Application of ebbeselenium in preparation of medicine for treating male delayed hypogonadism

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of ebbeselenium in preparation of a medicine for treating male delayed gonadal hypofunction. After the ebbeselenium is adopted for intervention, the autophagy activity of testicular interstitial cells of old mice is increased, lipid autophagy is enhanced, testosterone synthesis is promoted, the serum testosterone level is remarkably improved, and the symptoms of male delayed hypogonadism (LOH) are effectively relieved; ebbeselenium simultaneously shows a relatively good delaying effect on senescence of a plurality of organs. The ebbeselenium can improve the reproductive capacity, the behavior memory capacity, the athletic capacity and the metabolic function of old mice at the same time, and synergistic and effective treatment of low serum testosterone and LOH complications is achieved. Besides, by enhancing the molecular reduction effect of PRDX1 targeting ATG4B, the ebbeselenium regulates and controls the autophagy pathway of testicular interstitial cells and enhances the functions of the testicular interstitial cells so as to promote synthesis of testosterone, and a new treatment strategy is provided for treatment of LOH.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly relates to application of ebselen in preparation of a medicine for treating male late-onset hypogonadism. BACKGROUND

[0002] Male late-onset hypogonadism (LOH), also known as age-related testosterone deficiency syndrome, refers to a series of clinical and biochemical syndromes with low serum testosterone level as the main feature in men with age increasing. Starting from 40 years old, the serum testosterone of men decreases at a rate of about 1-2% per year. LOH not only causes male sexual dysfunction and decreased fertility, but also leads to muscle atrophy, osteoporosis and obesity and other systemic symptoms, and significantly increases the risk of major chronic diseases such as diabetes, cardiovascular and cerebrovascular diseases and Alzheimer's disease. More than 95% of testosterone in the human body is synthesized by Leydig cells, and is mainly regulated by the neuroendocrine pathway of the hypothalamic-pituitary-testicular axis.

[0003] Current research in the treatment or relief of male late-onset hypogonadism includes: 1. Hormone replacement therapy: (1) Testosterone replacement therapy (TRT): is the main method to treat late-onset hypogonadism (LOH), by injecting testosterone propionate, testosterone enanthate or testosterone undecanoate preparations, oral testosterone undecanoate, applying testosterone-containing gels or patches on the skin, subcutaneously implanting testosterone-containing pills or using nasal sprays, etc. to supplement exogenous testosterone to increase serum testosterone levels. Disadvantages: There are some obvious side effects, such as significantly increasing the risk of heart disease, stroke and prostate cancer; in addition, TRT also inhibits the feedback loop of the hypothalamic-pituitary-gonadal axis; the long-term efficacy is also unstable, and the safety risk will also increase. (2) Gonadotropin replacement therapy: by directly giving gonadotropins (such as human chorionic gonadotropin hCG and human menopausal gonadotropin hMG) to stimulate testicular Leydig cells to produce testosterone. Disadvantages: High treatment cost; complex operation. Long-term use will also inhibit the synthesis of original hormones and destroy endocrine stability. (3) GnRH replacement therapy: by pulsatile administration of GnRH to stimulate the pituitary to secrete gonadotropins, thereby promoting testicular Leydig cells to produce testosterone. Disadvantages: Requires complex drug delivery equipment, requires professional operation, high treatment cost; requires high patient compliance. 2. Surgical treatment: For some LOH patients due to testicular lesions, testicular resection or repair surgery may be required. Disadvantages: Surgical treatment is traumatic; recovery time is long; and the risk is high, which may permanently damage the fertility. 3. Stem cell therapy: (1) Testicular Leydig stem cell (SLCs) therapy: SLCs isolated from adult testicular tissue have the ability of self-renewal and can differentiate into testicular Leydig-like cells, which can activate testicular Leydig cells (LCs) regeneration through allogeneic transplantation. Disadvantages: SLCs are scarce; immune rejection is easy to occur after transplantation; surgical treatment of transplanted human SLCs may affect fertility; transplanted SLCs have a short survival time, requiring repeated and regular transplantation, and their colonization and function are uncertain. (2) Induced pluripotent stem cell (iPSC) therapy: peripheral blood mononuclear cells are induced into iPSCs using a multi-stage induction protocol, and gene editing technology is used to induce differentiation into testicular Leydig stem cells (SLCs). Induction success criteria: In addition to expressing CYP21B and most steroidogenesis genes, it can also respond to hCG / luteinizing hormone signals, and the concentration of testosterone and aldosterone / cortisol in the culture supernatant is verified. Disadvantages: The direction of induced differentiation is not unique, and multiple human testicular Leydig-like or adrenal-like cells may be produced; the product is not unique, and multiple steroid hormones may be produced; the survival time of induced SLCs is short; exogenous genome insertion may not be safe for future clinical applications.(3) Bone marrow mesenchymal stem cell (MSC) therapy: using different concentrations of gonadotropin / luteinizing hormone (HMG / LH), hCG, platelet-derived growth factor (PDGF) and interleukin-1α to induce human bone marrow MSCs to differentiate into testicular interstitial-like cells expressing steroidogenic enzyme 3β-HSD and capable of synthesizing testosterone. Disadvantages: the differentiation mechanism of bone marrow mesenchymal stem cells MSCs into testicular interstitial cells LCs is not clear; the safety of MSCs differentiated SLCs has not been verified. (4) Lifestyle intervention: including adjusting diet and living schedule, increasing exercise, and reducing body weight, etc. These methods can improve overall health and help alleviate LOH symptoms, but the effect varies from person to person, requires long-term adherence, and most of the effects are not obvious.

[0004] The above technical methods or drugs have certain potential in the treatment of LOH, but their actual application is limited by factors such as cost, technical requirements, individual differences of patients, and side effects. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art and provide a use of ebselen in the preparation of a drug for treating male late-onset hypogonadism.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0007] The present application provides a use of ebselen in the preparation of a product for treating male late-onset hypogonadism.

[0008] The inventors of the present application have found through extensive research and experiments that ebselen, which is easy to produce and obtain, can effectively treat or alleviate late-onset hypogonadism (LOH), and ebselen has a significant delaying effect on the aging of multiple organs.

[0009] Specifically, ebselen can significantly enhance the lipid autophagy activity of testicular interstitial cells (LCs), increase the level of testosterone, and effectively alleviate LOH-related symptoms; ebselen can also improve the reproductive, behavioral memory and motor ability, and metabolic function of old mice, achieving a synergistic and effective treatment of low serum testosterone and LOH complications.

[0010] The English name of ebselen is Ebselen, and the chemical name is 2-phenyl-1, 2-benziselenazol-3 (2H) -one, which is an organic selenium compound with antioxidant properties. The production cost of ebselen is low, which is conducive to rapid access and wide application, so that the whole treatment process is more economical; The side effects of ebselen are small, and the safety is high, and the test animals have no obvious abnormal reaction, normal diet, normal growth and development, and no abnormality in neural behavior, which can greatly improve the treatment comfort and compliance of patients; And ebselen does not need special low-temperature preservation, and can be administered by oral or intravenous injection, which is convenient and fast.

[0011] As a preferred embodiment of the application described herein, the product comprises a drug or a preparation.

[0012] As a preferred embodiment of the application described herein, the product is a preparation, and the ebselen is prepared into a preparation by dissolving with a solvent.

[0013] As a preferred embodiment of the application described herein, the solvent comprises dimethyl sulfoxide.

[0014] As a preferred embodiment of the application described herein, the dosage form of the product comprises an injection or an oral agent.

[0015] The present application can be orally or intravenously administered by a simple and convenient administration method, which is convenient to use and reduces the consumption of other medical resources.

[0016] Oral administration is the most common administration method, which has the advantages of simplicity, convenience and low cost, and is suitable for self-administration by patients. Ebselen, as a small molecule compound, can be absorbed into the body by oral administration to play a role, but at the same time, the influence of food intake, gastric emptying speed and other factors on the bioavailability of the drug should be considered. Ebselen is administered by intravenous injection, which is rapidly transported to all organs of the body through the blood system to improve the effect of the drug.

[0017] As a preferred embodiment of the application described herein, the preparation is applied to animal experiments by intraperitoneal injection, and the intervention dose of ebselen is 20 mg / kg / d.

[0018] As a preferred embodiment of the application described herein, the intervention time of ebselen is 1 month.

[0019] In some specific embodiments, the animal experiment is a 20-month-old normal old mouse and a galactoside-induced premature aging mouse model. Ebselen is prepared into a simple drug-physiological saline mixture, and the premature aging mouse model is intraperitoneally injected.

[0020] And, the primary cultured mesenchymal cells are intervened for 24 hours by ebselen, and the testosterone level produced is detected.

[0021] By simple administration and short experimental period, combined with in vivo and in vitro experiments, and using multiple models, it is found that the compound Ebselen promotes the molecular reduction of PRDX1-specific target autophagy molecule ATG4B. By regulating the autophagy pathway of Leydig cells LCs, the lipid autophagy homeostasis is maintained, the formation of testosterone is promoted, the serum testosterone level is improved, and the whole body metabolism and exercise capacity are improved, effectively treating or relieving late-onset hypogonadism (LOH); at the same time, Ebselen has obvious delaying effect on the aging of multiple organs.

[0022] As a preferred embodiment of the application described in the application, the preparation is applied to cell experiments, and the mass concentration of Ebselen in the preparation is 20 μM.

[0023] Ebselen can be applied to all age groups of patients, especially to the elderly, and has the effects of anti-aging, blood glucose reduction and blood lipid reduction, achieving the synergistic effect of one drug for multiple treatments. Ebselen has long stability at room temperature and can be stored at 4°C for a long time, which is suitable for different occasions and conditions, and is convenient for use and transportation.

[0024] As a preferred embodiment of the application described in the application, the intervention time of Ebselen is 24 hours.

[0025] In the technical solution of the application, Ebselen effectively protects the testis morphology of normal old mice and galactoside-induced early aging mice, promotes the survival of germ cells, increases the number and activity of sperm, and significantly improves the testosterone level in the testis and serum. Ebselen improves the memory and behavior exploration ability of old and early aging mice, which is manifested as an increase in the number of alternating behaviors in the Y maze test and an increase in the total distance and exploration behavior in the open field test. Ebselen increases the bone mineral density of old and early aging mice, and the ATP production capacity in skeletal muscle is stronger, and the exercise endurance is further improved. Ebselen improves the liver function of old and early aging mice, alleviates the accumulation of lipids in the liver, and reduces the levels of lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and total bilirubin in the serum; the abnormal increase of the concentrations of triglyceride, total cholesterol and blood glucose in the serum is obviously improved.

[0026] As a preferred embodiment of the application described in the application, Ebselen enhances the molecular reduction of peroxiredoxin 1 targeting ATG4B, promotes the lipid autophagy and testosterone synthesis of Leydig cells.

[0027] Experiments show that ebselen can promote the production of testosterone by enhancing the lipid autophagy function of Leydig cells through the following ways: enhancing the molecular reduction of PRDX1 targeting ATG4B, maintaining the enzyme activity of ATG4B, promoting the autophagy process, regulating the autophagy pathway of Leydig cells, and enhancing the lipid autophagy function of Leydig cells. Ebselen can also reduce oxidative stress and inflammation in cells, reduce oxidative damage, and has obvious anti-aging effect.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] The present application provides a use of ebselen in the preparation of a drug for treating male late-onset hypogonadism. After intervention with ebselen, the autophagy activity of Leydig cells in old mice increases, the lipid autophagy is enhanced, the synthesis of testosterone is promoted, and the serum testosterone level is significantly improved, effectively relieving the symptoms of male late-onset hypogonadism (LOH). Ebselen also shows good delaying effect on the aging of multiple organs. Ebselen can simultaneously improve the reproductive, behavioral memory and motor ability and metabolic function of old mice, achieving synergistic and effective treatment of low serum testosterone and LOH complications. In addition, ebselen can regulate the autophagy pathway of Leydig cells by enhancing the molecular reduction of PRDX1 targeting ATG4B, enhance the function of Leydig cells, and promote the synthesis of testosterone, providing a new treatment strategy for the treatment of LOH. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure (1) of the experimental results of Example 1 (wherein, Figure 1 In a, the body weight (body) and testis weight (testis) change curves of the control group (Ctrl) and Prdx1 conditional gene knockout group mice (cKO) are shown; Figure 1 In b, the cumulative number of offspring of Ctrl and cKO is shown. Figure 1 In c, the number of sperm of Ctrl and cKO is shown. Figure 1 In d, the sperm motility of Ctrl and cKO is shown. The sperm movement type is divided into: type a (fast forward movement), type b (slow or sluggish movement), and type c (non-forward movement). Figure 1 In e, the level of testosterone in the testis of Ctrl and cKO is shown. Figure 1 In f, the concentration of testosterone in the testis of 10-20 month-old Ctrl and cKO is shown. n=5 for each group. Statistical annotation: *, P<0.05, **, P<0.01, ***, P<0.001; n.s. represents no significant difference.

[0031] Figure 2 Figure (2) of the experimental results of Example 1 (wherein, Figure 2 In a, the ELISA detection of serum luteinizing hormone (LH) level of Ctrl and cKO is shown. Figure 2Medium b is the analysis of the expression levels of testosterone synthesis-related genes. Figure 2 Medium c-d is the total triglyceride (TG, c) and total cholesterol (TC, d) content of testicular tissue. Figure 2 Medium e-g is the total triglyceride (e), total cholesterol (f) and free cholesterol (FC, g) content in Leydig cells. Figure 2 Medium h is the ratio of free cholesterol to total cholesterol in Leydig cells. Figure 2 Medium i is the expression levels of Ldlr and Scarb1 genes in testis. Figure 2 Medium j is the detection of the expression levels of adult and embryonic Leydig cell markers in testis. Figure 2 Medium k-l is the Oil Red O staining showing lipid deposition in the Leydig compartment of testis. Classification by lipid droplet diameter: S-type, <0.5 μm; M-type, 0.5-1.0 μm; L-type, >1 μm. Figure 2 Medium m-n is the BODIPY staining (green) showing lipid deposition in the Leydig compartment of testis. Figure 2 Medium o-q is the co-localization analysis of BODIPY (green) and LC3 (red) in the Leydig compartment of testis. Figure 2 Medium r-s is the transmission electron microscopy showing the contact of lipid droplets (LD, yellow star) and autophagic vesicles (AV, blue triangle) in the Leydig compartment, Figure 2 Medium s is the statistics of AV-associated LD. n=5 per group. Statistical annotation: *, P<0.05, **, P<0.01, ***, P<0.001; n.s. represents no significant difference. Nuclei DAPI staining (blue). Figure 2 The scale bar of medium k is 10 μm, Figure 3 Medium m, Figure 3 The scale bar of medium o is 20 μm, Figure 3 The scale bar of medium r is 1 μm);

[0032] Figure 3 is the experimental result (3) figure of Example 1 (wherein, Figure 3 Medium a-b is the H&E staining of testis tissue of aged Ctrl (Ctrl), aged cKO (cKO), aged Ctrl and cKO intervened with ebselen (Ctrl+Ebs, cKO+Ebs) and PRDX1 conditional overexpression mice (cOE). d in figure a represents the diameter of seminiferous tubule, r represents the thickness of seminiferous epithelium. Figure 3 Medium c-d is the analysis of sperm number (c) and sperm motility (d) of aged mice. Figure 3 Medium e-f is the detection of the levels of testosterone (e) in testis tissue and serum testosterone (f) of aged mice. Figure 3 Medium g is the difference in the expression of LC3 protein in testis of young adult mice and aged mice. Figure 3 Medium h-j is the co-localization analysis of BODIPY (green) and LC3 (red) in the Leydig compartment of testis tissue of aged mice.Figure 3 k-l is the contact of lipid droplets (LD, yellow star) in the Leydig cell compartment with autophagic vesicles (AV, blue triangle). Figure 3 m-p is the evaluation of cognitive behavior and motor ability in old mice. n=5 per group. Statistical notation: *, P<0.05, **, P<0.01, ***, P<0.001; n.s. indicates no significant difference. Nuclei were stained with DAPI (blue). Figure 4 a, scale bar is 20 μm, Figure 4 h, scale bar is 50 μm, Figure 4 k, scale bar is 5 μm;

[0033] Figure 4 Figure (4) of Example 1 (wherein, Figure 4 a is the protein expression level of LC3 after treatment of primary Leydig cells (LCs) with ebselen. Figure 4 b-d are the co-fluorescence imaging of BODIPY 493 / 503 (green) and LC3 (red) after treatment of primary LCs with ebselen. Figure 4 e-f are micro-CT images and quantitative analysis of bone volume / tissue volume. Figure 5 g-h are the detection of lipid droplets (LDs) deposition in the liver by oil red O staining and quantitative analysis. Figure 5 i-o are the concentrations of lactate dehydrogenase (i), alanine aminotransferase (j), aspartate aminotransferase (k), total bilirubin (l), total cholesterol (m), triglyceride (n) and glucose (o) in serum. n=5 per group. Statistical notation: *, P<0.05, **, P<0.01, ***, P<0.001. n.s., no significant difference. Figure 5 b, scale bar is 10 μm;

[0034] Figure 5 Figure (5) of Example 1 (wherein, Figure 5 a-f are the immunofluorescence staining and quantitative analysis of γ-H2AX in the testis (a), brain (c) and liver (e) of old mice (b, d, f). Figure 5 g-j are the β-galactosidase staining and quantitative analysis (h, j) in the testis (g) and liver (i) of old mice. n=5 per group. Statistical notation: *, P<0.05, **, P<0.01, ***, P<0.001. n.s., no significant difference. Nuclei were stained with DAPI (blue). Scale bar: Figure 5 a, Figure 5 e, Figure 6 g, Figure 6 i, scale bar is 50 μm; Figure 6Scale bar 100 pm in c.

[0035] Figure 6 Figure (6) for the experimental results of Example 1, in which, Figure 6 Figure a-b are the testis morphology of mice in Ctrl, D-galactose-induced aging group and ebselen treatment group. In the figure, d represents the diameter of the seminiferous tubule, and r represents the thickness of the spermatogenic epithelium. The quantitative data are shown in Figure 6 Figure b. Figure 6 Figure c-d are the sperm number (c) and motility (d). Figure 6 Figure e-f are the testis and serum testosterone levels. Figure 6 Figure g is the Western blot (WB) analysis of LC3 levels in testis. Figure 6 Figure h-i are oil red O staining and quantitative analysis. Figure 6 Figure j-l are the co-fluorescence imaging of BODIPY493 / 503 (green) and LC3 (red) in testis. Figure 6 Figure m-n are transmission electron microscopy (TEM) images showing lipid droplets (LDs) (yellow star) and autophagic vesicles (AVs, blue triangle) in the interstitial area of testis. Figure 6 Figure o-r are Y maze test (o), exhaustive running test (p) and open field test (q) to evaluate memory, behavior and maximum exercise capacity; the trajectory image of open field test is shown in Figure 6 Figure r. Figure 6 Figure s-u are the immunofluorescence staining of γ-H2AX in liver (s), hippocampus (t) and testis (u). The quantitative analysis is shown in Figure 6 Figure w-y. Figure 6 Figure v is the β-galactosidase staining in testis. The quantitative analysis is shown in Figure 7 Figure z. n=5 in each group. Statistical annotation: *, P<0.05, **, P<0.01, ***, P<0.001. n.s., no significant difference. Scale bar 50 pm in (a), (r), (s), (t), Figure 7 Figure h, Figure 7 Figure j. Scale bar 10 pm in c. Figure 7 Figure m. Scale bar 1 pm in c. Figure 1 Figure u. Scale bar 100 pm in c.

[0036] Figure 1 Figure for the results of Comparative Example (Se-(methyl)selenocysteine hydrochloride as the comparative compound, and ebselen were used to treat aged primary leydig cells (LCs, cells extracted from 20-month-old aged mice) respectively. Figure 1 Figure a is the WB detection of autophagy levels of aged LCs under the treatment of 2 compounds. Figure 2b. Immunofluorescence detection of the co-localization of LC3 and LAMP1 under the treatment of the two compounds, i.e. autophagic flux. c. Testosterone levels in the supernatant of the cell culture medium after 24 h of treatment with the two compounds. **, P < 0.01, ***, P < 0.001 Figure 2 b. Scale bar, 1 μm. DETAILED DESCRIPTION

[0037] For the purpose of better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described below in conjunction with the drawings and specific embodiments.

[0038] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified, and the components of the raw materials used in each parallel experiment are the same.

[0039] In the following examples:

[0040] The mice were raised in a SPF environment, with free access to water and food, while avoiding wound infection. At the end of the drug intervention, the mice were euthanized, in accordance with the animal ethics standards, to minimize the pain and discomfort of the animals.

[0041] Example 1, application of ebselen in the preparation of a drug for treating male late-onset hypogonadism

[0042] (1) Extraction, culture and drug intervention of primary interstitial cells: 4-month-old and 20-month-old wild-type mice were taken, and after removing the testicular white membrane, the testicular tissue was subjected to collagenase IV enzymolysis treatment. The crude interstitial cells were purified by gradient Percoll. The purified primary cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. Ebselen was intervened at a dose of 20 μM, and the testosterone level in the culture medium and the autophagy pathway and lipid autophagy activity in the cells were detected after 24 h.

[0043] (2) Experimental animals, model construction and drug intervention: 2-month-old to 12-month-old Prdx1 conditional knockout mice and their control mice were not intervened, and were directly taken.

[0044] The 19-month-old control / Prdx1 conditional knockout mice were injected intraperitoneally with ebselen at a dose of 20 mg / kg / d for one month.

[0045] D-galactose-induced premature aging mouse model: 2-month-old wild-type C57 mice were injected intraperitoneally with D-galactose at a dose of 500 mg / kg / d. After 1 month, both ebselen (20 mg / kg / d) and D-galactoside (500 mg / kg / d) were injected simultaneously, and the intervention was continued for another month. All mice were raised in the SPF animal experiment center, with constant temperature (21±3) °C, humidity (60±5) %, and a light-dark cycle set to 12 hours of light + 12 hours of darkness.

[0046] (3) Observation index: observe the autophagy activity and lipid autophagy state of testicular interstitial cells, serum testosterone level, testicular tissue morphology, sperm number and motility, mouse memory, behavior and motor ability, bone density, skeletal muscle weight and ATP level in skeletal muscle, liver function, blood lipid and blood glucose level, etc.

[0047] (4) Data analysis: after collecting the data, appropriate statistical methods are used for analysis to compare the differences between the ebselen treatment group and the control group.

[0048] Experimental results:

[0049] (1) Deletion of Prdx1 gene in testicular interstitial cells causes testicular premature aging and significant decrease in testosterone level, which is highly consistent with the pathological characteristics of LOH.

[0050] The testicular weight of Prdx1 gene knockout (cKO) mice decreased significantly after 4 months of age, and the body weight began to decrease after 7 months of age. The reproductive ability of cKO mice began to decrease at 3 months of age, and completely lost at 6 months of age, which was about 5 months earlier than that of control mice (Ctrl). The sperm count of cKO mice decreased significantly at 10 months of age, but the sperm motility decreased significantly as early as 2 months of age. The testosterone level in the testis of cKO mice decreased significantly at 2 months of age, was comparable to that of Ctrl at 3 months of age, and decreased to the level of 20-month-old Ctrl mice at 10 months of age.

[0051] Mice at 3 months of age are equivalent to humans at 25 years of age, and mice at 9-10 months of age are equivalent to humans at 40 years of age, and mice at 20 months of age are equivalent to humans at 60 years of age. This shows that the change of testosterone in Prdx1 gene knockout mice in interstitial cells is similar to that of humans with LOH, and can be used as a model to explore the mechanism of testosterone level decrease and rescue scheme of LOH.

[0052] As Figure 2 ( Figure 3 in a to Figure 3 in f), the above results prove that dysfunction of PRDX1 in testicular interstitial cells can cause low testosterone, accompanied by decreased sperm motility and male sterility, which are highly consistent with the pathological characteristics of clinical LOH patients.

[0053] (2) Autophagy pathway and lipid autophagy process blockage in Leydig cells, causing testosterone synthesis disorder.

[0054] The present application then explores the potential mechanism of Prdx1 inactivation leading to testosterone deficiency. Serum luteinizing hormone (LH) levels show no significant difference between Ctrl and cKO groups, ruling out the possibility of impaired pituitary-gonadal axis function. qRT-PCR analysis shows that the key enzymes involved in testosterone synthesis are significantly reduced in cKO testes, indicating that the steroid synthesis pathway is not impaired. By detecting the lipid metabolism pathway, the present application finds that although the total triglyceride and total cholesterol content of the whole testis tissue shows no change, the levels of these two lipids in cKO Leydig cells are significantly increased. On the contrary, the free cholesterol, which is a direct substrate for testosterone synthesis, and its proportion are significantly reduced in cKO Leydig cells. And the mRNA expression of Ldlr and Scarb1, the key receptors that mediate the uptake of low-density lipoprotein and high-density lipoprotein, shows no difference between the two groups, suggesting that the cholesterol uptake pathway in cKO Leydig cells is not affected. By detecting the expression of embryonic (FLC) and adult (ALC) Leydig cell markers by qPCR, the influence of cKO Leydig cell differentiation is further ruled out. In summary, Prdx1 regulates the homeostasis of cholesterol and free cholesterol in Leydig cells, and too little free cholesterol triggers testosterone synthesis disorder, leading to LOH.

[0055] Further through oil red O and BODIPY staining, it is shown that the lipid droplets of triglycerides and cholesteryl esters abnormally accumulate in the Leydig area of cKO testes. Given the key role of autophagy in the conversion of cholesteryl esters to free cholesterol, the present application finds that the degree of co-localization of LC3 and lipids in the Leydig area of cKO testes is significantly reduced through BODIPY and autophagy marker protein LC3 co-localization analysis. Transmission electron microscopy directly shows that lipid droplets accumulate in large quantities in cKO Leydig cells, and there is little contact or coating phenomenon with autophagosomes. These evidences collectively indicate that the autophagy flow and lipid degradation process blockage in Leydig cells is one of the causes of LOH, and the results are as follows: Figure 3 ( Figure 4 in a to Figure 4 in s).

[0056] (3) Low testosterone levels accelerate testicular aging, causing LOH, and ebselen intervention can effectively improve the symptoms related to LOH.

[0057] Compared with old Ctrl mice, the testes of old cKO mice are significantly degenerated, including severe seminiferous tubule atrophy, thinning of the epithelial layer, reduction of germ cells, and increased germ cell apoptosis; the number and quality of sperm are significantly reduced, and the levels of testosterone in the testis and serum are significantly reduced. This shows that the deletion of PRDX1 exacerbates the testicular aging caused by age increase.

[0058] Ebselen, a mimic of PRDX1, effectively alleviated the atrophy of seminiferous tubules, improved sperm quantity and quality, and increased the level of testosterone in testis and serum. It indicated that PRDX1 overexpression significantly reversed the decrease of testosterone in Leydig cells and effectively alleviated the aging of male reproductive system. Similarly, PRDX1 overexpression not only reversed the aging phenotype of aged Ctrl, but also delayed the accelerated aging caused by Prdx1 deletion.

[0059] The level of LC3 was decreased in aged mice compared with young mice. Both ebselen intervention and PRDX1 overexpression significantly increased the level of LC3. TEM analysis confirmed the significant accumulation of lipid droplets and the decrease of lipid autophagy in the testis of aged cKO mice. Both ebselen treatment and PRDX1 overexpression significantly improved the activity of lipid autophagy, thereby increasing the level of testosterone, as shown in Figure 4 ( Figure 5 in a to Figure 5 in p).

[0060] 4) Ebselen can enhance the lipid autophagy of Leydig cells and improve the aging indicators of the body.

[0061] In vitro ebselen treatment also promoted the lipid autophagy in young and aged primary Leydig cells (LCs).

[0062] The aging indicators caused by low testosterone are closely related to LOH-related syndromes, including but not limited to memory, behavior and motor ability, osteoporosis, obesity and cardiovascular dysfunction. From the number of alternation behavior (measured by Y maze test), total distance and exploration behavior (measured by open field test), and fatigue time (measured by running fatigue test), ebselen effectively improved the memory, behavior and maximum motor ability parameters of aged Ctrl and cKO mice. It indicated that ebselen can effectively restore the testosterone level of aged mice and alleviate the symptoms related to LOH.

[0063] Ebselen also increased the bone density value of aged cKO mice. In aged cKO mice, there was a significant accumulation of lipids in the liver, which was significantly alleviated after ebselen treatment. The accumulation of lipids in the liver caused damage to liver function, and the serum lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and total bilirubin levels of aged cKO mice were increased, which were improved after ebselen intervention, indicating that ebselen treatment can improve liver function. In addition, the serum triglyceride, total cholesterol and glucose levels were significantly restored after ebselen intervention, indicating that ebselen can improve the blood lipid and blood glucose levels of aged mice, as shown in Figure 5 ( Figure 6 in a to Figure 6 in o).

[0064] (5) Ebselen intervention and PRDX1 overexpression can reduce the biochemical indicators of aging in multiple organs of old mice.

[0065] In old cKO mice, the levels of γ-H2AX in testis, brain and liver were significantly increased, and the corresponding overexpression of PRDX1 can significantly reduce the fluorescence level of γ-H2AX, which indicates that the DNA damage caused by aging in testis, brain and liver is significantly reduced. Similarly, ebselen treatment rescued the aging damage in old Ctrl and old cKO. β-galactosidase activity is a marker of aging, and β-GAL staining showed the same trend. It can be seen that ebselen intervention significantly delayed cell aging in multiple organs of old Ctrl and cKO mice, and the results are as follows Figure 6 ( Figure 7 in a to Figure 7 in j).

[0066] (6) Ebselen can also significantly alleviate the symptoms of LOH and organ aging caused by D-galactoside.

[0067] Using a β-galactoside-induced aging mouse model, further test the rescue of ebselen on aging. The results show that ebselen intervention significantly restores the sperm motility and testosterone levels of D-galactoside mice, which may be achieved by enhancing the lipophagy of testicular interstitial cells. In this model, the behavior and exercise-related indicators, cell aging in testis, brain and liver were significantly improved. These results consistently show that ebselen can effectively restore testosterone levels and alleviate LOH-related symptoms, and the results are as follows ​ (As ​ in a to ​ in z).

[0068] Comparative Example 1

[0069] Comparative Example 1 uses another selenium-containing compound, Se-(methyl) selenocysteine hydrochloride, chemical formula: C4HgNOzSe·HCI. Se-(methyl) selenocysteine is an effective chemotherapeutic drug for treating breast cancer, and is rich in selenium-enriched plants such as broccoli, garlic, onions, wild leeks, etc., and is also an important component of nutritional health products.

[0070] Se-(methyl)seleno-cysteine hydrochloride was purchased from Sigma (M6680). LCs were extracted from 20-month-old male mice and primary cultured. The cells were treated with 20 μΜ ebselen or 20 μΜ Se-(methyl)seleno-cysteine hydrochloride, respectively. The analysis was performed 24 h later. WB and IF showed that ebselen significantly increased the autophagy activity of aged LCs, but Se-(methyl)seleno-cysteine hydrochloride had weak effect. The level of testosterone in the supernatant of cell culture was detected, which showed that ebselen could significantly promote the synthesis and secretion of testosterone in aged LCs, but Se-(methyl)seleno-cysteine hydrochloride had weak effect. The above results suggest that the promotion of ebselen on testosterone synthesis and secretion is specific and effective, and the results are as follows ​ (As ​ in a-c).

[0071] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. Use of ebselen in the preparation of a product for treating male hypogonadism.

2. Use according to claim 1, wherein The product comprises a drug or a preparation.

3. The use according to claim 1, wherein The product is a preparation, and the ebselen is prepared into the preparation by dissolving with a solvent.

4. Use according to claim 3, wherein the compound is ###0002### The solvent comprises dimethyl sulfoxide.

5. The use according to claim 1, wherein the compound is ###0002### The dosage form of the product comprises an injection or an oral agent.

6. The use according to claim 3, wherein the compound is ###0002### The preparation is applied to an animal experiment by intraperitoneal injection, and the intervention dose of the ebselen is 20 mg / kg / d.

7. Use according to claim 6, wherein The intervention time of the ebselen is 1 month.

8. The use according to claim 1, wherein The preparation is applied to a cell experiment, and the mass concentration of the ebselen in the preparation is 20 μM.

9. Use according to claim 8, wherein the compound is ###0002### The intervention time of the ebselen is 24 hours.

10. The use according to claim 1, wherein The ebselen enhances the molecular reduction of peroxiredoxin 1 targeting ATG4B, promotes the lipid autophagy of testicular interstitial cells, and promotes the synthesis of testosterone.

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