Use of cytochrome P450 21A1 (Cyp21a1) inhibitor WIN51708 in the preparation of a drug for treating diabetes-related testicular lesions

Abstract

The application discloses application of a cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 in preparation of a medicine for treating diabetes-related testicular lesions, and particularly application of the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 in the following aspects: 1) as a molecular target, in preparation of a medicine for early-stage screening of diabetes-related testicular lesions; and 2) as a molecular target and its inhibitor WIN51708, in preparation of a medicine for treating diabetes-related testicular lesions. The application discloses, for the first time, that the protein expression amount of Cyp21a1 in a patient with diabetes-related testicular lesions is increased, so that the Cyp21a1 inhibitor WIN51708 is applied in the medicine for treating diabetes-related testicular lesions, and thus new ideas and drug resources are provided for the treatment of diabetes-related testicular lesions.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and male reproductive medicine, specifically relating to the application of WIN51708, a cytochrome P45021A1 (Cyp21a1) inhibitor, in the preparation of drugs for treating diabetes-related testicular lesions. In particular, it relates to the use of WIN51708 by directly binding to Cyp21a1, regulating the steroid hormone synthesis pathway, and improving insufficient testosterone synthesis and decreased sperm quality in diabetic states. Background Technology

[0002] Diabetes is a common contributing factor to male reproductive system diseases and infertility, and is inextricably linked to male reproductive health. As diabetes progresses, patients may experience testicular lesions and hormonal imbalances; however, research in this area remains very limited. Therefore, research on testicular lesions in diabetic patients has become an urgent clinical problem to be solved.

[0003] Cyp21a1 is one of the rate-limiting enzymes in the synthesis of adrenocortical steroid hormones. Its core function is to catalyze the 21-hydroxylation of steroid precursors. Abnormal expression of Cyp21a1 directly affects the balance of downstream steroid hormones (including testosterone, cortisol, and aldosterone), which is the core molecular basis for its regulation of testosterone synthesis in testicular interstitial cells. Cyp21a1 has been proven to play an important role in various diseases. WIN51708 is a non-peptide neurokinin-1 receptor (NK-1R) antagonist. The inventors further discovered through surface plasmon resonance technology that WIN51708 can directly bind to the Cyp21a1 protein, suggesting that in addition to its known NK-1R antagonistic activity, it may also have the potential to target Cyp21a1 and regulate steroid metabolism-related pathways. The inventors of this application have discovered that by inhibiting Cyp21a1 activity, WIN51708 can alter the metabolic distribution of steroid precursors, thereby affecting testosterone-related metabolism. Therefore, the inventors of this application intend to provide a new pharmaceutical use for the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708, specifically involving the application of the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 in the preparation of drugs for treating diabetes-related testicular lesions. Summary of the Invention

[0004] In view of the problems in the existing technology of diabetes-related testicular lesions, such as the lack of clear therapeutic targets, unclear mechanisms of existing intervention methods, and difficulty in effectively improving insufficient testosterone synthesis and decreased sperm quality, this invention provides the application of cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 in the preparation of drugs for treating diabetes-related testicular lesions.

[0005] The technical solution of this invention is based on the following findings: In the state of diabetic orchopathy, the expression level of Cyp21a1 in testicular tissue is increased, and the testosterone level is decreased; inhibiting Cyp21a1 can improve the testosterone synthesis capacity of Leydig cells; WIN51708 can directly bind to Cyp21a1 protein and has a clear SPR affinity parameter; further intervention with WIN51708 can improve testosterone levels and sperm quality in the diabetic orchopathy model. Therefore, this invention uses Cyp21a1 as a therapeutic target for diabetic orchopathy and uses WIN51708 to prepare a drug for treating diabetic orchopathy.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The first aspect of the present invention provides the use of WIN51708, a cytochrome P45021A1 (Cyp21a1) inhibitor, in the preparation of a drug for treating diabetes-related testicular lesions.

[0007] Optionally, the cytochrome P45021A1 (Cyp21a1) can be used as a molecular target in screening drugs, diagnostic drugs, efficacy evaluation drugs, or therapeutic drugs for diabetes-related testicular lesions.

[0008] Optionally, the application of cytochrome P45021A1 (Cyp21a1) as a molecular target in the early screening of diabetes-related testicular lesions includes: conducting early screening for diabetes-related testicular lesions in diabetic patients based on the higher expression level of Cyp21a1 in the testicular tissue of diabetic patients than in the testicular tissue of non-diabetic patients, and the decreased testosterone content in the testicular tissue of diabetic patients.

[0009] Optionally, the Cyp21a1 inhibitor WIN51708 is used to directly bind to the Cyp21a1 protein.

[0010] Optionally, the equilibrium dissociation constant KD between the Cyp21a1 inhibitor WIN51708 and the Cyp21a1 protein is 10^-6 M to 10^-5 M.

[0011] Optionally, the Cyp21a1 inhibitor WIN51708 is used to increase testosterone levels in a diabetes-associated testicular lesion model.

[0012] Optionally, the use of a specific knockdown virus of the cytochrome P45021A1 (Cyp21a1), or the use of the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 in a medicament for improving spermatogenesis in patients with diabetes-associated testicular lesions.

[0013] Optionally, the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 is used to promote testosterone synthesis in TM3 cells in vitro.

[0014] Optionally, the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 is administered via intraperitoneal injection at a dose of 2 mg / kg, once daily for 30 consecutive days, to improve sperm quality in mice with diabetic testicular lesions.

[0015] This invention provides the application of the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 in the preparation of a drug for treating diabetes-related testicular lesions, which has at least the following technical effects: First, this invention discovered that Cyp21a1 expression is elevated in the state of diabetes-associated testicular lesions, and identified Cyp21a1 as a potential therapeutic target for diabetes-associated testicular lesions.

[0016] Second, this invention demonstrates through SPR affinity testing that WIN51708 can directly bind to the Cyp21a1 protein, with a KD of 3.13 × 10⁻⁶. -6 M indicates that WIN51708 has a clear targeting effect on Cyp21a1.

[0017] Third, this invention further verifies that WIN51708 participates in the Cyp21a1 binding process in the 17-Hydroxyprogesterone related system, with a KD of 2.52 × 10⁻⁶. -5 M provides supplemental evidence for its regulation of Cyp21a1-mediated steroid metabolism pathways.

[0018] Fourth, this invention demonstrates through in vitro and in vivo experiments that inhibiting Cyp21a1 can improve insufficient testosterone synthesis and decreased sperm quality in diabetic-associated testicular lesions, providing a new pharmaceutical application for the treatment of diabetic-associated testicular lesions.

[0019] A second aspect of the present invention provides a kit comprising an antibody for detecting the expression of any of the above-described cytochrome P45021A1 (Cyp21a1) antibodies as a drug target for detecting diabetes-related testicular lesions; and cytochrome P45021A1 (Cyp21a1) protein as a drug target for detecting diabetes-related testicular lesions.

[0020] The kit provided by this invention utilizes the abnormally elevated expression of Cyp21a1 in diabetic testicular lesions, and uses Cyp21a1 as a detection target. It can be used for auxiliary screening, mechanism research or drug efficacy evaluation of diabetic testicular lesions.

[0021] Other features and advantages of the present invention will be further set forth in the following description, and some of these will become apparent by practicing the invention. The objectives and other advantages of the invention can be realized and obtained through the technical solutions described in the specification, claims, and drawings.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 This image shows the confirmation of diabetic phenotype and testicular lesion phenotype in mice from the db / m normal control group and the db / db diabetic group.

[0024] Figure 2 This figure shows the difference in Cyp21a1 expression levels in testicular tissue between mice in the db / m normal group and the db / db diabetic group.

[0025] Figure 3 The graph shows the difference in testosterone levels between the db / m normal group and the db / db diabetic group.

[0026] Figure 4 This figure shows the changes in Cyp21a1 expression and testosterone levels in TM3 cells after Cyp21a1 specific knockdown.

[0027] Figure 5 The figure shows the SPR affinity test results between Cyp21a1 inhibitors and Cyp21a1 protein.

[0028] Figure 6 The figure shows the SPR affinity test results of Cyp21a1 inhibitor with Cyp21a1 in the 17-Hydroxyprogesterone related system.

[0029] Figure 7 Figure showing the changes in testosterone levels in TM3 cells after treatment with different concentrations of Cyp21a1 inhibitor.

[0030] Figure 8 The graph shows the changes in testosterone levels, sperm density, and sperm motility in diabetic mice after intraperitoneal injection of Cyp21a1 inhibitor. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] The first aspect of this invention provides the application of a cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 in the preparation of a drug for treating diabetes-related testicular lesions.

[0033] The inventors of this invention have discovered for the first time that in diabetic dysentery disease, the expression level of Cyp21a1 in testicular tissue is increased, while the testosterone level is decreased. After using the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708, the testosterone synthesis capacity of Leydig cells can be improved. WIN51708 can directly bind to the Cyp21a1 protein and has a defined SPR affinity parameter. Further intervention with WIN51708 can improve testosterone levels and sperm quality in a diabetic dysentery disease model. Therefore, this invention uses Cyp21a1 as a therapeutic target for diabetic dysentery disease and uses WIN51708 to prepare a drug for treating diabetic dysentery disease.

[0034] In some possible embodiments, the cytochrome P45021A1 (Cyp21a1) is used as a molecular target in pre-screening drugs, diagnostic drugs, efficacy evaluation drugs, or therapeutic drugs for diabetes-related testicular lesions.

[0035] Here, both screening and diagnostic drugs can include drugs that detect the expression level of Cyp21a1 in the testicular tissue of diabetic patients or diabetic model animals, and these drugs can be combined with testosterone content, sperm quality, or pathological changes in testicular tissue to determine diabetes-related testicular lesions.

[0036] In some possible embodiments, the application of cytochrome P45021A1 (Cyp21a1) as a molecular target in the early screening of diabetes-related testicular lesions includes: screening for diabetes-related testicular lesions in diabetic patients based on the higher expression level of Cyp21a1 in the testicular tissue of diabetic patients compared to that in the testicular tissue of non-diabetic patients, and the decreased testosterone content in the testicular tissue of diabetic patients.

[0037] Here, when the expression level of Cyp21a1 in the testicular tissue of diabetic individuals is higher than that of non-diabetic controls, and is accompanied by decreased testosterone levels, decreased sperm quality, or impaired spermatogenesis, it can indicate that the individual has a risk of diabetes-related testicular lesions. Therefore, cytochrome P45021A1 (Cyp21a1) can be used as a molecular target for the early screening of diabetes-related testicular lesions.

[0038] Optionally, the Cyp21a1 inhibitor WIN51708 is used to directly bind to the Cyp21a1 protein.

[0039] Optionally, the equilibrium dissociation constant KD between the Cyp21a1 inhibitor WIN51708 and the Cyp21a1 protein is 10^-6 M to 10^-5 M.

[0040] Here, WIN51708 can directly bind to the Cyp21a1 protein. This direct binding result can be verified using SPR technology. The verification process includes: immobilizing the Cyp21a1 protein on a CM5 sensor chip, and then using WIN51708 as the analyte for multi-cycle kinetic analysis. The results show that the binding rate constant ka between WIN51708 and Cyp21a1 is 4.47 × 10⁻⁶. 3 1 / Ms, the dissociation rate constant kd is 1.40 × 10 -2 1 / s, equilibrium dissociation constant KD is 3.13 × 10 -6 M. The above results indicate that WIN51708 directly binds to the Cyp21a1 protein and exhibits a clear micromolar affinity.

[0041] In the Cyp21a1-17-Hydroxyprogesterone related system, WIN51708 can still participate in the Cyp21a1-related binding process. Using SPR detection, the binding rate constant ka of the 17-Hydroxyprogesterone+WIN51708 system with Cyp21a1 is 1.97 × 10⁻⁶. 3 1 / Ms, the dissociation rate constant kd is 4.97 × 10 -2 1 / s, equilibrium dissociation constant KD is 2.52 × 10 -5 M. This result suggests that WIN51708 may affect Cyp21a1-mediated steroid substrate-related binding or reaction processes.

[0042] Optionally, SPR detection includes the following steps: Activating the CM5 sensor chip with 0.4 MEDC and 0.1 MNHS; immobilizing Cyp21a1 protein at a concentration of approximately 12600 RU after diluting it to 20 μg / mL; using a reference channel as a blank control; preparing the WIN51708 analyte using PBST buffer containing 1% DMSO and setting a concentration gradient of 0.78–50.00 μM; injecting the sample at a flow rate of 20 μL / min, with a binding phase of 60 seconds and a dissociation phase of 90 seconds, recording the binding and dissociation curves, and calculating ka, kd, and KD. The method described in the appendix report uses a Biacore 8K instrument, with Cyp21a1 immobilized on a CM5 chip, and the WIN51708 concentration range of 0.78–50.00 μM.

[0043] In some possible embodiments, the Cyp21a1 inhibitor WIN51708 is used to increase testosterone levels in a diabetes-associated testicular lesion model.

[0044] Optionally, the use of a specific knockdown virus of the cytochrome P45021A1 (Cyp21a1), or the use of the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 in a medicament for improving spermatogenesis in patients with diabetes-associated testicular lesions.

[0045] In particular, the use of Cyp21a1-specific knockdown virus or the Cyp21a1 inhibitor WIN51708 can improve insufficient testosterone synthesis and decreased spermatogenesis in diabetic-associated testicular lesions.

[0046] Here, WIN51708 can be administered orally, by gavage, injection, local administration, local testicular delivery, sustained-release formulation delivery, or other pharmaceutically acceptable routes. Specifically, WIN51708 is used in intraperitoneal injection as a therapeutic agent to improve testosterone levels, sperm density, and sperm motility in individuals with diabetes-associated testicular disease.

[0047] In some possible implementations, the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 is used to promote testosterone synthesis in TM3 cells in vitro.

[0048] Here, WIN51708 can be used to promote testosterone synthesis in testicular interstitial cells in vitro, including TM3 cells.

[0049] In some possible implementation embodiments, the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 is used in an intraperitoneal injection (2 mg / kg) therapeutic drug, wherein the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 is administered via intraperitoneal injection at a dose of 2 mg / kg once daily for 30 consecutive days to improve sperm quality in mice with diabetic testicular lesions.

[0050] A third aspect of the present invention provides a kit comprising an antibody for detecting the expression of any of the above-described cytochrome P45021A1 (Cyp21a1) antibodies as a drug target for detecting diabetes-related testicular lesions; and cytochrome P45021A1 (Cyp21a1) protein as a drug target for detecting diabetes-related testicular lesions.

[0051] This kit contains antibodies, primers, probes, or combinations thereof used to detect Cyp21a1 expression levels. Specifically, by detecting Cyp21a1 expression in testicular tissue, testicular biopsy samples, cell samples, or animal model samples, the kit is used to assess the risk, severity, or response to WIN51708 treatment for diabetes-related testicular disease.

[0052] The following examples illustrate the application of WIN51708, a cytochrome P45021A1 (Cyp21a1) inhibitor provided by this invention, in the preparation of a drug for treating diabetes-related testicular lesions. It should be noted that, unless otherwise specified, the methods, reagents, and materials described in the following examples are all commercially available; and the experimental methods described are conventional methods unless otherwise specified.

[0053] Example 1: Establishment and phenotypic confirmation of a diabetes-related testicular lesion model In this embodiment, 8-week-old male db / db mice were purchased and used as the diabetic model animals, while db / m mice of the same strain or littermate background were used as normal control animals. db / db mice are spontaneously induced type 2 diabetes models and do not require further induction of the model through a high-fat diet combined with streptozotocin. During the experiment, the body weight and fasting blood glucose of mice in each group were monitored to confirm the stability of the diabetic model phenotype.

[0054] After the model was established, mouse testis, epididymis, and serum samples were collected. Serum was used to detect testosterone levels; testicular tissue was used for HE staining, immunohistochemistry, Western blot, qPCR, or other molecular detection; and epididymis was used for sperm quality analysis.

[0055] The experimental results showed that, compared with the normal group, diabetic mice exhibited testicular lesions, including decreased testosterone levels, decreased sperm quality, and testicular tissue damage.

[0056] Example 2: Increased Cyp21a1 expression in diabetes-associated testicular tissue To identify the molecular targets in diabetic testicular lesions, this study investigated the expression level of Cyp21a1 in the testicular tissues of normal and diabetic mice. Western blot, immunohistochemistry, and / or qPCR were used to detect Cyp21a1 protein and mRNA expression.

[0057] The results showed that Cyp21a1 expression was increased in the testicular tissue of diabetic mice compared with the normal group. Western blot analysis showed enhanced Cyp21a1 protein bands in the diabetic group; immunohistochemistry showed enhanced Cyp21a1 positive signal in the interstitial region and / or related steroid-producing cell regions of the testes in the diabetic group.

[0058] The above results indicate that Cyp21a1 is abnormally elevated in the state of diabetes-associated testicular lesions and can serve as a candidate molecular target for diabetes-associated testicular lesions.

[0059] Example 3: Decreased testosterone levels and abnormal steroid production pathways in diabetic-associated testicular lesions Serum and testicular tissue levels in normal and diabetic mice were detected using ELISA or chemiluminescence immunoassay. Results showed that testosterone levels were decreased in the diabetic group compared to the normal group.

[0060] Further detection of Leydig cell steroid production-related molecules such as Star, Cyp11a1, Hsd3b, Cyp17a1, and Hsd17b3 can be used to evaluate the extent of testosterone synthesis pathway impairment in diabetic patients.

[0061] The above results suggest that diabetic testicular disease not only manifests as decreased spermatogenesis, but is also accompanied by impaired steroid production in Leydig cells.

[0062] Example 4: Knockdown of Cyp21a1 improves testosterone synthesis in TM3 cells To verify whether Cyp21a1 has functional intervention value, TM3 testicular interstitial cells were treated with Cyp21a1-specific knockdown virus, and a negative control group was set up. After treatment, the expression level of Cyp21a1 and the testosterone content in the cell culture supernatant were measured.

[0063] The results showed that, compared with the control group, the Cyp21a1 knockdown group had decreased Cyp21a1 expression and increased testosterone synthesis capacity in TM3 cells. These results suggest that abnormally elevated Cyp21a1 levels may be involved in impaired testosterone synthesis in Leydig cells under diabetic conditions, and that inhibiting Cyp21a1 may help restore testosterone synthesis.

[0064] Example 5: SPR affinity assay of Cyp21a1 inhibitor and Cyp21a1 protein To verify whether Cyp21a1 inhibitors can directly act on the Cyp21a1 protein, this embodiment uses surface plasmon resonance technology to detect the binding ability between Cyp21a1 and the exemplary Cyp21a1 inhibitor WIN51708.

[0065] Cyp21a1 protein was immobilized on the surface of a CM5 chip at a concentration of 20 μg / mL, with an immobilization level of approximately 12600 RU. Seven concentration gradients of the analyte WIN51708 were set, ranging from 0.78 μM to 50.00 μM. A multi-cycle kinetic method was used to detect binding and dissociation processes, and a 1:1 Langmuir binding model was used for fitting.

[0066] .

[0067] The results showed that WIN51708 could directly bind to the Cyp21a1 protein, with an equilibrium dissociation constant KD of 3.13 × 10^-6 M, indicating that WIN51708 has a micromolar affinity for Cyp21a1. This result provides molecular-level evidence for the direct targeting of Cyp21a1 by Cyp21a1 inhibitors.

[0068] Example 6: SPR detection of Cyp21a1 inhibitor in 17-Hydroxyprogesterone-related systems To further evaluate the binding characteristics of Cyp21a1 inhibitors in steroid substrate-related systems, this embodiment uses SPR technology to detect the affinity of WIN51708 in the Cyp21a1-17-Hydroxyprogesterone-related system.

[0069] Cyp21a1 was immobilized on the surface of a CM5 chip at a concentration of 20 μg / mL, with an immobilization level of approximately 12600 RU. Seven concentration gradients were set for analyte 17-Hydroxyprogesterone + WIN51708, ranging from 1.56 μM to 100.00 μM, and detection was performed using a multi-cycle kinetic method.

[0070] .

[0071] The results showed that WIN51708 could still participate in the Cyp21a1 binding process in the 17-Hydroxyprogesterone-related system, with an equilibrium dissociation constant KD of 2.52 × 10^-5 M. This suggests that WIN51708 can influence the binding process of Cyp21a1-related steroid substrate systems, providing support for its intervention in Cyp21a1-mediated steroid metabolic pathways.

[0072] It should be noted that the above SPR results demonstrate that Cyp21a1 inhibitors have a direct binding relationship with Cyp21a1 protein, but whether it is competitive inhibition, non-competitive inhibition, or allosteric regulation still needs to be further confirmed by enzyme activity assays, substrate competition experiments, or kinetic enzymology experiments.

[0073] Example 7: Cyp21a1 inhibitor WIN51708 promotes testosterone synthesis in TM3 cells TM3 testicular interstitial cells were randomly divided into a blank control group, a model group, and a Cyp21a1 inhibitor WIN51708 treatment group. The model group was treated under high glucose conditions; the drug treatment groups were treated with different concentrations of Cyp21a1 inhibitor under model conditions. After treatment, cells and culture supernatants were collected, and cell viability, Cyp21a1 expression, testosterone content, and expression of steroid-related molecules were detected.

[0074] The results showed that, compared with the model group, treatment with Cyp21a1 inhibitors enhanced the testosterone synthesis capacity of TM3 cells. This indicates that Cyp21a1 inhibitors have a role in improving steroid production in Leydig cells in vitro.

[0075] Example 8: Cyp21a1 inhibitor WIN51708 improves testosterone levels and sperm quality in diabetic mice To evaluate the therapeutic effect of WIN51708 on diabetic testicular lesions, diabetic model mice were randomly divided into a model control group and a WIN51708 treatment group, with a normal control group also included. Mice in the WIN51708 treatment group received intraperitoneal injections of WIN51708 at a dose of 2 mg / kg once daily for 30 consecutive days. Mice in the model control group received an equal volume of the solvent intraperitoneally during the same period, while the normal control group received the same treatment.

[0076] After the last administration, serum, testis, and epididymal tissues were collected from mice in each group. Serum testosterone levels were detected using enzyme-linked immunosorbent assay (ELISA). The expression of Cyp21a1 and steroid-related molecules in testicular tissue was detected, and the density and motility of epididymal sperm were measured.

[0077] The results showed that, compared with the model control group, the serum testosterone level of mice in the WIN51708 treatment group was increased, and the sperm density and sperm motility were improved, suggesting that intraperitoneal injection of WIN51708 at 2 mg / kg per day for 30 consecutive days can improve the testosterone level and sperm quality of diabetic mice.

[0078] See Figure 1 Analysis revealed that compared to the db / db normal control group (NC), the db / db diabetic group (DM) mice had significantly higher body weight and fasting blood glucose levels. The oral glucose tolerance test (OGTT) showed significantly increased blood glucose levels and area under the curve (AUC) at all time points, along with elevated HOMA-IR, indicating significant glucose metabolism disorder and insulin resistance in the mice. Semen analysis showed significantly decreased sperm density and motility in the DM group. HE staining of testicular tissue revealed that the seminiferous tubules in the NC group were structurally intact and tightly packed, with relatively clear spermatogenic cell layers; while the seminiferous tubules in the DM group showed disordered structure, enlarged lumen, and loosely packed and fewer spermatogenic cells. Figure 1 The display shows 40× and 200× field of view. P < 0.0001. NC: normal control group (db / m); DM: diabetes group (db / db).

[0079] See Figure 2 Analysis revealed that, according to Western blot results, Cyp21a1 protein expression was significantly increased in the testicular tissue of db / db diabetic (DM) mice compared to the db / m normal group (NC), with β-actin used as an internal control. Immunohistochemical staining and mean optical density (AOD) analysis further indicated enhanced Cyp21a1 staining in the testicular tissue of the DM group, with expression levels significantly higher than those in the NC group. The figure shows 100× and 400× fields of view; the arrows indicate positively stained areas. P < 0.001, P < 0.0001. NC: normal db / m group; DM: db / db diabetes group.

[0080] See Figure 3 Analysis revealed that serum testosterone levels were significantly lower in the db / db diabetic group (DM) compared to the normal db / m group (NC), suggesting that diabetes may be accompanied by insufficient testosterone secretion. Data are presented as mean and error bars. NC: normal db / m group; DM: db / db diabetic group.

[0081] See Figure 4Analysis revealed that compared to the negative control group (sh-NC), Cyp21a1 mRNA expression was significantly reduced in the sh-Cyp21a1 group, indicating a good knockdown effect; simultaneously, testosterone levels were increased. These results suggest that reducing Cyp21a1 expression may be beneficial for testosterone synthesis or secretion in TM3 cells. (n=6) P < 0.001. sh-NC: negative control group; sh-Cyp21a1: Cyp21a1 knockdown group.

[0082] See Figure 5 Analysis revealed that the binding of WIN51708 to CYP21A1 protein was detected using surface plasmon resonance (SPR) technology. As the concentration of WIN51708 increased from 0.781 μM to 50 μM, the response signal gradually increased, exhibiting a concentration-dependent binding and dissociation process. Kinetic fitting results showed that the binding rate constant (ka) was 4.47 × 10⁻⁶. 3 M -1 ·s -1 The dissociation rate constant (kd) is 1.40 × 10⁻⁶. -2 s -1 The equilibrium dissociation constant (KD) is 3.13 × 10⁻⁶. -6 M indicates that WIN51708 can directly bind to the CYP21A1 protein. RU: Response unit.

[0083] See Figure 6 Analysis revealed that the binding of 17-Hydroxyprogesterone and WIN51708 to CYP21A1 protein was detected using surface plasmon resonance (SPR) technology. The response signal gradually increased in concentration-dependent manner as the analyte concentration increased from 1.56 μM to 100 μM. Kinetic fitting results showed that the binding rate constant (ka) was 1.97 × 10⁻⁶. 3 M -1 ·s -1 The dissociation rate constant (kd) is 4.97 × 10⁻⁶. -2 s -1 The equilibrium dissociation constant (KD) is 2.52 × 10⁻⁶. -5 M indicates that the system can bind to the CYP21A1 protein. RU: Response unit.

[0084] See Figure 7Analysis revealed that TM3 cells cultured under high glucose conditions were treated with 0, 1, 3, and 10 μM WIN51708, and the cell culture supernatant was collected at 0, 12, and 24 h after treatment to detect testosterone levels. Five independent replicates were set up for each treatment condition. The results showed that compared with the 0 μM group, the testosterone content in the culture supernatant of the WIN51708-treated groups was increased, exhibiting a concentration- and time-dependent trend within the concentration range of this experiment, suggesting that WIN51708 can promote testosterone synthesis or secretion in TM3 cells under high glucose conditions.

[0085] See Figure 8 Analysis revealed that, compared with the DMSO control group, diabetic mice treated with intraperitoneal injection of the Cyp21a1 inhibitor WIN51708 showed significantly increased serum testosterone levels, sperm density, and sperm motility. These results suggest that WIN51708 intervention can improve testosterone levels and sperm quality in diabetic mice. DMSO: solvent control group; WIN51708: Cyp21a1 inhibitor treatment group. P < 0.001.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a cytochrome P45021A1 (Cyp21a1) inhibitor, WIN51708, in the preparation of a drug for treating diabetes-related testicular lesions.

2. The application according to claim 1, characterized in that, The application of cytochrome P45021A1 (Cyp21a1) as a molecular target in early screening drugs, diagnostic drugs, efficacy evaluation drugs, or therapeutic drugs for diabetes-related testicular lesions.

3. The application according to claim 2, characterized in that, The application of cytochrome P45021A1 (Cyp21a1) as a molecular target in the early screening of diabetes-related testicular lesions includes: screening for diabetes-related testicular lesions in diabetic patients based on the higher expression level of Cyp21a1 in the testicular tissue of diabetic patients than in the testicular tissue of non-diabetic patients, and the decreased testosterone content in the testicular tissue of diabetic patients.

4. The application according to claim 1, characterized in that, The Cyp21a1 inhibitor WIN51708 is used to directly bind to the Cyp21a1 protein.

5. The application according to claim 4, characterized in that, The equilibrium dissociation constant KD between the Cyp21a1 inhibitor WIN51708 and the Cyp21a1 protein is 10^-6 M to 10^-5 M.

6. The application according to claim 1, characterized in that, The Cyp21a1 inhibitor WIN51708 was used to increase testosterone levels in a diabetes-associated testicular lesion model.

7. The application according to claim 1, characterized in that, The use of a specific knockdown virus of the cytochrome P45021A1 (Cyp21a1), or the use of the cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 in a medicament for improving spermatogenesis in patients with diabetes-associated testicular lesions.

8. The application according to claim 1, characterized in that, The cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 is used to promote testosterone synthesis in TM3 cells in vitro.

9. The application according to claim 1, characterized in that, The cytochrome P45021A1 (Cyp21a1) inhibitor WIN51708 was administered via intraperitoneal injection at a dose of 2 mg / kg once daily for 30 consecutive days to improve sperm quality in mice with diabetic testicular lesions.

10. A reagent kit, characterized in that, The invention includes an antibody for detecting the expression of the cytochrome P45021A1 (Cyp21a1) antibody as a drug target for detecting diabetes-related testicular lesions, as described in any one of claims 1 to 9; and the cytochrome P45021A1 (Cyp21a1) protein as a drug target for detecting diabetes-related testicular lesions.

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