Application of afzelin in preparation of anti-testis aging medicament

Abstract

The invention belongs to the technical field of medicines, and particularly discloses application of afzelin in preparation of an anti-testis aging medicament, and testis aging resistance is particularly improvement or recovery of an aged testis tissue structure. The invention also provides a medicament for resisting testis aging. The medicament contains afzelin. The invention provides a new application of afzelin, the afzelin can be used for saving senescence testicular tissue structure disorder, remarkably reducing the epididymal sperm deformation rate and increasing the number of supporting cells and undifferentiated spermatogonium cells, the new application value of afzelin is developed, and a theoretical basis is provided for developing afzelin into anti-reproductive aging drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to the use of afzelin in rescuing testicular aging. BACKGROUND

[0002] Aging is a conserved feature of organisms and a complex and pleiotropic biological process that affects multiple aspects of mammalian physiology, including reproductive development. It is generally believed that the negative effects associated with reproduction caused by increasing parental age are mainly attributed to females, and few studies have assessed the consequences of increasing paternal age. However, there is growing evidence that increasing male age has significant negative effects on spermatogenesis, sperm function, fertilization, pregnancy, and offspring health. Testicular premature aging is associated with the phenotype of testicular aging, thereby triggering a series of reproductive disorders and even infertility and health risks. This phenomenon has gradually attracted clinical attention. In addition to studying testicular aging, testicular premature aging, and potential mechanisms, it is necessary to explore how to use these studies to reduce the aging and premature aging of the reproductive system. There is currently a lack of effective and less side-effect intervention drugs. Such studies are helpful for preventing adverse reproductive outcomes, thereby contributing to human health reproduction.

[0003] It is a common method to use middle-aged animals or construct a model of reproductive premature aging to study the anti-reproductive aging effects of drugs, especially antioxidants. Afzelin, also known as kaempferol 3-O-rhamnoside, is a flavonoid compound found in Houttuynia cordata, which is widely used to prepare antibacterial and antipyretic agents, detoxifying agents, and treatments for inflammation, and has strong anti-inflammatory, anticancer, and antioxidant effects. A large number of studies have found that flavonoids have a protective effect on the reproductive system, such as quercetin, which can promote the proliferation of spermatogonia; and orange mulberry flavonoids, which can improve ovarian premature aging. Afzelin has been shown to reduce oxidative stress, damage to mitochondrial membrane potential, and mitochondrial permeability induced by ultraviolet light in human skin. Afzelin can also protect liver damage in mice caused by a high-fat diet and acetaminophen. Kaempferol can alleviate the decrease in developmental capacity during the aging process of porcine oocytes. However, there is no relevant study on the use of afzelin to rescue testicular aging, and therefore, it is necessary to further explore it. SUMMARY

[0004] The present application aims to provide a new use of afzelin, specifically the use of afzelin in the preparation of a testicular anti-aging drug. Another object of the present application is to provide a product for rescuing or delaying testicular aging, which can maximize the drug effect by rescuing the disordered structure of aging testicular tissue, sperm deformity, and reduced spermatogonia and Sertoli cells through the use of afzelin.

[0005] To achieve the above-mentioned objects, the present application adopts the following technical solutions: The use of afzelin in the preparation of an anti-testicular aging drug.

[0006] In particular, the application of aflatoxin in the preparation of a drug for improving or restoring the structure of the aging testis.

[0007] Further, the application of aflatoxin in the preparation of a drug for improving the treatment of testicular tissue structure disorder, reducing the rate of epididymal sperm deformity in aging individuals, increasing the number of Sertoli cells in the aging testis, and increasing the number of undifferentiated spermatogonia in the aging testis.

[0008] As described above, the effective dose of aflatoxin in the drug is 5 mg ~ 15 mg.

[0009] A drug for anti-aging of testis, the drug containing aflatoxin.

[0010] A drug for anti-aging of mouse testis, the drug containing an effective dose of aflatoxin ranging from 5 mg / kg body weight to 15 mg / kg body weight.

[0011] Further, the effective dose of aflatoxin in the drug ranges from 10 mg / kg body weight.

[0012] Further, the drug is in the form of a pharmaceutically acceptable preparation.

[0013] Further, the dosage form of the drug is granules, tablets, soft capsules, soft gelatin pills, drop pills, ointments, decoctions, oral liquids or injections.

[0014] The beneficial effects of the present application are: The present application provides a new use of aflatoxin, which is the first time that aflatoxin has been found to have the effect of rescuing testicular aging; aflatoxin can significantly reduce the rate of epididymal sperm deformity and increase the number of Sertoli cells and undifferentiated spermatogonia while rescuing the structure of the aging testis, The present application also provides an anti-reproductive aging drug containing aflatoxin. The present application develops the new application value of aflatoxin, which provides a theoretical basis for developing it as an anti-reproductive aging drug. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The results of identifying the testis phenotype of D-galactose-induced aging model mice by β-galactosidase; Figure 2 The results of identifying the testis phenotype of D-galactose-induced aging model mice by oil red O staining; Figure 3 H&E staining of testis tissue of aging model mice after intragastric administration of different concentrations of aflatoxin for 15 days; Figure 4Effects of different concentrations of afzelin on the quality of epididymal sperm in aging model mice after intragastric administration for 15 days Figure 5 Effects of different concentrations of afzelin on the undifferentiated spermatogonia in the testis of aging model mice after intragastric administration for 15 days Figure 6 Effects of different concentrations of afzelin on the Sertoli cells in the testis of aging model mice after intragastric administration for 15 days. DETAILED DESCRIPTION

[0016] The present application has been proved by experiments that afzelin can effectively rescue the testicular damage of aging mice induced by D-galactose, wherein it is found that the dose of 10 mg / kg is the most effective, which can not only rescue the testicular tissue structure disorder caused by aging, but also reduce the epididymal sperm abnormality rate and increase the number of Sertoli cells and undifferentiated spermatogonia. The present application provides a new application prospect for afzelin. It will also drive the extensive research of flavonoid antioxidants in the field of aging and reproductive aging.

[0017] The following examples are used to further illustrate the present application, but should not be construed as limiting the present application. Modifications or substitutions made to the present application without departing from the spirit and essence of the present application shall all belong to the scope of the present application.

[0018] Unless specifically indicated, the technical means used in the examples are conventional means familiar to those skilled in the art. Unless specifically indicated, the reagents used in the examples are analytical pure or above specifications, and commercially available products can be used. The mice used in the examples are 8-week-old C57BL / 6J male mice, which can be purchased from Jiangsu Lingfei Biological Technology Co., Ltd. Afzelin can be purchased from MedChemexpress Biological Technology Company, USA.

[0019] Example 1 1. Aging mouse modeling experiment First, 8-week-old C57BL / 6J male mice were subcutaneously injected with D-galactose at a dose of 150 mg / kg / day for 7 consecutive weeks, and the solvent was PBS. After the modeling was completed, β-galactosidase and oil red O staining were used to evaluate whether the modeling was successful.

[0020] A young group (subcutaneously injected with blank solvent PBS) and an aging model mouse group (subcutaneously injected with D-galactose at a dose of 150 mg / kg / day for 7 consecutive weeks) were set. After the modeling was completed, the mice were sacrificed by cervical dislocation, and the testis was taken and embedded in OCT, and frozen sections (thickness 8-10 μm) were prepared. By β-galactosidase and oil red O staining, it was found that a large number of X-Gal staining positive cells (aging cells) could be seen in the testicular interstitium as shown in Figure 1 , and red lipid droplets were colored as Figure 2As shown, it is illustrated that this aging model can be used.

[0021] 2. After the aging model mice were treated by aforboside by gavage for 15 days, testicular aging recovery test was performed.

[0022] The experimental mice were divided into four groups (n=3 / group): young mouse control group (given 10% anhydrous ethanol + 90% corn oil solvent); aging model mouse control group (given 10% anhydrous ethanol + 90% corn oil solvent); aging model mouse low-dose aforboside treatment group, given 5 mg / kg aforboside (dissolved in 10% anhydrous ethanol + 90% corn oil); aging model mouse high-dose aforboside treatment group, given 10 mg / kg aforboside (dissolved in 10% anhydrous ethanol + 90% corn oil). All groups were given drugs once a day by gavage, and the gavage volume was calculated according to the body weight and kept consistent. During the experiment, all mice were raised in standard SPF level animal room (22±2°C, 50%±10% humidity, 12 / 12 hour light-dark cycle), free to eat sterilized feed and drink water, using sterilized bedding and changing regularly, to ensure animal welfare and standardization of experimental environment.

[0023] After 15 days of gavage treatment, the mice were sacrificed by cervical dislocation, the testes were taken, and after the steps of fixation by 4% PFA, gradient dehydration by alcohol, clearing by xylene, paraffin embedding, sectioning, etc., the testicular tissue structure was observed by hematoxylin and eosin staining (H&E), and the testicular H&E staining chart is as shown in Figure 3 As shown in the figure, the green triangles represent apoptotic shrunken cells. It was found that compared with young mice, the testicular seminiferous tubules of the aging model mouse group atrophied and degenerated, most of the efferent ducts became narrow, the number of spermatogenic epithelial cells decreased and arranged in disorder, mature sperm could not be seen in the lumen, a small part of the peritubular muscle-like cells ruptured, and the cells in the tube flowed to the interstitium. In the aforboside treatment group, whether low-dose or high-dose, the abnormal seminiferous tubules were significantly reduced, the efferent ducts became larger, the gradient morphology of spermatogenesis was obvious, sperm could be seen in the lumen, and the structure was normal. However, in the aforboside-5mg / kg group, there were shrunken cells (green arrows) in the testicular seminiferous tubules.

[0024] After the experiment, the cauda epididymal sperm of all groups of mice was sampled by cutting the cauda epididymis and incubating in preheated physiological saline at 37°C to obtain sperm suspension. After sampling, morphological analysis was performed by sperm smear staining: the sperm suspension was spread on an adhesive glass slide, air-dried, fixed with methanol, and stained with H&E, and then observed under an oil immersion lens (100x) for at least 200 sperm per sample. The rate of abnormal sperm was calculated by the following formula: Abnormal sperm rate (%) = (number of abnormal sperm / total number of observed sperm) x 100%. The abnormality criteria include morphological abnormalities of the head (no hook, fragmentation), neck (bending, swelling) and tail (curling, breaking) of the sperm. The results of epididymal sperm smear are as shown in Figure 4The results of the abnormal sperm rate are shown in Table 1.

[0025] Table 1 Abnormal sperm rate of epididymal sperm after intragastrically administering different concentrations of aforboside to the aging model mice for 15 days Note: a, b, and c in the table represent significant differences between groups (p<0.05).

[0026] It was found that the abnormal sperm rate of the aging model mouse group was 14.63%. After intragastrically administering different concentrations of aforboside to the mice for 15 days, the abnormal sperm rate decreased, and the abnormal sperm rate of the aforboside-10 mg / kg group decreased by 65.87%, indicating that aforboside can significantly improve the sperm quality of aging mice and rescue the testicular aging induced by D-galactose.

[0027] The number of undifferentiated spermatogonia in each seminiferous tubule of each group was counted by immunofluorescence staining. Specifically, the mouse testicular tissue was paraffin-embedded, and paraffin sections (4 μm thick) were prepared. The undifferentiated spermatogonia-specific marker (such as LIN28A) was used as the primary antibody, and the fluorescent secondary antibody was stained, and the cell nucleus was stained with DAPI. High-resolution imaging of the seminiferous tubule cross-section was performed under a confocal microscope (≥5 fields of view were randomly selected for each mouse in each group). The number of undifferentiated spermatogonia was counted as follows: In a single section, the DAPI-labeled cell nucleus and the undifferentiated spermatogonia marker co-localization signal were identified, and the number of positive cells in each complete seminiferous tubule cross-section was counted; ≥15 seminiferous tubules were analyzed for each group (3 mice x 5 fields of view), and the data were expressed as mean ± standard deviation, and Excel software was used for analysis of differences between groups (a, b represent P <0.05). Representative images of undifferentiated spermatogonia immunofluorescence are shown in Figure 5 The number of LIN28A+ cells in each seminiferous tubule of different groups is shown in Table 2.

[0028] Table 2 Number of LIN28A+ cells in each seminiferous tubule of different groups Note: a, b, and c in the table represent significant differences between groups (p<0.05).

[0029] The results show that compared with the aging model mice, the number of Lin28 positive cells in the aforboside-5 mg / kg and aforboside-10 mg / kg groups increased by 36.10% and 42.91%, respectively, and there was no significant difference compared with the young group. This indicates that aforboside has a rescuing effect on testicular aging. Aforboside can remove excessive ROS, effectively reduce the oxidative stress level in the testis, reduce the oxidative damage to the DNA, protein, and lipid of spermatogonia, and maintain the normal metabolism and division ability of the cells.

[0030] The effect of afromontoside on the number of Sertoli cells in the testicular seminiferous tubules of the aging model mice was detected by immunofluorescence staining. The specific operation was the same as above, and SOX9 was the marker protein of Sertoli cells. The test results of Sertoli cell immunofluorescence representative pictures were as shown in Figure 6 Table 3 was the number of SOX9+ cells in each seminiferous tubule of different groups.

[0031] Table 3 was the number of SOX9+ cells in each seminiferous tubule of different groups. Group Number of SOX9 positive cells in each seminiferous tubule Modeling group of aged mice 11.70 ± 0.60 b ]] Young group 13.70 ± 0.22 a ]] Afuwaloside-5 mg / kg group 13.56 ± 1.49 ab ]] Afuwaloside-10 mg / kg group 14.09 ± 0.31 a ]]

[0032] Note: a, b, c in the figure represent significant differences between groups (p<0.05).

[0033] Compared with the aging model mice, the number of Sertoli cells in the afromontoside-10mg / kg group was significantly increased. Sertoli cells, as the only somatic cells in contact with spermatogenic cells, are the basis of testicular function and development. The significant increase in the number of Sertoli cells in aging mice by 10mg / kg afromontoside may activate the signal pathway promoting proliferation in Sertoli cells, accelerate cell division, and increase the number of Sertoli cells, which also helps to improve the regulation of spermatogenesis by the testicular microenvironment.

Claims

1. Application of afotoxin in the preparation of anti-testicular aging drugs.

2. Application of afotoxin in the preparation of pharmaceutical agents for improving or restoring the structure of aging testicular tissue.

3. The application according to claim 1, characterized in that, The applications are to improve the treatment of testicular tissue structure disorders, reduce the epididymal sperm abnormality rate in aging individuals, increase the number of Sertoli cells in aging testes, and / or increase the number of undifferentiated spermatogonia in aging testes.

4. The application according to claim 1, characterized in that, The effective dose of afotoxin in the drug is 5 mg to 15 mg.

5. The method according to claim 1, characterized in that, The dosage forms of the medicine are granules, tablets, soft capsules, soft pills, drop pills, ointments, powders, or injections.

6. An anti-testicular aging agent, said agent containing afotoxin.

7. A drug for inhibiting testicular aging in mice, said drug containing afotoxin.

8. The pharmaceutical preparation according to claim 7, characterized in that, The effective dose range of afotoxin in the drug is 5 mg / kg body weight to 15 mg / kg body weight.

9. The pharmaceutical preparation according to claim 7, characterized in that, The effective dose range of afotoxin in the drug is 10 mg / kg body weight.

10. The pharmaceutical preparation according to claim 7, characterized in that, The dosage forms of the medicine are granules, tablets, soft capsules, soft pellets, drop pills, ointments, powders, oral liquids, or injections.

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