Application of pterostilbene in preparation of medicine for preventing or treating dyszoospermia

Abstract

The invention relates to the technical field of non-targeted metabonomics, and discloses an application of pterostilbene in preparation of a medicine for preventing or treating dyszoospermia, and the medicine contains pterostilbene as an active component. In addition, the pterostilbene inhibits oxidative stress reaction by adjusting PI3K / AKT / mTOR signal channels and improves the pathological structure of testis tissues so as to prevent or treat cyclophosphamide-induced spermatogenesis dysfunction. According to the application, CTX is used for establishing a mouse dyszoospermia model, network pharmacological analysis and experimental verification are combined to discuss key action targets and pathways of PTS for preventing and treating dyszoospermia and a specific molecular mechanism, and meanwhile, metabonomics is combined to obtain the pterostilbene capable of preventing cyclophosphamide-induced male testicular dysfunction.

Description

Technical Field

[0001] This invention relates to the field of non-targeted metabolomics technology, and more specifically, to the application of pterostilbene in the preparation of drugs for the prevention or treatment of spermatogenesis disorders. Background Technology

[0002] Cyclophosphamide (CTX) has become one of the most commonly used antitumor drugs in humans and veterinary medicine, widely used in the treatment of various cancers, such as leukemia, breast cancer, and prostate cancer. CTX has also been a milestone in the treatment of kidney diseases, particularly lupus nephritis and systemic vasculitis. Its use is common in children with refractory nephrotic syndrome and young, infertile patients with systemic lupus erythematosus. CTX is cytotoxic to rapidly dividing cells; therefore, its interaction with rapidly proliferating tissues is central to its therapeutic properties and toxicity. Due to its effects on rapidly dividing cells, the reproductive system, including the testes and ovaries, is highly sensitive to CTX. Currently, reproductive disorders are a major challenge for young men undergoing CTX treatment. CTX application leads to a significant reduction in sperm count, sperm motility, and sperm performance, subsequently inducing testicular damage. Furthermore, CTX treatment increases the frequency of oligospermia and azoospermia, and its effects on the pituitary-gonadal axis are also believed to reduce the rates of spermatogenesis and oogenesis. CTX treatment significantly reduces sperm motility and causes histological changes in the testes. Gonadotropin secretion disorders and testicular damage have also been observed in patients receiving CTX treatment. Therefore, CTX-induced spermatogenesis disorders have become a significant clinical problem that urgently needs to be addressed. However, our understanding of the molecular mechanisms of CTX-induced testicular damage is currently limited, and effective drugs for preventing and alleviating CTX-induced reproductive damage are also lacking.

[0003] Pterostilbene (PTS) molecular formula C 16 H 16 O3 (oxygen 3) is a non-flavonoid polyphenol compound with various biological activities. PTS (polyphenolic sulfide) is mainly derived from plants such as sandalwood, blueberry, grape, and rosewood, and possesses good antioxidant, anti-inflammatory, and anti-tumor capabilities. Given the strong antioxidant activity of PTS, and the close relationship between reproductive damage and oxidative stress disorders, studying the protective effect of PTS against reproductive damage is of great practical significance. Summary of the Invention

[0004] In view of this, the present invention proposes the application of pterostilbene in the preparation of drugs for the prevention or treatment of spermatogenesis disorders.

[0005] The present invention proposes the application of pterostilbene in the preparation of drugs for the prevention or treatment of spermatogenesis disorders. The drugs contain pterostilbene as an active ingredient, and pterostilbene prevents or treats cyclophosphamide-induced spermatogenesis disorders by regulating the PI3K / AKT / mTOR signaling pathway, inhibiting oxidative stress response, and improving the pathological structure of testicular tissue.

[0006] Furthermore, in the preparation of the drug, pterostilbene alleviates oxidative damage to spermatogenic cells by reducing malondialdehyde content in testicular tissue and increasing the activity of superoxide dismutase, catalase, and glutathione.

[0007] Furthermore, in the preparation of the drug, pterostilbene is used to upregulate the mRNA and protein expression levels of estrogen receptor 1, epidermal growth factor receptor, CYP17A1, CYP19A1, PI3K, AKT, and mTOR, and to downregulate the expression levels of cyclooxygenase 2, CYP1A1, and CYP1B1.

[0008] Furthermore, in the preparation of the drug, pterostilbene improves the proportion of peripheral blood immune cells, increases the proportion of lymphocytes and decreases the proportion of granulocytes, thereby alleviating the inflammatory response and enhancing immune function.

[0009] Furthermore, the drug improves the pathological state of testicular tissue basement membrane thinning, seminiferous tubule morphology distortion, lumen dilation, and spermatogonia arrangement disorder induced by cyclophosphamide.

[0010] Furthermore, the drug restores or promotes the spermatogenesis process by increasing serum testosterone levels.

[0011] Furthermore, pterostilbene improves cyclophosphamide-induced metabolic abnormalities and restores normal reproductive function by regulating the levels of multiple oxidative stress-related metabolites, including stearic acid and cucurbitacin.

[0012] Furthermore, the drug is formulated into tablets, capsules, injections, or oral solutions by combining it with a pharmaceutically acceptable carrier, excipient, or solvent.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses network pharmacology to screen the core targets and enrichment pathways of pterostilbene in combating spermatogenic dysfunction, and performs molecular docking of pterostilbene with related core proteins. A mouse model of spermatogenic dysfunction was established using cyclophosphamide. Pterostilbene was administered by gavage, and changes in spermatogenic dysfunction-related indicators and pathological morphology were measured. RT-qPCR and Western blotting were used to detect the expression of core target proteins and related pathway proteins' mRNA and protein levels. Non-targeted metabolomics was used to detect changes in testicular tissue metabolites. KEGG pathway enrichment analysis showed that pterostilbene treatment of spermatogenic dysfunction was related to the AKT / mTOR signaling pathway. The results showed that pterostilbene administration reduced the testicular index in mice, increased serum testosterone levels, increased the peripheral blood lymphocyte ratio, and decreased the granulocyte ratio; it also restored pathological damage to testicular tissue to a certain extent, reduced MDA content in mouse testes, and significantly increased the activities of SOD, CAT, and GSH. Pterostilbene significantly reduced the expression of PTGS2, CYP1A1, and CYP1B1, while increasing the expression of ESR1, EGFR, CYP17A1, CYP19A1, PI3K, AKT, and mTOR. Metabolomics analysis revealed that the KEGG enrichment pathway showed multiple differentially expressed metabolites closely related to oxidative stress. Pterostilbene exhibited a significant protective effect against cyclophosphamide-induced spermatogenesis disorders, and it is possible that pterostilbene reduces cyclophosphamide-induced testicular spermatogenesis disorders by activating the PI3K / AKT / mTOR pathway to improve oxidative stress levels in mouse testicular tissue. This provides an important reference for the application of pterostilbene in the prevention of cyclophosphamide-induced male testicular dysfunction. Attached Figure Description

[0014] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is a graph showing the changes in the mouse testicular index according to an embodiment of the present invention; Figure 2 This is a diagram showing the analysis of complete blood cell counts in mouse testicular tissue according to an embodiment of the present invention. Figure 3 This is a graph showing the detection of serum testosterone levels in mice according to an embodiment of the present invention. Figure 4 This is a pathological section of mouse testicular tissue according to an embodiment of the present invention; Figure 5 This is a pathological section of testicular tissue from mice in the CTX group according to an embodiment of the present invention. Figure 6 This is a graph showing the changes in the content of SOD, MDA, CAT, and GSH in mouse testicular tissue according to an embodiment of the present invention. Figure 7This is a diagram showing the intersection of Pterostilbene and spermatogenic dysfunction, the target point of action, and the target point degree value in an embodiment of the present invention. Figure 8 This is a KEGG enrichment pathway diagram and related core target network diagram of an embodiment of the present invention; Figure 9 This is a GO function enrichment analysis diagram according to an embodiment of the present invention; Figure 10 This is a docking diagram of PTS with EGFR, ESR1, PTGS2, CYP1A1, and CYP1B1 molecules according to an embodiment of the present invention. Figure 11 This is a docking diagram of PTS with CYP17A1, CYP19A1, PIK3CA, ABCB1, and AR molecules according to an embodiment of the present invention. Figure 12 The expression changes of PTGS2, CYP1A1, and CYP1B1 mRNA in mouse testicular tissue according to an embodiment of the present invention; Figure 13 This invention relates to the changes in EGFR and ESR1 mRNA expression in mouse testicular tissue according to an embodiment of the present invention. Figure 14 The following are the changes in the expression of PI3K, AKT, and mTOR mRNA in mouse testicular tissue according to an embodiment of the present invention. Figure 15 This is a diagram showing the changes in protein expression of EGFR, p-EGFR, ESR1, and PTGS2 in mouse testicular tissue according to an embodiment of the present invention. Figure 16 The graph shows the expression results of PI3K, AKT, and mTOR proteins in mouse testes according to an embodiment of the present invention. Figure 17 This is a pre-evaluation diagram of the organization sample in an embodiment of the present invention; Figure 18 This is a discriminant analysis diagram using the orthogonal partial least squares method according to an embodiment of the present invention; Figure 19 Venn diagrams of the differential metabolites in each group according to embodiments of the present invention; Figure 20 This is a bar chart showing the difference multiples in an embodiment of the present invention; Figure 21 This is a differential metabolite volcano diagram of an embodiment of the present invention; Figure 22 This is a bar chart showing the enrichment of the differential metabolite KEGG in an embodiment of the present invention.

[0015] in, Figure 2 In the table, (A) represents the granulocyte ratio, (B) represents the lymphocyte ratio, and (C) represents the intermediate cell ratio. Figure 7In the diagram, A is the Venn diagram of the intersection of Pterostilbene and spermatogenic dysfunction targets; B is the network of targets for the anti-spermatogenic dysfunction effect of Pterostilbene; C is the degree value diagram of the targets for the anti-spermatogenic dysfunction effect of Pterostilbene. Figure 17 In the diagram, A: Correlation plot between samples; B: PCA analysis of all samples; Figure 18 In the diagram, A and B are OPLS-DA score plots, and C and D are model permutation test plots. A and C: N(Con) vs CTX; B and D: CTX vs PTS. Figure 20 Medium, A: N vs CTX; B: CTX vs PTS; Figure 21 Medium, A: N vs CTX; B: CTX vs PTS; Figure 22 Middle, A: Nvs CTX; B: CTX vs PTS. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] This invention uses CTX to establish a mouse model of spermatogenesis disorder. Network pharmacology analysis and experimental verification are used to explore the key targets and pathways of PTS in the prevention and treatment of spermatogenesis disorders, as well as the specific molecular mechanisms. Simultaneously, metabolomics is used to investigate the effects and mechanisms of pterostilbene on preventing cyclophosphamide-induced testicular dysfunction in men. The specific process is as follows: Materials and Methods 1.1 Network Pharmacology Research 1.1.1 Drug target screening The target sites of PTS were predicted using the TargetNet and pharmMapper databases. The names of the target protein were corrected using the UniProt database (https: / / www.uniprot.org / ) to finally obtain the potential targets of PTS.

[0018] 1.1.2 Disease Target Screening Using "Spermatogenic dysfunction" as the keyword, disease-related targets were screened in the OMIM database (https: / / omim.org / ) and the GeneCards database (https: / / www.genecards.org / ). Targets were selected based on a relevance score > 5 in the GeneCards database. After merging targets from the two disease databases, they were converted into Gene Symbols using the UniProt database.

[0019] PPI Network Construction The Venny 2.1.0 analysis tool (https: / / bioinfogp.cnb.csic.es / tools / venny / ) was used to draw a Venn diagram of drug-disease targets. The intersection points were taken as the potential targets for PTS treatment of spermatogenesis disorders. The diagram was then imported into the STRING database (https: / / cn.string-db.org / ) to obtain the target protein interaction network. The key protein targets were visualized and analyzed using Cytoscape 3.10.0 software to construct a pPI network.

[0020] GO and KEGG enrichment analysis Gene IDs of potential targets were obtained using R software and its backend database "org.Hs.eg.db"; and GO enrichment and KEGG pathway analysis were performed on these potential targets.

[0021] GO / KEGG enrichment analysis was performed using the R package Clusterprofile. GO enrichment analysis results, including biological processes (BP), cellular components (CC), molecular functions (MF), and KEGG pathway analysis results, were exported separately, with P < 0.05 used as the screening criterion.

[0022] 1.2 Molecular docking Select the core targets with higher degree values in the pPI network diagram as receptors, and select the corresponding active ingredients as ligands for relevant verification of molecular docking. First, download small molecule compounds in sdf format using the pubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), then convert the compounds into mol2 format through ChemBio3D, and then use AutoDock software to convert the mol2 format compounds into pdbqt format compounds. Next, obtain the crystal form corresponding to the target protein from the PDB database (http: / / www.rcsb.org / ), and then use AutoDockTools software for preprocessing such as dehydration and hydrogenation. Then, perform molecular docking with the help of AutoDockVina, visualize with DiscoveryStudio, and analyze the results.

[0023] 1.2 Animal experiments 1.2.1 Experimental animals [[ID=……]] Sixty male KM mice, weighing 22 - 26 g, were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (SCXK(Shandong)20220006). Under standard feeding conditions, with a 12-hour light and 12-hour dark mode, the temperature was maintained at (22 ± 2°C) and the humidity at (45 - 60%). Unrestricted food and water were provided. The animal research protocol was approved by the Laboratory Animal Ethics Committee of Jining Medical University.

[0024] 1.2.2 Reagents and instruments Pterostilbene reagent was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (S26817). Cyclophosphamide for injection was purchased from Aladdin Biochemical Technology Co., Ltd. (C10699). ESR-1 antibody (WL00940) and P-EGFR antibody (WL03432) were purchased from Wanlei Biotechnology Co., Ltd., EGFR antibody (51071-2-AP), PI3K antibody, AKT antibody and mTOR antibody were purchased from Proteintech China, horseradish peroxidase-labeled goat anti-mouse secondary antibody (A0216) was purchased from Beyotime Biotechnology Co., Ltd., and SOD, CAT, GSH and MDA biochemical detection kits were purchased from Nanjing Jiancheng Bioengineering Institute. Main instruments: Real-time PCR instrument (Bio-RAD, CFX96), high-speed refrigerated centrifuge (Thermo Fisher Scientific, Sorvall ST8R), fully automated biochemical analyzer (Nanjing Beiden Medical Co., Ltd., BS-280), high-speed refrigerated centrifuge (Thermo Fisher Scientific, Sorvall ST8R), ultra-sensitive multi-functional imaging system (Thermo Fisher Scientific, iBright™ FL1000), handheld tissue homogenizer (Shanghai Cebo Biotechnology Co., Ltd., CMY-10 / 20).

[0025] 1.2.3 Dosing and Experimental Design Mice were randomly divided into 5 groups: control group (Con group), testicular injury model group (CTX group), low-dose PTS group (PTS1, 10 mg / kg), medium-dose PTS group (PTS2, 20 mg / kg), and high-dose PTS group (PTS3, 40 mg / kg). From day 1 to day 14, the control group and model group were administered 0.2 mL of polyethylene glycol (PEG) by gavage, while PTS1, PTS2, and PTS3 were administered different concentrations of pterostilbene (dissolved in 0.2 mL of PEG) by gavage. From day 8 to day 12, the control group was administered 0.2 mL of 0.9% NaCl intraperitoneally daily, while the model group, PTS1, PTS2, and PTS3 were administered 0.2 mL of cyclophosphamide (80 mg / kg) intraperitoneally daily. After 14 days of administration, the mice were sacrificed, and testicular tissue was collected, rapidly frozen in liquid nitrogen, and stored at -80°C.

[0026] 1.2.4 Measurement of testicular organ index and complete blood cell count in mice After the last administration on day 14, the mice were weighed, sacrificed, and their testes were removed and weighed. The testicular index was calculated as: mouse testicular index = mouse testicular weight (mg) / mouse body weight (g).

[0027] Peripheral blood was collected from mice, and blood cell analysis was performed using a complete blood cell counter.

[0028] 1.2.5 Detection of serum testosterone levels in mice using ELISA Peripheral blood was centrifuged at 1000×g for 20 min, and the supernatant was collected. Serum testosterone levels were measured according to the ELISA kit instructions. 1.2.6 HE staining to observe morphological changes in mouse testes Testicular tissue was fixed in 4% paraformaldehyde for 24 hours and dehydrated using a gradient of ethanol. After clearing with xylene, the tissue was embedded in paraffin and sectioned. The tissue sections were stained with hematoxylin and eosin (H&E). After dehydration, the sections were mounted with neutral resin and observed and photographed under a microscope.

[0029] 1.2.7 Detection of SOD, CAT, GSH and MDA content Following the instructions, the activity and content of SOD, CAT, GSH and MDA in mouse testes were determined using a biochemical assay kit for SOD, CAT, GSH and MDA.

[0030] 1.2.8 Real-time quantitative polymerase chain reaction (qRT-PCR) Total RNA was extracted from testicular tissue using the Trizol method. RNA was reverse transcribed into cDNA using a reverse transcription kit and stored at -80℃. PCR reaction system: 15 μL of 2xAceQ qPCR SYBR Green Master Mix, 2 μL of upstream primer, 2 μL of downstream primer, and 1 μL of sample cDNA. Reaction parameters: Pre-denaturation temperature was set at 94℃ for 5 minutes. Subsequent cycles consisted of denaturation at 94℃ for 45 seconds, annealing at 55℃ for 45 seconds, extension at 72℃ for 90 seconds, for 38 cycles. Extension was performed at 65℃ for 5 seconds. Relative quantification (RQ) was used (RQ=2). -ΔΔCt The expression levels of the target gene relative to the internal reference gene were calculated, and the differences in mRNA expression were compared. The primer sequences used are shown in Table 1.

[0031] Table 1 Primer sequence listing

[0032] 1.2.9 Western blot analysis of protein expression Weigh 50 mg of testicular tissue into a 1.5 ml EP tube, add 400 μl of lysis buffer, and homogenize until a paste is formed. After standing for 20 min, centrifuge and collect the supernatant. Protein concentration is determined using BCA, followed by denaturation in a metal bath at 100 °C for 5 min. Store at -80 °C for long-term storage. Prepare a gel, load the sample, and perform electrophoresis. After electrophoresis, transfer the protein to a PVDF membrane and block. After blocking, incubate overnight with primary antibody. The next day, wash and incubate with the corresponding species' secondary antibody, then wash again and develop.

[0033] 1.3 Non-targeted metabolomics detection 50 mg of mouse testicular tissue was weighed and added to 1000 μL of extraction buffer containing an internal standard (methanol-acetonitrile-water volume ratio = 2:2:1, internal standard concentration 20 mg / L). The sample was then ground and sonicated using magnetic beads to ensure complete disruption and release of the tissue. The supernatant was then obtained through standing and centrifugation, and subsequently dried using vacuum drying to ensure sample stability and purity.

[0034] In the detection process, a WatersAcquity I-ClasspLUS ultra-high performance liquid chromatography-tandem Waters Xevo G2-XS QTOF mass spectrometer was strictly employed to ensure the accuracy and reliability of the results. Simultaneously, raw data from MassLynx V4.2 was used, and Progenesis QI software was employed for data processing to further explore and analyze various information within the samples. Metabolite identification was conducted based on the online METLIN database, public databases, and a self-built library to ensure the comprehensiveness and accuracy of the identification results.

[0035] After normalizing the original peak area information, subsequent analyses were performed. Principal component analysis and SPT Sarman correlation analysis were used to assess the reproducibility of within-group samples and quality control samples. Identified compounds were searched for classification and pathway information in the KEGG, HMDB, and LipidMaps databases. Based on the grouping information, fold differences were calculated and compared, and the p-value for the significance of difference for each compound was calculated using a t-test. OPLS-DA modeling was performed using the R package ropls, and 200 permutation tests were conducted to validate the model's reliability. The VIP value of the model was calculated using multiple cross-validation. A combined approach of fold difference, p-value, and VIP value from the OPLS-DA model was used to screen differentially expressed metabolites. The screening criteria were FC>1, p-value<0.05, and VIP>1. Hypergeometric distribution tests were used to calculate differentially expressed metabolites with significant enrichment in the KEGG pathway.

[0036] 1.4 Statistical Analysis GraphPadprism software was used for graphing and statistical analysis. Independent samples t-tests were used to compare two groups of data. One-way ANOVA was used to compare multiple groups of data. P < 0.05 was considered statistically significant.

[0037] 2. Experimental Results 2.1 Mouse testicular index analysis Compared with the Con group, the CTX group showed a significant increase in testicular index, while the PTS1, PTS2, and PTS3 testicular indices showed a significant decrease compared with the CTX group. Figure 1The graph showing the changes in the mouse testicular index (*p<0.05, **p<0.01) is shown.

[0038] 2.2 Detection and analysis of complete blood cells in mice Complete blood cell count analysis showed that compared with the Con group, the CTX group had an increased granulocyte ratio and a significantly increased lymphocyte ratio (P<0.05); compared with the CTX group, the PTS3 group had a significantly decreased granulocyte ratio (P<0.05) and a significantly increased lymphocyte ratio (P<0.05). The interstitial cell ratio also showed a certain upward trend after PTS administration. It can be inferred that CTX administration leads to a decrease in lymphocyte and interstitial cell ratios, triggering immunosuppression; the increased granulocyte ratio suggests that CTX induces inflammation, while high-dose PTS administration can effectively improve immunity and reduce inflammation levels. Figure 2 As shown (*p<0.05, **p<0.01).

[0039] 2.3 Detection of serum testosterone levels in mice After CTX administration, serum testosterone levels in mice showed a certain decreasing trend, while after administration of different doses of PTS, serum testosterone levels in mice all showed a certain rebound. Figure 3 As shown.

[0040] 2.4 Pathological morphological analysis of mouse testes Compared to the Con group, the CTX group showed significantly thinner testicular basement membrane, irregular and loose arrangement of spermatogonia and other cells within the seminiferous tubules, distorted seminiferous tubule shape, enlarged lumen, suspected swelling, reduced intertubular spaces, decreased interstitial cells, and the presence of syncytia. Testicular cells exhibited edema, accompanied by inflammatory cell infiltration. PTS treatment showed some degree of recovery in all groups. Compared to the CTX group, the PTS1 group showed an increase in interstitial cell number, significantly thickened tubule walls, relatively orderly and dense arrangement of spermatogenic cells, and significant recovery of intertubular spaces, but no significant change in the basement membrane. The PTS2 and PTS3 groups showed thickened basement membrane, thickened seminiferous tubule walls, smaller lumen, increased number of spermatogonia, primary spermatocytes, and spermatids, and significant recovery of intertubular spaces. Figure 4 (1000× abnormal syncytiosomes) Figure 5 As shown.

[0041] 2.5 Analysis of SOD, MDA, CAT and GSH activity in mouse testes Biochemical assays showed that, compared with the Con group, the CTX group mice had increased MDA content in the testes and decreased SOD, CAT, and GSH activities; while the PTS1, PTS2, and PTS3 groups, compared with the CTX group, had significantly decreased MDA content in testicular tissue and increased SOD, CAT, and GSH activities, indicating that PTS can effectively improve the state of excessive oxidative stress in the testes of mice induced by CTX. Figure 6As shown (*p<0.05, **p<0.01). The results indicate that PTS can effectively alleviate CTX-induced testicular damage, and this protective effect is related to the level of oxidative stress.

[0042] 2.6 Network Pharmacology 2.6.1 Screening of pTS targets for combating spermatogenesis disorders and construction of PPI network The 151 drug targets and 1088 spermatogenic dysfunction targets collected through screening were intersected using the Venny platform, yielding 23 intersecting targets, such as... Figure 7 As shown in (A), the pPI network of drug and disease targets was analyzed using the STRING database and visualized using CytoscaPTS. The network contains 22 nodes and 72 edges. Targets include ESR1, PTGS2, EGFR, CYP19A1, and CYP1A1, with these targets located at the core of the network. See [link to relevant documentation]. Figure 7 As shown in (B, C).

[0043] 2.6.2 Enrichment analysis of KEGG pathway and GO function KEGG and GO enrichment analyses were performed using the DAVID platform, and the results were sorted by p-value. KEGG enriched 71 pathways; the top 10 most relevant pathways were selected as core pathways, and a bubble diagram was constructed. A network diagram was also created with the core target proteins, as shown below. Figure 8 As shown. After performing genomic-based (GO) biological process enrichment analysis, BP yielded 739 results, CC yielded 36 results, and MF yielded 95 results. The results were sorted by p-value from smallest to largest, and the top 10 results were selected for visualization, as shown below. Figure 9 As shown.

[0044] PTS docking with key target molecules PTS was molecularly docked with 10 core targets: EGFR, ESR1, PTGS2, CYP1A1, CYP1B1, CYP17A1, CYP19A1, PIK3CA, ABCB1, and AR. The binding energies were -7.7, -6.4, -7.5, -7.9, -8.2, -7.2, -7.6, -7, -7.2, and -5 kcal·mol⁻¹, respectively. Lower binding energies indicate better docking performance. (See [link to documentation]). Figure 10 , Figure 11 .

[0045] 2.7 Changes in mRNA expression of core and pathway genes related to mouse testicular tissue Compared with the Con group, the CTX group showed a significant increase in PTGS2, CYP1A1, and CYP1B1 mRNA expression, and a significant decrease in CYP17A1 and CYP19A1 mRNA expression. After PTS administration, the PTS1, PTS2, and PTS3 groups all showed a significant decrease in PTGS2, CYP1A1, and CYP1B1 mRNA expression, and a significant increase in CYP17A1 and CYP19A1 mRNA expression. Compared with the Con group, the CTX group showed a significant downregulation of EGFR, ESR1, PI3K, AKT, and mTOR mRNA, while after PTS administration, the expression levels of EGFR, ESR1, PI3K, AKT, and mTOR mRNA significantly increased. Figure 12 , Figure 13 , Figure 14 As shown.

[0046] 2.8 Changes in the expression of core and pathway proteins related to mouse testicular tissue like Figure 15 As shown in Figure 16, compared with CTX, the expression of PTGS2 protein in the testicular tissue of mice treated with PTS (L group) decreased, while the expression of ESR1, EGFR, p-EGFR, pI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR proteins all increased significantly, consistent with the trend of PCR results. Figure 11 , 12 As shown (*p<0.05, **p<0.01, ***p<0.001).

[0047] 2.9 Changes in metabolites in mouse testicular tissue and their relationship with related pathways 2.9.1 Data Quality Assessment like Figure 17 As shown in Figure A, in the experimental data analysis, when the correlation coefficient between samples within a group is higher than that between samples between groups, it indicates that the reliability of the differentially expressed metabolites has been effectively enhanced. The results show strong correlation between samples. PCA analysis of the samples, such as... Figure 17 The results shown in B indicate that there are certain differences between the Con and CTX groups, and between the CTX and PTS groups. The PTS group shows a tendency to revert to the Con group, which suggests that the application of PTS can restore the metabolic disorders caused by CTX to a certain extent.

[0048] 2.9.2 Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) A comparison between the Con group and the CTX group revealed that Q2Y was 0.689, indicating an effective model. Figure 18 A and Figure 18 C). According to OPLS-DA analysis, comparing the CTX group and the PTS group, the Q2Y was 0.627, indicating an effective model. Figure 18 B and Figure 18 D).

[0049] 2.9.3 Differential Metabolite Analysis A total of 5346 differentially expressed metabolites were detected across all groups. Compared to the CTX group, 579 differentially expressed metabolites were detected in the Con group, with 393 upregulated and 186 downregulated. Compared to the CTX group, 586 differentially expressed metabolites were detected in the PTS group, with 290 upregulated and 296 downregulated. Among these, 26 differentially expressed metabolites were downregulated in the Con group compared to the CTX group and upregulated in the PTS group compared to the CTX group, while 93 differentially expressed metabolites were upregulated in the Con group compared to the CTX group and downregulated in the PTS group compared to the CTX group (as shown in Table 2). 24 differentially expressed metabolites showed changes in all three groups, such as… Figure 19 As shown.

[0050] Table 2

[0051] Figure 20 A represents the top 10 upregulated and downregulated metabolites in the Con group compared to the CTX group, with Hexadecanal showing the most significant downregulation and 1-(2-Hydroxyethoxy)methyl-5-methyluracil showing the most significant upregulation. Figure 20 B represents the top 10 upregulated and downregulated metabolites in the CTX group compared to the PTS group. 2,3-Undecanedione showed the most significant downregulation, while PS (20:5(5Z, 8Z, 11Z, 14Z, 17Z) / PGJ2) showed the most significant upregulation. Cucurbitacin and stearic acid are closely related to oxidative stress.

[0052] 3.9.1.1 Volcano plot analysis of differential metabolites Blue dots indicate downregulated differentially expressed metabolites, red dots indicate upregulated differentially expressed metabolites, and gray dots indicate detected metabolites with insignificant differential expression. Comparison between the Con group and the CTX group revealed downregulation of 5'-S-Methyl-5'-thioinosine and upregulation of Fluoroaceticacid, N2-Galacturonyl-L-lysine, 2-Fluoroadenosine, 1,2-Cyclohexaredicarboxylicacid, and 4-methionine. Figure 21As shown in A. Comparison between the CTX and PTS groups revealed that CDP-DG(PGJ2 / a-15:0), (R)-11,12,13-Tinor-1(5),6,9-guaiatrien-8-one, Guanadrel, and UDP-2,3-bis(3-hydroxytetraecanoyl)glucosamine were downregulated, while 2-(3-MethylpiPTSridin-1-yl)[1,3]thiazolo[4,5-d]pyrimidin-7(6H)-one was upregulated. Figure 21 As shown in B.

[0053] 3.9.1.2 Differential Metabolite Pathway Enrichment Analysis KEGG pathway enrichment analysis of the identified substances was performed, and the top 20 entries annotated with the most differentially expressed metabolites were selected to obtain a histogram of differentially expressed metabolites for each group. Comparison between the Con group and the CTX group revealed that the Histidine metabolism pathway had the highest enrichment of metabolites. Figure 22 A). Comparison between the CTX group and the PTS group revealed that the Neomycin, kanamycin, and gentamicin biosynthesis pathways accumulated the most metabolites, among which six KEGG pathways—histidine metabolism, choline metabolism, long-term depression, alcoholic liver disease, oxidative phosphorylation, and sphingolipid signaling—were closely related to oxidative stress. Figure 22 B).

[0054] In summary, this embodiment utilized network pharmacology to analyze the potential targets and signaling pathways of pterostilbene in combating spermatogenesis disorders. Screening revealed that core targets such as EGFR, ESR1, PTGS2, CYP1A1, CYP1B1, CYP17A1, CYP19A1, and PIK3CA are closely related to spermatogenesis disorders. KEGG enrichment analysis showed that the main signaling pathways for pterostilbene's effectiveness in combating spermatogenesis disorders include ovarian steroidogenesis, chemical carcinogenesis-receptor activation, and steroid hormone biosynthesis.

[0055] PTGS2, CYP1A1, CYP1B1, CYP17A1, and CYP19A1 are all important proteins involved in the ovarian steroid production pathway. Molecular docking results also showed that PTS docked well with the above-mentioned core targets, suggesting a potential direct interaction. Inducible prostaglandin intraperoxide synthase 2 / cyclooxygenase 2 (PTGS2) plays an important role in pathological processes related to inflammatory signal transduction. PTGS2 is tightly regulated; its expression and activation are directly induced by pro-inflammatory cytokines and growth factors, and it is involved in the control of testicular steroid production, spermatogenesis, and local immunity. The cytochrome P450 family is mainly expressed in testicular, adrenal, ovarian, and placental tissues, and CYP1A1, CYP1B1, CYP17A1, and CYP19A1 are important members. CYP17A1, the rate-limiting enzyme in androgen synthesis, can lead to hormonal imbalances and affect reproductive performance in animals if its gene mutations occur. CYP19A1, a key enzyme in estrogen synthesis, can also cause hormonal imbalances if its mutations occur. CYP1B1 regulates multiple metabolic pathways, including steroid hormone metabolism, fatty acid metabolism, vitamin metabolism, and melatonin metabolism, and is closely related to testosterone metabolism. CYP1A1 is highly expressed during inflammation, and previous studies have found a significant association between the CYP1A1 rs4646903 polymorphism and male infertility. This experiment found that the application of PTS can effectively reverse the increase in PTGS2, CYP1A1, and CYP1B1 induced by CTX, and enhance the expression of CYP17A1 and CYP19A1. This suggests that PTS may regulate the ovarian steroid production pathway, affecting reproductive hormone metabolism, and modulate inflammation levels to exert its anti-spermatogenic activity.

[0056] EGFR, ESR1, and PIK3CA are key proteins involved in the chemical oncogenesis-receptor activation pathway. Molecular docking results also showed that PTS docked well with EGFR, ESR1, and PIK3CA, suggesting a potential direct effect. Epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein receptor with tyrosine kinase activity. Activated by epidermal growth factor, it mainly participates in regulating cell proliferation and apoptosis. During spermatogenesis, EGF binds to EGFR, affecting the function of target cells (Sertoli and Leydig cells), thereby altering androgen synthesis or regulating the interaction between peritubular myoid cells and germ cells through paracrine mechanisms. Estrogen receptor 1 (ESR1), located in the cell nucleus, plays a crucial regulatory role in the development of the reproductive system, regulating spermatogenesis and maturation. This example demonstrates that the application of PTS can effectively reverse the decrease in EGFR and ESR1 in mouse testicular tissue induced by CTX, restoring testicular germ cell activity. PPI and KEGG pathway network analysis revealed that PIK3CA was closely associated with 8 of the top 10 enriched pathways, including the chemical oncogenic-receptor activation pathway, of which PI3K / Akt / mTOR is a crucial component. Among the factors influencing male fertility, the PI3K / Akt / mTOR signaling pathway plays a vital role in regulating oxidative stress-induced germ cell autophagy and apoptosis, and in controlling the number of spermatogenic cells, making it a potential clinical therapeutic target. EGFR is also an important upstream regulator. In this study, the expression of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR genes and proteins in the testicular tissue of a CTX-induced mouse reproductive disorder model was significantly decreased, indicating inhibition of this pathway. However, after PTS application, the protein levels of this pathway significantly increased, suggesting that the pathway effectively participates in the regulation of oxidative stress, apoptosis, and autophagy in damaged testicular tissue, thus restoring testicular damage. All of the above suggest that PTS may exert its anti-spermatogenic activity by regulating the chemical oncogenic receptor activation pathway. PTS exerts its effective anti-spermatogenic activity by adjusting the upstream EGFR and ESR1 activities and regulating the downstream PI3K / Akt / mTOR pathway, ultimately adjusting the proliferation, apoptosis and oxidative stress of germ cells.

[0057] Untargeted metabolomics aims to detect various small molecule metabolites. Changes in metabolite profiles can provide relevant information about physiological abnormalities caused by diseases, lifestyles, and toxic chemicals. This metabolomics analysis showed that a large number of differentially metabolites, such as stearic acid, cucurbitacin, and diacylglycerol, are closely related to oxidative stress. The differentially metabolites between the PTS and CTX groups were significantly enriched in oxidative phosphorylation and sphingolipid metabolism pathways. These metabolisms are all involved in the PI3K / Akt signaling pathway and are closely related to oxidative stress. This further clarifies at the metabolic level that PTS may participate in the protective effect against reproductive damage by regulating the process of oxidative stress.

[0058] We predict that PTS may exert its anti-spermatogenic effect through interactions with core targets EGFR, ESR1, PTGS2, CYP1A1, CYP1B1, CYP17A1, CYP19A1, and PIK3CA. Animal experiments have confirmed that PTS can effectively improve CTX-induced spermatogenic dysfunction in mice, and its regulatory mechanism is closely related to improving testicular oxidative stress and regulating the PI3K / AKT / mTOR signaling pathway. This example is the first of its kind, providing an important reference for the application of PTS in the prevention of CTX-induced testicular dysfunction in men.

[0059] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. The application of pterostilbene in the preparation of drugs for the prevention or treatment of spermatogenesis disorders, characterized in that, The drug contains pterostilbene as an active ingredient, and pterostilbene prevents or treats cyclophosphamide-induced spermatogenic dysfunction by regulating the PI3K / AKT / mTOR signaling pathway, inhibiting oxidative stress response, and improving the pathological structure of testicular tissue.

2. The application according to claim 1, characterized in that, In the preparation of the aforementioned drug, Pterostilbene alleviates oxidative damage to spermatogenic cells by reducing malondialdehyde content in testicular tissue and increasing the activity of superoxide dismutase, catalase, and glutathione.

3. The application according to claim 1, characterized in that, In the preparation of the aforementioned drug, pterostilbene is used to upregulate the mRNA and protein expression levels of estrogen receptor 1, epidermal growth factor receptor, CYP17A1, CYP19A1, PI3K, AKT, and mTOR, and to downregulate the expression levels of cyclooxygenase 2, CYP1A1, and CYP1B1.

4. The application according to claim 1, characterized in that, In the preparation of the aforementioned drug, pterostilbene improves the proportion of peripheral blood immune cells, increases the proportion of lymphocytes, and decreases the proportion of granulocytes, thereby alleviating the inflammatory response and enhancing immune function.

5. The application according to claim 1, characterized in that, The drug improves the pathological conditions induced by cyclophosphamide, including thinning of the testicular basement membrane, distortion of seminiferous tubule morphology, dilation of the lumen, and disordered arrangement of spermatogonia.

6. The application according to claim 1, characterized in that, The drug works by increasing serum testosterone levels to restore or promote the spermatogenesis process.

7. The application according to claim 1, characterized in that, Pterostilbene improves cyclophosphamide-induced metabolic abnormalities and restores normal reproductive function by regulating the levels of multiple metabolites associated with oxidative stress, including stearic acid and cucurbitacin.

8. The application according to claim 1, characterized in that, The drug is formulated into tablets, capsules, injections, or oral solutions by combining it with a pharmaceutically acceptable carrier, excipient, or solvent.

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