Application of combination of beta-nicotinamide mononucleotide and apigenin in preparation of product for delaying senescence of testicular interstitial cells

Abstract

The invention relates to the technical field of biological medicine, in particular to application of combination of beta-nicotinamide mononucleotide (NMN) and apigenin (APG) in preparation of a product for delaying senescence of testicular interstitial cells. Compared with single treatment, the combined use of NMN and apigenin can significantly improve the NAD < + > level of aged mice, promote the expression of testosterone synthesis related genes and proteins, promote the synthesis of testosterone in testicular interstitial cells, enhance the cell proliferation activity, and improve the mitochondrial function and antioxidant defense system, thereby delaying the aging of the testicular interstitial cells. The discovery of the invention not only contributes to understanding the regulation mechanism of testosterone synthesis in the aging process, but also provides technical support for developing a new anti-aging strategy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the combined use of β-nicotinamide mononucleotide (NMN) and apigenin (APG) in the preparation of products that delay the aging of testicular interstitial cells. Background Technology

[0002] With the increasing aging of the population, research on aging and related diseases has become particularly important. Testicular interstitial cells play a crucial role in male reproductive health; their functional decline leads to decreased testosterone levels, thereby affecting the quality of life of older men. Nicotinamide adenine dinucleotide (NAD) + Testosterone is a key coenzyme in cell metabolism and is closely related to testosterone synthesis. Its decline is associated with aging.

[0003] Traditional testosterone replacement therapy (TRT) is a common treatment for testosterone deficiency, increasing serum testosterone levels and alleviating related symptoms by supplementing with exogenous testosterone. However, the use of TRT is not without controversy, and its long-term safety and efficacy require further investigation. In particular, TRT may increase the risk of cardiovascular disease and prostate cancer, forcing clinicians to weigh the benefits and risks when recommending it. In recent years, with in-depth research into the mechanisms of testicular interstitial cell aging, new treatment strategies are constantly emerging. For example, antioxidants have been found to improve the steroid production function of testicular interstitial cells, while selective androgen receptor modulators (SARMs) offer a potential alternative therapy aimed at increasing serum testosterone levels while reducing the potential side effects of TRT. Furthermore, stem cell therapy provides a new perspective on restoring testicular interstitial cell function by activating or transplanting stem cells to promote endogenous testosterone production. However, the effectiveness of these methods in delaying testicular interstitial cell aging remains limited. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides the application of β-nicotinamide mononucleotide and apigenin in the preparation of products that delay the aging of testicular interstitial cells. This invention found that the combined application of NMN and APG significantly increased testosterone levels in the testes of aging mice compared to treatment alone, exhibiting a synergistic effect in delaying the aging of testicular interstitial cells.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of β-nicotinamide mononucleotide and apigenin in the preparation of products for delaying testicular interstitial cell aging and / or treating diseases related to testicular interstitial cell aging.

[0007] Preferably, the diseases associated with testicular interstitial cell aging include: androgen deficiency and / or decreased reproductive function.

[0008] Preferably, the method of delaying testicular interstitial cell senescence includes: promoting testosterone synthesis, increasing testicular interstitial cell volume, increasing mitochondrial membrane potential, and enhancing NAD+. + The expression levels of the functional gene or protein are adjusted by one or more of the following: upregulation of functional gene expression, upregulation of functional protein expression, downregulation of CD38 protein expression, downregulation of P38 protein expression, and downregulation of P21 protein expression; the functional gene or protein includes those involved in testosterone synthesis, NAD+ synthesis, and NAD+ synthesis. + Functional genes or proteins that are related to one or more of the following: synthesis, mitochondrial function, and antioxidant activity.

[0009] Preferably, the functional genes include one or more of Star, Scarb1, Cyp11a1, Cyp17a1, Hsd17b3, Hsd11b2, Insl3, Nr5a1, Hsd11b1, Nampt, and Nr3c1.

[0010] Preferably, the functional protein includes one or more of CYP11A1, CYP17A1, NAMPT, STAR, INSL3, SOD1, SOD2 and NRF2.

[0011] Preferably, the product alleviates the decline in mitochondrial function caused by aging by restoring the mitochondrial splitting and fusion function.

[0012] Preferably, based on mouse body weight, the unit dose of β-nicotinamide mononucleotide in the product is 450-550 mg / kg / d, and the unit dose of apigenin is 45-55 mg / kg / d.

[0013] Preferably, based on mouse body weight, the unit dose of β-nicotinamide mononucleotide in the product is 500 mg / kg / d, and the unit dose of apigenin is 50 mg / kg / d.

[0014] Preferably, the product includes a drug.

[0015] Preferably, the dosage form of the drug includes injections and / or oral formulations.

[0016] Beneficial effects:

[0017] This invention reveals that, compared to single treatments, the combined use of NMN and apigenin significantly increases NAD+ levels in aging mice, promotes the expression of testosterone synthesis-related genes and proteins, enhances testosterone synthesis in testicular interstitial cells, strengthens cell proliferation activity, improves mitochondrial function and the antioxidant defense system, thereby delaying the aging of testicular interstitial cells. This discovery not only contributes to understanding the regulatory mechanisms of testosterone synthesis during aging but also provides technical support for developing new anti-aging strategies. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 The level of testosterone in the testes of different groups of mice;

[0020] Figure 2 Immunohistochemical staining images of CYP11A1 and SOX9, as well as statistical graphs of the number and area of ​​CYP11A1-positive cells; where AE: double immunohistochemical staining images of CYP11A1 (testicular interstitial cell biomarker) and SOX9 (Supporting cell biomarker) in each group; arrows indicate CYP11A1+ testicular interstitial cells; scale bar = 10 μm; F: ratio of testicular interstitial cell number to Supporting cell number;

[0021] Figure 3 Immunohistochemical staining images of CYP11A1 and SOX9, and a statistical graph of the area of ​​CYP11A1-positive cells; where AE: double immunohistochemical staining images of CYP11A1 and SOX9 in each group; arrows indicate CYP11A1+ testicular interstitial cells; scale bar = 10 μm; 3F-H: area, cytoplasmic size, and nucleus size of testicular interstitial cells, respectively;

[0022] Figure 4 The expression levels of genes related to mouse testicular interstitial cells are shown below; where AK: mRNA expression levels of Lhcgr, Star, Scarb1, Cyp11a1, Cyp17a1, Hsd3b6, Hsd17b3, Hsd11b2, Insl3, Nr5a1, and Hsd11b1 (genes related to the testosterone synthesis pathway), respectively; L: mRNA expression level of Nampt (gene related to NAD+ synthesis); M and N: mRNA expression levels of Nr3c1 and Nr3c2 (genes related to the glucocorticoid pathway), respectively.

[0023] Figure 5The expression levels of proteins related to the testosterone synthesis pathway and NAD+ synthesis pathway in mouse testes are shown. Among them, A: Western blot map of proteins related to the testosterone synthesis pathway and NAD+ synthesis pathway in mouse testes; BF: Quantitative statistical graph of the expression of CYP11A1, CYP17A1, INSL3, STAR and NAMPT proteins in mouse testes, normalized, with ACTB as the housekeeping protein.

[0024] Figure 6 The combined application of NMN and APG in vivo restored the NAMPT protein level in testicular interstitial cells; AJ: Double immunofluorescence staining of testicular interstitial cells (3β-HSD6, green) and NAMPT-expressing cells (NAMPT, red) in paraffin sections of mouse testes; B, D, F, H, and J are magnified views of the yellow squares in A, C, E, G, and I, respectively; white arrows indicate NAMPT+ALCs; scale bar = 10 μm; K is a semi-quantitative statistical graph of NAMPT immunofluorescence staining;

[0025] Figure 7 To enhance the testosterone synthesis capacity of senescent MA10 cells in vitro using NMN and APG in combination; where A: in vitro drug administration instructions; B: testosterone levels in MA10 cell culture medium; C and D: cytotoxicity assays of NMN and APG on MA10 cells (CCK8); E: NAD+ levels in MA10 cells; FJ: β-gal cell staining diagram, with asterisks (*) and triangular arrows (△) used to indicate darker areas, indicating higher β-gal activity in these areas, thus reflecting the characteristics of cellular senescence;

[0026] Figure 8 The results show the expression levels of proteins related to anti-aging and testosterone synthesis in MA10 cells when NMN and APG are used in vitro. Among them, A: Western blot image of proteins related to aging and testosterone synthesis in MA10 cells in vitro; BG is a statistical graph of quantitative expression of P38, P21, INSL3, CYP11A1, CYP17A1 and STAR proteins in in vitro cells.

[0027] Figure 9 The combined use of NMN and APG was used to repair mitochondrial function and promote NAD+ synthesis in senescent MA10 cells; where A: Western blot; BG is a statistical graph of protein levels of SIRT1, PGC-1α, OPA1, MFN1, NAMPT and CD38; HJ is a statistical graph of protein levels of AMPK, pAMPK and pAMPK / AMPK.

[0028] Figure 10The in vitro exposure to NMN and APG alleviated the antioxidant levels in the testes of aging mice; where A: Western blot; BD: statistical graphs of protein levels of NRF2, SOD1, and SOD2;

[0029] Figure 11 The combined application of NMN and APG was used to block the decrease in mitochondrial membrane potential induced by H2O2 in MA10 cells; where AE: mitochondrial membrane potential graphs of 5 groups, and F: statistical graph of mitochondrial membrane potential.

[0030] Figure 12 A diagram illustrating the mechanism of action of the combined application of NMN and apigenin in the treatment of testicular interstitial cells in aging mice. Detailed Implementation

[0031] This invention provides the application of β-nicotinamide mononucleotide and apigenin in the preparation of products for delaying testicular interstitial cell aging and / or treating diseases related to testicular interstitial cell aging.

[0032] As one implementation, the diseases associated with testicular interstitial cell aging include: androgen deficiency and / or decreased reproductive function.

[0033] As one implementation method, the method of delaying testicular interstitial cell aging includes: promoting testosterone synthesis, increasing testicular interstitial cell volume, increasing mitochondrial membrane potential, and enhancing NAD+. + The expression levels of the functional gene or protein are adjusted by one or more of the following: upregulation of functional gene expression, upregulation of functional protein expression, downregulation of CD38 protein expression, downregulation of P38 protein expression, and downregulation of P21 protein expression; the functional gene or protein includes those involved in testosterone synthesis, NAD+ synthesis, and NAD+ synthesis. + The product contains one or more functional genes or proteins related to synthesis, mitochondrial function, and antioxidant activity. As one embodiment, the functional genes include one or more of the following: Star, Scarb1, Cyp11a1, Cyp17a1, Hsd17b3, Hsd11b2, Insl3, Nr5a1, Hsd11b1, Nampt, and Nr3c1. As one embodiment, the functional proteins include one or more of the following: CYP11A1, CYP17A1, NAMPT, STAR, INSL3, SOD1, SOD2, and NRF2. As one embodiment, the product alleviates age-related mitochondrial function decline by restoring mitochondrial fission and fusion function.

[0034] In one embodiment, based on mouse body weight, the unit dose of β-nicotinamide mononucleotide in the product is 450-550 mg / kg / day, and the unit dose of apigenin is 45-55 mg / kg / day. In another embodiment, based on mouse body weight, the unit dose of β-nicotinamide mononucleotide in the product is 500 mg / kg / day, and the unit dose of apigenin is 50 mg / kg / day. In one embodiment, the product comprises a drug. In one embodiment, the dosage form of the drug includes injections and / or oral dosage forms.

[0035] This invention has found that the combined use of NMN and apigenin can increase NAD+ levels in aging mice, promote the expression of testosterone synthesis-related genes and proteins, promote testosterone synthesis in testicular interstitial cells, enhance cell proliferation activity, improve mitochondrial function and antioxidant defense system, thereby delaying the aging of testicular interstitial cells. Figure 12 ).

[0036] This invention found that the combined use of NMN and APG significantly increased testosterone levels in the testes of aging mice compared to treatment alone. Figure 1 ), while not affecting the number of testicular interstitial cells ( Figure 2 This finding is closely related to the combined upregulation of NAD+ levels in testicular interstitial cells and the expression levels of genes and proteins related to testosterone synthesis by NMN and APG. At the molecular level, this invention found that the combined application of NMN and APG can significantly upregulate the gene expression of key enzymes in testosterone synthesis, such as CYP11A1, CYP17A1, and STAR. Figure 4 These enzymes play a crucial role in the steroid synthesis pathway, and their activity directly affects the efficiency of testosterone synthesis.

[0037] This invention found that the combined use of NMN and APG significantly promoted the synthesis of testosterone and the expression of NAD+ synthesis-related proteins in testicular interstitial cells. Figure 5 These proteins play a crucial role in the testosterone synthesis pathway, and changes in their expression levels directly reflect the regulation of testosterone synthesis capacity. Western blotting results showed significant differences in the expression levels of CYP11A1, CYP17A1, and STAR proteins between the young group (Group A) and the aging group (Group B), consistent with the decline in testosterone synthesis capacity during aging. However, when aging mice received NMN (Group C) or APG (Group D) treatment alone, the expression levels of these proteins recovered somewhat, but to a limited extent compared to the combined treatment group (Group E). These results indicate that the combined application of NMN and APG has a synergistic effect in treating testicular interstitial cell aging, and is more effective than single treatment in promoting the expression of testosterone synthesis-related proteins, thereby enhancing testosterone synthesis capacity.

[0038] In in vitro experiments, this invention used H2O2 to treat the MA10 cell line to simulate an in vitro aging cell model, further confirming the effectiveness of the combined application of NMN and APG in delaying aging. This invention found that the combined treatment could reverse the expression of aging marker proteins P38 and P21, while simultaneously promoting the expression of testosterone synthesis-related proteins. Figure 8 These results further demonstrate the potential role of NMN and APG in delaying aging and promoting testosterone synthesis.

[0039] The combined use of NMN and APG can restore mitochondrial membrane potential. Figure 11 Mitochondrial membrane potential is a key indicator of cellular energy metabolism, and its decline is closely related to cellular senescence and dysfunction. The combined application of NMN and APG may help maintain cellular energy supply by restoring mitochondrial membrane potential, thereby slowing down the aging process.

[0040] The results of this invention show that the combined application of NMN and APG significantly upregulated the expression of NRF2 protein, as well as the antioxidant enzymes SOD1 and SOD2. Figure 10 It may reduce oxidative stress by enhancing the antioxidant capacity of cells, thereby delaying cell aging.

[0041] In summary, this invention reveals that the combined application of NMN and APG delays the aging of testicular interstitial cells by increasing NAD+ levels in aging mice, promoting the expression of testosterone synthesis-related genes and proteins, enhancing cell proliferation activity, improving mitochondrial function, and strengthening the antioxidant defense system. These findings not only contribute to understanding the regulatory mechanisms of testosterone synthesis during aging but also provide technical support for developing new anti-aging strategies.

[0042] To further illustrate the present invention, the application of the combined use of β-nicotinamide mononucleotide and apigenin provided by the present invention in the preparation of products that delay the aging of testicular interstitial cells is described in detail below with reference to embodiments and accompanying drawings, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0043] Preparation Example

[0044] This invention utilizes the C57BL / 6 mouse model, purchased from the Shanghai Laboratory Animal Center. A total of 24 19-month-old male mice were randomly divided into four groups: an aging group (Group B), an NMN treatment group (Group C), an APG treatment group (Group D), and a combined NMN and APG treatment group (Group E), with six mice in each group. Additionally, six 4-month-old male C57BL / 6 mice were purchased as a control group for aging mice (Group A). ​​All experimental animals were housed in an environment with a temperature of 21-25℃ and a relative humidity of 45-55%, following a diurnal light-dark cycle (12 hours of light, 12 hours of darkness). Water and standard feed were freely available to all animals. Animal experiments were conducted strictly in accordance with the ethical and operational guidelines of the "Guidelines for the Care and Use of Laboratory Animals," and have been approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University.

[0045] The reagent sources or primer sequences used in the following examples are shown below:

[0046] β-Nicotinamide mononucleotide (NMN, CAS: 1094-61-7, purity ≥95%) and apigenin (APG, CAS: 520-36-5, purity ≥98.0%) were both purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0047] Table 1. Primer information used for qPCR

[0048]

[0049]

[0050] Table 2 Materials and reagents for testosterone concentration determination

[0051] Main materials and reagents company Standard products Waters Technology Co., Ltd. sample bottle Waters Technology Co., Ltd. Testosterone-d5(T-d5) Zhenzhun Biotechnology (Shanghai) Co., Ltd. Acetonitrile Wenzhou Jinshan Chemical Reagent and Instrument Company

[0052] Table 3 Antibody information from Western blot and immunohistochemistry.

[0053]

[0054]

[0055] Note: In Table 3, WB represents the dilution concentration of Western blot; HS represents the dilution concentration of histochemical staining; ND represents no detection.

[0056] Example

[0057] 1. Animal experiments

[0058] (1) Grouping and administration of C57BL / 6 mice

[0059] ① Twenty-four 19-month-old male C57BL / 6 mice that had adapted to the environment were randomly divided into four groups of six each. At the same time, six 4-month-old male C57BL / 6 mice were placed in a separate group. The following conditions were provided: a temperature of 23±2℃ and a relative humidity of 55±5%. The animal room was kept under a 12-hour light / 12-hour dark cycle to ensure that the mice had free access to sufficient and fresh water and food.

[0060] ②NMN was dissolved in 0.9% sodium chloride solution, and APG was dissolved in 0.5% sodium carboxymethyl cellulose (preliminary experiments confirmed that oral administration of 0.5% sodium carboxymethyl cellulose had no significant biological effect on mice);

[0061] ③ The treatments for different groups were as follows: Mice in the control and aging groups were intraperitoneally injected with 0.9% sodium chloride (solvent control); mice in the NMN treatment group were intraperitoneally injected with NMN 500 mg / kg / day; mice in the APG treatment group were administered APG 50 mg / kg / day by gavage; and mice in the NMN and APG combined treatment group were intraperitoneally injected with NMN 500 mg / kg / day and administered APG 50 mg / kg / day by gavage. All five groups were administered the medications continuously for 23 days. The mice's weight and mental state were recorded daily.

[0062] ④ At a fixed time (10:00) every day, administer the medication by gavage for 23 consecutive days, and observe and record the weight and activity of the mice.

[0063] (2) Sampling and freezing

[0064] ① After administration, the mice were euthanized using CO2 asphyxiation.

[0065] ② Collect testicular samples from each experimental mouse and place them in a clean 1.5 mL EP tube;

[0066] ③ Place one testis from each male mouse into a clean EP tube containing Bouin's solution, ensuring complete immersion and fixation for 1-2 days for use as a sample for immunohistochemical analysis. Place the other testis into another clean EP tube and store it in a -80°C cryogenic freezer for subsequent detection and analysis of testosterone, gene, and protein expression levels.

[0067] (3) The testosterone level in mouse testes was determined by mass spectrometry.

[0068] (4) After dehydrating and embedding the testicular tissue, tissue chips and paraffin sections were prepared. The paraffin sections of the testicular tissue were subjected to immunohistochemical staining and immunofluorescence staining.

[0069] (5) RNA was extracted from testicular tissue and reverse transcribed for qPCR. The results of qPCR were analyzed using the relative cyclic threshold method. The mRNA levels of the target gene in the testicular interstitial cells of each sample were standardized by using the internal reference gene Gapdh through the double standard curve method, so as to standardize the data.

[0070] (6) Perform Western blot experiments on testicular tissue.

[0071] 2. Cell experiments

[0072] Cell culture of mouse testicular interstitial cells, CCK8 cell viability assay and MMP assay

[0073] (1) Cell Culture

[0074] ① Gelatin coating of T25 culture flasks: Add 1 mL of 1% gelatin to a T25 culture flask and spread it evenly at the bottom of the flask. Then place it in an incubator at 37°C for 30 min and wash it 1-2 times with PBS.

[0075] ② Cell thawing: After removing the MA10 cell line (USA Standard Biological Collection Center) cryovial from the liquid nitrogen tank, immediately place it in 37°C hot water, shaking the cryovial continuously until thawed. Add 1 mL of culture medium, balance the mixture using a centrifuge, centrifuge at 1000 rpm / min at room temperature for 5 min, discard the supernatant, resuspend the cells in an appropriate amount of culture medium, and then transfer the cells to a culture flask for culture. Passage the cells when the cells in the culture flask reach 80% confluence.

[0076] ③ Passaging: Discard the old culture medium for adherent cells. Before digestion, wash the cells once in the culture flask with autoclaved PBS. After discarding the PBS, add 1 mL of trypsin digestion solution diluted 5 times. Shake the culture flask from side to side to allow the trypsin to cover the entire culture surface. Place the flask in a 37°C cell culture incubator for 3 min. Observe the cells carefully during this period. After observing obvious cell shrinkage under a microscope, add fresh culture medium (DMEMF12 medium + 15% HS + 1% penicillin-streptomycin mixture) to terminate the digestion.

[0077] ④ Plating: After collecting cells, centrifuge at 1000 rpm / min at room temperature for 5 min, discard the supernatant, add culture medium to resuspend and plate the cells. Cells intended for experiments should be plated and cultured overnight. Once the cells reach the appropriate growth stage, administer the drug treatment. A portion of cells should be preserved by cryopreservation. Resuspend the cells in cryopreservation solution and transfer them to cryovials (usually 1 mL / tube, no less than 2 × 10⁻⁶). 6 (each cell), placed in a cryopreservation box and stored at -80°C overnight, then transferred to liquid nitrogen;

[0078] ⑤ Induction of senescence: H2O2 was added to the already plated MA10 cells and senescence was induced within 4 hours;

[0079] ⑥ Drug administration: After the plated cells have grown to a suitable size, they are treated with NMN 500μM, APG 10μM, and a combination of NMN 500μM and APG 10μM (referred to as groups FH, respectively). Untreated cells are retained as a positive control group (group E). Untreated MA10 cells are used as a negative control (group A). ​​Untreated MA10 cells are treated with NMN 500μM, APG 10μM, and a combination of NMN 500μM and APG 10μM (referred to as groups BD, respectively). See the grouping diagram below. Figure 7 In the middle A group, NAD+ levels and β-galactosidase staining were performed on different groups of cells using an NAD+ detection kit (Shanghai Beyotime Biotechnology Co., Ltd.) and a cell senescence β-galactosidase staining kit (Shanghai Beyotime Biotechnology Co., Ltd.).

[0080] (2) CCK8 cytotoxicity assay: MA10 cells were seeded at 2000 per well in 96-well plates. The cytotoxicity of MA10 cells was detected using the CCK8 reagent kit. The working solution was prepared at a ratio of CCK8 reagent to (serum-free) medium of 1:10, added to the cells, and incubated in the dark for 2 hours. The absorbance at 450 nm was detected using an ELISA reader.

[0081] (3) MMP detection

[0082] ① All reagents were placed in an incubator for rewarming before the experiment;

[0083] ② Digest the cells and collect them into a 1.5 mL EP tube. Centrifuge at 1000 rpm / min for 5 min, discard the supernatant, and wash with PBS 1-2 times.

[0084] ③ Set up a positive control group, add 1 μL CCCCP to a final concentration of 50 μM, and incubate at 37℃ for 5 min;

[0085] ④ After the positive tubes have finished incubating, add 1 μL of JC-1 (final concentration 2 μM) to each tube and incubate at 37°C for 15 min in a 5% CO2 incubator;

[0086] ⑤ Centrifuge at 1000 rpm / min for 5 min, discard the supernatant, wash with PBS 1-2 times, and finally resuspend in PBS;

[0087] ⑥ Filter the resuspended cells through a 200nm nylon cloth into a flow cytometer, and then perform analysis.

[0088] ⑦ Flow cytometer testing and data statistics: The positive control and each sample tube were tested on the flow cytometer in sequence, the experimental data were saved, the flow cytometer data results were analyzed and plotted using FlowJo VX 10 software, and then the statistical analysis was performed using GraphPad software based on the above specific data results.

[0089] Statistical analysis

[0090] Statistical results are expressed as mean ± standard error, with a sample size of n = 6. A t-test was used to compare statistical significance between two groups; for data from three or more groups, ANOVA was used for comparison, followed by Dunnett's test for pairwise comparisons to detect differences between the experimental and control groups. #, ##, and ### indicate statistically significant differences between the aging group and the young group (p < 0.05, p < 0.01, and p < 0.001, respectively); *, **, and *** indicate statistically significant differences between the treatment group and the aging group (p < 0.05, p < 0.01, and p < 0.001, respectively).

[0091] result

[0092] 1. General zoological parameters and serum and testicular testosterone levels

[0093] This invention recorded the body weight, testicular weight, and epididymal weight of mice in different groups. The results showed that the relative testicular and epididymal weights were significantly increased in aged mice treated with NMN and APG (Table 1). Specifically, in terms of relative epididymal weight, the younger group was significantly higher than the aged group, while NMN and APG treatment alone did not significantly increase the relative epididymal weight of aged mice. However, combined NMN and APG treatment significantly increased the relative epididymal weight of aged mice. Regarding relative testicular weight, the younger group also showed a significant increase compared to the aged group, and the combined treatment group also showed a significant increase, while the single treatment groups did not reach statistical significance.

[0094] Table 4. General zoological parameters of mice in each group after NMN and APG treatment.

[0095]

[0096] Further analysis showed that the level of testosterone in the testes ( Figure 1 Significant differences were observed between the young and aging groups. Compared to the aging group, the combined treatment with NMN and APG significantly increased testosterone levels, while single treatments showed no significant effect. This indicates that the combined application of NMN and APG effectively restored the testosterone synthesis capacity of testicular interstitial cells in aging mice, while single treatments had limited effects.

[0097] 2. The combined use of NMN and APG does not affect the number of interstitial cells in the testes of aging mice.

[0098] This invention performed immunohistochemical staining to determine the number of testicular interstitial cells and Sertoli cells. Figure 2 In a study involving multiple inflammatory bowel disease (CIBG), the number of testicular interstitial cells and testicular Sertoli cells was assessed by detecting CYP11A1 (a biomarker for testicular interstitial cells) and SOX9 (a biomarker for testicular Sertoli cells). Results showed that combination therapy did not significantly alter the number of testicular interstitial cells. Figure 2 (F).

[0099] 3. The combined use of NMN and APG restored the markers of testicular interstitial cells in aging mice.

[0100] This invention further analyzed the cell morphology of senescent testicular interstitial cells, including cell size, cytoplasmic size, and nuclear size. It was observed that the combined application of NMN and APG significantly increased the cell and cytoplasmic size of senescent testicular interstitial cells, while the nuclear size remained unchanged across groups. Figure 3 ).

[0101] 4. The regulatory effect of the combined use of NMN and APG on the expression of related genes in testicular interstitial cells of aging mice.

[0102] To gain a deeper understanding of the molecular mechanisms by which the combined treatment of NMN and APG alleviates the effects of aging, this invention conducted a detailed analysis of the expression levels of genes related to several key signaling pathways using qPCR technology. These included the testosterone synthesis pathway (involving Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b6, Hsd17b3, and Insl3), the NAD+ synthesis pathway (Nampt), and glucocorticoid-related pathways (Hsd11b2, Nr3c1, and Nr3c2). The results are shown in […]. Figure 4 The results showed that, compared with the young group, the aging group exhibited significant differences in the expression of Lhcgr, Star, Cyp11a1, Cyp17a1, Insl3, and Nampt genes. Compared with the aging group, the NMN treatment group showed upregulated Insl3 gene expression, and the APG treatment group showed significant upregulated Hsd11b2 and Insl3 gene expression levels. The NMN and APG combined treatment group showed significant upregulated expression of genes such as Star, Scarb1, Cyp11a1, Cyp17a1, Hsd17b3, Hsd11b2, Insl3, Nr5a1, Hsd11b1, Nampt, and Nr3c1. These findings suggest that the combined treatment of NMN and APG plays a positive role in regulating the expression of genes related to testicular interstitial cells in aging mice. The combined use of NMN and APG can significantly enhance the expression of multiple key genes and is expected to promote testosterone synthesis, NAD+ synthesis, and the function of the glucocorticoid pathway. These molecular regulatory effects help maintain or restore the normal function of testicular interstitial cells and further improve age-related testicular damage and functional decline.

[0103] 5. Expression levels of proteins related to testosterone and NAD+ synthesis in mouse testicular interstitial cells

[0104] To investigate the effects of combined NMN and APG treatment on protein expression in testicular interstitial cells of aging mice, this invention employed Western blot technology to detect relevant proteins. Figure 5 The results showed that, compared with the younger group, the aging group exhibited significant differences in the expression of CYP11A1, CYP17A1, NAMPT, STAR, and INSL3 proteins. Within the treatment groups, the NMN treatment group showed significant differences in CYP11A1 and INSL3 protein expression compared to the aging group; the APG treatment group also showed a significant difference in CYP11A1 protein expression; and the combined NMN and APG treatment group showed significantly increased expression of CYP11A1, CYP17A1, NAMPT, STAR, and INSL3 proteins compared to the aging group, bringing them close to the levels of the younger group.

[0105] 6. The combined use of NMN and APG promotes NAMPT protein expression in testicular interstitial cells of aging mice.

[0106] NAMPT protein expression levels are crucial for maintaining normal NAD+ levels and cellular function. To further verify changes in NAMPT protein expression in mouse testicular interstitial cells, this invention performed a dual fluorescence staining experiment using paraffin-embedded mouse testicular sections with 3β-HSD6 (a biomarker of mouse testicular interstitial cells, green) and NAMPT (red). Figure 6 Subsequently, the optical density of each group of fluorescence images was measured and analyzed. The results showed that the combined use of NMN and APG significantly promoted the expression of NAMPT protein in senescent testicular interstitial cells.

[0107] 7. In vitro, the combined use of NMN and APG enhances the testosterone synthesis capacity of H2O2-induced senescent MA10 cells.

[0108] This invention uses H2O2 to treat MA10 cells for 4 hours to simulate an in vitro testicular interstitial cell senescence model. H2O2 induced senescence in both the senescent and normal cell groups, and in the culture medium, testosterone (… Figure 7 (B) and β-gal ( Figure 7 Differences were observed in both NMN and APG on FJ (French FJ), indicating successful aging modeling. Furthermore, this invention used CCK8 assays to detect the toxic effects of NMN and APG on MA10 cells; the results showed that 500 μM NMN (French FJ) toxicity was significantly reduced. Figure 7 (C) and 10 μM APG ( Figure 7(D) does not cause cytotoxicity. Compared with H2O2-induced senescence cells, the APG treatment group and the NMN and APG combined treatment group significantly improved the testosterone synthesis capacity of H2O2-induced senescent MA10 cells. Figure 7 (B) This study aimed to weaken the H2O2-induced senescence effect in MA10 cells. Finally, the NAD+ levels in each group of cells were measured. Figure 7 The results showed that the combination of NMN and APG significantly increased NAD+ levels in H2O2-induced senescent MA10 cells.

[0109] 8. In vitro, the combined use of NMN and APG blocked H2O2-induced senescence in MA10 cells and upregulated the expression of testosterone synthesis-related proteins.

[0110] The expression of P38 and P21 is associated with the senescence of testicular interstitial cells, and the expression of INSL3 (insulin-like factor 3) is closely related to the senescence process of testicular interstitial cells. In vitro, the expression levels of senescence-related proteins (P38, P21, and INSL3) and testosterone synthesis-related proteins (CYP11A1, CYP17A1, and STAR) after MA10 cell treatment were detected by Western blotting. The results are shown in [Figure number missing]. Figure 8 The results showed that, compared with the H2O2 treatment group, the APG treatment group significantly downregulated P38 expression and upregulated CYP11A1 and STAR protein expression levels; the NMN and APG combination group significantly downregulated P38 and P21 expression levels and upregulated INSL3, CYP11A1, CYP17A1, and STAR expression levels. The combination of NMN and APG significantly blocked the H2O2-induced protein expression imbalance in MA10 cells, suggesting that the combination of NMN and APG has a mitigating effect on H2O2-induced MA10 cell senescence and has a better therapeutic effect than APG alone.

[0111] 9. In vitro combined use of NMN and APG repairs mitochondrial function and NAD+ synthesis-related protein imbalance induced by H2O2-induced senescence in MA10 cells.

[0112] Decreased SIRT1 and PGC1α levels are associated with the induction of oxidative stress. This invention uses Western blot to detect SIRT1 and PGC1α levels. Compared with the H2O2-induced aging group, the combined use of NMN and APG significantly upregulated SIRT1 (…). Figure 9 (B), PGC1α ( Figure 9 (C), OPA1 ( Figure 9 (middle D) and MFN1 ( Figure 9 The levels of NMN and APG suggest that the combined use of NMN and APG can alleviate age-related mitochondrial dysfunction by restoring mitochondrial fission and fusion functions; simultaneously, the NMN and APG combined treatment group upregulated NAMPT (NAMPT). Figure 9 (F), lower CD38 ( Figure 9 The expression of G protein suggests that it promotes NAD+ synthesis.

[0113] 10. In vitro NMN and APG promote the expression of antioxidant proteins in aging MA10 cells.

[0114] This invention detected the levels of the antioxidant defense systems SOD1, SOD2, and NRF2 in cellular proteins. The results showed that, compared with the aging group, the aging group had a significantly reduced level of antioxidant proteins. Figure 10 Furthermore, the combined application of NMN and APG reversed the effects of antioxidant protein in the aging group, increasing its levels. This indicates that the combined application of NMN and APG can also alleviate oxidative stress by upregulating the expression of antioxidant proteins in aging testicular interstitial cells and enhancing the cells' antioxidant defense capabilities.

[0115] 11. In vitro, NMN and APG reversed the mitochondrial membrane potential of testicular interstitial cells in aging mice.

[0116] This invention uses a mitochondrial membrane potential kit for staining, followed by flow cytometry detection. Figure 11 (Middle AE). Experimental results showed that the combined application of NMN and APG reversed the mitochondrial membrane potential level in the aging group ( Figure 11 (F).

[0117] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of the combined use of β-nicotinamide mononucleotide and apigenin in the preparation of products for delaying testicular interstitial cell senescence and / or treating diseases related to testicular interstitial cell senescence.

2. The application according to claim 1, characterized in that, The diseases associated with testicular interstitial cell aging include androgen deficiency and / or decreased reproductive function.

3. The application according to claim 1, characterized in that, The method of delaying testicular interstitial cell aging includes: promoting testosterone synthesis, increasing testicular interstitial cell volume, increasing mitochondrial membrane potential, and increasing NAD+. + The expression levels of the functional gene or protein are adjusted by one or more of the following: upregulation of functional gene expression, upregulation of functional protein expression, downregulation of CD38 protein expression, downregulation of P38 protein expression, and downregulation of P21 protein expression; the functional gene or protein includes those involved in testosterone synthesis, NAD+ synthesis, and NAD+ synthesis. + Functional genes or proteins that are related to one or more of the following: synthesis, mitochondrial function, and antioxidant activity.

4. The application according to claim 3, characterized in that, The functional genes include one or more of Star, Scarb1, Cyp11a1, Cyp17a1, Hsd17b3, Hsd11b2, Insl3, Nr5a1, Hsd11b1, Nampt, and Nr3c1.

5. The application according to claim 3, characterized in that, The functional proteins include one or more of CYP11A1, CYP17A1, NAMPT, STAR, INSL3, SOD1, SOD2, and NRF2.

6. The application according to claim 1, characterized in that, The product alleviates age-related decline in mitochondrial function by restoring the mitochondrial splitting and fusion functions.

7. The application according to claim 1, characterized in that, Based on mouse body weight, the unit dose of β-nicotinamide mononucleotide in the product is 450-550 mg / kg / day, and the unit dose of apigenin is 45-55 mg / kg / day.

8. The application according to claim 7, characterized in that, Based on mouse body weight, the unit dose of β-nicotinamide mononucleotide in the product is 500 mg / kg / day, and the unit dose of apigenin is 50 mg / kg / day.

9. The application according to claim 1, 7, or 8, characterized in that, The products include pharmaceuticals.

10. The application according to claim 9, characterized in that, The dosage forms of the drug include injections and / or oral formulations.

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