Application of 12-Ketolithocholic Acid in Improving Declining Sperm Quality

Abstract

This invention belongs to the field of pharmaceutical treatment for male infertility, and discloses the application of 12-keto-lithocholic acid (12-keto-LCA) in improving declining sperm quality, specifically its application in the preparation of drugs for improving declining sperm quality. This invention provides a novel use for 12-keto-LCA, revealing for the first time that 12-keto-LCA can support the spermatogenesis process, especially improving the reproductive function of obese men, thereby increasing testosterone secretion, repairing spermatogenesis, improving sperm quality and embryonic development, and improving the decline in sperm quality in mammals. This invention reveals that 12-keto-LCA can effectively alleviate male spermatogenesis disorders, especially the damage to male reproductive function caused by obesity, providing a new avenue for developing drugs to improve the reproductive function of obese men.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical treatment for male infertility, and more specifically, relates to the application of 12-ketolithocholic acid in improving decreased sperm quality. Background Technology

[0002] Decreased sperm quality, including reduced sperm count, decreased sperm motility, and increased sperm abnormality rate, is one of the main causes of male infertility. With the increasing global obesity rate, obesity has become a significant risk factor for declining male sperm quality.

[0003] Currently, clinical treatments for declining sperm quality are limited, relying heavily on empirical drug therapy and surgery. These methods may have significant side effects and uncertain efficacy. Therefore, finding safe and effective new intervention strategies is crucial for the prevention and treatment of obesity-related sperm quality decline.

[0004] In recent years, the scientific community has gradually revealed the important role of the "gut-testis axis," that is, the gut, as a core metabolic and immune organ, can remotely regulate testicular function through the circulatory system and its secreted active substances, providing a new approach to improve reproductive health by intervening in active molecules from the gut.

[0005] 12-Keto-LCA is a secondary bile acid produced by gut microbiota metabolism and is an endogenous signaling molecule that can simultaneously regulate host metabolism and immunity. Currently, there are no reports indicating that 12-keto-LCA can be used in the field of reproductive health or to improve declining sperm quality. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, the present invention aims to provide an application of 12-keto-LCA in improving declining sperm quality. It offers a novel use for 12-keto-LCA, which can support the spermatogenesis process, particularly improving reproductive function in obese men. This includes increasing testosterone secretion, repairing spermatogenesis, improving sperm quality and embryonic development, and thus improving the decline in sperm quality in mammals. The present invention reveals that 12-keto-LCA can effectively alleviate male spermatogenesis disorders, especially the damage to male reproductive function caused by obesity, providing a new avenue for developing drugs to improve the reproductive function of obese men.

[0007] To achieve the above objectives, according to one aspect of the present invention, the use of 12-keto-LCA in the preparation of a medicament for improving decreased sperm quality is provided.

[0008] As a further preferred embodiment of the present invention, the decline in sperm quality specifically refers to obesity-related decline in sperm quality.

[0009] As a further preferred embodiment of the present invention, the decrease in sperm quality specifically refers to a decrease in the sperm quality of mammals.

[0010] As a further preferred embodiment of the present invention, the structural formula of the 12-keto-LCA is as follows:

[0011] .

[0012] As a further preferred embodiment of the present invention, the 12-keto-LCA has at least one of the following functions:

[0013] a) Increase sperm concentration;

[0014] b) Improve sperm motility, wherein improving sperm motility includes at least one of increasing the percentage of progressively motile sperm or increasing the percentage of total motile sperm;

[0015] c) Reduce sperm abnormality rate;

[0016] d) Improve embryonic developmental potential;

[0017] e) Improve the structure of the seminiferous tubules in the testes;

[0018] f) Increase the number of spermatogenic cells;

[0019] g) Increase serum testosterone levels.

[0020] As a further preferred embodiment of the present invention, the spermatogenic cells include DAZL. + Cells, SYCP3 + Cells, TNP1 + Cells, WT1 + Cells and PGK2 + One or more of them in the cell.

[0021] As a further preferred embodiment of the present invention, the dose of 12-keto-LCA applied per kg of organism per day is 10 mg.

[0022] As a further preferred embodiment of the present invention, the drug is in the form of an oral preparation, injection, granule, pill, capsule, powder, sustained-release, controlled-release preparation, or targeted preparation.

[0023] According to another aspect of the invention, the invention provides a medicament for improving decreased sperm quality, characterized in that it comprises 12-keto-LCA and a pharmaceutically acceptable carrier.

[0024] Compared with existing technologies, the technical solutions conceived in this invention, through rigorous animal experiments, have pioneered the demonstration that 12-keto-LCA can directly act on the spermatogenesis process, significantly improving sperm quality. This opens up a new application direction for using 12-keto-LCA to improve male sperm quality (especially in obese men) and alleviate spermatogenesis disorders. As illustrated in the following examples, in vivo experiments have verified that daily administration of 12-keto-LCA to mice with impaired reproductive function due to obesity, after a certain period of continuous intervention, can significantly increase sperm count, motility, and the rate of normal morphology.

[0025] Compared with existing technologies, this invention, using 12-keto-LCA, can fundamentally improve sperm count, motility, and normal morphology rate. Due to the endogenous nature of 12-keto-LCA, this invention has the significant advantage of high safety. 12-keto-LCA can be used to develop safe and effective drugs for the prevention and adjunctive treatment of spermatogenesis disorders in obese men, with very broad application prospects.

[0026] Based on the innovative concept of the "gut-testis axis," this invention systematically studies the direct effects of 12-keto-LCA on the reproductive system in an obesity model. Taking the following examples, although ursodeoxycholic acid (UDCA) and 12-keto-LCA are closely related in structure and origin (UDCA and 12-keto-LCA are both bile acid metabolites and secondary bile acids), they show significant functional differences in improving obesity-induced sperm quality decline: only 12-keto-LCA shows better improvement efficacy and can significantly improve obesity-related sperm quality decline, while the effect of UDCA is relatively limited.

[0027] In summary, this invention is the first to reveal a novel use of 12-keto-LCA in improving declining sperm quality (especially obesity-related sperm quality decline), providing a new direction for the development of drugs related to the reproductive health of obese men. Attached Figure Description

[0028] Figure 1 A comparison chart of testicular organ coefficients and epididymal organ coefficients for mice in each group.

[0029] Figure 2 This is a comparison chart of serum testosterone levels in mice from different groups.

[0030] Figure 3 Comparison of H&E staining of seminiferous tubules in testicular tissue of mice in different groups.

[0031] Figure 4 A comparison chart showing the proportion of seminiferous tubules containing mature sperm in each group of mice.

[0032] Figure 5 DAZL in the seminiferous tubules of mice in each group + (Spermatogonia markers), SYCP3 + (Spermatocyte markers), TNP1 + (Sperm cell markers), WT1 + (Supporting cell markers) and PGK2 + Comparison of immunofluorescence staining of (mature sperm markers).

[0033] Figure 6 DAZL in the seminiferous tubules of mice in each group + (Spermatogonia markers), SYCP3 + (Spermatocyte markers), TNP1 + (Sperm cell markers), WT1 + (Supporting cell markers) and PGK2 + Comparison of average fluorescence density values ​​(markers of mature sperm).

[0034] Figure 7 This is a comparison chart of sperm motility in different groups of mice.

[0035] Figure 8 Comparison of sperm morphology in different groups of mice.

[0036] Figure 9 Comparison of mouse embryos in different groups at the two-cell stage and blastocyst stage.

[0037] Figure 10 A comparative graph showing the quantitative analysis of forward motility, sperm abnormality rate, two-cell stage rate, and blastocyst rate in mice of different groups. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0039] The 12-keto-lithocholic acid (12-keto-LCA) used in the following examples was purchased from MedChemExpress, catalog number: HY-135772, and its chemical structure is as follows:

[0040]

[0041] Example 1:

[0042] Thirty-two 40-week-old SPF-grade male C57BL / 6J mice were acclimatized for one week and then randomly assigned to:

[0043] a) Control group, namely, the "Control diet" (CD) group;

[0044] b) High-fat group, i.e., the "High-fat diet" (HFD) group;

[0045] c) High-fat + ursodeoxycholic acid group, i.e., "HFD + UDCA" group;

[0046] d) High-fat + 12-keto-lithocholic acid group, i.e., "HFD + 12-keto-LCA" group;

[0047] The control group mice were fed a normal diet (product number: AIN-93M; purchased from: ResearchDiets), while the other three groups were fed a high-fat diet (45% fat energy ratio, product number: D12451, purchased from: ResearchDiets). The “HFD+UDCA” group and the “HFD+12-keto-LCA” group were administered 10 mg / kg UDCA (product number: HY-B0172, purchased from: MedChemExpress) and 10 mg / kg 12-keto-LCA (product number: HY-135772, purchased from: MedChemExpress) daily by gavage, respectively. The control group and the high-fat group were administered the same volume of solvent orally daily by gavage. Both UDCA and 12-keto-LCA were dissolved in a mixed solvent consisting of 10 wt% DMSO, 40 wt% PEG 300, 5 wt% Tween-80, and 45 wt% physiological saline. This mixed solvent formulation ensures both complete drug dissolution and stability while minimizing irritation to the gastrointestinal tract of mice. It exhibits good biocompatibility and simultaneously meets the requirements for solubility and animal tolerance.

[0048] Twelve weeks later, mice were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital, followed by blood collection via the abdominal aorta and sacrifice. The testes and bilateral epididymis were rapidly separated. Testicular tissue was preserved in testicular fixation solution for subsequent pathological analysis; whole blood samples were centrifuged (12,000 rpm, 15 min), and the supernatant was collected for testing testosterone levels.

[0049] Bilateral epididymal tails of mice were harvested and placed in 12-well plates, then cut into small pieces using sterile scissors. 1 mL of preheated HTF capacitation medium was added, and the plates were incubated at 37°C with 5% CO2 for 5 min to allow for complete sperm release. Finally, the proportion of forward-moving sperm was recorded using a computer-aided sperm analysis system (CASA; SAS, China).

[0050] Bilateral epididymal tails of mice were collected and placed in 12-well plates, then cut into small pieces using sterile scissors. 1 mL of preheated HTF capacitation medium was added, and the plates were incubated at 37°C with 5% CO2 for 5 min. A suitable amount of sperm suspension was collected and smeared. After air-drying, the smears were fixed with methanol and stained with 1% eosin solution for 1 h. Finally, sperm morphology was assessed: at least 500 sperm were observed from each mouse, excluding sperm with head / tail defects, blurred features, or damage, and the morphological abnormality rate was calculated.

[0051] Bilateral epididymal tails of mice were harvested and placed in 12-well plates, then cut into small pieces using sterile scissors. 1 mL of preheated HTF capacitation medium was added, and the plates were incubated at 37°C with 5% CO2 for 1 h. Ampoules of oviducts from superovulated female mice at 5 weeks gestation were placed in pre-equilibrated M2 medium, and the oocyte complex was mechanically dissected to release it. The ampoules were then cultured at 37°C with 5% CO2 for 15 min. Capacitated sperm were then added to the oocyte-containing medium and co-cultured at 37°C with 5% CO2 for 3–4 h. Fertilized zygotes were transferred to HTF medium, residual granulosa cells were mechanically removed, and the embryos were cultured stably for 30 min. Finally, the obtained single-cell embryos were cultured in vitro, and the rates of development to the 2-cell stage and blastocyst stage were recorded at 24 h and 84 h post-culture.

[0052] The experimental results showed that, compared with the high-fat group mice, the testicular organ coefficient of the high-fat + 12-ketolithocholic acid group mice was 0.5165±0.0599. vs. 0.3940±0.0602, P <0.01), epididymal organ coefficient (0.3684±0.0452) vs. 0.2837±0.0507, P <0.05) and serum testosterone levels (3.4627±0.6690). vs. 2.0620±0.427, P <0.0001) significantly increased (e.g. Figure 1 , Figure 2(As shown). H&E staining results showed that, compared with the high-fat group mice, the high-fat + 12-ketolithocholic acid group mice had a significantly increased number of spermatogenic cells in the seminiferous tubules, with more compact and orderly cell arrangement, and a significant increase in mature sperm in the tubules (38.2300±4.6000). vs. 24.6880±5.5816, P <0.05, such as Figure 3 , Figure 4 As shown in the figure); in the high-fat group, there were detached and disordered spermatogenic cells in the seminiferous tubules, while this was significantly improved in the high-fat + 12-keto-lithocholic acid group; from the basement membrane to the lumen, the high-fat + 12-keto-lithocholic acid group showed a more complete and orderly layer of spermatogenic cells (spermatogonia-spermatocytes-spermatids-spermocytes), indicating that the seminiferous tubule structure was significantly improved by 12-keto-LCA intervention. Immunofluorescence staining results showed that, compared with HFD mice, the high-fat + 12-keto-lithocholic acid group had DAZL in the seminiferous tubules. + (Spermatogonia marker, 0.8722±0.0706) vs. 0.6549±0.0787, P <0.05), SYCP3 + (Spermatocyte marker, 0.9093±0.0584) vs. 0.7233±0.0798, P <0.05), TNP1 + (Sperm cell markers, 0.9401±0.7974) vs. 0.7974±0.0423, P <0.05), WT1 + (Supporting cell markers, 0.9000±0.0662) vs. 0.7573±0.0889, P <0.05) and PGK2 + (Mature sperm marker, 0.9248±0.0453) vs. 0.7323±0.1216, P The expression level of <0.05 was significantly increased (e.g., Figure 5 , Figure 6 (As shown). However, the above indicators in the high-fat + ursodeoxycholic acid group of mice showed no statistically significant differences compared to the high-fat group (e.g., ...). Figure 1 , Figure 2 , Figure 4 , Figure 6 As shown, P >0.05).

[0053] Furthermore, compared to the high-fat group, the HFD+12-keto-LCA group showed increased sperm forward motility (23.8360±2.1298). vs. 18.8400±2.3182, P <0.05) and a decrease in sperm abnormality rate (33.20000±3.7283). vs. 45.7400±3.7334, P <0.05) (e.g. Figure 7 , Figure 8 , Figure 10 (As shown). In in vitro fertilization experiments, compared with the high-fat group, the high-fat + 12-ketolithocholic acid group showed a significantly increased rate of single-cell embryos developing into blastocysts (45.7506±6.7513). vs. 29.7326 ±8.2345, P <0.05, such as Figure 9 , Figure 10 As shown in the figure). Key indicators such as sperm count, abnormal sperm rate, and sperm motility all indicate that 12-keto-LCA intervention can effectively improve sperm quality. However, there were no statistically significant differences in these indicators between the high-fat + ursodeoxycholic acid group and the high-fat group. P >0.05).

[0054] The above embodiments of the present invention construct a multi-level, multi-omics systematic evaluation system, wherein:

[0055] At the histopathological level: H&E staining analysis directly confirmed that 12-keto-LCA has a repairing effect on the seminiferous tubule structure, providing a morphological basis for functional improvement.

[0056] At the cell biology level: Immunofluorescence staining analysis was performed using a set of specific molecular markers (including DAZL, SYCP3, TNP1, WT1, PGK2, etc.) to systematically evaluate the effects of 12-keto-LCA on the spermatogenic cell lineage. The results showed that 12-keto-LCA effectively promoted the proliferation and differentiation of cells at all stages from spermatogonia to mature spermatozoa, with a significantly wider range and stronger effect than UDCA, demonstrating its comprehensive regulatory capacity at the cellular level.

[0057] Functional validation: By detecting sperm motility and morphological parameters, and combining this with in vitro fertilization experiments, the impact of 12-keto-LCA on embryonic developmental potential was further evaluated. This series of experiments constructed a complete chain of evidence from tissue structure to cell function and then to embryonic developmental potential, systematically elucidating the significant effect of 12-keto-LCA in improving spermatogenesis, demonstrating the technical advantages of this invention.

[0058] In this embodiment of the invention, a 12-week intervention period was chosen for the animal model. This duration was determined based on the physiological characteristic that the complete spermatogenesis cycle in mice is approximately 35 days. The 12-week intervention period covers at least three complete spermatogenic epithelial cycles, thereby ensuring a systematic assessment of the complete biological process from spermatogonial stem cell proliferation and differentiation to mature sperm formation and embryonic developmental potential. This design not only fully validates the long-term, stable improvement effect of 12-keto-LCA on sperm quality but also complies with the ethical and economic principles of animal experiments.

[0059] The above experiments show that after 12 weeks of intervention, 12-keto-LCA can effectively improve reproductive dysfunction in mice induced by a high-fat diet, that is, it can effectively alleviate reproductive dysfunction in obese mice. Its effects are manifested in increasing testosterone levels, repairing the structure of the seminiferous epithelium, improving sperm quality, and ultimately enhancing embryonic developmental potential. Its improvement effect is more significant than that of UDCA. Therefore, based on this invention, the preparation of a drug using 12-keto-LCA to improve spermatogenesis disorders and enhance sperm quality has important value for the prevention or adjunctive treatment of male infertility caused by obesity and other related factors. Furthermore, the optimal effective dose of 10 mg / kg / day of 12-keto-LCA (i.e., 10 mg of 12-keto-LCA per kg of organism per day) is the one that strikes a balance between effectively improving spermatogenesis and animal safety and tolerability.

[0060] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of 12-ketolithocholic acid in the preparation of a medicament for improving obesity-related decline in sperm quality, said improvement in obesity-related decline in sperm quality being achieved by at least one of the following effects: a) increasing sperm concentration; b) increasing sperm motility, said increasing sperm motility including at least one of increasing percentage of progressively motile sperm, increasing percentage of total motile sperm; c) decreasing sperm abnormality rate; d) improving embryo development potential; e) improving testicular seminiferous tubule structure; f) increasing spermatogenic cell number; g) increasing serum testosterone level.

2. The use according to claim 1, characterized in that, Said decline in sperm quality is specifically a decline in sperm quality in a mammal.

3. The use according to claim 1, characterized in that, The 12-ketolithocholic acid has the following structural formula: 。 4. The use according to claim 1, characterized in that, The spermatogenic cells comprise DAZL + cells, SYCP3 + cells, TNP1 + cells, WT1 + cells, and PGK2 + one or more of the cells.

5. The use according to claim 1, characterized in that, The dose of 12-ketolithocholic acid to be administered per kg of organism per day is 10 mg.

6. The use according to claim 1, characterized in that, The medicament is in the form of an oral preparation, an injection, granules, pills, capsules or powder.

7. The use according to claim 1, characterized in that, The medicament is in the form of a sustained-release, controlled-release or targeted preparation.

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