Compounds for increasing the level of testosterone secretion and uses thereof

Abstract

The present application discloses a compound for improving the secretion level of testosterone and its use. In the present application, the over-sulfur glutathione or the composition containing the same can be used for preparing a medicine for preventing and / or treating a disease related to testosterone deficiency; and for preventing and / or treating the disease related to testosterone deficiency, reducing the side effects caused by directly supplementing testosterone.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to compounds that increase testosterone secretion levels and their uses. Background Technology

[0002] Testosterone is secreted by interstitial cells in the testes and is crucial for gonadal development, spermatogenesis, maintenance of secondary sexual characteristics, and gonadal function. Testosterone production is regulated by the hypothalamic-pituitary-testicular (HPT) axis. Luteinizing hormone (LH) secreted by the pituitary gland binds to LH receptors on the cell membrane, activating G protein-coupled receptors to promote the production of 3',5'-cyclic adenosine monophosphate (cAMP) and the phosphorylation of cAMP-dependent protein kinase A (PKA). PKA activation promotes the expression of downstream steroid synthases, among which steroidogenic acute regulatory protein (StAR) transfers cholesterol to the outer mitochondrial membrane, initiating testosterone production in interstitial cells (LCs). This is the rate-limiting step in testosterone synthesis. Cholesterol on the outer mitochondrial membrane is transferred to the inner mitochondrial membrane by cholesterol side-chain cleavage enzyme (Cyp11a1), where it is catalyzed to produce pregnenolone. Pregnenolone is then catalyzed by 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase / 17,20 lyase (Cyp17a1), and type 3 17β-hydroxysteroid dehydrogenase (17β-HSD3) on the smooth endoplasmic reticulum to ultimately produce testosterone. Simultaneously, testosterone exerts negative feedback through the hypothalamus, inhibiting the release of LH from the pituitary gland.

[0003] Hypogonadism (testosterone deficiency) caused by low testosterone levels is a common clinical condition in men. A significant proportion of middle-aged and older men experience male menopause syndrome due to a partial decrease in testosterone levels. Current treatment typically involves testosterone supplementation therapy (TST), which exogenously supplements testosterone to achieve normal physiological concentrations in the serum, thereby alleviating the physiological changes and clinical symptoms caused by partial testosterone deficiency. While TST alleviates the symptoms of testosterone deficiency to some extent, its long-term safety remains unknown. In some known clinical cases, men receiving TST have a higher incidence of stroke, heart disease, and prostate cancer. Furthermore, because testosterone is a steroid, orally administered testosterone preparations are inactivated in the liver, significantly reducing their therapeutic effect. Therefore, there is an urgent need in this field to study new methods to promote the body's own production of testosterone, starting from the mechanism of testosterone synthesis, and to develop new drugs for treating hypogonadism and male menopause syndrome, thereby increasing the body's own testosterone secretion and reducing side effects. Summary of the Invention

[0004] The purpose of this invention is to provide a compound that increases testosterone secretion levels and its uses.

[0005] Another object of the present invention is to provide a method for increasing testosterone secretion levels.

[0006] To address the aforementioned technical problems, the first aspect of the present invention provides the use of perthioglutathione for the preparation of pharmaceuticals or compositions, said pharmaceuticals or compositions being used for one or more purposes selected from the group consisting of:

[0007] (i) Prevention and / or treatment of testosterone deficiency-related disorders; and / or

[0008] (ii) Increase serum testosterone levels; and / or

[0009] (iii) Increase the expression levels of steroid synthases associated with testosterone synthesis; and / or

[0010] (iv) Increase the expression level of EHD3; and / or

[0011] (v) Increase NR5A1 expression levels; and / or

[0012] (vi) Used to suppress weight gain.

[0013] In some preferred embodiments, the testosterone deficiency-related diseases include hypogonadism, male menopausal syndrome, partial androgen deficiency syndrome in middle-aged and elderly men, prostate cancer, systemic lupus erythematosus, osteoporosis, sarcopenia, atherosclerosis, Alzheimer's disease, diabetes, metabolic syndrome, infertility, or obesity.

[0014] In some preferred embodiments, the testosterone deficiency-related disease is caused by a high-fat diet.

[0015] In some preferred embodiments, the weight gain is caused by a high-fat diet.

[0016] In some preferred embodiments, the steroid synthase associated with testosterone synthesis is selected from at least one of StAR, 3β-HSD, Cyp11a1, and Cyp17a1.

[0017] In some preferred embodiments, the perthioglutathione increases testosterone secretion levels by enhancing the expression levels of steroid synthases associated with testosterone synthesis.

[0018] In some preferred embodiments, the perthioglutathione enhances the expression level of steroid synthases associated with testosterone synthesis by activating EHD3.

[0019] In some preferred embodiments, the composition is selected from pharmaceutical compositions.

[0020] In some preferred embodiments, the pharmaceutical composition contains a safe and effective amount of GSSH and a pharmaceutically acceptable carrier or excipient.

[0021] In some preferred embodiments, the pharmaceutical composition is a unit dosage form.

[0022] In some preferred embodiments, the composition is a health supplement composition.

[0023] In some preferred embodiments, the composition comprises a gastrointestinal dosage form or a parenteral dosage form.

[0024] In some preferred embodiments, the excipient is a solid filler, diluent, solvent, or encapsulating material.

[0025] In some preferred embodiments, the carrier is alumina, aluminum stearate, lecithin, serum protein, buffering substances such as phosphate, glycine, sorbic acid, potassium sorbate, a mixture of glycerides of saturated vegetable fatty acids, water, salt or electrolyte, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene block polymer, polyethylene glycol, and lanolin.

[0026] In some preferred embodiments, the composition is administered via enteral (oral, sublingual, rectal), parenteral (intravenous, subcutaneous, intramuscular, intraperitoneal), pulmonary absorption, or absorption via the conjunctiva, nasopharynx, oral cavity, rectum, urethra, or bladder; for example, intraperitoneal injection.

[0027] In some preferred embodiments, the safe and effective amount refers to 20 to 2000 mg / kg, preferably 50 to 1000 mg / kg, and more preferably 100 to 800 mg / kg; for example: 100 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, or 800 mg / kg.

[0028] In some preferred embodiments, the safe and effective dose includes 50-200 mg / kg (mice) or 500-1000 mg / kg (humans).

[0029] In some preferred embodiments, the composition is applied once daily for at least 10 days, preferably at least 20 days, more preferably at least 3 days, and most preferably at least 40 days.

[0030] A second aspect of the present invention provides a method for increasing testosterone secretion levels, the method comprising the steps of: administering to a subject a safe and effective amount of superthione glutathione; or

[0031] Administering a safe and effective amount of a composition containing supersulfurized glutathione to subjects.

[0032] In some preferred embodiments, the method is an in vitro, non-therapeutic method.

[0033] In some preferred embodiments, the method involves the superthioglutathione increasing testosterone secretion levels by enhancing the expression of steroid synthases associated with testosterone synthesis.

[0034] In some preferred embodiments, the method involves the superthione glutathione enhancing the expression of steroid synthases associated with testosterone synthesis by activating EHD3-mediated endocytosis.

[0035] In some preferred embodiments, the perthioglutathione activates EHD3-mediated endocytosis by activating NR5A1.

[0036] A third aspect of the present invention provides a method for controlling weight, the method comprising the steps of: administering a safe and effective amount of superthione glutathione to a subject; or

[0037] Administering a safe and effective amount of a composition containing supersulfurized glutathione to subjects.

[0038] A fourth aspect of the present invention provides an EHD3 agonist, said EHD3 agonist comprising perthioglutathione.

[0039] A fifth aspect of the present invention provides a method for activating EHD3-mediated endocytosis, the method comprising the steps of: administering a safe and effective amount of superthionin to a subject; or

[0040] Administering a safe and effective amount of a composition containing supersulfurized glutathione to subjects.

[0041] Compared with the prior art, the present invention has at least the following advantages:

[0042] (1) Embodiments of the present invention provide new uses for superthion glutathione (GSSH) or compositions containing the same for the prevention and / or treatment of testosterone deficiency-related diseases, such as infertility and obesity caused by testosterone deficiency.

[0043] (2) The embodiments of the present invention provide a method for treating testosterone deficiency-related diseases by administering glutathione (GSSH) or a composition containing it to the subject to promote the secretion of testosterone and increase the testosterone content in the body. This method does not require direct supplementation of testosterone and has few side effects.

[0044] (3) The embodiments of the present invention provide a method for controlling weight, which can effectively reduce weight fluctuations caused by a high-fat diet.

[0045] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0046] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0047] Figure 1 This is an HE staining image of mouse testicular tissue according to an embodiment of the present invention. LFD represents a section of testicular tissue from a mouse fed a normal diet, and HFD represents a section of testicular tissue from a mouse fed a high-fat diet.

[0048] Figure 2This is a mouse testis coefficient diagram according to an embodiment of the present invention, where LFD represents the testis coefficient of mice fed a normal diet and HFD represents the testis coefficient of mice fed a high-fat diet.

[0049] Figure 3 The results of ELISA for serum testosterone concentration in mice according to embodiments of the present invention are shown. LFD represents the serum testosterone concentration in mice fed a normal diet, and HFD represents the serum testosterone concentration in mice fed a high-fat diet; the scale bar is 100 μm. P<0.01, P<0.001;

[0050] Figure 4 This is a graph showing the changes in body weight of mice in the LFD, HFD, GSSH, and GSH groups before and after drug administration, according to an embodiment of the present invention.

[0051] Figure 5 This is an RT-qPCR result of steroid synthase expression in mouse testicular tissue after GSSH treatment according to an embodiment of the present invention. LFD represents testicular tissue of mice fed a normal diet after intraperitoneal injection of physiological saline, HFD represents testicular tissue of mice fed a high-fat diet after intraperitoneal injection of physiological saline, GSSH represents testicular tissue of mice fed a high-fat diet after intraperitoneal injection of GSSH, and StAR, 3β-HSD, Cyp11a1 and Cyp17a1 are steroid synthases in the process of testosterone synthesis. P<0.05, P<0.01;

[0052] Figure 6 This is a Western blot result of the expression of EHD3, NR5A1, StAR and 3β-HSD in mouse testicular tissue after GSSH treatment according to an embodiment of the present invention.

[0053] Figure 7 This is a grayscale statistical image of the expression of EHD3, NR5A1, StAR and 3β-HSD in the testicular tissue of mice after GSSH treatment according to the embodiments of the present invention; wherein LFD represents the testicular tissue of mice fed a normal diet after intraperitoneal injection of physiological saline, HFD represents the testicular tissue of mice fed a high-fat diet after intraperitoneal injection of physiological saline, and GSSH represents the testicular tissue of mice fed a high-fat diet after intraperitoneal injection of GSSH. P<0.05;

[0054] Figure 8This is a schematic diagram of the immunofluorescence results of Clathrin in the testicular tissue of an obese mouse model after GSSH administration according to an embodiment of the present invention. LFD represents a section of testicular tissue from a mouse fed a normal diet, HFD represents a section of testicular tissue from a mouse fed a high-fat diet, GSSH represents testicular tissue from a mouse fed a high-fat diet after intraperitoneal injection of GSSH, Clathrin represents the green fluorescently labeled key protein of endocytosis Clathrin, and DAPI represents cell nuclear staining; the scale bar is 20 μm. Detailed Implementation

[0055] Through extensive and in-depth research, the inventors unexpectedly discovered that glutathione persulfate (GSSH) can activate NR5A1, the upstream transcription factor of the endocytic cell transport regulator protein EHD3. Under the positive regulation of NR5A1, the activity of the EHD3 promoter increases, promoting EHD3-mediated endocytosis, increasing intracellular cAMP levels, upregulating the expression of cAMP synthesis-related enzymes and steroid synthases (including StAR, 3β-HSD, Cyp11a1, and Cyp17a1), promoting testosterone synthesis and secretion, and increasing serum testosterone concentration, thereby preventing and / or treating testosterone deficiency-related diseases. Based on this, embodiments of the present invention provide the use of glutathione persulfate for the preparation of pharmaceuticals or compositions, said pharmaceuticals or compositions for one or more uses selected from the group consisting of:

[0056] (i) Prevention and / or treatment of testosterone deficiency-related disorders; and / or

[0057] (ii) Increase serum testosterone levels; and / or

[0058] (iii) Increase the expression levels of steroid synthases associated with testosterone synthesis; and / or

[0059] (iv) Increase the expression level of EHD3; and / or

[0060] (v) Increase NR5A1 expression levels; and / or

[0061] (vi) Used to control weight (e.g., to suppress weight gain caused by a high-fat diet).

[0062] supersulfurized glutathione

[0063] The "persulfurized glutathione (GSSH)" used in this invention is a persulfated product of glutathione (GSH), as shown in the following formula. The persulfurized glutathione in this invention can be obtained through biosynthesis; for details, please refer to the following literature:

[0064] .

[0065] In this invention, the perthioglutathione can be derivatized or modified using conventional methods, such as forming polymorphs or salts. These derivatives can typically be obtained using conventional techniques, and their effects and efficacy can be obtained by comparing them with GSSH in cell experiments or equivalence studies, all within the scope that can be understood and expected by those skilled in the art.

[0066] Composition

[0067] The term "composition" as used in this invention refers to a combination of superthione glutathione or superthione glutathione derivatives and other biosafety-compliant ingredients. Preferably, the term "composition" as used in this invention is a "pharmaceutical composition" or a "composition with health-promoting functions".

[0068] The "pharmaceutical composition" used in this invention includes, in addition to superglutathione or superglutathione derivatives as active ingredients, pharmaceutically acceptable carriers and excipients. In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acids in the form of glycerides, water, salts or electrolytes such as lysozyme sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin. In some embodiments, pharmaceutically acceptable excipients include, but are not limited to, binders, fillers, disintegrants, lubricants, the base portion of ointments or creams, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants, diluents, or encapsulating materials.

[0069] The "composition with health-promoting functions" used in this invention may include, in addition to superglutathione or superglutathione derivatives as active ingredients, a carrier, excipients and food additives.

[0070] As used in this invention, "active ingredient" refers to a compound, alone or in combination with one or more pharmaceutically acceptable excipients, that is applied to a subject to treat, prevent, or alleviate one or more symptoms of a condition, disorder, or disease. "Active ingredient" as used herein may be GSSH or a derivative thereof as described in this invention.

[0071] The concentration of the active ingredient in the "pharmaceutical composition" used in this invention will depend on the rates of absorption, distribution, inactivation, and excretion of the drug, as well as other factors known to those skilled in the art. It should be noted that the dosage values ​​will also vary depending on the severity of the condition to be alleviated. It should also be understood that, for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the individual administering or supervising the administration of the composition, and the concentration ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions. The active ingredient may be administered once or divided into many smaller doses administered at different time intervals.

[0072] Treatment

[0073] The application of "thioglutathione or its derivatives" or "compositions" in this invention is directed at mammals, including but not limited to primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a specific embodiment, the subject is a human.

[0074] To suit the characteristics of the recipients and the needs of treatment, the "supersulfurized glutathione or its derivatives" or "composition" in this invention are preferably administered in the form of a pharmaceutical preparation, which may include: powder, granule, liquid, decoction, pill, tablet, injection or ointment, such as an injection.

[0075] The method of administering "thioglutathione or its derivatives" or "composition" to the subject in this invention is not limited. Optional administration methods include: enteral administration (oral, sublingual, rectal administration), parenteral injection (intravenous, subcutaneous, intramuscular, intraperitoneal injection), absorption via the lungs, or absorption via the conjunctiva, nasopharynx, oral cavity, rectum, urethra, or bladder. In one embodiment, the subject is administered via intraperitoneal injection.

[0076] In this invention, "thioglutathione or its derivatives" or "composition" refers to a unit dosage form.

[0077] In this invention, the safe and effective amount of "thioglutathione or its derivatives" or "composition" applied to the target organism is defined as 20 to 2000 mg / kg, preferably 50 to 1000 mg / kg, and more preferably 100 to 800 mg / kg; for example: 100 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, or 800 mg / kg. All of the aforementioned safe and effective amounts refer to the content of the active ingredient. In some embodiments, the safe and effective amount is 50-200 mg / kg (mice) or 500-1000 mg / kg (humans).

[0078] In this invention, the "thioglutathione or its derivatives" or "composition" is applied once daily for at least one day, preferably at least 10 days, more preferably at least 20 days, more preferably at least 3 days, and most preferably at least 40 days.

[0079] Indications

[0080] In this invention, the inventors unexpectedly discovered that superthioned glutathione can significantly increase serum testosterone levels. Therefore, the inventors use superthioned glutathione (“superthioned glutathione or its derivatives”) or “compositions” for the prevention and / or treatment of testosterone deficiency-related diseases, and provide methods for preventing and / or treating these diseases. In non-limiting embodiments, the testosterone deficiency-related diseases are selected from at least one of hypogonadism, male menopausal syndrome, partial androgen deficiency syndrome in middle-aged and elderly men, prostate cancer, systemic lupus erythematosus, osteoporosis, sarcopenia, atherosclerosis, Alzheimer's disease, diabetes, metabolic syndrome, infertility, and obesity, preferably hypogonadism and obesity.

[0081] Furthermore, the inventors experimentally verified that superthione glutathione can significantly increase the body's testosterone levels by activating NR5A1 to promote EHD3-mediated endocytosis and activating the expression of steroid synthases related to testosterone synthesis (including StAR, 3β-HSD, Cyp11a1, and Cyp17a1). Therefore, the inventors believe that superthione glutathione plays a significant promoting role in EHD3-mediated endocytosis and can therefore be used as an EHD3 agonist to increase the expression levels of EHD3 and NR5A1.

[0082] On the other hand, the inventors discovered that supersulfur-glutathione has a significant beneficial effect in inhibiting weight gain induced by a high-fat diet, and therefore can be used to control weight and inhibit weight gain (e.g., inhibiting weight gain induced by a high-fat diet).

[0083] the term

[0084] Unless otherwise specified, “testosterone deficiency” as used herein refers to a level of testosterone in the body that is below normal, which can generally be determined by examining serum testosterone concentration.

[0085] Unless otherwise specified, the term "male menopause syndrome" as used in this article refers to the symptoms experienced by men over the age of 50, including anxiety, depression, memory loss, poor concentration, fatigue, insomnia, hot flashes, night sweats, and decreased sexual function.

[0086] Unless otherwise specified, the term “partial androgen deficiency syndrome in middle-aged and older men (PADAM)” as used in this article refers to partial androgen deficiency after the age of 40, which leads to a mismatch between libido and ability, manifested as a decline in energy, mental capacity, physical strength, and sexual function, as well as a decrease in muscle mass and an increase in body fat.

[0087] Unless otherwise specified, “obesity” as used in this article refers to a body mass index (BMI) of 30 or higher, where BMI (kg / m2) = weight (kg) / height squared.

[0088] Unless otherwise specified, the term “steroid synthases associated with testosterone synthesis” as used herein refers to steroid synthases involved in the molecular mechanisms of testosterone synthesis disclosed in this invention or those involved in the molecular mechanisms of testosterone synthesis recognized in the art, such as StAR, 3β-HSD, Cyp11a1, and Cyp17a1.

[0089] Unless otherwise specified, “StAR” as used in this article refers to the acute regulatory steroid protein.

[0090] Unless otherwise specified, “3β-HSD” as used herein refers to 3β-hydroxysteroid dehydrogenase.

[0091] Unless otherwise specified, “Cyp11a1” as used herein refers to cholesterol cleavage enzyme on the cholesterol side chain.

[0092] Unless otherwise specified, “Cyp17a1” as used in this article refers to the gene encoding 17α-hydroxylase.

[0093] Unless otherwise specified, “EHD3” as used in this article refers to a member of the EHD protein family, located in early exosomes.

[0094] Unless otherwise specified, “NR5A1” as used in this article refers to the gene encoding steroidogenic factor 1 (SF1), which is primarily expressed in the adrenal glands, gonads, and ventral nucleus of the hypothalamus.

[0095] Unless otherwise specified, “weight control” as used herein refers to controlling significant weight fluctuations under a high-fat diet, which means that the subject’s daily calorie intake significantly exceeds the calories required to maintain normal life activities, such as more than 1, 2, 3, 4 or 5 times the calories required to maintain normal life activities.

[0096] Unless otherwise specified, the term "high-fat diet" as used herein refers to the consumption of high-fat foods, which are defined as foods containing at least 60% saturated and unsaturated fatty acids.

[0097] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0098] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available. The main reagents and their sources in some embodiments are as follows:

[0099]

[0100] Example 1: Construction and Administration of Obese Mouse Model

[0101] (1) Construction and validation of obese mouse model

[0102] Eighty 3-week-old male C57BL / 6 mice were purchased from the Animal Experiment Center of Nanjing Medical University. Twenty mice were fed a normal diet and were designated as the LFD group. Their weight was recorded. The remaining mice were fed a high-fat diet containing 60% fat for 10 weeks. Mice that were underweight (<30g) were discarded by weighing. The remaining mice were randomly divided into three groups according to their weight: the HFD group, the GSSH group, and the GSH group, with 15 mice in each group.

[0103] Mice were allowed free access to food for 10 weeks. Five mice from each of the LFD and HFD groups were used to validate the success rate of the obesity model. The results are as follows: Figure 1 As shown, a high-fat diet significantly disrupted testicular tissue morphology, resulting in significantly loose seminiferous tubules, a reduced number of interstitial cells, and a significantly lower testicular coefficient compared to the control group (e.g., Figure 2 Next, the serum testosterone levels in mice were measured, and the results were as follows: Figure 3 As shown, the average serum testosterone level in mice on a normal diet was 39.13 ng / ml, while the testosterone concentration in the high-fat diet group was significantly reduced to 18.95 ng / ml. This indicates that the obese mouse model was successfully established, and that a high-fat diet can significantly reduce serum testosterone levels in mice.

[0104] Compared to the HFD group, the GSSH group showed significantly reduced weight fluctuations and a total weight 17% lighter than the HFD group, demonstrating that GSSH can effectively control weight fluctuations caused by a high-fat diet.

[0105] (2) Drug administration to mice

[0106] Mice in the LFD and HFD groups were injected intraperitoneally with PBS, mice in the GSSH group were injected intraperitoneally with 200 mg / kg superthione glutathione, and mice in the GSH group were injected intraperitoneally with 200 mg / kg glutathione (GSSH; the GSSH and GSH used in this experiment were donated by Professor Xu Defeng of Changzhou University, with a purity of about 95%; GSSHH and GSH can also be synthesized using conventional methods). The drugs were administered for a total of 40 days.

[0107] Obese mice were intraperitoneally injected with GSSH and fed a high-fat diet. Serum testosterone concentrations were measured using an ELISA kit (Testosterone ELISA kit, BD Biosciences, USA) to analyze the effects of GSSH and GSH on serum testosterone concentrations in mice. The specific steps are as follows:

[0108] (a) GSSH was administered intraperitoneally to obese mice, and blood was collected from the orbital cavity at the same time. (b) The cells were incubated overnight at 4°C, centrifuged at 1000 rpm for 10 min at 4°C, and the supernatant serum was collected. Cell debris was removed by centrifugation at 2000 rpm for 10 min.

[0109] (c) Dilute the standard according to the instructions, and allow all reagents in the kit to equilibrate at room temperature for 15 min in advance;

[0110] (d) Add 50 μL of Primary Antibody Solution to all test wells and incubate on a horizontal shaker at room temperature for 1 h;

[0111] (e) Wash the plate with 400 μL of 1×Wash Buffer, repeat 4 times, and pat dry.

[0112] (f) Add 100 μL of Calibrator Diluent RD5-48 to each well;

[0113] (g) Add 100 μL of standard and test sample to each well, and make 3 replicates for each sample;

[0114] (h) Add 50 μL of Testosterone Conjugate to each well and seal the plate with a sealing film;

[0115] (i) Incubate at room temperature on a horizontal shaker for 3 h;

[0116] (j) Repeat step 5;

[0117] (k) Add 200 μL of Substrate Solution to each well and incubate at room temperature in the dark for 30 min;

[0118] (l) Add 50 μL of Stop Solution to each well. The color in the well should change from blue to yellow.

[0119] (m) Set the wavelength to 450 nm and the calibration wavelength to 540 nm on the microplate reader to obtain the absorbance value for each well;

[0120] (n) Use the four-parameter Logistic curve fitting to generate a testosterone standard curve, and use the absorbance value corresponding to the sample well to calculate the corresponding testosterone concentration.

[0121] The results showed that the mean testosterone concentration in the HFD group (10.27 ng / ml) was significantly lower than that in the LFD group (17.61 ng / ml). The mean testosterone concentration in the GSH group (11.18 ng / ml) was not statistically different from that in the HFD group. However, the mean testosterone concentration in the GSSH group (22.37 ng / ml) was significantly higher than that in the HFD group. These results indicate that GSSH significantly promoted and increased serum testosterone levels in mice. After administration, body weight was measured and recorded. The changes in body weight before and after administration in the LFD, HFD, GSSH, and GSH groups are shown in the table below. Figure 4 In the figure, ns indicates no statistical difference, and asterisks indicate statistical differences. The results showed that there was no significant difference in body weight before and after administration in the LFD group; the body weight of mice in the HFD group continued to increase after administration, and the increase was significant compared with the body weight before administration; there was no significant difference in body weight before and after administration in the GSSH group, indicating that GSSH inhibited the body weight gain caused by a high-fat diet; while the body weight of mice in the GSH group continued to increase after administration, and the increase was significant compared with the body weight before administration.

[0122] Example 2: Detection of steroid synthase expression in testicular tissues of mice in the LFD, HFD, and GSSH groups using RT-qPCR technology.

[0123] Testicular tissues were collected from mice in the LFD, HFD, and GSSH groups. RNA was extracted, reverse transcribed into cDNA, and the expression of steroid synthases StAR, 3β-HSD, Cyp11a1, and Cyp17a1 in different treatment groups was detected using RT-qPCR. The specific experimental steps for RNA extraction, cDNA reverse transcription, and RT-qPCR are as follows:

[0124] (1) RNA extraction using the Trizol method

[0125] (a) Add 1 mL of Trizol to the cells in a 12-well plate and repeatedly pipette to induce cell division.

[0126] Dissolve and collect the cells into 1.5 mL EP tubes without RNase;

[0127] (b) Add 200 μL of chloroform, shake vigorously for 15 s, and let stand at room temperature for 10 min;

[0128] (c) Centrifuge at 12000 rpm at 4℃ for 15 min, and transfer the supernatant to a new EP tube, being careful not to aspirate any protein;

[0129] (d) Add 500 μL of isopropanol, gently invert 10 times, and let stand at room temperature for 10 min;

[0130] (e) Centrifuge at 12000 rpm at 4℃ for 10 min. At this time, a white precipitate appears at the bottom of the EP tube. Gently aspirate the supernatant.

[0131] (f) Add 1 mL of 75% ethanol to wash the precipitate, and centrifuge at 7500 rpm for 5 min at 4℃;

[0132] (g) Repeat step 6;

[0133] (h) Thoroughly remove the upper layer of liquid, and evaporate excess alcohol in the laminar flow hood until the white precipitate becomes transparent, according to...

[0134] Add an appropriate amount of Nuclease-free H2O to the precipitate, mix gently, place on ice, and take 1 μL to measure the concentration.

[0135] (i) Take 1 μg of RNA and reverse transcribe it into cDNA. Store the remaining RNA at -80℃.

[0136] (2) Reverse transcription PCR

[0137] (a) Take 1 μg of RNA for reverse transcription, and the system is shown in Table 1 below:

[0138] Table 1

[0139]

[0140] After gently mixing, incubate at 42°C for 2 min;

[0141] (b) Add 5×Hiscript III Enzyme Mix to the mixture from the previous step, as shown in Table 2 below:

[0142] Table 2

[0143]

[0144] After gently mixing, run the PCR program as follows: 37°C, 5 min; 85°C, 5 s; 12°C, ∞. Store the cDNA at -20°C or continue to the next experiment.

[0145] (3) Real-time PCR

[0146] (a) The reverse-transcribed cDNA was diluted 5-fold with RNase-free ddH2O. The reaction volume (20 μL) is shown in Table 3 below:

[0147] Table 3

[0148]

[0149] After gently mixing, aliquot the mixture into 8-tube RT-qPCR tubing and follow the reaction conditions shown in Table 4 below:

[0150] Table 4

[0151]

[0152] (b) Each sample was replicated in 3 wells, with β-Actin as an internal control, using 2 -ΔΔCT The fold change in relative gene expression was calculated using a method. The primer sequences used in this chapter's experiments are shown in Table 5 below:

[0153] Table 5

[0154]

[0155] The expression of steroid synthase in the testicular tissues of mice in the LFD, HFD, and GSSH groups was detected, and the results are as follows: Figure 5 As shown, a high-fat diet significantly inhibited the expression of steroid synthases StAR, 3β-HSD, Cyp11a1, and Cyp17a1, while the expression of steroid synthases in the GSSH group mice was significantly upregulated compared with that in the HFD group.

[0156] Example 3: Western blot detection of EHD3, NR5A1, StAR and 3β-HSD protein expression in testicular tissue of mice in different treatment groups

[0157] Mouse testicular tissue was collected, homogenized, and proteins were extracted. Western blot analysis was used to detect the expression of EHD3, NR5A1, StAR, and 3β-HSD proteins in different treatment groups. The Western blot experimental procedure is as follows:

[0158] (1) Preparation of polyacrylamide gel

[0159] (a) Clean the glass plate with degreased cotton balls under tap water, rinse it with ddH2O after cleaning, put it in the oven to dry, install the glass plate with the glue applicator, and check for leaks with ddH2O.

[0160] (b) Prepare separating gels of appropriate concentrations according to the protein molecule size, as shown in Table 6:

[0161] Table 6

[0162]

[0163] (c) Discard the ddH2O used for leak testing, absorb excess water with absorbent paper, mix the separating adhesive thoroughly and quickly pour it into the glass plate, then seal it with ddH2O.

[0164] (d) After 30 min, a clear boundary line appears between ddH2O and the separating gel. Pour off the upper layer of ddH2O, absorb excess water with absorbent paper, mix the concentrated gel thoroughly, and quickly pour it into a glass plate (be careful not to create air bubbles). Then insert the pore-forming device and wait for the gel to solidify before use.

[0165] (2) Preparation of protein samples

[0166] (a) When collecting mouse testicular tissue protein samples, rinse with pre-cooled PBS to remove blood, peel off the white membrane layer with tweezers, add an appropriate amount of tissue lysis buffer according to the tissue size, grind the tissue with a tissue grinder, and sonicate; when collecting adherent cell protein samples, discard the culture medium, rinse twice with pre-cooled PBS, add an appropriate amount of lysis buffer, scrape off the cells with a cell scraper, and transfer them to a 1.5 mL EP tube with a pipette;

[0167] (b) Centrifuge at 12000 rpm at 4℃ for 5 min, and transfer the supernatant to a new EP tube to prepare for protein concentration measurement.

[0168] (3) Protein quantification

[0169] (a) Take a 96-well plate and set up 7 protein standard concentration gradients using protein standards, as shown in Table 7:

[0170] Table 7

[0171]

[0172] (b) Add 98 μL of ddH2O and 2 μL of the protein sample to be tested to the sample well;

[0173] (c) Prepare the colorimetric reagent (Regent A 98 μL + Regent B 2 μL), mix thoroughly, and add 100 μL / well to the wells of the standard and the sample to be tested;

[0174] (d) Place the 96-well plate in a constant temperature shaker at 37℃ × 150 rpm × 30 min;

[0175] (e) Remove the 96-well plate and measure the absorbance at 562 nm using a microplate reader;

[0176] (f) Based on the known concentration and absorbance value of the standard, use Excel to create a standard curve of protein concentration, obtain the regression equation, and calculate the corresponding protein concentration based on the obtained absorbance value using the equation;

[0177] (g) Dilute the protein to 2 μg / μL and heat at 100°C for 5 min to denature the protein. The protein sample is then stored at -80°C or used for the next step of the experiment.

[0178] (4) Protein electrophoresis

[0179] (a) After assembling the prepared protein gel and electrophoresis apparatus, add 1×Running buffer;

[0180] (b) Use a pipette to add the protein sample and protein marker sequentially according to the experimental requirements;

[0181] (c) Turn on the power supply and set it to a constant voltage of 90 V. After the sample moves from the stacking gel to the separating gel, adjust the voltage to a constant voltage of 120 V.

[0182] (d) Stop electrophoresis when the blue dye in the sample has moved to the bottom of the protein gel after about 90 min.

[0183] (5) Transfer membrane

[0184] (a) After electrophoresis, transfer the membrane and form a "sandwich" structure in the following order: negative electrode of transfer clamp - sponge - filter paper - protein gel - PVDF membrane - filter paper - sponge - positive electrode of transfer clamp. Avoid air bubbles in each layer.

[0185] (b) Place the assembled transfer clamp into the transfer tank, add 1×Transfer buffer pre-cooled at 4°C, and place an ice pack in the tank;

[0186] (c) Turn on the power supply and set the transfer conditions to 200 mA (constant current) for 90 min. The entire transfer process is carried out on ice.

[0187] (6) Closed

[0188] After the transfer is complete, carefully remove the PVDF membrane and place it in pre-prepared 5% skim milk, then seal it at room temperature for 1 hour.

[0189] (7) Antibody hybridization

[0190] (a) Refer to the antibody instructions and dilute the primary antibody with 5% skim milk or 1×TBST;

[0191] (b) After the sealing is completed, take out the PVDF membrane and put it into a clean plastic sealant, add the diluted primary antibody, seal it with a sealing machine, and incubate at 4°C overnight;

[0192] (c) After the primary antibody incubation is completed, rinse the PVDF membrane with 1×TBST on a shaker 3 times, 10 min each time;

[0193] (d) Prepare the corresponding secondary antibody using 5% skim milk or 1×TBST, depending on the species of origin of the primary antibody;

[0194] (e) After rinsing the PVDF membrane, place it in a clean plastic sealing film, add the diluted secondary antibody, seal it with a sealing machine, and incubate at room temperature for 1 h;

[0195] (f) Rinse the PVDF membrane with 1×TBST on a shaker 3 times, 10 min each time, in preparation for exposure.

[0196] The names, molecular weights, and dilution ratios of the antibodies used in the experiment are shown in Table 8.

[0197] Table 8

[0198]

[0199] (8) Exposure and development

[0200] Prepare the ECL developer (Regent A:Regent B = 1:1), take out the PVDF film, remove as much excess liquid as possible, and evenly drop the ECL solution onto the film for exposure and development.

[0201] The inventors previously proposed a mechanism by which EHD3, under the positive regulation of the upstream transcription factor NR5A1, mediates cellular endocytosis, upregulates the expression of steroid synthases, and ultimately promotes testosterone synthesis. The above experiments validated this mechanism by analyzing the expression of EHD3, NR5A1, and the steroid synthases StAR and 3β-HSD. Results are as follows... Figure 6 As shown, the protein expression levels of EHD3, NR5A1, StAR, and 3β-HSD were significantly reduced in high-fat diet-induced obese mouse models. A grayscale analysis of the Western blot results is presented (see...). Figure 7 Compared to the LFD group, the HFD group showed a 50% downregulation of EHD3 expression, a 54% decrease in NR5A1 expression, a 30% decrease in StAR expression, and a 34% decrease in 3β-HSD expression. In contrast, the GSSH group exhibited significantly increased protein expression levels of EHD3, NR5A1, StAR, and 3β-HSD compared to the HFD group. Specifically, EHD3 expression was 1.63 times higher than in the HFD group, NR5A1 expression was 1.62 times higher, StAR expression was 1.43 times higher, and 3β-HSD expression was 1.86 times higher. These results further validate that EHD3, under the regulation of NR5A1, regulates testosterone secretion by affecting the expression of steroid synthases.

[0202] Example 4: Immunofluorescence staining of mouse testicular tissue

[0203] In GSSH-treated obese mouse models, the expression of Clathrin, a key protein in endocytosis, was detected using immunofluorescence staining. The results are as follows: Figure 8 As shown, there was a significant Clathrin protein fluorescence signal in the interstitial cells of the LFD group mice, and the fluorescence signal was mainly expressed in the perinuclear cytoplasm. In the testicular tissue of the HFD group mice, due to the reduction in the number of interstitial cells caused by the high-fat diet, the number of interstitial cells with Clathrin protein fluorescence signal was also significantly reduced. In the testicular tissue of the GSSH treatment group mice, although the number of interstitial cells was not significantly reduced compared with the LFD group, the number of interstitial cells with Clathrin fluorescence signal did not increase significantly.

[0204] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. Use of glutathionylsulfoxide, characterized in that, Used to prepare a drug or composition for the prevention and / or treatment of obesity caused by a high-fat diet; Or, to prevent, alleviate and / or treat testosterone deficiency in men caused by obesity.

2. The use according to claim 1, characterized in that, The perthioglutathione increases testosterone secretion by enhancing the expression level of steroid synthases associated with testosterone synthesis.

3. The use according to claim 1, characterized in that, The perthioglutathione enhances the expression level of steroid synthases associated with testosterone synthesis by activating EHD3.

4. The use according to claim 2 or 3, characterized in that, The steroid synthase associated with testosterone synthesis is selected from at least one of StAR, 3β-HSD, Cyp11a1, and Cyp17a1.

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