Use of an agent that inhibits expression of PPARa in the manufacture of a medicament for the treatment of male infertility

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a reagent for inhibiting PPAR alpha expression in preparation of a medicine for treating male infertility diseases, wherein a PPAR alpha inhibitor (GW6471) or an active oxygen scavenger (NAC) is given to male mice exposed to cadmium water, it is found that the number of sperm in a Cd+GW6471 group is higher than that in a Cd+NAC group, the serum testosterone level in the Cd+GW6471 group is also higher than that in the Cd+NAC group, and the results of testicular tissue pathological staining also show that GW6471 can more significantly restore the decrease in the number of mature spermatogenic tubules caused by Cd, GW6471 restores the decrease in the number of SYCP3 positive spermatocytes caused by Cd, so that the sperm production efficiency is restored, and therefore the application of the reagent for inhibiting PPAR alpha expression in preparation of the medicine for treating male infertility diseases is proposed.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of reagents that inhibit PPARα expression in the preparation of drugs for treating male infertility. Background Technology

[0002] Cadmium (Cd) is a proven environmental toxin linked to male infertility. Extensive epidemiological and toxicological evidence suggests that cadmium exposure leads to decreased sperm quality, testosterone imbalance, and pathological damage to testicular tissue. Despite these associations, mechanistic insights remain fragmented; decades of research have yielded only piecemeal conclusions, such as oxidative stress or DNA damage responses, without establishing a systematic causal network. This fragmentation hinders the identification of key intervention targets because independent research data makes it difficult to determine treatment priorities, and the role of PPARα in male infertility has not yet been reported using current technology. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides the application of reagents that inhibit PPARα expression in the preparation of drugs for treating male infertility.

[0004] The application of agents that inhibit PPARα expression in the preparation of drugs for treating male infertility. The amino acid sequence of PPARα is: MVDTESPLCPLSPLEADDLESPLSSEEFLQEMGNIQEISQSIGEDSSGSFGFTEYQYLGSCPGSDGSVITDTLSPASSPSSVTYPVVPGSVDESPSGALNIECRICGDKASGYHYGVHACEGCKGFFRRTIRLKLVYDKCDRSCKIQKKNRNKCQYCRFHKCLSVGMSHNAIRFGRMPRSEKAKLKAEILTCEHDLIEDSETADLKSLAKRIYEAY LKNFNMNKVKARVILSGKASNNPPFVIHDMETLCMAEKTLVAKLVANGIQNKEAEVRIFHCCQCTSVETVTELTEFAKAIPGFANLDLNDQVTLLKYGVYEAIFAMLSSVMNKDGMLVAYGNGFITREFL KSLRKPFCDIMEPKFDFAMKFNALELDDSDISLFVAAIICCGDRPGGHLLNVIEKMQEGIVHVLRLHLQSNHPDDIFLFPKLLQKMADLRQLVTEHAQLVQIIKKTESDAALHPLLQEIYRDMY, recorded as SEQ ID NO.1.

[0005] This invention administers PPARα inhibitors or reactive oxygen species (ROS) scavengers to male mice exposed to cadmium-contaminated water. The results showed that the sperm count in the PPARα inhibitor group was higher than that in the ROS scavenger group, and the serum testosterone level in the PPARα inhibitor group was also higher. Testicular tissue pathological staining also indicated that the PPARα inhibitor more significantly restored the reduction in the number of mature seminiferous tubules caused by Cd, and restored the reduction in the number of SYCP3-positive spermatocytes caused by cadmium, thus restoring spermatogenesis efficiency. Therefore, the application of reagents that inhibit PPARα expression in the preparation of drugs for treating male infertility is proposed.

[0006] Preferably, the reagent for inhibiting PPARα expression is GW6471.

[0007] Preferably, the drug uses GW6471 as its active ingredient.

[0008] Preferably, the drug also includes a medically acceptable carrier.

[0009] Preferably, the medically acceptable carrier is a diluent.

[0010] Preferably, the infertility is cadmium-induced infertility.

[0011] Preferably, the drug is used to increase sperm count.

[0012] Preferably, the drug is used to reverse the reduction in the number of mature seminiferous tubules caused by Cd.

[0013] Preferably, the drug is used to increase testosterone levels.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] This invention administers a PPARα inhibitor (GW6471) or a reactive oxygen species scavenger (NAC) to male mice exposed to cadmium water. The results showed that the sperm count in the Cd+GW6471 group was higher than that in the Cd+NAC group, and the serum testosterone level in the Cd+GW6471 group was also higher. Testicular histopathological staining also indicated that GW6471 significantly restored the reduction in the number of mature seminiferous tubules caused by Cd, and restored the reduction in the number of SYCP3-positive spermatocytes caused by cadmium, thus restoring spermatogenesis efficiency. Therefore, the application of reagents that inhibit PPARα expression in the preparation of drugs for treating male infertility is proposed. Attached Figure Description

[0016] Figure 1The study shows that cadmium exposure primarily leads to male infertility by reducing testosterone levels. A shows the key events (KEs) of cadmium-induced male infertility further screened based on the AOP-Wiki. B shows an undirected network of nine adverse outcome pathways (AOPs) for cadmium-induced male infertility, where each node represents an AOP, and an edge between two nodes indicates that the two AOPs share at least one key event. C shows a single-cell atlas of testicular tissue. D shows changes in the transcriptome of single cells after cadmium treatment. E shows a Venn diagram of the intersection of differentially expressed genes in the testicular interstitial cell transcriptome with key genes screened from the CTD database. F shows a volcano plot of the testicular interstitial cell transcriptome. G shows GO enrichment analysis based on downregulated intersection genes.

[0017] Figure 2 The association between cadmium exposure and serum testosterone levels is shown in Figure A, which shows a flowchart of the participants included in the final analysis (N=2920) from the National Health and Nutrition Examination Survey (NHANES) in the United States, covering the periods of 1999–2004 and 2011–2016. Figure B shows a violin plot of blood cadmium versus testosterone concentration. Figure C shows the nonlinear relationship between blood cadmium concentration and serum testosterone levels. Figure D shows the further nonlinear relationship after stratification by BMI.

[0018] Figure 3The discovery and validation of key molecular initiation events are shown. AB displays subnetworks of the undirected network of nine types of male infertility AOPs induced by cadmium. These are divided into two categories: testosterone reduction caused by PPARα activation and testosterone reduction caused by increased reactive oxygen species. A represents testosterone reduction caused by PPARα activation, B represents testosterone reduction caused by increased reactive oxygen species, C is a flowchart of the animal experiment, and DF shows representative images and quantitative analysis of epididymal sperm counts (n=17 mice). D represents representative images, E describes sperm count, and F describes sperm motility. G represents serum testosterone levels detected by enzyme-linked immunosorbent assay (ELISA), n=7 mice; HK represents representative images and quantitative analysis of testicular CYP11A1, 3β-HSD, and STAR protein expression detected by Western blotting, n=4 mice, where H is a representative image, I is a quantitative analysis of CYP11A1, J is a quantitative analysis of 3β-HSD, K is a quantitative analysis of STAR; LM represents representative images and quantitative analysis of the number of seminiferous tubules at different stages, n=5 mice, where L is a representative image. Images, M for quantitative analysis, NO for representative images and quantitative analysis of SYCP3-positive testicular cells detected by immunohistochemistry, n=4 mice, where N is a representative image and O is a quantitative analysis, PR for representative images and quantitative analysis of PPARα and HO-1 protein expression in the testes detected by Western blotting, n=4 mice, where P is a representative image and R is a quantitative analysis, SU for TM3 cells treated with cadmium chloride (20 μmol) for 0, 6, 12, and 24 hours, as detected by Western blotting. Representative images and quantitative analysis of PPARα and CYP11A1 protein expression in cells were detected by the qPCR method. S represents a representative image, T represents the quantitative analysis of CYP11A1, U represents the quantitative analysis of PPARα, and V represents the expression of PPARα and CYP11A1 promoter binding sites in C57BL / 6J mice treated with 200 mg / L cadmium chloride. The results are shown in the qPCR quantitative image. *p<0.05, **p<0.01, ***p<0.001, intergroup comparisons.

[0019] Figure 4 The results show that GW6471 has superior binding properties and conformational regulation capabilities compared to cadmium ions. Among them, A shows the molecular docking simulation of PPARα with cadmium ions, B shows the molecular docking simulation of PPARα with GW6471, C shows the RMSD value of the complex system, D shows the rotation radius diagram of the experimental system, EF is the protein free energy speciation diagram, E is the protein free energy speciation diagram of PPARα with cadmium ions, and F is the protein free energy speciation diagram of PPARα with GW6471. Detailed Implementation

[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0021] The experiments involved in this invention were conducted according to the following methods:

[0022] 1. Animal experiments

[0023] All experiments used male C57BL / 6J mice (SPF grade). These mice were housed in an SPF-grade animal facility under standard environmental conditions (temperature 20-25°C, humidity 50-60%, 12-hour light / dark cycle) with free access to food and water. After a one-week acclimatization period, the 5-week-old mice were randomly divided into six groups:

[0024] Control group: Normal drinking water;

[0025] Group Cd: Drinking water contains 200 mg / L of cadmium chloride;

[0026] N-acetylcysteine ​​group: daily intraperitoneal injection of 300 mg / kg NAC;

[0027] GW6471 group: 20 mg / kg of GW6471 was injected intraperitoneally every other day;

[0028] Cd+NAC group: drinking water containing 200 mg / L cadmium chloride, and intraperitoneal injection of 300 mg / kg NAC daily;

[0029] Cd+GW6471 group: drinking water containing 200 mg / L cadmium chloride, and intraperitoneal injection of 20 mg / kg GW6471 every other day.

[0030] The drugs in each group were dissolved and diluted using 10% DMSO as a diluent to prepare injection solutions.

[0031] After 35 days of treatment (one spermatogenesis cycle), the mice were anesthetized with tribromoethanol and then euthanized for sample collection. Blood, testes, and epididymis were collected for subsequent analysis. The Cd, Cd+NAC, and Cd+GW6471 groups were given cadmium-containing water throughout the experiment, while NAC and GW6471 compounds were administered via intraperitoneal injection, with dosage and method of administration performed as specified. All procedures were performed in accordance with the institution's animal care guidelines.

[0032] All animal experiments were approved by the Experimental Animal Ethics Committee of Anhui Medical University (Ethics Approval Number: LLSC20241672).

[0033] 2. Cell Culture

[0034] The mouse TM3 testicular interstitial cell line is a well-established model for studying testosterone synthesis. This cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dubick Modified Eagle Medium / Nutrient M12 (DMEM / F-12) containing 5% horse serum and 2.5% fetal bovine serum, and cultured under humid conditions at 37°C and saturated with 5% carbon dioxide. To investigate the reproductive toxicity mechanism caused by cadmium, cells were treated with 20 μmol of cadmium for periods ranging from 0 to 24 hours.

[0035] 3. Sperm count

[0036] Sperm count and motility were assessed using an automated semen analyzer (Hamilton Thorne, USA). The left epididymis of mice was cut into 70 pieces and placed in preheated saline. The saline was then incubated at 37°C for 5 minutes until all sperm were released. The final step involved diluting the sperm suspension with preheated medium and placing 20 μL of the suspension onto a glass slide for parameter evaluation.

[0037] 4. Immunoblotting

[0038] Total protein was extracted from mouse testes using RIPA homogenate buffer and quantified using a BCA protein assay kit. Proteins were separated by electrophoresis on 7%–15% SDS-PAGE gels and transferred to PVDF membranes (IPVH00010, Milwaukee). The membranes were blocked with 5% non-lipoic whey protein for 1.5 hours, then coated with primary antibody at 4°C and incubated overnight, followed by incubation with secondary antibody for 2 hours. Finally, images were captured using a Bio-Rad ChemiDoc™ MP imaging system, and quantification was performed using ImageJ software.

[0039] 5. Immunohistochemistry

[0040] After fixation with 4% mDF / PFA, the tissue was cut into 5 mm thick paraffin sections, followed by dewaxing, rehydration, infiltration, antigen retrieval, peroxidase blocking, and inhibition. The sections were then incubated overnight at 4°C with SYCP3 (1:200) antibody. The next day, after incubation with HRP-labeled homologous secondary antibody, the sections were bound with DAB solution. Cell nuclei were stained with hematoxylin. Photographs were taken using a microscope (Olympus BX53F).

[0041] 6. Chromatin Immunoprecipitation

[0042] Following a standardized chromatin immunoprecipitation (ChIP) procedure, mouse testicular tissue was cross-linked with 1% formaldehyde, homogenized, and then the reaction was terminated with glycine. The tissue was then washed with cold PBS and lysed sequentially using two different lysis buffers. Subsequently, the chromatin was broken down to an average length of 200-500 base pairs by sonication (using a Bioruptor instrument, set to medium intensity, 10 cycles of 30 seconds ON / 30 seconds OFF). Immunoprecipitation was performed using a specific antibody (ThermoFisher, MA1-822) against PPARα, with normal mouse IgG used as a negative control, and the immunoprecipitation reaction was incubated overnight at 4°C. The antibody-chromatin complex was captured with magnetic beads and then washed with low-salt, high-salt, lithium chloride buffer, and TE buffer. The complex was then reverse cross-linked and eluted, followed by purification of the associated DNA via phenol-chloroform extraction and ethanol precipitation. Finally, the DNA was analyzed by qPCR using a covering... Cyp11a1 Specific primers for gene promoter regions are used to quantify enriched DNA fragments, thereby analyzing the precise binding sites of PPARα.

[0043] 7. Detection of cadmium content in the testes and blood

[0044] Quantitative analysis of cadmium concentration in testicular and blood samples was performed using inductively coupled plasma mass spectrometry (ICP-MS). For testicular analysis, 1 gram of tissue was weighed and homogenized, then digested with 8 mL of concentrated nitric acid (HNO3) using a digestion apparatus. Blood samples (100 μL) were digested directly with 8 mL of nitric acid. After digestion, all samples were diluted to a constant volume (10 mL), filtered through a 0.45 μm membrane filter, and analyzed using ICP-MS. Quantitative analysis was performed using an external calibration curve matched to the matrix.

[0045] 8. Molecular model construction

[0046] (1) Molecular docking

[0047] Protein-ligand docking was performed to characterize binding interactions. For the cadmium-PPARα complex, metal binding sites were predicted using the MIB2 server (default parameters). The highest-scoring conformation (score: 3.955) was selected, revealing coordination bonds between cadmium²⁺ and residues N415, D371, and S373. For GW6471-PPARα, calculations were performed using AutoDock Vina (v1.1.2) with a grid centered on the cadmium binding site. The lowest-energy conformation (binding energy: -7.2 kcal / mol) showed hydrogen bonding and hydrophobic interactions within the same binding pocket. All structures were visualized using PyMOL 3.0.

[0048] (2) Molecular dynamics simulation

[0049] The top-ranked docking complex underwent 100-nanosecond molecular dynamics simulations using AMBER24. Modeling of the PPARα-GW6471 system employed the ff19SB force field to simulate the protein, the gaff2 parameters to simulate the ligand, and previous results for the Cd ion parameters. All systems were solvated in an OPC water model, neutralized with counterions, and subjected to energy minimization. Production simulations were performed under NPT ensemble conditions (310 K, 1 Pa) with a time step of 2 nanoseconds and SHAKE bond constraints. Trajectory analysis included calculating the root mean square deviation (RMSD) to assess backbone stability, the radius of rotation (Rg) to assess structural compactness, calculating the binding free energy using the last 40 nanoseconds of the simulation data via the MM / PBSA method, and projecting the free energy landscape (FEL) onto the RMSD and Rg coordinates.

[0050] 9. Statistical Analysis

[0051] Descriptive analyses were performed to statistically analyze blood cadmium exposure and serum testosterone concentrations during the NHANES survey period. RCS simulated the nonlinear dose-response relationship and quantitatively determined the inflection point. Stratified analyses assessed the effect of body mass index category (overweight / obese vs. normal weight) on the effect. For in vivo data, results are presented as mean ± standard error (SEM), with the number of independent replicates (n) indicated in the legend. Bonferroni correction was used for post-hoc comparisons following one-way ANOVA. All analyses were performed within R 4.4.1, and statistical significance was defined as p < 0.05.

[0052] result

[0053] 1. Decreased testosterone levels are a key event in cadmium-induced male infertility.

[0054] This invention identified eight cell types in the testes of C57BL / 6J mice based on single-cell transcriptome data. Figure 1 C). This invention obtained transcriptome data of testicular cells from a cadmium-exposed group (drinking water containing cadmium chloride) and a control group. Differentially expressed genes in testicular interstitial cells were cross-referenced with key genes identified in the previous step, yielding 64 genes ( Figure 1 DE). Figure 1 F shows the expression of these 64 cross-expression genes in differentially expressed genes in testicular interstitial cells. The results indicate that most cross-expression genes are downregulated. Among them, the downregulated gene with the largest change in differential expression is... Cyp11a1 . Cyp11a1This gene is involved in the synthesis of steroid hormones and is a key gene regulating testosterone synthesis, suggesting that decreased testosterone levels may be a central key event in cadmium-induced male infertility. GO analysis of the downregulated intergrowth genes revealed significant enrichment in pathways such as testosterone synthesis, cholesterol synthesis, and cellular responses to testosterone stimulation. Figure 1 G).

[0055] In conclusion, the above data indicate that decreased testosterone levels are a key event in cadmium-induced male infertility.

[0056] 2. Critical event verification based on the NHANES database

[0057] To verify the relationship between cadmium and testosterone from an epidemiological perspective, this invention employed a cross-sectional survey method. 2920 eligible adult male participants from the NHANES study (1999–2004 and 2011–2016) were included. Figure 2 A). It should be noted that, since previous studies have shown that age 44 is a turning point in aging, this invention excluded participants over the age of 44 to avoid age-related decline in testosterone levels. The average age of the participants was 30 years, and 62.95% of the subjects were overweight or obese. Figure 2 B shows the distribution of blood cadmium concentration and serum testosterone levels in the study population across different NHANES cycles. Blood cadmium concentrations ranged from 0.07 to 9.17 μg / L, while serum testosterone levels ranged from 2.7 to 5000 ng / dL. The RCS curves showed a significant non-linear association between blood cadmium levels and serum testosterone concentrations (overall P < 0.05, non-linear P < 0.05). Figure 2 C indicates that low-dose cadmium exposure initially increases testosterone levels, with a peak response at approximately 1.17 (1.15–1.19) μg / L; beyond this threshold, serum testosterone concentrations gradually decrease with increasing cadmium exposure.

[0058] In addition, stratified analysis was performed. Notably, the inflection point of the RCS curve appeared earlier in participants with normal body mass index (BMI). Figure 2 (D) These data indicate that serum testosterone levels decrease with increasing cadmium exposure after exceeding a certain threshold. This also highlights the importance of testosterone reduction in cadmium-induced male infertility.

[0059] 3. Activation of PPARα is a key molecular initiation event in cadmium-induced male infertility.

[0060] To clarify the key molecular initiation events leading to cadmium-induced testosterone reduction, this invention further performed subnetwork analysis on the AOP network using the MCODE algorithm in Cytoscape software. The analysis results show that, based on the subnetwork analysis, the potential key pathways leading to cadmium-induced testosterone reduction and male infertility can be divided into PPARα activation and increased reactive oxygen species (ROS). Figure 3 AB).

[0061] Then, an in vivo intervention experiment was designed for further investigation. Male mice exposed to cadmium-contaminated water were given either a PPARα inhibitor (GW6471) or a reactive oxygen species scavenger (NAC). All animals were euthanized 35 days after exposure. Figure 3 C). To compare the intervention effects, a relatively high dose (200 mg / L) was selected for exposure. Subsequent ICP-MS analysis of blood cadmium levels showed that the average exposure concentration in mice was 6.68 μg / L, which is within the range of cadmium levels in the blood of NHANES individuals (0.07–9.17 μg / L).

[0062] Sperm analysis showed that both interventions effectively restored cadmium-induced sperm count reduction. Figure 3 Interestingly, the sperm count in the Cd+GW6471 group was higher than that in the Cd+NAC group (DF). Figure 3 D). Furthermore, serum testosterone levels in the Cd+GW6471 group were higher than in the Cd+NAC group, therefore the GW6471 intervention group was more effective than the NAC intervention group. Figure 3 G). Western blotting results of testicular tissue showed that a specific testosterone synthase (CYP11A1) was involved in this process. Figure 3 HK). Histopathological staining of testicular tissue also showed that GW6471 could more significantly restore the reduction in the number of mature seminiferous tubules caused by Cd. Figure 3 LM). Previous studies have found that Cd-induced sperm quality decline is associated with meiotic disorders. Here, both interventions effectively reversed the reduction in the number of SYCP3-positive spermatocytes caused by cadmium, indicating that spermatogenesis efficiency was restored. Figure 3 NO). As expected, compared with the Cd group, the protein level of PPARα was significantly reduced in the Cd+GW6471 group, while the protein level of the oxidative stress effector HO-1 was significantly reduced in the Cd+NAC group. Figure 3 PR). Furthermore, 24-hour cadmium exposure increased PPARα protein levels in TM3 cells but decreased CYP11A1 protein levels. Figure 3 SU). The results of the ChIP-qPCR experiment showed that PPARα and Cyp11a1 Multiple promoter subregions combined ( Figure 3 The above results suggest that PPARα activation may be a key molecular initiation event in cadmium-induced male infertility.

[0063] 4. Verification of molecular initiation events

[0064] The above results indicate that PPARα activation is a more critical molecular initiation event compared to the oxidative stress pathway. To further verify the reliability of this critical molecular initiation event, this invention combines bioinformatics methods to verify the direct interaction between GW6471 and PPARα and its potential ability to competitively inhibit cadmium binding.

[0065] Molecular docking analysis showed that cadmium ions bind to residues N415, D371, and S373 of PPARα via metal-coordinate bonds. The binding sites were predicted by MIB2 and 26, with a score of 3.955. Figure 4 A). However, GW6471 forms a more stable complex at the same position through hydrogen bonding and hydrophobic interactions, with a binding energy of -7.2 kcal / mol ( Figure 4 B). Molecular dynamics simulations further show that the RMSD value of the GW6471 complex ( Figure 4 C) and radius of rotation (Rg) Figure 4 D) was significantly lower than that of the cadmium ion complex, indicating that its binding significantly improved the overall conformational stability of the protein. Analysis of the binding free energy showed that the binding free energy of GW6471 (-24.82 kcal / mol) was significantly lower than that of the cadmium ion (-9.21 kcal / mol), while the free energy spectrogram ( Figure 4 As shown in E and F, the GW6471 complex forms a deep and narrow energy trap, while the cadmium ion complex has a shallow and wide energy trap. This indicates that GW6471 achieves stronger binding affinity through a variety of non-covalent interactions (van der Waals forces, hydrogen bonds).

[0066] Overall, GW6471 forms a more stable interaction network by competitively occupying cadmium binding sites. It not only directly inhibits the binding of cadmium to PPARα, but also indirectly regulates the functional activity of PPARα by inducing protein conformational contraction and enhancing the flexibility of local residues. This dual mechanism explains the effect of GW6471 in reversing cadmium toxicity at the molecular level, providing a theoretical basis for targeted interventions against cadmium-induced male infertility.

[0067] The Action-Oriented (AOP) research model has been widely used in toxicology due to its ability to construct mechanistic knowledge systems, enabling predictive risk assessments of endocrine disruptors and carcinogens. However, its application to cadmium-induced reproductive toxicity has been slow: while individual effect indicators such as germ cell apoptosis have been proposed, a standardized network has not yet been established to clarify causal or temporal relationships related to cadmium. Crucially, the lack of a validated AOP network makes it impossible to distinguish between direct causes and indirect responses to testicular injury. For example, pathways like testosterone synthesis are frequently mentioned but lack cross-system validation. This limitation hinders the process of translating mechanistic data into public health interventions.

[0068] To resolve this impasse, a multidimensional approach spanning different biological scales is crucial. For example, network toxicology combines omics and literature data to map gene-phenotype networks, revealing key nodes affected by toxins. Epidemiological research resources like NHANES provide population-level validation of mechanistic associations, alleviating concerns about extrapolation. Furthermore, in vivo models can validate causal relationships of key effector elements through targeted interventions at critical AOP nodes, while molecular modeling can resolve molecular interactions at the atomic scale. These approaches work together to overcome the limitations of single-system studies.

[0069] This invention combines network toxicology, single-cell transcriptomics, NHANES analysis, in vivo intervention, and molecular simulation to: (1) construct the first standardized AOP network for cadmium-induced male infertility; (2) identify key AOPs through cross-system validation; and (3) validate therapeutic targets within this pathway. In summary, this invention employs a comprehensive multi-method framework to systematically map and validate key AOPs in cadmium-induced male infertility, thereby establishing a new paradigm for precision intervention in environmental toxicology.

[0070] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0071] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0072] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a reagent that inhibits PPARα expression in the preparation of drugs for treating male infertility, characterized in that, The amino acid sequence of PPARα is shown in SEQ ID NO.

1. The reagent used to inhibit PPARα expression is GW6471, and the infertility is cadmium-induced infertility.

2. The application according to claim 1, characterized in that, The drug uses GW6471 as its active ingredient.

3. The application according to claim 2, characterized in that, The drug also includes a medically acceptable carrier.

4. The application according to claim 3, characterized in that, The medically acceptable carrier is a diluent.

5. The application according to claim 1, characterized in that, The drug is used to increase sperm count.

6. The application according to claim 1, characterized in that, The drug is used to reverse the reduction in the number of mature seminiferous tubules caused by Cd.

7. The application according to claim 1, characterized in that, The drug is used to increase testosterone levels.

Images