Application of hederagenin in preparation of product preparation for treating hidden testis

Abstract

The invention discloses application of hederagenin in preparation of a product preparation for treating hidden testis, and belongs to the technical field of biological medicine. By establishing a cryptorchis mouse model, the influence of the hederagenin as a main active component on a testis tissue structure, spermatogenic cell proliferation and apoptosis and sperm parameters is evaluated. By designing experimental groups of different administration dosages of hederagenin and combining with comprehensive analysis of data such as mouse sperm quantity, organ index and tissue slice, the treatment effect of 80mg / kg of hederagenin is most prominent, the testis tissue structure of cryptorchis mouse can be improved, the expression of spermatogenesis related markers is promoted, and the clinical application prospect is broad. Therefore, the effects of repairing testis and improving spermatogenesis capability are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of hederagenin in the preparation of a product preparation for treating cryptorchidism. Background Art

[0002] With the continuous development of social industry, environmental pollution is becoming more and more serious, and humans are increasingly affected by biochemical products, which has led to a gradual decline in the quality of male sperm. According to research data, the proportion of azoospermia, oligospermia and asthenospermia has increased in male infertility patients; at the same time, sperm density, vitality and survival rate have also shown a downward trend. Some experts pointed out that infertility has become the third largest disease endangering human health after tumors and cardiovascular diseases. The impact of lifestyle on male fertility cannot be ignored. Modern society has a fast pace and high work pressure. Many men are in a state of tension and anxiety for a long time. This mental stress can cause endocrine system disorders, thereby affecting the production and quality of sperm. In addition, irregular work and rest habits can also disrupt the biological clock and further damage the health of sperm.

[0003] In addition to clear causes such as genetic factors, gonad infection, acquired testicular damage, and immune factors, about 70% of male infertility has no clear cause. Male infertility can be mainly divided into congenital, acquired, and idiopathic. The most common cause of male congenital infertility is primary testicular failure. The treatment of congenital infertility includes removing sperm from the testicles to obtain sperm for immediate use in assisted reproductive technology (ART) cycles or cryopreservation for later use. Acquired infertility is defined as the absence of sperm in the ejaculate due to obstruction of sperm transport. Some acquired diseases can be improved through drug therapy, acupuncture, and intensive exercise. Idiopathic infertility is also called unexplained infertility, which refers to a type of infertility for which the cause of the infertility cannot be found using existing diagnostic methods.

[0004] Hederagenin is a flavonoid compound extracted from ivy leaves, which has anti-inflammatory and vascular protective effects. The chemical structure characteristics of hederagenin give it a variety of pharmacological activities. Its molecular structure contains multiple hydroxyl groups and sugar groups, which give it good hydrophilicity and biological activity. In addition, hederagenin also has a strong anti-inflammatory function, which can control the discharge of inflammatory mediators, thereby alleviating the inflammatory response. In addition, hederagenin also has an antioxidant function, which can eliminate free radicals in the human body and protect cells from damage by antioxidant reactions. In addition to tolerance and antioxidant effects, hederagenin also produces immunomodulatory effects. It can regulate the activity of immune cells and enhance the body's immune function. In addition, some studies have also shown that hederagenin also has a variety of biological activities such as anti-tumor and anti-viral. Although hederagenin is rarely used in reproductive health, its antioxidant properties make it a potential candidate for studying the mechanism of spermatogenesis. However, there is no clear technical disclosure on the effect of hederagenin on spermatogenesis in cryptorchid mice. Summary of the invention

[0005] The purpose of the present invention is to provide an application of hederagenin in the preparation of a product preparation for the treatment of cryptorchidism. A cryptorchid mouse model is established to evaluate the effects of hederagenin as the main active ingredient on testicular tissue structure, spermatogenic cell proliferation and apoptosis, and sperm parameters. This study will lay a solid foundation for a more in-depth exploration of the molecular mechanism of spermatogenesis in the future and may bring new insights into the field of reproductive medicine.

[0006] The present invention is achieved through the following technical solutions:

[0007] Application of hederagenin in the preparation of product preparations for the treatment of cryptorchidism.

[0008] Application of hederagenin in the preparation of product preparations for treating spermatogenesis disorders.

[0009] Application of hederagenin in the preparation of product preparations for maintaining and restoring spermatogenesis in cryptorchid testes.

[0010] Application of hederagenin in the preparation of preparations for improving cryptorchid testicular structure and testicular repair products.

[0011] A hederagenin product preparation, including any one of a product preparation for treating cryptorchidism, a product preparation for treating spermatogenesis disorder, a product preparation for promoting cryptorchid testicular spermatogenesis, a product preparation for protecting the seminiferous tubule structure of cryptorchid testicles and a product preparation for testicular repair, which takes hederagenin as the main active ingredient.

[0012] Preferably, the product preparation is used to increase sperm count.

[0013] Preferably, the product preparation is used to improve the testicular organ coefficient and protect the testicular seminiferous tubule structure.

[0014] Preferably, the product preparation also contains pharmaceutically acceptable excipients to prepare a pharmaceutically corresponding dosage form.

[0015] Preferably, the dosage of the hederagenin is 20 to 100 mg / kg.

[0016] Preferably, the dosage of the hederagenin is 80 mg / kg.

[0017] Compared with the prior art, the present invention has at least the following technical effects:

[0018] (I) The present invention provides an application of hederagenin in the preparation of a product preparation for the treatment of cryptorchidism. A cryptorchid mouse model is established to evaluate the effects of hederagenin as the main active ingredient on testicular tissue structure, spermatogenic cell proliferation and apoptosis, and sperm parameters. This study will lay a solid foundation for a more in-depth exploration of the molecular mechanism of spermatogenesis in the future and may bring new insights to the field of reproductive medicine.

[0019] (ii) The present invention uses biological experiments to measure the organ coefficient (i.e., the ratio of organ weight to the total body weight of the animal), which is a common method for evaluating the effects of a particular treatment or product on organ health and function. The higher organ coefficient of H80 indicates that it has better testicular repair ability.

[0020] (III) In the experiment, the present invention successfully established the CO cryptorchidism model, and no sperm was detected in the cryptorchidism group mice, which verified the validity of the model. In the BO normal control group, mice showed a higher sperm count, which provided a strong basis for subsequent comparison of results.

[0021] Regarding the increase in sperm count in the mice treated with hederagenin, although they did not reach the level of the BO normal control group, they still showed a certain degree of positive effect. Especially in the H80 group, they showed a significant effect in increasing sperm count, which exceeded the performance of other experimental groups. It was further pointed out that the product had a certain promoting effect on spermatogenesis.

[0022] (IV) In the experiment of the present invention, the testicular tissue of the CO cryptorchidism model mice showed obvious pathological changes, such as enlarged seminiferous tubule cavity, disordered cell arrangement and thinning of the tubule wall, which showed that the high temperature environment caused by the operation had a negative impact on the spermatogenesis process. However, the testicular tissue of the mice treated with H80 showed a healthier seminiferous tubule structure, with more orderly cell arrangement, smaller tubule cavity, and mature sperm, which showed that these compounds could protect the spermatogenesis process to a certain extent. The results highlight the importance of product dosage for spermatogenesis.

[0023] (V) The present invention designs experimental groups with different dosages of hederagenin, and combines comprehensive analysis of data such as mouse sperm count, organ index, and tissue sections to find that the 80 mg / kg dosage has the most prominent therapeutic effect and the most significant repair ability for the testicles.

[0024] The above conclusions can lay a foundation for a deeper understanding of the molecular mechanism of spermatogenesis in the future and generate new insights into the field of reproductive medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the left testicle organ coefficient of each mouse group;

[0026] Figure 2 Schematic diagram of the right testicular organ coefficient of each mouse group;

[0027] Figure 3 This is a schematic diagram showing the effect of hederagenin on serum hormone levels in cryptorchid mice;

[0028] Figure 4 Schematic diagram of the sperm count of mice in each group, with a scale of 20×10;

[0029] Figure 5 Schematic diagram of sperm counting of mice in each group;

[0030] Figure 6 Schematic diagram of HE staining microscope photography of each group of mice, with a scale of 20×10. DETAILED DESCRIPTION

[0031] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The product preparations or instruments used without indicating the manufacturer are all conventional product preparations that can be purchased commercially.

[0032] Experimental example:

[0033] 1. Experimental purpose: Based on the establishment of cryptorchid mouse model, the testicular tissue structure, spermatogenic cell proliferation and apoptosis, and sperm parameters were analyzed to comprehensively evaluate the effect of hederagenin on spermatogenesis in cryptorchid mice.

[0034] 2. Experimental methods:

[0035] 2.1 Materials and Equipment

[0036] 2.1.1 Experimental materials: The main experimental materials are shown in Table 1

[0037] Table 1 Main experimental materials

[0038]

[0039]

[0040] 2.1.2 Solution formulation

[0041] (1) Harris hematoxylin solution: 1 g of hematoxylin, 10 mL of anhydrous ethanol, 200 mL of distilled water, 1 to 2 drops of glacial acetic acid, and 1 g of Harris hematoxylin crystals. Dissolve them in 95% alcohol to a volume of about 10 mL. Add 1 to 2 drops of glacial acetic acid and adjust the pH to an acidic reaction. Filter the solution to remove insoluble matter.

[0042] (2) Water-soluble eosin stain: eosin powder: 1 g, 70% ethanol 95 mL, distilled water 5 mL, glacial acetic acid 10 mL. Add eosin powder to 70% ethanol and stir evenly. Add distilled water and glacial acetic acid and continue stirring until completely dissolved. Filter the solution to remove impurities. Store the solution in a brown bottle and protect from light.

[0043] (3) Fixative: 10 mL of paraformaldehyde solution (usually 37%) and 90 mL of phosphate buffered saline (PBS).

[0044] 2.1.3 Experimental equipment: The instruments required for this experiment are shown in Table 2.

[0045] Table 2 Instrument table

[0046]

[0047]

[0048] 2.2 Experimental procedures

[0049] 2.2.1 Establishment of cryptorchid mouse model

[0050] In this experiment, cryptorchidism surgery was used to construct a model in 10-day-old Kunming mice.

[0051] Anesthetized 10-day Kunming mice were placed in a wide-mouth bottle filled with ether and fixed on the operating table in a supine position. After wiping the abdomen of the mouse with 75% alcohol, the abdominal cavity of the mouse was incised, and the size of the wound was about 1cm±0.5mm. Then the fat head connected to the testicle and epididymis was sutured into the abdominal cavity, so that the testicle and epididymis were indirectly fixed in the abdominal cavity. Finally, the wound was sutured and disinfected. The mice were observed and recorded regularly after the operation.

[0052] 2.2.2 Dosage Grouping

[0053] Experimental grouping:

[0054] The mice were randomly divided into four groups, namely BO group (normal control group), CO group (cryptorchidism control group) and Hx (hederma saponin group, i.e. experimental group) group (x represents dosage, which are 20 mg / kg, 40 mg / kg and 80 mg / kg respectively).

[0055] The mice in the BO and CO groups received the same soybean oil injection treatment.

[0056] In the BO and CO groups, the amount of soybean oil gavaged into mice was 10 μL / g, which was dynamically adjusted according to the body weight of the mice.

[0057] Experimental operation process:

[0058] During the 35-day experimental period, the experimental mice were subcutaneously injected every day. The BO and CO groups were injected with soybean oil, while the Hx group was injected with hederagenin solution according to the corresponding dose gradient. The body weight of the mice was measured and the data were recorded at a fixed time every week.

[0059] Data processing method: All experimental data were statistically analyzed using GraphPad Prism 7.04 software.

[0060] 2.2.3 Dissection and sperm counting

[0061] Experimental steps:

[0062] On the 35th day after the end of the experiment, blood samples were collected from the eyeballs of the experimental mice, and then they were euthanized, and the weight of both testicles was weighed and recorded. One testicle was quickly frozen in liquid nitrogen and transferred to an ultra-low temperature freezer for subsequent histological analysis; the other testicle was placed in an EP tube containing warm phosphate buffered saline (PBS) for processing.

[0063] Sperm count: The collected paratesticular epididymal tissue was sheared and counted using a Myron sperm counting instrument. The data were analyzed using GraphPad Prism 7.04 software.

[0064] 2.2.4 HE staining

[0065] The testis is the reproductive gland of male animals, which has the function of producing gametes and endocrine. The testis is oval, with a layer of tunica vaginalis on the outside and a dense connective tissue on the deep part. It thickens at the posterior edge of the testis and enters the testis to form the testicular septum. The radial branches of the mediastinal connective tissue penetrate into the testicular substance and divide the substance into testicular lobules. Each lobule contains one or several winding and tortuous seminiferous tubules. The seminiferous tubules are composed of supporting cells and various germ cells and have the function of producing sperm. The interstitial cells between the seminiferous tubules have the function of producing androgens. The seminiferous tubules merge into straight seminiferous tubules at the tips of each lobule, enter the ductal network in the mediastinal connective tissue, and finally 10 to 30 testicular efferent ducts are separated from the testicular network and merge into the epididymal duct of the epididymal caput. Testicular cells are divided into spermatogenic cells and somatic cells. Somatic cells include interstitial cells, myoid cells and supporting cells, which constitute the microenvironment of the testis and are crucial for regulating normal spermatogenesis.

[0066] HE staining solution (also known as hematoxylin-eosin staining) is one of the commonly used staining methods in the field of cell biology and histology, mainly used to observe the morphology and structure of cells and tissues. The function of hematoxylin is to bind to the DNA and RNA in the cell nucleus to make the cell nucleus appear blue-purple. The function of eosin is to bind to the proteins and sugars in the cytoplasm to make the cytoplasm appear red or pink, thereby achieving the purpose of distinguishing the cell nucleus and cytoplasm, and clearly observing the structural details inside the cell. When studying testicular tissue, HE staining can help researchers observe the morphological structures of seminiferous tubules, supporting cells, interstitial cells, etc. The specific process is as follows:

[0067] (1) Fixation: Place the tissue sample in a 4% paraformaldehyde solution for fixation, usually for 24 hours.

[0068] (2) Dehydration and transparency: The fixed tissue samples need to be dehydrated in a series of alcohol solutions with increasing concentrations, generally including 70%, 80%, 90%, 95% and 100% alcohol. The soaking time of each concentration depends on the actual situation. The dehydrated tissue samples need to be transparentized with xylene.

[0069] (3) Wax immersion: Place the transparent tissue sample into melted paraffin, then cool and solidify to form a paraffin block.

[0070] (4) Sectioning: Use a microtome to cut the paraffin block into thin slices, usually with a thickness of 4-5 μm.

[0071] (5) Paste: Paste the cut tissue sections on a glass slide and gently dry them.

[0072] (6) Dewaxing: Remove the paraffin on the tissue sections, usually using xylene for dewaxing.

[0073] (7) Hydration: Dewaxed tissue sections need to be hydrated through a gradient of ethanol and water.

[0074] (8) Hematoxylin staining: Soak the tissue sections in hematoxylin solution for staining for about 5-10 minutes.

[0075] (9) Wash with distilled water: Rinse tissue sections with distilled water to remove excess hematoxylin dye.

[0076] (10) Eosin staining: Immerse the tissue sections in eosin staining solution for about 1-2 minutes.

[0077] (11) Wash with distilled water: Rinse tissue sections with distilled water to remove excess eosin dye.

[0078] (12) Differentiation: Place tissue sections in a 1% hydrochloric acid alcohol solution for differentiation. The time can be adjusted as needed, usually from a few seconds to tens of seconds.

[0079] (13) Dehydration: Tissue sections were dehydrated using a 95% and 100% ethanol series.

[0080] (14) Transparentization: Place tissue sections in xylene for transparentization.

[0081] (15) Sealing: Add sealing glue to the dehydrated and transparent slices and cover them with a coverslip.

[0082] (16) Microscopic observation: Use a microscope to observe and photograph tissue sections for further analysis and recording.

[0083] 3 Results Analysis

[0084] 3.1 Mouse body weight analysis

[0085] During the administration, the body weight of the mice was accurately measured and recorded every week.

[0086] Combined with the data in Table 3, the results show that the weight gain of mice in the H20 and H40 groups was particularly significant, significantly higher than that of the other groups. In contrast, the average weight of mice in the CO group was relatively low, significantly lower than that of the H20 and H40 groups.

[0087] The data showed that the Hx group had a significant effect on the recovery of physical signs of mice.

[0088] However, the weight gain of mice in the H80 group was significantly lower than that in the CO group. The results suggest that there is an optimal dose of the Hx group in promoting weight recovery in mice.

[0089] In addition, it was observed that the body weight of mice in the BO group was lower than that of mice in the H20 and H40 groups.

[0090] The experimental results are shown in Table 3.

[0091] Table 3 Effect of hederagenin on body weight of cryptorchid mice

[0092] Mouse grouping Dosage Weight(g) CO - 43.904±3.24257059 BO - 43.946±0.68797093 H20 20mg / kg 44.532±5.05195962 H40 40mg / kg 44.028±3.10687882 H80 80mg / kg 43.224±2.39885473

[0093] In the above Table 3, the body weight of mice was measured every week (n=5). Among them, BO is the normal mouse at 45 days old that was subcutaneously injected with soybean oil; CO is the cryptorchid mouse at 45 days old that was subcutaneously injected with soybean oil; H20, H40, and H80 are the body weights of cryptorchid mice at 45 days old that were subcutaneously injected with different doses of hederagenin.

[0094] 3.2 Mouse testis analysis

[0095] 3.2.1 Testicular coefficient is one of the important indicators for measuring spermatogenesis, which is usually obtained by calculating the ratio of testicular weight to body weight. The testicular coefficient reflects the relative size and spermatogenesis of the testicles and is of great significance for evaluating male reproductive health.

[0096] Table 4 Effect of hederagenin on the organ coefficient of left testis in cryptorchid mice ( n=8)

[0097] Mouse grouping Dosage Left testicle (g) CO - 0.001690±0.000904188 BO - 0.003276±0.000445021 H20 20mg / kg 0.000911±0.000298250 H40 40mg / kg 0.000707±0.000523542 H80 80mg / kg 0.001938±0.000657917

[0098] Table 5 Effect of hederagenin on the organ coefficient of right testis in cryptorchid mice ( n=8)

[0099] Mouse grouping Dosage Right testicle (g) CO - 0.001909±0.001116987 BO - 0.003437±0.000400973 H20 20mg / kg 0.000911±0.000298250 H40 40mg / kg 0.000952±0.000261105 H80 80mg / kg 0.001752±0.000762030

[0100] The testicular organ coefficient shown in Tables 4 and 5 was calculated by the ratio of testicular weight to the body weight of the experimental mice.

[0101] The testicular organ coefficient can be used as a direct indicator of spermatogenesis ability. By analyzing the testicular organ coefficient, the development of the testicles under normal circumstances can be obtained.

[0102] like Figure 1 Shown is a schematic diagram of the left testicle organ coefficient of each mouse group.

[0103] like Figure 2 Shown is a schematic diagram of the right testicular organ coefficient of each mouse group.

[0104] The results show that Figure 1 and Figure 2 Among them, H80 had the highest testicular organ coefficient, proving that H80 has a strong ability to repair testicles.

[0105] The repair results of other experimental groups in this experiment were weaker, which proved that the product has a gradient property and the higher the concentration, the better the effect.

[0106] 3.2.2 Effects of hederagenin on serum hormone levels in cryptorchid mice

[0107] Spermatogenesis refers to the process by which sperm is produced by the seminiferous tubules in the testicles through a series of complex biological processes. This process is regulated by a variety of hormones, including gonadotropins (such as follicle-stimulating hormone FSH and luteinizing hormone LH) and sex hormones (such as testosterone). Normal spermatogenesis is essential for maintaining male fertility. The testis coefficient can be used as a direct indicator of spermatogenesis. Under normal circumstances, a high testis coefficient indicates that the testicles are well developed, the seminiferous tubules are active, and sperm production is normal.

[0108] Spermatogenesis refers to the process by which sperm is produced by the seminiferous tubules in the testicles through a series of complex biological processes. The spermatogenesis process is regulated by multiple hormones such as gonadotropins and sex hormones.

[0109] Male reproduction is regulated by a variety of reproductive hormones in the hypothalamus-pituitary-testis axis, and changes in reproductive hormone levels can lead to damage or repair of male reproductive function. In this study, the ELISA method was used to determine the levels of testosterone (T), follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), total superoxide dismutase (T-SOD), estrogen (E), malondialdehyde (MDA), luteinizing hormone (LH) and other hormones in the serum of each group of mice.

[0110] The results showed that cryptorchidism surgery and different doses of hederagenin had a certain effect on hormone levels in cryptorchid mice.

[0111] Testosterone (T) levels are closely related to spermatogenesis in male mice, showing a positive correlation: the higher the testosterone level, the more active the spermatogenesis of the mice.

[0112] according to Figure 3 The data shown in A show that compared with the BO group (normal control group), the testosterone (T) level in the serum of male mice in the CO group (cryptorchidism control group) was significantly reduced (P < 0.01). This result clearly shows that cryptorchidism can lead to a decrease in the testosterone (T) level in the body.

[0113] Further observations revealed that as the concentration of hederagenin increased, the testosterone content showed an upward trend, especially at a concentration of 80 mg / kg, the best effect was achieved. However, when the concentration of hederagenin increased to 80 mg / kg, the testosterone content was still lower than that of the CO group, indicating that within the dose range of 20-80 mg / kg, hederagenin intervention can effectively increase the testosterone (T) level of cryptorchid mice, and this enhancement effect is dose-dependent. When the dose of hederagenin reaches or exceeds 80 mg / kg, it may still have an enhancement effect on testosterone levels.

[0114] On the other hand, low concentrations of follicle-stimulating hormone (FSH) promote the formation of seminiferous tubules, but too high a concentration may suppress testosterone levels, thereby adversely affecting spermatogenesis.

[0115] Figure 3 The data in B revealed that compared with the BO group, the level of follicle stimulating hormone (FSH) in the serum of male mice in the CO group was slightly lower, but it did not reach a significant difference (P>0.05). This result suggests that cryptorchidism may lead to a decrease in the level of follicle stimulating hormone (FSH) in the body. It is speculated that this may be because the mouse body attempts to further regulate the level of follicle stimulating hormone (FSH) by increasing the formation of seminiferous tubules in order to cope with the damage caused by cryptorchidism surgery. Compared with the CO group, the FSH concentration in the I20, I40, and I80 groups (representing the 20mg / kg, 40mg / kg, and 80mg / kg doses of hederagenin treatment groups, respectively) that received hederagenin intervention was significantly increased (P<0.01). This finding suggests that within the dose range of 20-80mg / kg, hederagenin may promote the formation of seminiferous tubules by increasing the level of follicle stimulating hormone (FSH), thereby helping to repair the damage to the testicles caused by cryptorchidism surgery.

[0116] Gonadotropin-releasing hormone (GnRH) plays an important role in promoting testosterone secretion and spermatogenesis.

[0117] like Figure 3 As shown in Figure C, compared with the BO group (normal control group), the gonadotropin-releasing hormone (GnRH) content in the serum of male mice in the CO group (cryptorchidism control group) was reduced, but it did not reach a significant difference (P>0.05). This result indicates that cryptorchidism surgery does not significantly affect the level of gonadotropin-releasing hormone (GnRH) in the body.

[0118] Further analysis found that compared with the CO group, the GnRH levels in the I20 and I40 groups (representing the 20mg / kg and 40mg / kg hederagenin treatment groups, respectively) that received hederagenin intervention were significantly reduced (P < 0.01), while no significant changes were observed in the I80 group. This finding suggests that within the dose range of 20-80mg / kg, hederagenin intervention may increase the gonadotropin-releasing hormone (GnRH) level of cryptorchid mice through some mechanism, thereby promoting the secretion of testosterone (T), which helps to repair the damage caused by cryptorchid surgery to the body. However, it is worth noting that in the lower dose (20mg / kg and 40mg / kg) of hederagenin treatment groups, the GnRH level was significantly reduced, which may be related to the specific mechanism of action of hederagenin and its effect differences at different doses, which requires further research to clarify.

[0119] High levels of estradiol (E) have an adverse effect on the quantity and quality of sperm, thereby reducing male fertility. According to the data shown in Figure D, compared with the BO group (normal control group), the estradiol (E) level in the serum of male mice in the CO group (cryptorchidism control group) did not increase significantly, and this increase did not reach a significant difference (P>0.05). This result still suggests that the spermatogenesis process of cryptorchidism mice may be seriously affected.

[0120] Further analysis found that compared with the CO group, the levels of estradiol (E) in I20, I40, and I80 (representing 20 mg / kg, 40 mg / kg, and 80 mg / kg doses of hederagenin treatment groups, respectively) treated with hederagenin showed a certain degree of downward trend, and this trend was significantly different (P<0.05). In addition, in the higher dose hederagenin treatment group (80 mg / kg), a significant decrease in estradiol (E) levels was observed. However, estradiol (E) levels were still higher than those in the CO group. This result suggests that within the dose range of 20-80 mg / kg, hederagenin intervention may have the potential to reduce estradiol (E) levels in mice, thereby helping to improve spermatogenesis. However, it is worth noting that this reducing effect seems to have a dose effect, that is, as the dose of hederagenin increases, its effect of reducing estradiol (E) levels continues to increase, and at higher doses (>80 mg / kg), there may be a more significant effect of reducing estradiol (E) levels.

[0121] Malondialdehyde (MDA) is a signature product of lipid peroxidation. When a large number of oxygen free radicals are generated in the body, they react with the lipid part of sperm cells to produce malondialdehyde, which has a clear toxic effect on sperm cells. According to the data shown in Figure E, compared with the BO group (normal control group), the malondialdehyde (MDA) level in the serum of male mice in the CO group (cryptorchidism control group) increased, and this increase reached a statistically significant difference (P < 0.01), indicating that cryptorchidism surgery will lead to an increase in malondialdehyde (MDA) levels. This change may be that the mouse body attempts to reduce the level of malondialdehyde (MDA) through some mechanism to cope with the oxidative stress damage caused by cryptorchidism surgery. At the same time, a decrease in malondialdehyde (MDA) levels usually means a decrease in the products of lipid peroxidation, which may be due to the increase in the activity of the body's antioxidant enzymes, which effectively limits the occurrence of oxidative damage. However, after comparing the intervention groups with different doses of hederagenin: compared with the BO group, the MDA levels in the serum of mice in the I40 and I80 groups treated with 40 mg / kg and 80 mg / kg hederagenin increased. The I20 group did not show significant changes in MDA levels. This result shows that within a certain low-dose range, hederagenin can reduce the malondialdehyde (MDA) level in mice, but this increase is not accompanied by a significant improvement in the spermatogenesis process, and there is an obvious dose effect. The effect of hederagenin on MDA levels is not always positive, and its effect is strictly regulated by dose.

[0122] Luteinizing hormone (LH) plays a vital role in the reproductive system. It not only promotes the secretion of testosterone (T), but also works synergistically with follicle-stimulating hormone (FSH) to ensure the normal progress of spermatogenesis.

[0123] according to Figure 3 The data shown in F show that compared with the BO group (normal control group), the luteinizing hormone (LH) level in the serum of male rats in the CO group (cryptorchidism control group) did not decrease. Although this trend did not reach a statistically significant difference (P>0.05), it still indicates that cryptorchidism surgery may have a certain inhibitory effect on the secretion of LH.

[0124] Further comparison of different doses of hederagenin intervention groups: Compared with the CO group, the serum LH level of mice in the I80 group treated with 80 mg / kg hederagenin was significantly reduced (P < 0.05), suggesting that hederagenin may have an inhibitory effect on the secretion of LH at a specific dose. Moreover, when the dose of hederagenin was increased to 80 mg / kg, the LH level of mice in the I80 group still showed a downward trend. In other experimental groups, the LH level was higher than that in the control group.

[0125] This result shows that the effect of hederagenin on LH levels is dose-dependent, that is, it may show different effects at different doses. Low doses of hederagenin may inhibit the secretion of LH, while high doses of hederagenin may promote the secretion of LH, but this promoting effect did not reach statistical significance.

[0126] In summary, this study measured the serum reproductive hormone levels of mice by ELISA method, and found that cryptorchidism surgery reduced testosterone (T) levels and affected spermatogenesis. The effects of hederagenin intervention on reproductive hormones are complex. Low doses (20-40 mg / kg) reduce T levels, while high doses increase T levels; low doses (20-40 mg / kg) increase FSH levels, while high doses reduce them; low doses reduce GnRH, while high doses have no significant changes; it can reduce estradiol (E) levels, but there is a dose effect; the effects on malondialdehyde (MDA) levels are inconsistent, low doses may reduce, while high doses have no significant changes; the effects on luteinizing hormone (LH) are also dose-dependent, with low doses inhibiting and high doses promoting but not reaching significance. Therefore, the mechanism of action and dose effect of hederagenin in the reproductive system need to be further explored. Cryptorchidism surgery can destroy the reproductive internal environment of mice, interfere with the secretion of multiple reproductive hormones in the male hypothalamus-pituitary-testis axis, destroy the spermatogenesis process, and change the levels of hormones related to mouse reproduction. Hederagenin can significantly improve the changes in hormone levels caused by cryptorchidism surgery and is beneficial to spermatogenesis in mice. There is a dose effect, and 20 mg / kg may be the optimal concentration of hederagenin to promote spermatogenesis.

[0127] 3.3 Mouse sperm count analysis

[0128] Normal sperm should be tadpole-shaped, consisting of a head, body, and tail. Through HE staining, you can see that the acrosome is light red, the nucleus is located at the base of the acrosome, which is purple-red, and the tail is light purple-red. There are many types of abnormal sperm morphology, such as large head, small head, cone head, amorphous head, vacuolar head, double head, acrosome-free head, etc. These abnormal morphologies may be related to infection, trauma, androgen changes or chemical products and genetic factors.

[0129] During the mouse dissection, each experimental group was observed and recorded in detail. The specific operation steps included: placing the bilateral epididymis of each group of mice in a 1.5mL centrifuge tube containing 200μL phosphate buffered saline (PBS) and carefully processing it until the tissue fragments were no longer significantly visible. Subsequently, 10μL of supernatant was extracted and added to a sperm counting plate for accurate sperm count determination.

[0130] It is worth noting that no sperm could be detected in the mice of the CO cryptorchidism group, which confirmed the successful establishment of the cryptorchidism azoospermia model.

[0131] In contrast, the BO normal group showed a higher sperm count, which was significantly better than the other experimental groups.

[0132] Furthermore, it was found that the sperm count of each group of mice treated with ivy sapogenin increased. Although it was still lower than that of the BO normal control group, this difference revealed that the substance had a positive effect on mouse spermatogenesis to some extent.

[0133] like Figure 4 The figure shows the analysis of sperm count in each group of mice. A: BO normal control group; B: CO cryptorchidism control group; C: Hederagen H20 experimental group; D: Hederagen H40 experimental group; E: Hederagen H80 experimental group. Scale: Microscope 20×10. In the figure, the red arrow marks the sperm.

[0134] like Figure 5 Shown is a schematic diagram of sperm counts in each group of mice.

[0135] like Figure 4 and Figure 5 The results showed that the sperm count in the hederagenin H80 group was particularly outstanding, exceeding that of other experimental groups.

[0136] The above conclusions not only demonstrate the effectiveness of the cryptorchidism model, but also prove the potential promoting effect of ivy sapogenin on spermatogenesis.

[0137] 3.4 Mouse HE staining

[0138] During the dissection, special attention was paid to changes in the mouse testicles.

[0139] The testicles of mice in each group were placed in Bouin's fixative, then embedded in paraffin and stained with HE.

[0140] like Figure 6 The figure shows the schematic diagram of HE staining microscope photography of each group of mice. A: BO normal control group; B: CO cryptorchidism control group; C: Hederagenin H20 experimental group; D: Hederagenin H40 experimental group; E: Hederagenin H80 experimental group. The scale bar is microscope 20×10.

[0141] Depend on Figure 6 It can be seen that by observing and taking pictures under a microscope, subtle differences in tissues can be captured.

[0142] Observation of the testicular tissue sections of each group of mice under a light microscope revealed that:

[0143] The testes of mice in the CO cryptorchidism group showed obvious abnormalities, including enlarged lumina of the seminiferous tubules, disordered cell arrangement, thinning of the tubular wall, and obvious germ cell fragments.

[0144] This suggests that the high temperature environment caused by the surgery interfered with the normal spermatogenesis process, resulting in impaired sperm production.

[0145] However, in mice treated with hederagenin H80, the structure of the seminiferous tubules was relatively healthy, the cells were arranged more orderly, the lumen was smaller, and they contained mature sperm, showing a certain degree of protective effect.

[0146] Nevertheless, compared with the BO normal control group, the lumen of the seminiferous tubules in the treatment group was still enlarged and the number of sperm was lower, revealing the role of ivy sapogenin in maintaining or restoring spermatogenesis in mice.

[0147] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of hederagenin in the preparation of product preparations for the treatment of cryptorchidism.

2. Application of ivy sapogenin in the preparation of products for treating spermatogenesis disorders.

3. Application of ivy sapogenin in the preparation of product preparations for maintaining and restoring spermatogenesis in cryptorchid testes.

4. Application of ivy sapogenin in the preparation of preparations for improving cryptorchid testicular structure and testicular repair products.

5. A hederagenin product preparation, characterized in that: Including any one of the product preparations for treating cryptorchidism with ivy sapogenin as the main active ingredient, product preparations for treating spermatogenesis disorders, product preparations for promoting spermatogenesis in cryptorchidism, product preparations for protecting the structure of the seminiferous tubules in cryptorchidism and product preparations for testicular repair.

6. A hederagenin product preparation according to claim 5, characterized in that: The use of the product preparation in increasing sperm count.

7. A hederagenin product preparation according to claim 5, characterized in that: The product preparation is used in improving the testicular organ coefficient and protecting the testicular seminiferous tubule structure.

8. A hederagenin product preparation according to claim 5, characterized in that: The product preparation also contains pharmaceutically acceptable excipients to prepare pharmaceutically corresponding dosage forms.

9. A hederagenin product preparation according to claim 8, characterized in that: The dosage of the hederagenin is 20-100 mg / kg.

10. A hederagenin product preparation according to claim 9, characterized in that: The dosage of the hederagenin is 80 mg / kg.

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