Application of L-phenylalanyl-L-lysine in improving spermatogenesis function

Abstract

The invention discloses an application of L-phenylalanyl-L-lysine in improving a spermatogenic function. The L-phenylalanyl-L-lysine is proposed for the first time to be capable of remarkably increasing sperm concentration, improving sperm malformation, improving sperm motility and effectively improving the spermatogenic function. Meanwhile, L-phenylalanyl-L-lysine is used for treating oligoasthenozoospermia, can promote thickening of spermatogenic tubule spermatogenic cell layers, increase spermatogenic cell areas, improve sperm differentiation and formation processes, relieve spermatogenic dysfunction of testicular tissues and increase the total number of sperms, is remarkable in treatment effect and high in safety, and has excellent clinical transformation prospects.

Description

Technical Field

[0001] This invention relates to small molecule bioactive peptides, and more particularly to the application of L-phenylalanyl-L-lysine in improving spermatogenesis. Background Technology

[0002] Infertility has become a major challenge affecting the health of people of reproductive age worldwide, ranking third globally in incidence, after cancer and cardiovascular disease. Globally, approximately 17.5% of couples of reproductive age suffer from infertility, with male factors accounting for about half of these cases. In recent years, male sperm quality has shown a continuous deterioration trend. Studies show that between 1973 and 2018, global male sperm concentration and total sperm count decreased by more than 50% and 60%, respectively, and this trend continues. Simultaneously, the incidence of male infertility is rising year by year, threatening not only individual fertility but also placing an increasingly heavy burden on social healthcare systems.

[0003] Among the many causes of male infertility, oligospermia and asthenospermia are particularly common. Their causes are complex, involving multiple factors such as genetics, environment, immunity, and medication. Currently, there is a lack of definitively effective treatments, making the development of novel therapeutic drugs particularly urgent. Recent research has further revealed a close interaction network between testicular function, gut microbiota, and body metabolism, forming the so-called "gut-testis axis." For example, alginate oligosaccharides (AOS) can improve the blood and testicular metabolic environment and promote spermatogenesis recovery by regulating the gut microbiota, increasing beneficial bacteria, and inhibiting harmful bacteria. Animal experiments have shown that mice treated with AOS showed significantly improved sperm quality and motility through fecal microbiota transplantation. Furthermore, factors such as alcohol intake and long-term exposure to nanoplastics can also trigger gut microbiota dysbiosis and related lipid metabolism abnormalities, thereby impairing the spermatogenesis process. Multiple studies have confirmed that there is a significant correlation between specific gut microbiota and host metabolites and reproductive hormone levels, indicating that the microbiota-metabolism-testis network plays a key role in regulating sperm quality. This provides new ideas and targets for the prevention and treatment of male infertility from the perspective of gut microbiota and metabolism. However, specific key compounds that can be used to improve spermatogenesis still need to be discovered. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide an application of L-phenylalanyl-L-lysine in improving spermatogenesis by increasing sperm count and quality.

[0005] Technical solution: The application of L-phenylalanyl-L-lysine described in this invention in improving spermatogenesis.

[0006] Preferably, the CAS number of the L-phenylalanyl-L-lysine is 6456-72-0.

[0007] Preferably, the improvement in spermatogenesis includes improving sperm quality and / or increasing sperm count.

[0008] More preferably, the improvement of sperm quality includes any one or more of the following: improving sperm motility, increasing sperm activity rate, and reducing sperm abnormality rate.

[0009] Preferably, the application is in the preparation of food additives or health foods that regulate male fertility.

[0010] Preferably, the application is in the preparation of infertility treatment drugs.

[0011] More preferably, the infertility is any one or more of oligospermia, asthenospermia, teratospermia, and spermatogenesis dysfunction.

[0012] Preferably, the drug contains L-phenylalanyl-L-lysine or a pharmaceutically acceptable salt, solvate, or hydrate thereof as an active ingredient.

[0013] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0014] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention is the first to propose that L-phenylalanyl-L-lysine can significantly improve spermatogenesis, including increasing sperm concentration, improving sperm abnormalities, and improving sperm motility; 2. L-phenylalanyl-L-lysine can be used to treat oligoasthenospermia, promote the thickening of the spermatogenic cell layer in the seminiferous tubules, increase the number of spermatogenic cells, improve the sperm differentiation and formation process, effectively alleviate testicular tissue spermatogenesis dysfunction, and increase the total number of sperm. The therapeutic effect is significant and the safety is high, with excellent clinical translation prospects. Attached Figure Description

[0016] Figure 1 Flowchart of an experiment on the use of L-phenylalanyl-L-lysine to treat oligoasthenospermia in mice; Figure 2 The graph shows the statistical results of body weight changes in mice treated with L-phenylalanyl-L-lysine for oligoasthenospermia. Figure 3The graph shows the statistical results of organ index in mice used to treat oligoasthenospermia in mice with L-phenylalanyl-L-lysine. Figure 4 Microscopic images of mouse testes stained with hematoxylin and eosin for the treatment of oligoasthenospermia with L-phenylalanyl-L-lysine, with a scale bar of 100 μm; Figure 5 The graph shows the statistical results of sperm quality parameters in mice treated with L-phenylalanyl-L-lysine for oligoasthenospermia. In the graph, A represents sperm concentration, B represents sperm abnormality rate, C represents sperm motility, and D represents sperm activity rate. Figure 6 A graph showing the statistical results of mouse weight changes in the in vivo safety evaluation of L-phenylalanyl-L-lysine. Figure 7 A graph showing the statistical results of organ index in mice during the in vivo safety evaluation of L-phenylalanyl-L-lysine. Figure 8 The figure shows the statistical results of the analysis of mouse sperm quality parameters in the in vivo safety evaluation of L-phenylalanyl-L-lysine. In the figure, A is sperm concentration, B is sperm abnormality rate, C is sperm motility, and D is sperm activity rate. Figure 9 The images are hematoxylin-eosin stained microscopic images of major organs in mice during the in vivo safety evaluation of L-phenylalanyl-L-lysine, with the scale bar at 100 μm. Figure 10 The graph shows the statistical results of serum liver and kidney function measurements in mice during the in vivo safety evaluation of L-phenylalanyl-L-lysine. In the graph, A represents the statistical results of alanine aminotransferase measurement, B represents the statistical results of aspartate aminotransferase measurement, C represents the statistical results of AST / ALT transaminase ratio measurement, D represents the statistical results of alkaline phosphatase measurement, E represents the statistical results of total bilirubin measurement, F represents the statistical results of creatinine measurement, G represents the statistical results of urea measurement, and H represents the statistical results of urea nitrogen measurement. Detailed Implementation

[0017] The technical solution of the present invention will be further described below.

[0018] Example 1: L-phenylalanyl-L-lysine for the treatment of oligoasthenospermia in mice The experimental procedure is as follows Figure 1 As shown.

[0019] 1. Construction of a mouse model of oligoasthenospermia Male Kunming white mice aged 6-8 weeks were purchased from the Animal Experiment Center of Xinjiang Medical University, weighing 22-25g. After 2 weeks of acclimatization feeding, they were randomly divided into 5 groups, including: Group A: Normal control (Ctrl); Group B: Cyclophosphamide model group / Oligospermia-asthenospermia model group (CTX); Group C: Cyclophosphamide + L-phenylalanyl-L-lysine low-dose treatment group (CTX+LM); Group D: Cyclophosphamide + L-phenylalanyl-L-lysine high-dose treatment group (CTX+HM); Group E: Cyclophosphamide + L-carnitine treatment group (CTX+ZK); 6 animals in each group. Among them, the BE group was injected intraperitoneally with cyclophosphamide (30 mg / kg) for 7 consecutive days to establish the oligospermia-asthenospermia model, while the A group was injected intraperitoneally with an equal volume of physiological saline daily.

[0020] After modeling, the following drug administration protocol was adopted: The experimental period lasted for 28 days. Groups A and B were given normal saline by gavage, Group C was given 10 mg / kg L-phenylalanine (low dose) by gavage, Group D was given 30 mg / kg L-phenylalanine (high dose) by gavage, and Group E was given 400 mg / kg L-carnitine by gavage (positive drug control group).

[0021] 2. Mouse weight monitoring During the treatment period, the body weight of mice in each group was recorded every 4 days; the statistical results are as follows: Figure 2 As shown, there were no significant differences among the groups.

[0022] 3. Determination of organ indices in mice After the final gavage, the mice were fasted overnight. The following morning, the weight of each group of mice was recorded. They were then euthanized by cervical dislocation, and the heart, liver, spleen, lung, kidney, and testis tissues were removed and weighed. Organ index (%) = organ wet weight (g) / fasting body weight (g) × 100%. The results are as follows: Figure 3 Compared with the control group, the organ indices in the CTX group showed a decreasing trend in liver tissue, but this was not statistically significant. The organ indices in other groups showed an increasing trend, with the CTX+ZK group showing a significant increase. The organ indices of other organs did not change significantly.

[0023] 4. Histopathological observation of testicular tissue After cervical dislocation, testicular tissue was removed from each group of mice and fixed in testicular tissue fixative (purchased from Sewell, catalog number G1121) for 48 hours. After fixation, the tissue was dehydrated, embedded, sectioned, and baked in a 60°C oven overnight. The sections were then dewaxed, hydrated, and stained with hematoxylin and eosin (HE). After staining, the testicular tissue sections were morphologically observed under a microscope.

[0024] HE staining results of testicular tissue from each group of mice are as follows: Figure 4 As shown, both high-dose (CTX+HM) and low-dose (CTX+LM) L-phenylalanyl-L-lysine can improve the structural damage of mouse testicular tissue.

[0025] 5. Statistics on mouse sperm concentration, abnormality rate, motility rate, and viability. (1) Sperm collection: After the mice were euthanized by cervical dislocation, the abdominal skin was disinfected with 75% alcohol, the abdominal cavity was cut open to expose the reproductive system, the bilateral epididymal tails were separated and cut into pieces, and placed in 2 mL of preheated physiological saline. The mixture was incubated in a 37°C water bath for 15 minutes to allow the sperm to fully detach. After mixing evenly, the mixture was filtered through a 200-mesh filter to remove tissue fragments and obtain a sperm suspension.

[0026] (2) Sperm concentration, motility, and viability analysis: 8 μl of sperm suspension was placed in the central counting cell of the Ruby MACRO sperm counting plate for counting. The total number of sperm in 50 cells was counted, divided by 5 to obtain the number of sperm per row (n), and then the sperm concentration was calculated. Sperm concentration = n × 10 6 / mL. Sperm quality was simultaneously graded according to the following standards: Grade A: rapid, linear forward movement; Grade B: slow, linear forward movement or sluggish forward movement; Grade C: no forward movement or stationary trembling; Grade D: no motility. Sperm viability and motility were calculated using the following formula: Sperm motility (%) = [(Number of Grade A sperm + Number of Grade B sperm + Number of Grade C sperm) ÷ Total number of sperm] × 100%; Sperm motility (%) = (Number of Grade A sperm + Number of Grade B sperm) ÷ Total number of sperm × 100%.

[0027] (3) Sperm malformation rate analysis: Take an appropriate amount of sperm suspension and drop it onto one side of a glass slide. Take another clean glass slide and prepare a sperm smear using the push-slide method. Place the sperm smear in the air to dry naturally. After the smear is dry, fix it with methanol for 5 minutes, remove it and let it dry naturally. Stain the smear with 0.5% eosin solution for 1 hour, rinse it with distilled water, let it dry naturally, and mount it with neutral resin. Under low magnification, identify areas with clear backgrounds and minimal sperm overlap. Each mouse serves as an observation unit. Count sperm from four arbitrary viewing angles and record the number of abnormal sperm and the total sperm count among intact sperm. Examine at least 300 intact sperm per mouse, and count at least 1500 sperm per group. Abnormalities are mainly manifested in the sperm head, and can be broadly categorized as: hookless, banana-shaped, amorphous, double-headed, double-tailed, folded tail, and swollen head. Sperm with a head but no tail (indistinct outline), sperm whose heads overlap with other sperm or fragments, and sperm damaged by human factors are not counted. Calculate the sperm abnormality rate using the following formula: Sperm deformity rate (%) = number of deformed sperm / total number of sperm × 100%.

[0028] The results are as follows Figure 5As shown, compared with the modeling group, the high-dose L-phenylalanine treatment group significantly improved sperm concentration, sperm motility and sperm activity rate, and could significantly improve sperm abnormality rate.

[0029] Example 2: In vivo safety evaluation of L-phenylalanyl-L-lysine Male Kunming mice aged 6-8 weeks were purchased from the Animal Experiment Center of Xinjiang Medical University, weighing 22-25g. After 2 weeks of acclimatization feeding, the mice were randomly divided into two groups (n=7): Group A: blank control group (Ctrl) and Group B: L-phenylalanyl-L-lysine treatment group (LL-30). The administration regimens were as follows: Group A mice were continuously administered physiological saline by gavage for 28 days, 0.3 mL per day; Group B mice were continuously administered L-phenylalanine-L-lysine (30 mg / kg) by gavage for 28 days.

[0030] 1. Mouse weight monitoring From the start of drug administration, the body weight of mice in each group was recorded every 4 days; the statistical results are as follows: Figure 6 As shown, there were no significant differences among the groups.

[0031] 2. Determination of organ index in mice After the last administration (day 28), mice were fasted overnight. The fasting weight of the mice was recorded the following morning (day 29). They were then euthanized by cervical dislocation, and the heart, liver, spleen, lung, kidney, and testis tissues were removed and weighed. Organ index = organ wet weight (g) / fasting weight (g) × 100%. Results are as follows: Figure 7 The indices of organs such as the heart, liver, and spleen in the drug group did not show significant differences compared with those in the control group.

[0032] 3. Sperm quality testing According to the method described in Example 1, the sperm concentration, abnormality rate, motility, and viability of mice in different treatment groups were measured, and the results are as follows: Figure 8 As shown, compared with the control group, the sperm concentration of mice in the drug group was slightly increased, but the overall sperm quality did not change significantly.

[0033] 4. Histopathological evaluation of mouse organs According to the method described in Example 1, the heart, liver, spleen, lungs, kidneys, and testes were dehydrated, embedded, sectioned, and stained with hematoxylin and eosin (HE). Subsequently, histopathological examination of the stained sections was performed, and the results are as follows: Figure 9 As shown, no obvious signs of tissue damage were observed in the major organs of mice in both the control and treatment groups, demonstrating the safety of the drug.

[0034] 5. Determination of liver and kidney biochemical indicators in mouse serum Blood was collected using the orbital blood sampling method. After standing at room temperature for 2 hours, the serum was separated by centrifugation at 3000 rpm for 15 minutes and stored at -20℃ for later use. Serum physiological and biochemical indicators were measured by Wuhan Saiweier Biotechnology Co., Ltd. using dedicated testing equipment.

[0035] The results are as follows Figure 9 As shown, there were no significant changes in relevant indicators in the LL-30 group compared with the control group, indicating that 30 mg / kg of L-phenylalanyl-L-lysine did not cause significant damage to the liver and kidney function of mice, demonstrating good biocompatibility.

Claims

1. Use of L-phenylalanyl-L-lysine in improving spermatogenesis function.

2. Use according to claim 1, characterized in that, The improvement of spermatogenesis function includes improving sperm quality and / or increasing sperm quantity.

3. Use according to claim 2, characterized in that, The improvement of sperm quality includes any one or more of improving sperm motility, increasing sperm progressive motility, and reducing sperm abnormality rate.

4. Use according to claim 1, characterized in that, The use is use in preparing a food additive or health food for regulating male fertility function.

5. The use according to claim 1, characterized in that, The use is use in preparing a drug for treating infertility.

6. Use according to claim 5, characterized in that, The infertility is any one or more of oligospermia, asthenospermia, teratospermia, and spermatogenic dysfunction.

7. Use according to claim 5, characterized in that, The drug contains L-phenylalanyl-L-lysine or a pharmaceutically acceptable salt, solvate, or hydrate thereof as an active ingredient.

8. Use according to claim 5, characterized in that, The drug further contains a pharmaceutically acceptable excipient.

9. Use according to claim 8, characterized in that, The pharmaceutically acceptable excipient includes any one or more of excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, pH buffering substances, solubilizers, co-solvents, or solvents.

10. Use according to claim 5, characterized in that, The dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, pellets, injection solutions, infusion solutions, suspensions, patches, suppositories, transdermal patches, microemulsion liposomes, and nanoparticles.

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