A composition comprising quercetin and methods for delaying testicular aging thereof

Abstract

The application discloses a composition containing quercetin and a method for delaying testicular aging, and relates to the technical field of cell biology, and the composition is composed of active ingredients and solvents and dispersion media, the active ingredients include quercetin and quercetin-3-O-glucuronide, the solvent and the dispersion medium are 1% carboxymethyl cellulose sodium solution, 1% carboxymethyl cellulose sodium solution is selected as the dispersion medium and the solvent carrier, the application activates the AMPK signal pathway in testicular supporting cells through the quercetin composition, up-regulates key glycolytic enzymes, enhances lactic acid production and ATP synthesis, and maintains the energy homeostasis of spermatogenic microenvironment, down-regulates aging markers, delays testicular aging, and quercetin is mainly converted into Q3G form to play a role after metabolism in the body, both of which can connect energy sensing, metabolic reprogramming and reproductive function maintenance through the AMPK-glycolysis metabolic axis, so as to improve the natural aging testicular function damage.

Description

Technical Field

[0001] This invention relates to the field of cell biology, specifically to a composition containing quercetin and a method for delaying testicular aging. Background Technology

[0002] With improved living standards and medical conditions, the testes, as an important male reproductive organ, undergo progressive degeneration in function and structure with age, manifesting as a decline in hormone secretion and a weakening of spermatogenesis. Natural aging refers to the degenerative changes in the body's tissues and organs that occur over time, and is positively correlated with age. Aging is a complex process involving multiple aspects such as genomic instability, telomere loss, epigenetic changes, and loss of protein homeostasis. Testicular aging is characterized by the gradual decline in the structure and function of the testes with increasing age. Current clinical treatment for male testicular aging and hypogonadism is still mainly based on testosterone replacement therapy, which carries significant risks of toxic side effects and adversely affects male fertility. Although there are monomeric components in the field of traditional Chinese medicine such as icariin, oleanolic acid, and wolfberry polysaccharide that have certain effects in delaying reproductive aging, their specific targets and molecular mechanisms have not been fully elucidated, and they mostly focus on anti-oxidative stress or single signaling pathways, lacking effective regulation of testicular energy metabolism. Summary of the Invention

[0003] This invention provides a composition containing quercetin and a method for delaying testicular aging, which can effectively solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a composition containing quercetin, the composition comprising an active ingredient, a solvent, and a dispersion medium; The active ingredients include quercetin and quercetin-3-O-glucuronic acid, quercetin: Que, quercetin-3-O-glucuronic acid: Q3G; Que is a natural flavonol compound found in fruits, vegetables, and various medicinal plants. Q3G is a metabolite of quercetin in the body. It is a metabolite produced by glucuronidation of quercetin in the small intestine and liver after oral administration. It is also the main circulating form of quercetin in plasma and various tissues. The solvent and dispersion medium are 1% sodium carboxymethyl cellulose solution, which is a high molecular weight polysaccharide excipient. 1% sodium carboxymethyl cellulose solution is selected as the dispersion medium and solvent carrier.

[0005] According to the above technical solution, the quercetin-containing composition is formulated with different concentrations, and the following three concentration specifications are provided: Low-dose quercetin suspension: Add 45 mg of quercetin powder to every 1 mL of 1% sodium carboxymethyl cellulose solution; High-dose quercetin suspension: Add 90 mg of quercetin powder to every 1 mL of 1% sodium carboxymethyl cellulose solution; Positive control NAD + Suspension: Add 100 mg of nicotinamide adenine dinucleotide (NAD) to each 1 mL of 1% sodium carboxymethyl cellulose solution. + .

[0006] According to the above technical solution, the quercetin-containing composition is prepared differently in the in vitro cell experiment section: Quercetin and Q3G were both dissolved in complete culture medium to prepare stock solutions or working solutions of corresponding concentrations, and different concentration gradients were screened. In in vitro experiments, the drug used to induce senescence of testicular Sertoli cells was doxorubicin (Doxo), which was selected by CCK-8 screening and used to establish a Doxo-induced TM4 cell senescence model.

[0007] A method for delaying testicular aging using a quercetin-containing composition, the method comprising an in vivo experimental procedure and an in vitro experimental procedure; The specific in vivo experimental procedure is as follows: Step 11, Experimental Animals and Grouping; Step 12, Sample Collection and Processing; Step 13, General phenotypic and morphological observation; Step 14, serum reproductive hormone level detection; Step 15, sperm quality analysis; Step 16, Detection of cellular senescence levels; Step 17: Observe using a transmission electron microscope; Step 18, Protein expression level detection; Step 19, Immunofluorescence staining; The specific in vitro experimental procedure is as follows: Step 21, cell culture and drug concentration screening; Step 22, Construction of Doxo-induced TM4 cell senescence model; Step 23, Experimental grouping and intervention; Step 24, detection of cell senescence-related factors; Step 25, Seahorse XF energy metabolism analysis; Step 26, AMPK and glycolysis-related factors detection; Step 27, AMPK knockdown verification experiment.

[0008] According to the above technical solution, in step 11, SD rats are selected as experimental animals, and they are divided into different experimental groups according to their age: Six-month-old young SD rats were used as the normal control group (Control). Sixteen-month-old aged SD rats were used to establish a natural aging model. The aged rats were further randomly divided into: a model control group (Model), a low-dose quercetin group (L-Que), a high-dose quercetin group (H-Que), and a positive control group (NAD). + Each group received the drug via gavage once daily for 60 consecutive days to simulate the effects of long-term intervention on testicular aging. In step 12, after the drug intervention was completed, the rats were anesthetized after fasting, and blood was collected through the apex of the heart. After the blood was allowed to stand and coagulate, it was centrifuged and the upper light yellow serum was collected and stored for later use. The rat was dissected and both testes were removed. The adipose tissue, connective tissue and vascular membrane around the testes were removed. The fresh weight of the testes was weighed and the testicular index was calculated. One testis was quick-frozen and stored, while the other testis was fixed in paraformaldehyde solution. At the same time, epididymal tissue was taken, and an incision was made in physiological saline to release sperm and prepare a sperm suspension. In step 13, during the intervention period, the growth status of rats in each group was observed and recorded in detail daily; In terms of testicular tissue pathology, testicular tissue fixed in paraformaldehyde solution was punctured by a needle tip and then fixed again to allow the fixative to penetrate. Subsequently, the tissue underwent gradient dehydration and embedding. The embedded tissue blocks were cut into paraffin sections using a microtome, baked in an oven to remove the wax, and then stored for later use. Paraffin sections were dewaxed with xylene, rehydrated with graded ethanol, stained with hematoxylin and eosin, and observed under an optical microscope.

[0009] According to the above technical solution, in step 14, the levels of reproductive hormones in the serum of rats in each group, including testosterone, luteinizing hormone and follicle-stimulating hormone, are detected by enzyme-linked immunosorbent assay (ELISA). A standard curve was constructed with the concentration of the standard as the x-axis and the optical density as the y-axis. The hormone concentration in each serum sample was calculated by fitting a four-parameter logistic curve. In step 15, the epididymal tail is placed in physiological saline, an incision is made to release sperm, allowing the sperm to swim out fully, tissue fragments are filtered out, a sperm suspension is obtained, and the suspension is dropped onto a glass slide to evaluate the sperm motility parameters. Sperm morphology observation was performed using the Papanicolaou staining method. Sperm suspension was dropped onto a glass slide to make a uniform smear, which was then air-dried and fixed with a fixative. The Papanicolaou staining procedure was followed to observe the morphological changes of the sperm. The 16th method assesses the level of cellular senescence in testicular tissue by SA-β-Gal staining. Frozen sections are prepared first, followed by SA-β-Gal staining, and finally the sections are mounted with water-soluble mounting medium. The staining results were observed under an optical microscope. The level of cell senescence was assessed by blue-green positive signals, and the area of ​​the positively stained region was quantitatively statistically analyzed using ImageJ software.

[0010] According to the above technical solution, in step 17, testicular tissue samples are taken, cut into small pieces, and fixed in pre-cooled electron microscopy fixative. After the samples are processed by the standard transmission electron microscopy sample preparation procedure of post-fixation, gradient acetone dehydration, embedding, sectioning, and uranium-lead double staining, the ultrastructural changes of mitochondria in testicular supporting cells are observed under a transmission electron microscope to evaluate the morphology and size of mitochondria, membrane structure integrity, mitochondrial cristae arrangement density, and matrix state. In step 18, the expression levels of multiple proteins in testicular tissue were detected by Western Blot. After protein extraction, grinding and centrifugation, the supernatant was collected and stored for later use. The supernatant is the total protein of the tissue. Protein concentration was determined using the BCA method: the protein stock solution was diluted with ultrapure water, a standard curve was prepared using bovine serum albumin, LBCA working solution and standard or sample to be tested were added to each well, after incubation, the optical density value was measured, the protein concentration of each sample was calculated, and the loading amount was calculated based on the protein concentration. SDS-PAGE gel electrophoresis was performed using a one-step PAGE gel rapid preparation kit to prepare 10% separating and compression gels. During electrophoresis, the protein is first compressed into a line in a compression gel, and then separated according to molecular weight in a separating gel. After electrophoresis, the protein is transferred to a PVDF membrane using a wet transfer method. After activating the PVDF membrane with methanol, the transfer sandwich is assembled in sequence, placed in the transfer tank, and ice-free rapid transfer buffer is poured in. The membrane is transferred at a constant current of 400 mA for 1.5 hours. After the transfer was completed, the PVDF membrane was removed, blocked with protein blocking buffer, washed with Tris buffer, and then incubated overnight on a shaker at 4°C with primary antibody. The next day, after recovering the primary antibody, the membrane was washed three times with Tris-buffered saline solution for 5 minutes each time. Horseradish peroxidase-labeled secondary antibody was added and incubated at room temperature. After washing with Tris-buffered saline solution, chemiluminescence imaging was performed. The PVDF membrane was placed in the chemiluminescence solution and incubated using the Omni-ECL universal chemiluminescence detection kit. The membrane was exposed and photographed in a Western blot chemiluminescence analyzer, and the gray values ​​of each band were analyzed using ImageJ software. The ratio of the gray values ​​of the target protein to the internal control protein was used as the relative expression level for statistical analysis. In step 19, the expression and co-localization of HK2 and WT1 proteins in testicular tissue were detected by immunofluorescence staining. After paraffin sections were repaired, endogenous peroxidase activity was blocked with hydrogen peroxide solution, followed by permeabilization with Triton X-100, blocking with goat serum blocking solution, and primary antibody was added to cover the sections and incubated overnight. The next day, the cells were washed with phosphate buffer, and the corresponding fluorescently labeled secondary antibody was added. The cells were incubated in the dark. After rinsing with phosphate buffer in the dark, the cell nuclei were stained with DAPI solution. After rinsing in the dark, the cells were mounted with anti-fluorescence quenching mounting medium and observed and photographed under a laser confocal microscope. WT1 is mainly located in the nucleus of supporting cells and serves as a specific marker for supporting cells. The fluorescence signal intensity of HK2 reflects glycolytic activity. The fluorescence intensity and co-localization of the two were quantitatively analyzed using ImageJ software.

[0011] According to the above technical solution, in step 21, the mouse testicular support cell line TM4 is selected for in vitro cell experiments. TM4 cells are cultured and passaged in a carbon dioxide incubator using a complete culture medium containing 10% fetal bovine serum. The safe and effective concentrations of quercetin and Q3G were screened using the CCK-8 assay. TM4 cells in logarithmic growth phase were seeded into 96-well plates and cultured until the cells adhered to the plate. The medium was then replaced with complete medium containing a series of concentrations of quercetin or Q3G, with replicates for each concentration. 10 μL of CCK-8 reagent was added to each well. After incubation, the optical density was measured, and the cell viability of each concentration group was calculated. The lowest concentration with higher cell viability than the control group and the ability to promote cell proliferation was identified as the optimal concentration. In step 22, TM4 cells in the logarithmic growth phase were seeded in a culture plate and cultured. The culture medium was then replaced with a complete medium containing an appropriate concentration of doxorubicin and treated for 24 hours to induce cell senescence. Subsequently, the doxorubicin-containing medium was discarded and replaced with a complete medium or a complete medium containing quercetin and Q3G for subsequent intervention experiments. The successful construction of the senescence model was verified by Western blotting, which showed a significant increase in the expression levels of senescence-related proteins p16, p21, and γ-H2AX.

[0012] According to the above technical solution, the in vitro experiment is divided into blank control group: TM4 group, model group: TM4 plus Doxo group, quercetin intervention group: TM4 plus Doxo plus Que group, and Q3G intervention group: TM4 plus Doxo plus Q3G group. In step 24, cells from each group after intervention were collected, total cellular protein was extracted, and the protein expression levels of aging-related factors p16, p21, p53 and γ-H2AX were detected by Western Blot. At the same time, the intracellular distribution and expression levels of aging-related factors were observed under a laser confocal microscope using immunofluorescence staining. In the 25th case, the energy metabolism status of each group of live cells was detected in real time using the Seahorse XF cell energy metabolism analyzer, and TM4 cells were seeded in Seahorse XF dedicated cell culture plates. During testing, the Seahorse XF analytical medium was replaced, and the measurements were performed in real time using the Seahorse XF analyzer. For oxygen consumption rate detection, oligomycin, FCCP and antimycin A were injected into each well sequentially during the measurement process, and the cell indicators were calculated by the changes in the oxygen consumption rate curve. In terms of extracellular acidification rate detection, cellular indicators are calculated by measuring the extracellular acidification rate curve to reflect changes in cellular glycolysis flux.

[0013] According to the above technical solution, in step 26, the expression levels of AMPK and glycolysis-related factors in each group of cells are detected by Western Blot method. The ratio of p-AMPK to total AMPK reflects the phosphorylation activation level of AMPK, and the expression levels of each key glycolysis enzyme reflect the glycolysis activity of the cells. Simultaneously, immunofluorescence staining was used to detect the expression distribution and fluorescence intensity of HK2, PKM2, and LDHC in cells; In the second part, to verify the key mediating role of AMPK in quercetin regulation of glycolysis and delay of testicular Sertoli cell senescence, a stable cell line with AMPKα gene knockdown was constructed. Transcriptomic sequencing was performed on TM4-shNC and TM4-shAMPKα cells to screen differentially expressed genes and conduct GO function and KEGG pathway enrichment analysis to observe the effect of AMPK knockdown on cellular energy metabolism-related pathways. Subsequently, TM4-shAMPKα cells were treated with quercetin and Q3G, respectively, and the expression levels of glycolysis-related factors were detected by Western blotting to verify the regulatory role of quercetin on glycolysis in the absence of AMPK.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The core mechanism of delaying testicular aging through a quercetin-containing composition lies in the construction of an AMPK-glycolysis metabolic axis. After quercetin enters the body, it is absorbed orally and metabolized into the active metabolite Q3G. It accumulates in testicular tissue and activates the AMPK signaling pathway in Sertoli cells. Activated AMPK acts as an energy sensor for cells. On the one hand, it directly phosphorylates and activates downstream metabolic enzymes, enhancing glucose uptake and glycolysis. On the other hand, it upregulates the expression levels of key glycolysis enzymes such as HK2, PKM2, and LDHC, promoting the conversion efficiency of glucose to lactate and increasing the production of ATP and lactate. Lactic acid, as the preferred energy substrate for spermatogenic cells, is transported from Sertoli cells to germ cells via monocarboxylic acid transporters, providing sufficient energy for spermatogenesis and maturation. Meanwhile, AMPK activation also upregulates the expression of mitochondrial SIRT3, improves mitochondrial functional integrity, promotes pyruvate entry into the tricarboxylic acid cycle for oxidative phosphorylation, further enhances the cell's energy supply capacity, and the improvement in energy metabolism further feedback downregulates the expression of aging markers p16, p21, p53 and γ-H2AX, inhibits the cellular senescence program, maintains the number and functional integrity of supporting cells, improves the permeability and selectivity of the blood-testis barrier, and ultimately achieves a comprehensive protective effect on testicular morphology, hormone secretion, spermatogenesis and energy metabolism, thereby achieving the overall effect of delaying testicular aging.

[0015] 2. By combining in vivo animal experiments and in vitro cell experiments, the specific molecular mechanism by which quercetin-containing compositions delay testicular aging was effectively elucidated. Testicular tissue in a naturally aging state exhibits typical aging phenotypes, including decreased serum testosterone levels, compensatory increases in gonadotropin levels, atrophy of seminiferous tubules, reduction in spermatogenic cell layers, decreased sperm quality, and ultrastructural damage and dysfunction of Sertoli cell mitochondria. At the molecular level, this is manifested by increased expression of aging markers p16, p21, p53, and γ-H2AX, decreased AMPK phosphorylation levels, and downregulation of key glycolytic enzymes HK2, PKM2, LDHC, and SIRT3, leading to insufficient energy supply to the spermatogenic microenvironment.

[0016] In summary, by activating the AMPK signaling pathway in testicular Sertoli cells through the quercetin composition, key enzymes in glycolysis are upregulated, lactate production and ATP synthesis are enhanced, and energy homeostasis in the spermatogenic microenvironment is maintained. Furthermore, aging markers are downregulated, thus delaying testicular aging. Quercetin, after being metabolized in vivo, is mainly converted to the Q3G form to exert its effects. Both quercetin and Q3G can connect energy sensing, metabolic reprogramming, and reproductive function maintenance through the AMPK-glycolysis metabolic axis, thereby improving testicular function damage caused by natural aging. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0018] In the attached diagram: Figure 1 This is a schematic diagram illustrating the effect of the present invention on the energy metabolism of testicular supporting cells; Figure 2 This is a schematic diagram illustrating the effect of quercetin of the present invention on senescent testicular supporting cells; Figure 3 This is a flowchart of the in vivo experiment of the present invention; Figure 4 This is a technical roadmap for the in vivo experiments of this invention; Figure 5 This is a flowchart of the in vitro experiments of the present invention; Figure 6 This is a technical roadmap for the in vitro experiments of this invention. Detailed Implementation

[0019] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0020] Example: Figure 1-2 As shown, the present invention provides a technical solution, a composition containing quercetin, which is composed of an active ingredient, a solvent, and a dispersion medium; The active ingredients include quercetin and quercetin-3-O-glucuronic acid. Quercetin: Quercetin, Que; Quercetin-3-O-glucuronic acid: Q3G. Quercetin is a natural flavonol compound, chemically named 3,3,4,5,7-pentahydroxyflavone. It is widely found in fruits, vegetables, and various medicinal plants and is the core active ingredient of the composition, with a purity ≥98%. Q3G is the main metabolite of quercetin in the body. Q3G is the main metabolite produced by glucuronidation of quercetin in the small intestine and liver after oral administration. It is also the main circulating form of quercetin in plasma and various tissues. Q3G is characterized by its resistance to self-oxidation, lack of free radical production, and good cyclic stability. After long-term supplementation with quercetin, it dominates in plasma, liver, and lung tissue. The solvent and dispersion medium is 1% sodium carboxymethyl cellulose solution (CMCNa). Sodium carboxymethyl cellulose is a commonly used high molecular weight polysaccharide excipient with good biocompatibility and thickening and suspending ability. It can uniformly disperse the hydrophobic drug quercetin in the solution, ensuring the consistency of the dosage and the stability of the formulation. Because quercetin has poor water solubility, a 1% sodium carboxymethyl cellulose solution was selected as the dispersion medium and solvent carrier to ensure its uniform dispersion in the composition and facilitate oral administration.

[0021] Based on the above technical solution, and according to the experimental design requirements, the following three concentration specifications of the quercetin-containing composition were prepared: Low-dose quercetin suspension: Add 45 mg of quercetin powder to each 1 mL of 1% sodium carboxymethyl cellulose solution, stir thoroughly to ensure that the quercetin is evenly dispersed in the solvent to form a uniform suspension, and administer it to the low-dose group of animals by gavage at a dose of 45 mg per kilogram of body weight per day. High-dose quercetin suspension: Add 90 mg of quercetin powder to each 1 mL of 1% sodium carboxymethyl cellulose solution, stir thoroughly to ensure that the quercetin is evenly dispersed in the solvent to form a uniform suspension, and administer it to the high-dose group of animals by gavage at a dose of 90 mg per kilogram of body weight per day. Positive control NAD + Suspension: Add 100 mg of nicotinamide adenine dinucleotide (NAD) to each 1 mL of 1% sodium carboxymethyl cellulose solution. + The specification is 100mg per tablet, NAD. + As a positive control drug, the dosage was 100 mg per kilogram of body weight per day, which was used as a positive reference to evaluate the effect of quercetin in delaying testicular aging.

[0022] Based on the above technical solutions, the preparation methods of the quercetin-containing compositions differ in the in vitro cell experiments: Quercetin and Q3G were both dissolved in complete culture medium to prepare stock solutions of corresponding concentrations. Different concentration gradients were screened by CCK-8 cell viability assay. Finally, the optimal intervention concentration of quercetin was determined to be 20 μM and the optimal intervention concentration of Q3G was determined to be 30 μM. In addition, the drug used to induce senescence of testicular Sertoli cells in in vitro experiments was doxorubicin (Doxo). After determining its induction concentration and treatment time through CCK-8 screening, it was used to establish a Doxo-induced TM4 cell senescence model.

[0023] A method for using a quercetin-containing composition to delay testicular aging, the method comprising an in vivo experimental procedure and an in vitro experimental procedure; like Figure 3-4 As shown, the specific in vivo experimental procedure is as follows: Step 11, Experimental Animals and Grouping; Step 12, Sample Collection and Processing; Step 13, General phenotypic and morphological observation; Step 14, serum reproductive hormone level detection; Step 15, sperm quality analysis; Step 16, Detection of cellular senescence levels; Step 17: Observe using a transmission electron microscope; Step 18, Protein expression level detection; Step 19, Immunofluorescence staining; like Figure 5-6 As shown, the specific in vitro experimental procedure is as follows: Step 21, cell culture and drug concentration screening; Step 22, Construction of Doxo-induced TM4 cell senescence model; Step 23, Experimental grouping and intervention; Step 24, detection of cell senescence-related factors; Step 25, Seahorse XF energy metabolism analysis; Step 26, AMPK and glycolysis-related factors detection; Step 27, AMPK knockdown verification experiment.

[0024] Based on the above technical solution, 11, healthy male SD rats were selected as experimental animals. The breeding environment was controlled at room temperature of 22±2℃ and humidity of 55%, with a daily 12-hour light cycle and good ventilation. The experimental rats could eat and drink freely. After one week of acclimatization feeding, the rats were divided into different experimental groups according to their age: Six-month-old young SD rats were selected as the normal control group (Control). Rats of this age are in the peak of physiological function and represent the normal young testicular state. Sixteen-month-old aged SD rats were used to construct a natural aging model. The aged rats were further randomly divided into the following four groups: The model control group was given an equal volume of 1% sodium carboxymethyl cellulose solvent; The low-dose quercetin group L-Que was given quercetin suspension at 45 mg / kg / d by gavage; The high-dose quercetin group (H-Que) was given quercetin suspension at 90 mg / kg / d by gavage. Positive control group NAD + Administer NAD at 100 mg / kg / day + Suspension administered via gavage; Each group of drugs was administered by gavage once daily for 60 consecutive days to simulate the effects of long-term intervention on testicular aging. 12. After the drug intervention, the rats were fasted for 12 hours to eliminate food interference. After the fasting period, they were anesthetized and 3 mL of blood was collected through the apex of the heart. The blood was allowed to stand at 4°C for 30 minutes. After the blood coagulated, it was centrifuged at 4°C for 15 minutes at a speed of 3000 rpm. The upper light yellow serum was collected and stored at -80°C for later use. Bilateral testes were removed from the dissected rats. The surrounding adipose tissue, connective tissue, and vascular membrane were removed. The fresh weight of the testes was measured on an electronic analytical scale, and the testicular index was calculated. The formula for calculating the testicular index is: Testicular index = Fresh weight of both testes / Body weight of the rat after the last administration. It reflects the relative weight and functional status of the testes. One testis was flash-frozen and then stored at -80°C for subsequent tissue protein extraction and Western blotting. The other testis was fixed in 4% paraformaldehyde solution for morphological and histopathological observation. At the same time, epididymal tissue was taken, and an incision was made in physiological saline to release sperm into the saline to prepare a sperm suspension for sperm quality analysis. 13. During the intervention period, observe and record the growth status of rats in each group daily, including the luster and smoothness of their fur, changes in fur color, hair loss, hair piloerection, mental state, spontaneous activity, and whether their movements are slow. For histopathological examination of testicular tissue, testicular tissue fixed in 4% paraformaldehyde solution was punctured through the capsule with a needle and then fixed for another 12 hours to allow the fixative to fully penetrate. Subsequently, the tissue underwent gradient dehydration and embedding. The specific process is as follows: The cells were sequentially rinsed with running water for 6 hours, soaked in phosphate buffer for 4 hours, then subjected to gradient dehydration with 50% ethanol for 1 hour, 75% ethanol for 2 hours, 80% ethanol for 1.5 hours, 95% ethanol for 1.5 hours, and 100% ethanol I and II for 40 minutes each. After clearing with xylene I and II for 10 minutes each, the cells were finally embedded in paraffin I and II for 1.5 hours each. The embedded tissue blocks were cut into paraffin sections with a thickness of 10 micrometers using a microtome. After dewaxing, the sections were baked in an oven at 65°C for 2 hours and then stored at 4°C for later use. Paraffin sections were dewaxed with xylene, rehydrated with graded ethanol, and then stained with hematoxylin and eosin. The staining steps are as follows: After baking the slide for 30 minutes, dewax with xylene I and II for 10 minutes each, rehydrate with graded ethanol, stain with hematoxylin solution for 4 minutes, rinse with running water, differentiate with hydrochloric acid alcohol differentiation solution for 3-5 seconds, blue with tap water for 10 minutes, counterstain with eosin solution for 30 seconds, rinse with running water, and mount with neutral resin. The structural integrity of the seminiferous tubules in the testis, the clarity of the seminiferous epithelium, the regularity of the arrangement of spermatogenic cells at each level, the normality of sperm morphology in the lumen, and the state of interstitial cells and interstitial blood vessels were observed under an optical microscope.

[0025] Based on the above technical solution, 14, the levels of reproductive hormones in the serum of rats in each group, including testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH), were detected by enzyme-linked immunosorbent assay (ELISA). The ELISA kit instructions were followed, and the specific steps are as follows: Thawed serum samples and a series of concentration standards were added to the wells of a pre-coated ELISA plate. 50 μL of working solution A was added to each well. The plate was incubated at 37°C for 1 hour and then washed 3 times with washing buffer for 2 minutes each time. Then add 100 μL of working solution B, incubate at 37°C for 30 minutes, wash the plate 5 times for 2 minutes each time, add 90 μL of substrate solution, and develop color in the dark for 15 minutes. Finally, 50 μL of stop solution was added to terminate the reaction, and the optical density value of each well was measured at a wavelength of 450 nm. A standard curve was constructed with the concentration of the standard as the x-axis and the optical density as the y-axis. The hormone concentration in each serum sample was calculated by fitting a four-parameter logistic curve. 15. Place the epididymal tail in 2 ml of preheated 37°C physiological saline, make an incision to release the sperm, incubate at 37°C for 15 minutes to allow the sperm to swim out fully, filter through a 200-mesh filter to remove tissue debris, and obtain a sperm suspension. Take 10 μL of the sperm suspension and drop it onto a glass slide. Use an automated rat sperm analysis system to evaluate the sperm motility parameters. The main analytical indicators include the proportion of immotile sperm and the proportion of rapidly forward-moving sperm. Sperm morphology was observed using Papanicolaou staining. A 10 μL sperm suspension was dropped onto a glass slide to prepare a uniform smear. After air-drying, the smear was fixed with a fixative and then the Papanicolaou staining procedure was followed. After staining with nuclear staining solution for 1 minute, rinse with running water for 1 minute. After staining with plasma staining solution for 2 minutes, add color-enhancing solution twice, each time for 10 seconds. After rinsing with running water, observe the morphological changes of sperm under an optical microscope, focusing on whether there are abnormal sperm with broken heads, broken tails, or curled tails, and count them. 16. The level of cellular senescence in testicular tissue was assessed by SA-β-Gal staining. The preparation process of frozen sections was as follows: testicular tissue was fixed overnight with 4% paraformaldehyde solution, rinsed with running water for 6 hours, dehydrated with 15% sucrose solution for 24 hours until the tissue settled, and dehydrated with 30% sucrose solution for 24 hours until the tissue settled. Finally, it was embedded with OCT embedding agent, cut into frozen sections with a thickness of 10 micrometers, and stored at -20°C for later use. The SA-β-Gal staining procedure is as follows: After thawing frozen sections at room temperature for 30 minutes, rinse three times with phosphate buffer for 5 minutes each time. After fixing with SA-β-Gal staining fixative for 30 minutes, rinse three times with phosphate buffer for 5 minutes each time. Prepare the staining working solution, place the sections in the staining working solution, and incubate overnight at 37°C. The next day, rinse with phosphate buffer and mount with water-soluble mounting medium. The staining working solution is: 10 μL of β-galactosidase staining solution A, 10 μL of solution B, 930 μL of solution C, and 50 μL of X-Gal staining solution, mixed thoroughly. The staining results were observed under an optical microscope. The level of cell senescence was assessed by blue-green positive signals, and the area of ​​the positively stained region was quantitatively statistically analyzed using ImageJ software.

[0026] Based on the above technical solution, 17, fresh testicular tissue samples were taken, quickly cut into small pieces of 1 cubic millimeter, and immediately placed in pre-cooled electron microscopy fixation solution for fixation. After the samples were processed by the standard transmission electron microscopy sample preparation procedure of post-fixation, gradient acetone dehydration, embedding, sectioning, and uranium-lead double staining, the ultrastructural changes of mitochondria in testicular supporting cells were observed under a transmission electron microscope to evaluate the morphology and size of mitochondria, membrane structure integrity, mitochondrial cristae arrangement density, and matrix state. 18. The expression levels of multiple proteins in testicular tissue were detected by Western blotting. During protein extraction, 20 mg of testicular tissue was placed in a pre-cooled grinding tube, and 1 ml of total protein lysis buffer was added. 10 μL of phosphatase inhibitor, 1 μL of protease inhibitor and 10 μL of 100 mmol / L benzyl sulfonyl fluoride were added to the total protein lysis buffer. The tissue was ground for 30 seconds at a time on a cryogenic grinder for a total of 15 times. Each grinding was placed at -20°C at an interval. Then, the tissue was centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected as the total protein of the tissue and stored at -80°C for later use. Protein concentration was determined using the BCA method: 5 μL of protein stock solution was diluted with 95 μL of ultrapure water. A standard curve was prepared using bovine serum albumin. 200 μL of BCA working solution and 20 μL of standard were added to each well. The working solution was mixed with solution A: solution B in a 50:1 ratio. After incubation at 37°C for 30 minutes, the optical density was measured at 562 nm. The protein concentration of each sample was calculated. The loading amount was calculated based on the protein concentration. Each sample was adjusted to the same concentration and volume with ultrapure water and 5 times loading buffer. After mixing, the samples were denatured in a metal bath at 100°C for 7 minutes and stored at -20°C for later use. SDS-PAGE gel electrophoresis was performed using a one-step PAGE gel rapid preparation kit to prepare 10% separating and compression gels. Separating gel: 2.7 ml separating gel solution, 2.7 ml separating gel buffer, and 60 μL modified coagulant were mixed. Compression gel: 0.75 ml compression gel solution, 0.75 ml compression gel buffer, and 15 μL modified coagulant were mixed. During electrophoresis, the protein was first electrophoresed at 80V for 20 minutes to compress it into a line in the compression gel, followed by electrophoresis at 150V for 60 minutes to separate the protein according to its molecular weight in the separating gel. After electrophoresis, the protein was transferred to a PVDF membrane using a wet transfer method. After activating the PVDF membrane with methanol, the transfer sandwich was assembled in the following order: sponge, filter paper, gel, PVDF membrane, filter paper, sponge. The sandwich was then placed in the transfer tank and ice-free rapid transfer buffer was poured in. The membrane was transferred at a constant current of 400mA for 1.5 hours. After the transfer was completed, the PVDF membrane was removed, blocked with protein blocking buffer for 30 minutes, washed three times with Tris buffer for 5 minutes each time, and then the primary antibody was added and incubated overnight on a shaker at 4°C. Primary antibodies include: p16 antibody, p21 antibody, p53 antibody, γ-H2AX antibody, StAR antibody, CYP11A1 antibody, CYP17A1 antibody, HSD17B3 antibody, ZO-1 antibody, WT1 antibody, Sox9 antibody, AMPK antibody, p-AMPK antibody, HK2 antibody, PKM2 antibody, PFKM antibody, LDHC antibody, SIRT3 antibody, β-actin antibody, and GAPDH antibody; The next day, after recovering the primary antibody, the membrane was washed three times with Tris-buffered saline solution for 5 minutes each time. Horseradish peroxidase-labeled secondary antibody (anti-rabbit IgG) was added and incubated at room temperature for 1 hour. After washing the membrane three times with Tris-buffered saline solution for 5 minutes each time, chemiluminescence development was performed using the Omni-ECL universal chemiluminescence detection kit. E-ECL Substrate and E-CL Oxodant were mixed at a 1:1 ratio. The PVDF membrane was placed in the chemiluminescence solution and incubated. The membrane was exposed and photographed in a Western blot chemiluminescence analyzer, and the gray values ​​of each band were analyzed using ImageJ software. The ratio of the gray values ​​of the target protein to the internal control protein was used as the relative expression level for statistical analysis. 19. The expression and co-localization of HK2 and WT1 proteins in testicular tissue were detected by immunofluorescence staining. Paraffin sections were baked, dewaxed, rehydrated, and subjected to microwave antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution, followed by permeabilization with 0.3% Triton X-100 for 30 minutes, and then blocked with 5% goat serum blocking solution for 1 hour. Primary antibodies were then added to cover the sections and incubated overnight at 4°C. The primary antibodies were HK2 antibody and WT1 antibody, both diluted 1:100. The next day, rinse three times with phosphate buffer for 5 minutes each time, add the corresponding fluorescently labeled secondary antibody at a dilution of 1:100, incubate at 37°C in the dark for 1 hour, rinse three times with phosphate buffer in the dark for 5 minutes each time, stain the cell nuclei with DAPI solution for 5 minutes, rinse in the dark, mount with anti-fluorescence quenching mounting medium, and observe and photograph under a laser confocal microscope. WT1 is mainly located in the nucleus of supporting cells and serves as a specific marker for supporting cells. The fluorescence signal intensity of HK2 reflects glycolytic activity. The fluorescence intensity and co-localization of the two were quantitatively analyzed using ImageJ software.

[0027] Based on the above technical solution, 21, the mouse testicular support cell line TM4 was selected for in vitro cell experiments. TM4 cells were cultured and passaged in a complete culture medium containing 10% fetal bovine serum at 37°C and 5% carbon dioxide. The safe and effective concentrations of quercetin and Q3G were screened using the CCK-8 assay. TM4 cells in logarithmic growth phase were seeded at a density of 5000-8000 cells per well in 96-well plates. After 24 hours of culture to allow cell adhesion, the medium was replaced with complete medium containing a series of concentrations of quercetin or Q3G: quercetin: 0, 10, 20, 40, 60, 80, 100 μM gradient; Q3G: 0, 10, 20, 30, 40, 50, 60 μM gradient. Each concentration was used in quadruplicate wells. After 24 hours of treatment, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 3 hours. The optical density was measured at 450 nm, and cell viability was calculated for each concentration group. The lowest concentration with cell viability greater than 80% of the control group and promoting proliferation was determined to be the optimal concentration. The optimal intervention concentration for quercetin was determined to be 20 μM, and for Q3G, 30 μM. 22. TM4 cells in the logarithmic growth phase were seeded in culture plates and cultured to 70%-80% confluence. The culture medium was then replaced with a complete medium containing an appropriate concentration of doxorubicin and treated for 24 hours to induce cell senescence. Subsequently, the doxorubicin-containing medium was discarded and replaced with a complete medium for subsequent intervention experiments. The successful construction of the senescence model was verified by Western blotting, which showed a significant increase in the expression levels of senescence-related proteins p16, p21, and γ-H2AX.

[0028] Based on the above technical solutions, 23, the in vitro experiments are divided into the following four groups: Blank control group: TM4 group, which consists of normally cultured TM4 cells without any treatment; Model group: TM4 plus Doxo group, which is doxorubicin-induced senescent TM4 cells, given an equal volume of complete culture medium solvent; Quercetin intervention group: TM4 plus Doxo plus Que group, which was treated with 20μM quercetin for 24 hours after induction with doxorubicin; Q3G intervention group: TM4 plus Doxo plus Q3G group, which was treated with 30μMQ3G for 24 hours after doxorubicin induction; 24. Collect cells from each group after intervention, extract total cellular protein, and use Western blotting to detect the protein expression levels of aging-related factors p16, p21, p53 and γ-H2AX. The operation steps are the same as in the in vivo experimental section. At the same time, use immunofluorescence staining to observe the intracellular distribution and expression levels of aging-related factors under a laser confocal microscope. 25. The energy metabolism status of each group of live cells was detected in real time using the Seahorse XF Cell Energy Metabolism Analyzer. TM4 cells were seeded at an appropriate density in Seahorse XF cell culture plates and processed according to the above grouping. During testing, the Seahorse XF analytical medium was replaced, and the measurements were performed in real time using the Seahorse XF analyzer. For oxygen consumption rate detection, oligomycin, FCCP and antimycin A were injected into each well sequentially during the measurement process. Oligomycin inhibits ATP synthase, FCCP is a decoupling agent that maximizes the operation of the electron transport chain, and antimycin A inhibits electron transport chain complex III. The basal respiratory capacity, maximum respiratory capacity and ATP production of cells were calculated by the changes in the oxygen consumption rate curve. In terms of extracellular acidification rate detection, the glycolysis level, glycolysis capacity and glycolysis reserve of cells are calculated by measuring the extracellular acidification rate curve, reflecting the changes in cellular glycolysis flux.

[0029] Based on the above technical solution, 26, the expression levels of AMPK and glycolysis-related factors in each group of cells were detected by Western Blot, including p-AMPK, AMPK, HK2, PKM2, LDHC and SIRT3. The ratio of p-AMPK to total AMPK was used to reflect the phosphorylation activation level of AMPK, and the expression levels of each key glycolysis enzyme were used to reflect the glycolysis activity of the cells. Simultaneously, immunofluorescence staining was used to detect the expression distribution and fluorescence intensity of HK2, PKM2, and LDHC in cells, with the same procedure as before; 27. To verify the key mediating role of AMPK in quercetin-regulated glycolysis and delayed senescence of testicular Sertoli cells, a stable cell line with AMPKα gene knockdown was constructed. The specific method was as follows: A lentiviral vector was constructed using a short hairpin RNA sequence targeting AMPKα. The vector was then transfected into 293T cells to package the lentivirus. TM4 cells were subsequently infected with the lentivirus. The TM4-shAMPKα cell line, which stably expresses shAMPKα, was established by selection with puromycin. TM4-shNC cells transfected with the empty vector shNC were used as a negative control. Western blotting confirmed that the AMPK protein expression level was significantly reduced, thus confirming the successful construction of the knockdown cell line. Transcriptomic sequencing was performed on TM4-shNC and TM4-shAMPKα cells to screen differentially expressed genes and conduct GO function and KEGG pathway enrichment analysis to observe the effect of AMPK knockdown on cellular energy metabolism-related pathways. Subsequently, TM4-shAMPKα cells were treated with quercetin and Q3G, respectively. Western blotting was used to detect changes in the expression levels of glycolysis-related factors such as HK2, PKM2, LDHC, and SIRT3 to verify whether quercetin can still play a regulatory role in glycolysis under AMPK deficiency.

[0030] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition containing quercetin, characterized in that: The composition consists of an active ingredient, a solvent, and a dispersion medium; The active ingredients include quercetin and quercetin-3-O-glucuronic acid, quercetin: Que, quercetin-3-O-glucuronic acid: Q3G; Que is a natural flavonol compound found in fruits, vegetables, and various medicinal plants. Q3G is a metabolite of quercetin in the body. It is a metabolite produced by glucuronidation of quercetin in the small intestine and liver after oral administration. It is also the main circulating form of quercetin in plasma and various tissues. The solvent and dispersion medium are 1% sodium carboxymethyl cellulose solution, which is a high molecular weight polysaccharide excipient. 1% sodium carboxymethyl cellulose solution is selected as the dispersion medium and solvent carrier.

2. The composition containing quercetin according to claim 1, characterized in that: The quercetin-containing composition is formulated with different concentrations and prepared in three concentration specifications: low-dose quercetin suspension, high-dose quercetin suspension, and positive control NAD. + Suspension.

3. The composition containing quercetin according to claim 1, characterized in that: The quercetin-containing composition was prepared differently in the in vitro cell experiment section: Quercetin and Q3G were both dissolved in complete culture medium to prepare stock solutions or working solutions of corresponding concentrations, and different concentration gradients were screened. In in vitro experiments, the drug used to induce senescence of testicular Sertoli cells was doxorubicin (Doxo), which was selected by CCK-8 screening and used to establish a Doxo-induced TM4 cell senescence model.

4. A method for using a quercetin-containing composition to delay testicular aging, characterized in that: This method includes in vivo experimental procedures and in vitro experimental procedures; The specific in vivo experimental procedure is as follows: Step 11, Experimental Animals and Grouping; Step 12, Sample Collection and Processing; Step 13, General phenotypic and morphological observation; Step 14, serum reproductive hormone level detection; Step 15, sperm quality analysis; Step 16, Detection of cellular senescence levels; Step 17: Observe using a transmission electron microscope; Step 18, Protein expression level detection; Step 19, Immunofluorescence staining; The specific in vitro experimental procedure is as follows: Step 21, cell culture and drug concentration screening; Step 22, Construction of Doxo-induced TM4 cell senescence model; Step 23, Experimental grouping and intervention; Step 24, detection of cell senescence-related factors; Step 25, Seahorse XF energy metabolism analysis; Step 26, AMPK and glycolysis-related factors detection; Step 27, AMPK knockdown verification experiment.

5. A method for delaying testicular aging using a quercetin-containing composition according to claim 4, characterized in that: In the 11th experiment, SD rats were selected as experimental animals and divided into different experimental groups according to their age: Six-month-old young SD rats were used as the normal control group (Control). Sixteen-month-old aged SD rats were used to establish a natural aging model. The aged rats were further randomly divided into: a model control group (Model), a low-dose quercetin group (L-Que), a high-dose quercetin group (H-Que), and a positive control group (NAD). + Each group received the drug via gavage once daily for 60 consecutive days to simulate the effects of long-term intervention on testicular aging. In step 12, after the drug intervention was completed, the rats were anesthetized after fasting, and blood was collected through the apex of the heart. After the blood was allowed to stand and coagulate, it was centrifuged and the upper light yellow serum was collected and stored for later use. The rat was dissected and both testes were removed. The adipose tissue, connective tissue and vascular membrane around the testes were removed. The fresh weight of the testes was weighed and the testicular index was calculated. One testis was quick-frozen and stored, while the other testis was fixed in paraformaldehyde solution. At the same time, epididymal tissue was taken, and an incision was made in physiological saline to release sperm and prepare a sperm suspension. In step 13, during the intervention period, the growth status of rats in each group was observed and recorded in detail daily; In terms of testicular tissue pathology, testicular tissue fixed in paraformaldehyde solution was punctured by a needle tip and then fixed again to allow the fixative to penetrate. Subsequently, the tissue underwent gradient dehydration and embedding. The embedded tissue blocks were cut into paraffin sections using a microtome, baked in an oven to remove the wax, and then stored for later use. Paraffin sections were dewaxed with xylene, rehydrated with graded ethanol, stained with hematoxylin and eosin, and observed under an optical microscope.

6. A method for delaying testicular aging using a quercetin-containing composition according to claim 4, characterized in that: In step 14, the levels of reproductive hormones in the serum of rats in each group, including testosterone, luteinizing hormone and follicle-stimulating hormone, were detected by enzyme-linked immunosorbent assay (ELISA). A standard curve was constructed with the concentration of the standard as the x-axis and the optical density as the y-axis. The hormone concentration in each serum sample was calculated by fitting a four-parameter logistic curve. In step 15, the epididymal tail is placed in physiological saline, an incision is made to release sperm, allowing the sperm to swim out fully, tissue fragments are filtered out, a sperm suspension is obtained, and the suspension is dropped onto a glass slide to evaluate the sperm motility parameters. Sperm morphology observation was performed using the Papanicolaou staining method. Sperm suspension was dropped onto a glass slide to make a uniform smear, which was then air-dried and fixed with a fixative. The Papanicolaou staining procedure was followed to observe the morphological changes of the sperm. The 16th method assesses the level of cellular senescence in testicular tissue by SA-β-Gal staining. Frozen sections are prepared first, then SA-β-Gal staining is performed, and finally the sections are mounted with water-soluble mounting medium. The staining results were observed under an optical microscope. The level of cell senescence was assessed by blue-green positive signals, and the area of ​​the positively stained region was quantitatively statistically analyzed using ImageJ software.

7. A method for delaying testicular aging using a quercetin-containing composition according to claim 4, characterized in that: In step 17, testicular tissue samples were taken, cut into small pieces, and fixed in pre-cooled electron microscopy fixative. After the samples were processed by the standard transmission electron microscopy sample preparation procedure of post-fixation, gradient acetone dehydration, embedding, sectioning, and uranium-lead double staining, the ultrastructural changes of mitochondria in testicular supporting cells were observed under a transmission electron microscope to evaluate the morphology and size of mitochondria, membrane structure integrity, mitochondrial cristae arrangement density, and matrix state. In step 18, the expression levels of multiple proteins in testicular tissue were detected by Western Blot. After protein extraction, grinding and centrifugation, the supernatant was collected and stored for later use. The supernatant is the total protein of the tissue. Protein concentration was determined using the BCA method: the protein stock solution was diluted with ultrapure water, a standard curve was prepared using bovine serum albumin, LBCA working solution and standard or sample to be tested were added to each well, after incubation, the optical density value was measured, the protein concentration of each sample was calculated, and the loading amount was calculated based on the protein concentration. SDS-PAGE gel electrophoresis was performed using a one-step PAGE gel rapid preparation kit to prepare 10% separating and compression gels. During electrophoresis, the protein is first compressed into a line in a compression gel, and then separated according to molecular weight in a separating gel. After electrophoresis, the protein is transferred to a PVDF membrane using a wet transfer method. After activating the PVDF membrane with methanol, the transfer sandwich is assembled in sequence, placed in the transfer tank, and ice-free rapid transfer buffer is poured in. The membrane is transferred at a constant current of 400 mA for 1.5 hours. After the transfer was completed, the PVDF membrane was removed, blocked with protein blocking buffer, washed with Tris buffer, and then incubated overnight on a shaker at 4°C with primary antibody. The next day, after recovering the primary antibody, the membrane was washed three times with Tris-buffered saline solution for 5 minutes each time. Horseradish peroxidase-labeled secondary antibody was added and incubated at room temperature. After washing with Tris-buffered saline solution, chemiluminescence imaging was performed. The PVDF membrane was placed in the chemiluminescence solution and incubated using the Omni-ECL universal chemiluminescence detection kit. The membrane was exposed and photographed in a Western blot chemiluminescence analyzer, and the gray values ​​of each band were analyzed using ImageJ software. The ratio of the gray values ​​of the target protein to the internal control protein was used as the relative expression level for statistical analysis. In step 19, the expression and co-localization of HK2 and WT1 proteins in testicular tissue were detected by immunofluorescence staining. After paraffin sections were repaired, endogenous peroxidase activity was blocked with hydrogen peroxide solution, followed by permeabilization with Triton X-100, blocking with goat serum blocking solution, and primary antibody was added to cover the sections and incubated overnight. The next day, the cells were washed with phosphate buffer, and the corresponding fluorescently labeled secondary antibody was added. The cells were incubated in the dark. After rinsing with phosphate buffer in the dark, the cell nuclei were stained with DAPI solution. After rinsing in the dark, the cells were mounted with anti-fluorescence quenching mounting medium and observed and photographed under a laser confocal microscope. WT1 is mainly located in the nucleus of supporting cells and serves as a specific marker for supporting cells. The fluorescence signal intensity of HK2 reflects glycolytic activity. The fluorescence intensity and co-localization of the two were quantitatively analyzed using ImageJ software.

8. A method for delaying testicular aging using a quercetin-containing composition according to claim 4, characterized in that: In the 21st case, the mouse testicular support cell line TM4 was selected for in vitro cell experiments. TM4 cells were cultured and passaged in a carbon dioxide incubator using a complete culture medium containing 10% fetal bovine serum. The safe and effective concentrations of quercetin and Q3G were screened using the CCK-8 assay. TM4 cells in logarithmic growth phase were seeded into 96-well plates and cultured until the cells adhered to the plate. The medium was then replaced with complete medium containing a series of concentrations of quercetin or Q3G, with replicates for each concentration. 10 μL of CCK-8 reagent was added to each well. After incubation, the optical density was measured, and the cell viability of each concentration group was calculated. The lowest concentration with higher cell viability than the control group and the ability to promote cell proliferation was identified as the optimal concentration. In step 22, TM4 cells in the logarithmic growth phase were seeded in a culture plate and cultured. The culture medium was then replaced with a complete medium containing an appropriate concentration of doxorubicin and treated for 24 hours to induce cell senescence. Subsequently, the doxorubicin-containing medium was discarded and replaced with a complete medium or a complete medium containing quercetin and Q3G for subsequent intervention experiments. The successful construction of the senescence model was verified by Western blotting, which showed a significant increase in the expression levels of senescence-related proteins p16, p21, and γ-H2AX.

9. A method for delaying testicular aging using a quercetin-containing composition according to claim 4, characterized in that: The in vitro experiments were divided into two groups: a blank control group (TM4 group), a model group (TM4 plus Doxo group), a quercetin intervention group (TM4 plus Doxo plus Que group), and a Q3G intervention group (TM4 plus Doxo plus Q3G group). In step 24, cells from each group after intervention were collected, total cellular protein was extracted, and the protein expression levels of aging-related factors p16, p21, p53 and γ-H2AX were detected by Western Blot. At the same time, the intracellular distribution and expression levels of aging-related factors were observed under a laser confocal microscope using immunofluorescence staining. In the 25th case, the energy metabolism status of each group of live cells was detected in real time using the Seahorse XF cell energy metabolism analyzer, and TM4 cells were seeded in Seahorse XF dedicated cell culture plates. During testing, the Seahorse XF analytical medium was replaced, and the measurements were performed in real time using the Seahorse XF analyzer. For oxygen consumption rate detection, oligomycin, FCCP and antimycin A were injected into each well sequentially during the measurement process, and the cell indicators were calculated by the changes in the oxygen consumption rate curve. In terms of extracellular acidification rate detection, cellular indicators are calculated by measuring the extracellular acidification rate curve to reflect changes in cellular glycolysis flux.

10. A method for delaying testicular aging using a quercetin-containing composition according to claim 4, characterized in that: In the second part, the expression levels of AMPK and glycolysis-related factors in each group of cells were detected by Western blotting. The ratio of p-AMPK to total AMPK was used to reflect the phosphorylation activation level of AMPK, and the expression levels of each key glycolysis enzyme were used to reflect the glycolysis activity of the cells. Simultaneously, immunofluorescence staining was used to detect the expression distribution and fluorescence intensity of HK2, PKM2, and LDHC in cells; In the second part, to verify the key mediating role of AMPK in quercetin regulation of glycolysis and delay of testicular Sertoli cell senescence, a stable cell line with AMPKα gene knockdown was constructed. Transcriptomic sequencing was performed on TM4-shNC and TM4-shAMPKα cells to screen differentially expressed genes and conduct GO function and KEGG pathway enrichment analysis to observe the effect of AMPK knockdown on cellular energy metabolism-related pathways. Subsequently, TM4-shAMPKα cells were treated with quercetin and Q3G, respectively, and the expression levels of glycolysis-related factors were detected by Western blotting to verify the regulatory role of quercetin on glycolysis in the absence of AMPK.

Images