously, it activates the functional potential of the mitochondrial respiratory chain. Under cellular stress, mitochondria meet additional energy demands by increasing their maximum oxygen consumption capacity, thus enhancing stress reserves and demonstrating a key mechanism for cellular anti-aging and function maintenance. Increased ATP production indicates that Biochanin A promotes the efficient conversion of energy from oxidative phosphorylation into ATP in mitochondria, improving energy deficiency in senescent cells and providing sufficient energy substrates for anti-aging activities such as cell repair and metabolism.

[0082] Based on the construction of a decidualization model and the screening of the target compound Biochanin A from a library of natural flavonoids, this invention, through aging assays, CCK8 assays, apoptosis assays, and detection of the expression of classic aging markers P21, P53, IL-1α, IL-1β, and CXCL2, found that Biochanin A effectively alleviates the aging of decidual stromal cells without cytotoxic effects.

[0083] Furthermore, this application used proteomics sequencing to discover a potential close relationship with mitochondrial energy metabolism pathways. By detecting the mRNA expression levels of mitochondrial-related differentially expressed proteins SNPH, DNAJC6, NDUFA5, and COX2, changes in mitochondrial oxygen consumption, and changes in mitochondrial morphology, it was found that Biochanin A significantly alleviates decidual stromal cell senescence by regulating mitochondrial energy metabolism pathways, which has important theoretical significance and clinical value.

[0084] 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. Application of Biochanin A in the preparation of drugs for inhibiting or improving the senescence of uterine decidual stromal cells.

2. The application according to claim 1, characterized in that, The drug is used to prevent or treat pregnancy-related diseases caused by decidual stromal cell aging.

3. The application according to claim 1, characterized in that, Biochanin A inhibits the senescence process of decidual stromal cells by downregulating the expression of cell cycle arrest proteins P21 and / or P53.

4. The application according to claim 1, characterized in that, Biochanin A exerts its anti-aging effect by inhibiting the expression of aging-related secretory phenotypic factors.

5. The application according to claim 1, characterized in that, Biochanin A alleviates decidual stromal cell senescence by improving mitochondrial energy metabolism.

6. The application according to claim 5, characterized in that, The improvement of mitochondrial energy metabolism function includes: increasing at least one of the following: mitochondrial maximum respiratory capacity, residual respiratory capacity, and ATP production efficiency.

7. The application according to claim 5 or 6, characterized in that, Biochanin A improves mitochondrial energy metabolism by upregulating the expression of mitochondrial function-related proteins.

8. The application according to claim 7, characterized in that, The mitochondrial function-related protein is COX2.

9. A pharmaceutical composition, characterized in that, Containing a therapeutically effective amount of Biochanin A as the active ingredient, along with a pharmaceutically acceptable carrier or excipient, it is used to inhibit or improve the senescence of uterine decidual stromal cells.

10. A method for screening candidate compounds with inhibitory activity against decidual cell senescence, characterized in that, The method includes: evaluating the effect of candidate compounds on improving mitochondrial energy metabolism function in uterine decidual stromal cells; wherein the effect on improving mitochondrial energy metabolism function includes enhancing mitochondrial respiration capacity and / or upregulating the expression of mitochondrial function-related proteins.

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