Use of natural product composition for the preparation of a medicament for preventing or treating azoospermia

Abstract

The application discloses application of a natural product composition in preparation of medicines for preventing or treating azoospermia. The natural product composition is composed of astragalin and curcumin. The natural product composition composed of astragalin and curcumin can inhibit spermatogenic cell apoptosis by synergistically regulating Nrf2 / HO-1 / HIF-1a, and improve rat azoospermia. Compared with western medicines, the natural product composition has a wider treatment window and smaller toxic side effect, and has application prospects in the medicines for preventing or treating azoospermia.

Description

Technical Field

[0001] This invention relates to the application of a natural product composition in the preparation of drugs for the prevention or treatment of azoospermia, and belongs to the field of medicine. Background Technology

[0002] Epidemiological statistics show that by the end of the 20th century, infertility continued to affect 15% of couples worldwide, remaining a sociological and medical problem related to global human development. Research indicates that male factors account for approximately 40% of these cases, including asthenospermia and azoospermia, primarily related to sperm concentration, morphology, and motility disorders. Azoospermia (AZS) is one of the most common types of male infertility, defined as a sperm motility rate of less than 42% or a forward-moving sperm count of less than 30%. Metabolic syndrome, drug-induced azoospermia, and oxidative stress are among the main pathogenic mechanisms of azoospermia. However, the mechanisms of asthenospermia are complex and remain unclear.

[0003] Under physiological conditions, the body's oxidation and antioxidant systems are in a state of equilibrium. However, factors such as metabolism or drugs can disrupt this balance, causing a sharp increase in the content of reactive oxygen species in the body. Since sperm are susceptible to oxidative attacks and lipid peroxidation of the plasma membrane, oxidative stress is often the main cause of azoospermia.

[0004] Nrf2 is an essential node in the body's antioxidant system. It can dissociate from keap1 and enter the nucleus, acting as a transcription factor to upregulate the body's antioxidant system. Studies have reported that Nrf2 can target and regulate spermatogenic cell apoptosis in rats with asthenospermia, thus treating asthenospermia. In related studies, mice with low Nrf2 expression experienced spermatogenic cell and spermatid apoptosis in a Nrf2 / HO-1 / HIF-1α-dependent manner, leading to azoospermia. Therefore, targeting Nrf2 to regulate spermatogenic cell apoptosis is a feasible therapeutic strategy.

[0005] Therefore, there is an urgent need for more effective treatment strategies to prevent the occurrence and progression of azoospermia. Unlike synthetic small molecule drugs with single targets, traditional Chinese medicine and natural products have the advantages of multiple targets and low toxicity, and have synergistic effects on complex reproductive diseases through multiple pathways, making them a better choice for inhibiting the progression of azoospermia. Summary of the Invention

[0006] The purpose of this invention is to provide an application of a natural product composition in the preparation of a drug for the prevention or treatment of azoospermia.

[0007] The technical solution for achieving the objective of this invention is as follows:

[0008] The use of a natural product composition in the preparation of a drug for the prevention or treatment of azoospermia, wherein the natural product composition comprises astragalin (AG) and curcumin (CM).

[0009] Furthermore, in the natural product composition, the molar ratio or mass ratio of astragaloside and curcumin is 1:1.

[0010] The dosage form of the drug for preventing or treating azoospermia described in this invention is a pharmaceutically permissible oral or injectable dosage form.

[0011] Furthermore, medications for the prevention or treatment of azoospermia also include acceptable excipients.

[0012] Furthermore, the dosage of both astragaloside and curcumin was ≥20 mg / kg. -1 .

[0013] This invention, through experiments, reveals that at the cellular level, both CM and AG can dose-dependently inhibit Gc-1 cell apoptosis, and the combined use of CM and AG produces a synergistic inhibitory effect. At the animal level, CM and AG synergistically improve sperm concentration, sperm viability, sperm motility, and serum sex hormone levels in cyclophosphamide (CP)-stimulated SD rats, and significantly reduce oxidative stress and spermatogenic cell apoptosis levels in the testes of SD rats. Furthermore, CM and AG synergistically upregulate Nrf2 and HO-1 expression levels and downregulate HIF-1α expression levels in the testicular tissue of SD rats. Therefore, the natural product composition consisting of astragaloside and curcumin can inhibit spermatogenic cell apoptosis and improve azoospermia in rats by synergistically regulating Nrf2 / HO-1 / HIF-1α. Compared with Western medicine, it has a wider therapeutic window and fewer toxic side effects, showing promising application potential in the prevention or treatment of azoospermia. Attached Figure Description

[0014] Figure 1 This is a schematic diagram showing the results of CM and AG on H2O2-induced CCK-8 mice spermatogonia in Example 1, and a schematic diagram showing the results of their combined use index.

[0015] Figure 2 The graph shows the effect of CM and AG on improving sperm parameters in azoospermic rats in Example 2.

[0016] Figure 3 Figure 2 shows the effect of CM and AG on the morphological improvement of testicular tissue in azoospermic rats. Figure A shows the H&E staining results, B shows the number of spermatogenic cells, and C shows the number of interstitial cells.

[0017] Figure 4 The image shows the effect of CM and AG in Example 3 on improving oxidative stress in the testicular tissue of azoospermic rats.

[0018] Figure 5 The effect of CM and AG in Example 3 on improving apoptosis of testicular tissue cells in azoospermic rats;

[0019] Figure 6 This is a schematic diagram of the HIF-1α immunohistochemical results of CM and AG on testicular tissue of azoospermic rats in Example 4;

[0020] Figure 7 This is a schematic diagram showing the regulatory effects of CM and AG on the Nrf2 / HO-1 / HIF-1α pathway in the testicular tissue of azoospermic rats in Example 4.

[0021] Figure 8 Figure 4 shows the docking results of CM and AG with Nrf2 molecules in Example 4. Figure A shows the CM structure, Figure B shows the AG structure, Figure C shows the docking results of the Kelch domain of CM and Keap1, and Figure D shows the docking results of the BTB domain of AG and Keap1. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the technical solution of the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods; unless otherwise specified, the materials and reagents used in the following embodiments can be obtained from publicly available commercial channels.

[0023] In the following examples, curcumin (CAS: 458-37-7) and astragaloside (CAS: 480-10-4) were purchased from Nanjing Yuanzhi Biotechnology Co., Ltd., the experimental cells were mouse spermatogonial cell line (Gc-1), and the experimental animals were SPF-grade SD rats.

[0024] Example 1: CM and AG synergistically inhibit H2O2-induced apoptosis in spermatogenic cells.

[0025] Gc-1 cells were divided into 14 groups, with three parallel wells in each group. 5 x 10⁶ cells were seeded per well. 3Cells were inoculated into 96-well plates and cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. After inoculation, the three groups of cells were treated as follows: Group 1 was the blank control group (Con group); Group 2 was the H2O2 group (H2O2 concentration 200 μM / mL); Groups 3-6 and 7-10 were the CM group (2.5 μM, 5 μM, 10 μM, and 20 μM) and AG group (2.5 μM, 5 μM, 10 μM, and 20 μM), respectively, with an H2O2 concentration of 200 μM / mL; Groups 10-14 were the combined CM and AG groups (CM+AG=2.5 μM + 2.5 μM, CM+AG=5 μM + 5 μM, CM+AG=10 μM + 10 μM, CM+AG=20 μM + 2.5 μM, CM+AG=5 μM + 5 μM, CM+AG=10 μM + 10 μM, CM+AG=20 μM + 2.5 μM, CM+AG=2.5 ...10 μM + 10 μM, CM+AG=20 μM + 2.5 μM, CM+AG=2 20 μM), each group was treated for 24 h. After the above 14 groups of cells were treated as described above, the culture medium was replaced with fresh one, CCK-8 was added, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated, and the results are as follows. Figure 1 As shown in AC. The overall results were imported into Compusyn software for coupled exponential fitting, and the results are as follows. Figure 1 As shown in DG.

[0026] Depend on Figure 1 The results of AC showed that both CM and AG could inhibit H2O2-induced apoptosis in Gc-1 cells in a dose-dependent manner, and the combined use had a more significant inhibitory effect on apoptosis. Figure 1 DG's results indicate that the combined use of CM and AG at a molar ratio of 1:1 can produce a synergistic effect on cells.

[0027] Example 2: CM and AG synergistically improve sperm motility and testicular damage in azoospermic rats.

[0028] Seventy-five male SD rats (SPF grade, weighing 210±5 g) were selected and acclimatized for one week. After fasting for 12 hours, CP was dissolved in 0.9% physiological saline at a dose of 35 mg / kg. -1 ·d -1 Sixty rats were randomly assigned to receive intraperitoneal injection of a specific dose for five consecutive days, while the remaining 15 rats received the same dose of 0.9% saline as a blank control group (Con group). These 60 rats were then randomly divided into four groups of 15 each: the model group (CP group), the curcumin group (CM group), the astragaloside group (AG group), and the combination group (CM+AG group). Each group was administered sodium carboxymethyl cellulose (CMC-Na) solution by gavage at a dose of 20 mg / kg. -1 ·d -1 Curcumin, 20 mg·kg -1 ·d -1 Astragaloside and 20 mg·kg-1 ·d -1 Curcumin + 20 mg·kg -1 ·d -1 Rats were treated with astragaloside solution for 14 consecutive days. During the experiment, all rats had free access to water and food, and were administered the solution via gavage at fixed times daily. The experiment ended after 21 days of intervention. After anesthesia, blood was collected from the abdominal aorta, and bilateral testis and epididymal tissues were harvested. The left testis was fixed in 4% paraformaldehyde, and the right testis was cryopreserved at -80°C. Both epididymis were placed in preheated Hams' F10 medium in a water bath and incubated at 37°C for 30 min until complete sperm dissociation. Sperm motility and kinematic parameters were detected using a WLJY-9000 Weili digital color sperm quality detection system. Serum samples were collected, and rat testosterone expression was detected using an ELISA kit. Testicular tissue was harvested, and the H&E method was used to assess testicular damage. The tissues were then observed and photographed under an upright optical microscope.

[0029] Depend on Figure 2 It was found that the CP group had significantly lower sperm motility, sperm count, and sperm survival rate than the Con group, while the CM and AG groups showed significantly higher levels. The results of sperm motility rate (VAP, VCL, VSL, STR, and LIN) examinations also showed the same trend. This indicates that both CM and AG can improve sperm quality in azoospermic rats. The sperm quality of rats in the CM+AG group was significantly better than that of the single-treatment groups, indicating that CM and AG can synergistically improve sperm quality in azoospermic rats. Sex hormone detection results showed that CP significantly reduced testosterone expression in rats, while CM and AG reversed this phenomenon. CM and AG synergistically increased testosterone expression in azoospermic rats. Figure 3 The H&E results showed that the seminiferous tubules in the CP group rats were significantly atrophied compared to the Con group, with increased intertubular spaces and a significant decrease in the number of spermatogenic cells and spermatids. After drug intervention, these conditions were significantly alleviated. The intertubular spaces narrowed, the number of spermatogenic cells recovered, and their arrangement became more compact. Cell counting results revealed that CM and AG synergistically improved the number of spermatogenic cells and interstitial cells in the testicular tissue of azoospermic rats.

[0030] Example 3: CM and AG synergistically improve oxidative stress and spermatogenic cell apoptosis in testicular tissue of azoospermic rats.

[0031] The testicular tissue from Example 2 was ground using a tissue homogenizer before being analyzed for ROS, MDA, SOD, GSH, and GSH-px. All assays were performed according to the instructions of commercially available kits and were conducted using a multi-functional microplate reader.

[0032] In Example 2, after dewaxing, the paraffin sections of testicular tissue were stained according to the instructions of the TUNEL staining kit and mounted with anti-fluorescence quenching mounting solution. The staining results were immediately observed and photographed under an inverted fluorescence microscope, and the positive area (green fluorescence) was analyzed using ImageJ.

[0033] Depend on Figure 4 It was found that after CP intervention, the ROS and MDA levels in the testicular tissue of rats increased significantly, while the SOD level decreased significantly. After CM and AG intervention, the ROS and MDA levels in the testicular tissue decreased significantly, while the SOD level increased significantly, indicating that both CM and AG can inhibit oxidative stress in the testes of azoospermic rats. However, the inhibitory effect of single administration on oxidative stress was significantly weaker than that of combined administration, indicating that CM and AG can synergistically improve oxidative stress in the testicular tissue of azoospermic rats.

[0034] Figure 5 TUNEL staining results showed that CM and AG synergistically improved spermatogenic cell apoptosis in the testicular tissue of azoospermic rats.

[0035] Example 4: CM and AG synergistically regulate Nrf2 / HO-1 / HIF-1α in azoospermic rats to inhibit spermatogenic cell apoptosis.

[0036] In Example 2, testicular tissue fixed in 4% paraformaldehyde fixative for 72 h was embedded in paraffin and sectioned to 5 μm. The paraffin sections were dewaxed using a gradient elution with ethanol, underwent antigen retrieval and DNA denaturation, and were then incubated with 3% hydrogen peroxide solution in the dark for 30 min to block endogenous peroxidase. Finally, they were incubated with 3% BSA solution at room temperature for 30 min as blocking agent. The paraffin sections were then incubated overnight at 4°C with a prepared rabbit-derived HIF-1α mixed primary antibody in a humidified chamber. A mixed secondary antibody was prepared using HRP-labeled goat anti-rabbit IgG. Sections incubated with the primary antibody were placed in a humidified chamber, and the mixed secondary antibody was added to the section rings. After incubation at room temperature for 1 h, DAB staining was performed. After staining, the cell nuclei were counterstained with hematoxylin, mounted with glycerol, and the staining was observed under a microscope. The positive area was analyzed using ImageJ.

[0037] In Example 2, testicular tissue was washed twice with PBS, and cells were lysed with RIPA lysis buffer containing 1 mmol / L LMSF (100 μL RIPA lysis buffer was added per 10 mg of tissue). The tissue was homogenized for 5 min, then lysed on ice for 30 min. After BCA quantification, loading buffer was added, and the mixture was denatured in a 100°C metal bath for 10 min. Protein expression was detected using Western blotting. 30 μg of protein was loaded onto a 10% SDS-PAGE gel, and electrophoresed at constant voltage for 80V-30 min and 120V-55 min. The target protein was then cut and transferred to a PVDF membrane. The PVDF membrane was blocked by incubation with 5% BSA on a shaker at room temperature for 2 h, and then incubated overnight at 4°C with a primary antibody (1:1000). The next day, the membrane was washed with TBST and incubated at room temperature with a horseradish peroxidase-labeled secondary antibody (1:2000) for 2 h. After washing, the membrane was imaged using ECL chemiluminescence solution on a gel imaging system.

[0038] The structures of CM and AG were obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and the protein structure of Nrf2 was obtained from the PDB database (RCSB PDB, https: / / www.rcsb.org / ). Binding sites of two different domains of Keap1, the negative regulator of Nrf2, including the BTB domain and the Kelch domain, were used. Autodocktools 1.5.6 was used for protein structure processing, and Autodocking vina 1.2.0 was used for molecular docking. The docking results were imported into Pymol for visualization.

[0039] Figure 6 A is a schematic diagram of the immunohistochemical results of HIF-1α in rat testicular tissue, and B is a schematic diagram of the positive area quantification results. CM and AG can synergistically reduce the expression of HIF-1α in the testicular tissue of azoospermic rats.

[0040] Figure 7 A is a schematic diagram of WB results, B is a quantitative map of Nrf2 expression, C is a quantitative map of HO-1 expression, and D is a quantitative map of HIF-1α expression. The results show that CM and AG can synergistically restore the Nrf2 / HO-1 / HIF-1α pathway in the testicular tissue of azoospermic rats.

[0041] Figure 8Figure 1 shows the structural formulas of CM (A) and AG (B). Figure 2 shows the docking diagram of the Kelch domain of CM and Keap1, with a binding energy of -8.3 kJ / mol. Figure 3 shows the docking diagram of the BTB domain of AG and Keap1, with a binding energy of -6.2 kJ / mol. These results indicate that the synergistic effect of CM and AG may be achieved by inhibiting Keap1 expression and activity through different binding sites, and synergistically upregulating Nrf2.

Claims

1. The use of a natural product composition in the preparation of a drug for the prevention or treatment of azoospermia, characterized in that, The natural product composition consists of astragaloside and curcumin, with a mass ratio of astragaloside to curcumin of 1:

1.

2. The application according to claim 1, characterized in that, The medications mentioned for the prevention or treatment of azoospermia are available in oral or injectable form.

3. The application according to claim 1, characterized in that, Drugs for the prevention or treatment of azoospermia also include excipients.

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