Use of purine nucleoside phosphorylase inhibitors to improve reproductive function in a subject

Abstract

This application provides the use of purine nucleoside phosphorylase inhibitors in improving the reproductive function of subjects. This application provides the use of purine nucleoside phosphorylase inhibitors in the preparation of drugs for antagonizing hydrosalpinx toxicity or improving the reproductive function of subjects. This application also provides the use of purine nucleoside phosphorylase inhibitors in the preparation of reagents for embryo culture and gamete treatment. This application utilizes purine nucleoside phosphorylase inhibitors to inhibit PNP activity, antagonizing hydrosalpinx toxicity caused by purine metabolism disorders, thereby improving the reproductive function of subjects.

Description

Technical Field

[0001] This application relates to the fields of reproductive medicine technology and biomedicine, specifically to the application of a purine nucleoside phosphorylase inhibitor in improving the reproductive function of subjects. Background Technology

[0002] The fallopian tube (FT) is an important organ of the female reproductive system, connecting the ovary and uterus. It is a tubular organ responsible for gamete transport, fertilization, and preimplantation embryonic development, and plays a vital physiological role. Hydrosalpinx refers to a chronic inflammatory condition caused by the distal end of the fallopian tube becoming blocked due to infection, adhesions, or other factors, leading to fluid accumulation and dilation within the lumen. It is a significant contributing factor to female infertility and the reduced pregnancy rate in in vitro fertilization and embryo transfer (IVF-ET) treatments.

[0003] Hydrosalpinx severely impacts IVF-ET pregnancy rates, specifically by reducing pregnancy and embryo implantation rates and increasing the risk of early pregnancy failure. Related research suggests two main mechanisms by which hydrosalpinx leads to infertility. First, mechanical obstruction and impaired ciliary movement within the fallopian tubes disrupt the fertilization process and the transport of sperm and embryos. Second, cytotoxic components in the fallopian tube fluid or their reflux into the uterine cavity can adversely affect the receptivity of the endometrium, ultimately leading to undesirable reproductive outcomes.

[0004] Currently, the main treatment for hydrosalpinx is laparoscopic salpingectomy or tubal reconstruction. These are invasive surgeries. Although they remove the source of toxicity, they increase the patient's pain and financial burden, reduce the patient's chances of natural pregnancy, and affect ovarian blood supply and ovarian reserve function. Tubal reconstruction also carries the possibility of recurrence of hydrosalpinx.

[0005] Therefore, clarifying the mechanism of hydrosalpinx toxicity and screening drugs to antagonize hydrosalpinx toxicity and improve patients' reproductive function has important biological and medical value. Summary of the Invention

[0006] In view of the above, in order to at least partially solve at least one of the aforementioned technical problems, this application provides the use of a purine nucleoside phosphorylase (PNP) inhibitor in the preparation of a medicament for antagonizing hydrosalpinx toxicity in a subject or improving the subject's reproductive function.

[0007] This application also provides the use of purine nucleoside phosphorylase inhibitors in the preparation of reagents for embryo culture and gamete treatment.

[0008] This application improves the reproductive function of subjects by using a purine nucleoside phosphorylase inhibitor to inhibit PNP activity and antagonize the toxicity of hydrosalpinx caused by purine metabolism disorders. Attached Figure Description

[0009] The above and other objects, features and advantages of this application will become clearer from the following description of embodiments with reference to the accompanying drawings, in which:

[0010] Figure 1 The results of orthogonal partial least squares discriminant analysis (OPLS-DA) of the metabolomics of fallopian tube flushing fluid in the hydrosalpinx group (HSPX group) and healthy fallopian tube controls (HFT group) are shown in the figure, where each point represents a single sample.

[0011] Figure 2 Volcano analysis plots of differential metabolites in the fallopian tube lumen fluid of the HSPX and HFT groups are shown; where P-correction value <0.05, fold change ≥1.5 or ≤0.67; the horizontal axis is the logarithm of the fold change base 2 (log2(Fold Change)), and the vertical axis is the negative logarithm of the false detection rate (-log2(Fold Change)). 10 (FDR)).

[0012] Figure 3 The diagram shows the KEGG pathway analysis of the upregulated metabolite in the fallopian tube lumen fluid of the HSPX and HFT groups. In this diagram, p < 0.05, the x-axis represents the mapped percentage, and the y-axis represents the negative logarithm of the p-value (-log). 10 (P-values)).

[0013] Figure 4 The results of ELISA measurements of inosine, adenosine, and hypoxanthine concentrations in the fallopian tube fluid of the HFT and HSPX groups are shown in the figure. In the figure, A represents inosine, B represents adenosine, and C represents hypoxanthine. The data are presented as mean ± standard deviation. The p-value was calculated using an unpaired two-tailed Student's t-test. **P < 0.01, a two-tailed unpaired t-test was used.

[0014] Figure 5Histological staining and electron micrographs of the HFT and HSPX groups are shown. Among them, a) is the hematoxylin-eosin (H&E) staining result of the ampulla of the HFT group (1:20); b) is the H&E staining result of the ampulla of the HSPX group (1:20); c) is the scanning electron micrograph of the ampulla of the HFT group (50 μm) and a magnified view (10 μm); d) is the scanning electron micrograph of the ampulla of the HSPX group (50 μm) and a magnified view (10 μm); and e) is the staining image of activated caspase-3, phosphorylated histone H2AX (γH2AX) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end marker (TUNEL) in the fallopian tubes of the HFT and HSPX groups. The scale bar is 100 μm.

[0015] Figure 6A A dot plot showing the expression of representative metabolic pathways (including purine metabolism, oxidative phosphorylation, glycolysis, and tricarboxylic acid cycle) in 11 major oviduct cell subtypes, where color represents expression level.

[0016] Figure 6B The graph shows the results of the PNP expression level analysis in non-ciliated secretory epithelial cells (NCSE). *P<0.05, and a two-tailed unpaired t-test was used.

[0017] Figure 6C The image shows the immunofluorescence (red) and immunohistochemical localization analysis results of purine nucleoside phosphorylase (PNP) in the fallopian tube epithelium, with a scale bar of 100 μm.

[0018] Figure 7A Representative images of embryos treated with 1.5‰ dimethyl sulfoxide (DMSO) or 25 μM inosine at the blastocyst stage (96 h) are shown, with scale bars at 100 µm.

[0019] Figure 7B The statistical results of the developmental rates of mouse embryos at different developmental stages in the control group and the inosine group are shown in the figure. The experiment was independently repeated three times, with more than 30 embryos in each group. The data are expressed as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, and a two-tailed unpaired t-test was used.

[0020] Figure 8The figures show the results of the test on the effect of furodixine on mouse embryonic development. A represents representative images of embryos treated with 1.5‰ dimethyl sulfoxide (DMSO), 25 μM inosine, and 25 μM inosine combined with different concentrations of furodixine at the blastocyst stage (96 h). B shows a comparison of the developmental rates of mouse embryos at different developmental stages in the DMSO and furodixine groups. This experiment was independently repeated three times, and the data are expressed as mean ± standard deviation. C shows the developmental rates of mouse embryos at different developmental stages in the DMSO, 25 μM inosine (Ino), and 25 μM inosine (Ino) combined with different concentrations of furodixine (Foro) groups. This experiment was independently repeated three times. Data are expressed as mean ± standard deviation; D is the RT-qPCR result of PNP mRNA levels in late 2-cell embryos treated with DMSO, inosine, or inosine combined with furodixine, with glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as the internal reference gene. Each sample contained 5 embryos, and the experiment was repeated three times. The relative PNP mRNA level in the DMSO group was defined as 1.0. Data are expressed as mean ± standard deviation, *P<0.05, and a two-tailed unpaired t-test was used; E is a representative fluorescence image of reactive oxygen species (ROS) in mouse 2-cell embryos after treatment with DMSO, inosine, inosine, and furodixine; and F is the result of quantitative analysis of fluorescence intensity of E.

[0021] Figure 9 The results show the effects of designed interfering RNA targeting the PNP gene on mouse embryonic development. In the figure, A is a flowchart, B is the qPCR experimental results, C is the PNP expression level results, and D is the development rate statistics of different groups. Detailed Implementation

[0022] The embodiments of this application will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of this application. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of this application for ease of explanation. However, it will be apparent that one or more embodiments may be implemented without these specific details. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this application.

[0023] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0024] When using expressions such as "at least one of A, B, and C," they should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art.

[0025] In this application, the term "early embryo" refers to an embryo that develops before implantation, that is, an embryo in the developmental stage from the formation of a fertilized egg to the implantation of the embryo into the mother's uterus (including fertilized eggs, 2-cell, four-cell, 8-cell, morula, and blastocyst).

[0026] The term "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, to the extent of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio.

[0027] The term "prevention" refers to the reduction of symptoms after the onset of a disease by exposing (e.g., administering medication) a subject to a drug, composition, etc. based on this application before the onset of the disease, compared to the absence of exposure, and does not imply the necessity of completely suppressing the disease.

[0028] In realizing the concept of this application, it was discovered that although related technologies consider hydrosalpinx fluid to be toxic to embryos, reducing pregnancy and implantation rates, the following shortcomings exist in addressing this issue: 1. Lack of targeted "antidote" drugs, with limited and invasive treatment options: Currently, the effective clinical means of eliminating hydrosalpinx toxicity is prophylactic salpingectomy or tubal reconstruction, but such invasive surgery reduces the ability to conceive naturally; 2. Lack of understanding of the metabolic toxicity mechanism, preventing molecular-level rescue: Related technologies mainly attribute the toxicity of hydrosalpinx fluid to pathogenic microorganisms such as Chlamydia trachomatis, accumulation of inflammatory factors (such as IL-6, TNF-α), or oxidative stress, neglecting changes in the metabolic microenvironment. Because the key metabolic toxic molecules leading to embryonic developmental arrest have not been identified, targeted antagonists cannot be developed clinically.

[0029] This application found that significantly elevated purine metabolites (e.g., inosine, adenosine, hypoxanthine, etc.) in hydrosalpinx fluid lead to embryonic (e.g., early embryonic) developmental arrest and abnormal zygotic genome activation (ZGA). Furthermore, by using purine nucleoside phosphorylase inhibitors (e.g., forodesine) to inhibit PNP enzyme activity, downstream conversion of purine metabolites can be blocked and reactive oxygen species (ROS) can be reduced. This research aims to regulate the microenvironmental disturbances in hydrosalpinx fluid caused by the accumulation of purine metabolites (e.g., inosine, adenosine, hypoxanthine, etc.), reduce the toxicity of hydrosalpinx fluid, reverse embryonic (e.g., early embryonic) developmental arrest caused by the accumulation of purine metabolites (e.g., inosine, adenosine, hypoxanthine, etc.), restore the developmental potential of embryos (e.g., early embryos), improve blastocyst formation rate and blastocyst quality, solve the problems of infertility and assisted reproductive technology failure caused by hydrosalpinx, improve patients' reproductive function, and provide a new technical route for solving embryo toxicity problems while preserving reproductive organs.

[0030] In some aspects, this application provides the use of a purine nucleoside phosphorylase inhibitor in the preparation of a medicament for antagonizing hydrosalpinx toxicity in a subject.

[0031] Specifically, the purine nucleoside phosphorylase inhibitors of this application also include small interfering RNA (siRNA) of the purine nucleoside phosphorylase (PNP) gene. That is, the ability to reduce the activity of purine nucleoside phosphorylase or the ability to reduce the expression level of purine nucleoside phosphorylase can be understood as purine nucleoside phosphorylase inhibitors.

[0032] In one or more embodiments of this application, the hydrosalpinx toxicity is caused by purine metabolites.

[0033] In one or more embodiments of this application, the purine metabolite is at least one of inosine, adenosine, and hypoxanthine, such as inosine, adenosine, or hypoxanthine.

[0034] In one or more embodiments of this application, the purine metabolite is at least two of inosine, adenosine, and hypoxanthine, such as inosine and adenosine, inosine and hypoxanthine, or adenosine and hypoxanthine.

[0035] In one or more embodiments of this application, the purine metabolites are inosine, adenosine, and hypoxanthine.

[0036] In one or more embodiments of this application, the hydrosalpinx toxicity is caused by the accumulation (increased concentration) of purine metabolites.

[0037] In one or more embodiments of this application, the hydrosalpinx toxicity is caused by the accumulation (increased concentration) of inosine, adenosine, and / or hypoxanthine.

[0038] In one or more embodiments of this application, the hydrosalpinx toxicity is caused by inosine accumulation (increased concentration).

[0039] In some respects, this application provides the use of a purine nucleoside phosphorylase inhibitor in the preparation of a medicament for improving the reproductive function of a subject.

[0040] In one or more embodiments of this application, the improvement of the subject's reproductive function includes one or more of the following: (1) improving fallopian tube function; (2) increasing endometrial receptivity; (3) inhibiting abnormal activation of the zygote genome in embryonic cell development; (4) improving embryonic cytoskeleton stability; (5) increasing the developmental potential of fertilized eggs; (6) increasing the embryo's tolerance to metabolic stress; (7) reducing embryonic damage; (8) increasing embryonic developmental potential; (9) inhibiting reactive oxygen species bursts in embryonic cells; (10) preventing or reversing embryonic developmental arrest; (11) increasing embryo formation rate; (12) improving embryo quality; (13) increasing natural conception rate; (14) reducing infertility rate; (15) increasing embryo implantation rate; (16) increasing pregnancy rate; and (17) reducing miscarriage rate.

[0041] In one or more embodiments of this application, the improvement of fallopian tube function includes one or more of the following: maintaining the number of fibroblasts in the fallopian tube; maintaining the length of fibroblasts in the fallopian tube; reducing adhesions between ciliated cells in the fallopian tube; reducing mucus coverage on ciliated cells in the fallopian tube; reducing apoptosis of non-ciliated secretory epithelial cells in the fallopian tube; alleviating or improving fallopian tube edema; and reducing inflammatory cell infiltration in the fallopian tube.

[0042] In one or more embodiments of this application, the subject is a mammal, such as a human, mouse, rat, monkey, dog, pig, horse, or rabbit. In one or more specific embodiments of this application, the subject is a human.

[0043] In one or more embodiments of this application, the subject suffers from hydrosalpinx.

[0044] In one or more embodiments of this application, the embryo is an in vivo embryo or an embryo produced by in vitro fertilization.

[0045] In one or more embodiments of this application, the embryo produced by in vitro fertilization is an embryo produced in vitro through assisted reproductive treatment, such as an embryo produced in vitro through in vitro fertilization and embryo transfer (IVF-ET) treatment.

[0046] In one or more embodiments of this application, the embryo is an early embryo, such as a 2-8 cell (2-cell, 4-cell, or 8-cell) stage embryo, a morula, or a blastocyst.

[0047] In one or more embodiments of this application, the subject's hydrosalpinx leads to decreased reproductive function.

[0048] In one or more embodiments of this application, hydrosalpinx in the subject results in: (1) decreased fallopian tube function; (2) decreased endometrial receptivity; (3) abnormal activation of the zygote genome in embryonic cell development; (4) decreased embryonic cytoskeleton stability; (5) decreased fertilized egg developmental potential; (6) decreased embryonic tolerance to metabolic stress; (7) embryonic damage; (8) decreased embryonic developmental potential; (9) bursts of reactive oxygen species in embryonic cells; (10) embryonic developmental arrest; (11) decreased embryo formation rate; (12) decreased embryo quality; (13) decreased natural conception rate; (14) infertility; (15) decreased embryo implantation rate; (16) decreased pregnancy rate; and (17) increased miscarriage rate.

[0049] In one or more embodiments of this application, the dosage form of the drug is selected from intrauterine irrigation solution, suppository, injection dosage form, ointment, paste or gel.

[0050] In one or more embodiments of this application, the dosage forms of various drugs meet different needs, improve patient compliance, optimize drug efficacy and safety, and can meet different clinical needs and patient preferences, as well as adapt to different routes of administration.

[0051] In one or more embodiments of this application, the drug may further include excipients, such as at least one of preservatives, solubilizers, stabilizers, diluents, and lubricants. These excipients can improve the physicochemical properties of the drug, enhance its efficacy and safety, increase the acceptability of the formulation, meet diverse clinical needs and patient preferences, and contribute to the stable release and effective delivery of the drug, thereby improving the safety of the formulation. By rationally selecting and using these excipients, the dosage form of the drug can be optimized, and its efficacy improved.

[0052] In some respects, this application provides the use of purine nucleoside phosphorylase inhibitors in the preparation of reagents for embryo culture and gamete treatment.

[0053] In one or more embodiments of this application, the embryo is a mammalian embryo.

[0054] In one or more embodiments of this application, the gametes are mammalian gametes.

[0055] In one or more embodiments of this application, the mammal is a human, mouse, rat, monkey, dog, pig, horse, or rabbit.

[0056] In one or more embodiments of this application, the concentration of the purine nucleoside phosphorylase inhibitor in the reagent is 1-60 μM, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 μM, or any number between any two of the above values ​​(e.g., 5-20 μM, 10-45 μM, or 20-30 μM).

[0057] In one or more embodiments of this application, the concentration of the purine nucleoside phosphorylase inhibitor in the reagent is 5-50 μM, for example 5, 6, 7, 8, 9, 10, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 μM, or any number between any two of the above values.

[0058] In one or more embodiments of this application, the reagent for embryo culture may be an embryo culture medium.

[0059] In one or more embodiments of this application, the reagent for gamete treatment may be a gamete treatment solution.

[0060] In one or more embodiments of this application, the purine nucleoside phosphorylase inhibitor is selected from: forodesine or a pharmaceutically acceptable salt thereof, ulodesine or a pharmaceutically acceptable salt thereof, or siRNA targeting the PNP gene.

[0061] In one or more embodiments of this application, when the active pharmaceutical ingredient is a pharmaceutically acceptable salt, such as a pharmaceutically acceptable acid addition salt or base addition salt, it can be prepared in situ by reacting with a pH adjuster in solution to produce the corresponding free active ingredient, such as a free basic active ingredient or a free acidic active ingredient.

[0062] In one or more embodiments of this application, salting of PNP inhibitors can improve their stability, bioavailability, or efficacy. Salting can improve the pharmacokinetic properties of PNP inhibitors, such as prolonging half-life, enhancing targeting, or reducing degradation, thereby improving their effectiveness and convenience in practical applications.

[0063] In one or more embodiments of this application, the pharmaceutically acceptable salt may be an inorganic acid salt, such as a hydrochloride, sulfate, phosphate, nitrate, borate, or carbonate.

[0064] The solution of this application will be further explained below with reference to specific embodiments. Unless otherwise stated, the reagents and detection methods used are all commercially available reagents and conventional detection methods in the art.

[0065] Example 1: Identification of the metabolically toxic molecule inosine

[0066] 1. Sample Source

[0067] We collected tubal irrigation fluid from patients who underwent tubal resection for medical reasons at the Department of Obstetrics and Gynecology and Reproductive Medicine Center of Peking University Third Hospital.

[0068] Patient inclusion criteria for the hydrosalpinx group: premenopausal patients who were initially diagnosed with hydrosalpinx by ultrasound examination, and subsequently underwent laparoscopic examination under general anesthesia for salpingectomy or tuboplasty, and whose postoperative diagnosis and pathological results were reported as hydrosalpinx.

[0069] The criteria for inclusion of patients in the control group were: premenopausal patients who underwent total hysterectomy and bilateral salpingo-oophorectomy due to benign indications (non-fallopian tube factors, such as uterine fibroids, adenomyosis, uterine prolapse, endometriosis, cervical fibroids, etc.), were diagnosed with benign lesions after the operation, and whose pathology report showed no significant lesions in the fallopian tubes or benign residual cysts.

[0070] Fallopian tube flushing fluid was collected from 49 patients with hydrosalpinx (Hydrosalpinx group, HSPX group) and 51 control patients who underwent salpingectomy for benign diseases (Healthy FT control, HFT group). Immediately after salpingectomy, the fallopian tube lumen was flushed with 10 mL of sterile saline to collect the fluid. All collected fluids were combined, aliquoted into 1.5 mL microcentrifuge tubes, and stored at -80°C.

[0071] 2. Detection Method

[0072] First, non-targeted metabolomics detection was performed using liquid chromatography-mass spectrometry (LC-MS / MS). Subsequently, the independent sample size was expanded (15 samples per group), and the concentrations of inosine, adenosine, and hypoxanthine in the fallopian tube fluid were specifically detected using an ELISA kit.

[0073] 3. Experimental Results

[0074] 3.1 Non-targeted metabolomics screening of differentially metabolized molecules

[0075] according to Figure 1 It can be seen that there are significant differences in the orthogonal partial least squares discriminant analysis (OPLS-DA) clusters. The results show that the HSPX group and the HFT group are completely separated in terms of metabolic expression profile, indicating that hydrosalpinx fluid has unique pathological metabolic characteristics compared with normal fallopian tube fluid.

[0076] according to Figure 2 It can be seen that, in the differential metabolite screening, among the 126 differential metabolites identified, inosine, adenosine, and hypoxanthine were all significantly upregulated in the HSPX group.

[0077] according to Figure 3 It can be seen that in the pathway enrichment analysis, purine metabolism is the metabolic pathway with the most significant differences, and inosine, adenosine and hypoxanthine are the core differential substances in this pathway.

[0078] 3.2 Validation of key metabolic molecules using ELISA

[0079] according to Figure 4 It can be seen that the mean concentrations of inosine (approximately 22 μM), adenosine (approximately 62 μM), and hypoxanthine (approximately 1.6 μM) in the HSPX group were significantly higher than those in the HFT group, and the P value < 0.01 indicates that there is a significant difference between the two groups.

[0080] Example 2: Validation of genes related to the inosine metabolism pathway

[0081] 1. Source Positioning

[0082] The morphology of hydrosalpinx tissue and normal fallopian tube tissue was observed by scanning electron microscopy, H&E staining, and immunofluorescence staining. The results are as follows: Figure 5 As shown.

[0083] according to Figure 5It can be seen that there are significant differences between hydrosalpinx and normal fallopian tube tissue. In the HSPX group, there is extensive damage and shedding of the mucosal epithelium, a decrease in ciliated cells and secretory cells, and edema and extensive infiltration of inflammatory cells (e.g. Figure 5 (As shown in a and b in the figure); in the HSPX group, the number of fallopian tube ciliated cells decreased, their length shortened, and the ciliated cells adhered to each other and were covered with mucus (as shown in the figure). Figure 5 (As shown in c and d in the figure); and the non-ciliated secretory epithelial cells (NCSE) in the HSPX group showed damage and a higher degree of apoptosis compared to other cell types (e.g., ...). Figure 5 (as shown by d and e in the figure).

[0084] 2. Experimental Results

[0085] Using single-cell transcriptome sequencing data combined with a metabolic pathway activity scoring algorithm, the expression of representative metabolic pathways (including purine metabolism, oxidative phosphorylation, glycolysis, and the tricarboxylic acid cycle) in 11 major oviduct cell subtypes was assessed. A higher metabolic pathway activity score indicated a higher expression level. Specifically, inosine is one of the key metabolites in the purine metabolism pathway. Analysis revealed high expression of purine metabolism pathway-related genes (such as...) in NCSE cells of oviduct epithelial cells. Figure 6A As shown), the expression of the key enzyme regulating inosine (PNP) in the fallopian tube NCSE cells of the HSPX group was higher than that of the fallopian tube NCSE cells of the HFT group, as verified by immunofluorescence and immunohistochemical staining (e.g. Figure 6B and 6C (As shown in the image). The results demonstrated a correlation between elevated inosine levels in hydrosalpinx fluid and pathological damage to NCSE epithelial cells in hydrosalpinx. Inosine specifically reflects the hydrosalpinx condition, indicating that the elevated inosine levels within the lumen are primarily due to the leakage of purine metabolites caused by widespread damage and apoptosis of NCSE cells, rather than simply increased purine synthesis by NCSE cells. Increased NCSE cell apoptosis and abnormal PNP expression in the hydrosalpinx tissue confirmed, at the gene expression level, that the abnormal expression of the key downstream metabolic enzyme PNP in hydrosalpinx epithelial cells is the cause of inosine accumulation in the hydrosalpinx fluid.

[0086] Example 3: Verification of Inosine-Induced Early Embryonic Developmental Arrest in Mice at the 2-4 Cell Stage

[0087] 1. Sample Source and Experimental Methods

[0088] This experiment used C57BL / 6J mice, housed in a temperature-controlled environment with a 12-hour light cycle, maintained temperature between 18 and 23 degrees Celsius, and humidity between 40% and 60%, with free access to food and water. Six- to eight-week-old female mice were intraperitoneally injected with 7.5 IU of pregnant mare serum gonadotropin (PMSG), followed by an injection of 7.5 IU of human chorionic gonadotropin (hCG) 46 hours later. Following hCG injection, female mice were housed overnight with adult male mice for mating. The following morning, female mice with vaginal plugs were used to collect fertilized eggs. Fertilized eggs were retrieved from the oviducts, and cumulus cells were removed using 0.4% hyaluronidase solution. Fertilized eggs were cultured in mouse embryo culture medium supplemented with inosine at different concentration gradients (5, 12.5, 25, and 50 μM). Developmental progress was assessed at the 2-cell (2C), 4-cell (4C), 8-cell (8C), morula (morula), 96-hour blastocyst, and 108-hour blastocyst stages. The experiment was independently repeated three times. The control group consisted of fertilized eggs cultured in mouse embryo culture medium supplemented with 1.5‰ dimethyl sulfoxide (DMSO), with all other culture conditions and methods identical to the experimental group.

[0089] 2. Experimental Results

[0090] Figure 7A and Figure 7B The test results showed that 5 μM inosine had no significant effect on mouse embryonic development, while 25 μM inosine resulted in a lower blastocyst rate than the control group. Figure 7A This concentration is similar to that of inosine in the effusion. However, the use of 50 μM inosine blocked the development of a large number of mouse embryos at the 2- to 4-cell stage. Figure 7B The above results indicate that inosine primarily affects early developmental processes, and its effects are concentration-dependent and cumulative.

[0091] Example 4. Test on the effect of furazolidone on mouse embryonic development

[0092] 1. Sample Source and Experimental Methods

[0093] The mouse embryos used in the initial stage of this experiment were obtained in the same manner as in Example 3. Different concentration gradients (5, 12.5, 25, and 50 μM) of furofisin were added to the mouse embryo culture medium. Cultures were started from the fertilized egg stage, and developmental progress was assessed at the 2-cell (2C), 4-cell (4C), 8-cell (8C), morula, and 96-hour blastocyst stages to verify the effect of furofisin on mouse embryonic development. The experiment was independently repeated three times. Embryos treated with 1.5‰ dimethyl sulfoxide (DMSO) served as a control.

[0094] Subsequently, 25 μM inosine and different concentration gradients (5, 12.5, 25, and 50 μM) of furodixine were added to mouse embryo culture medium. Developmental progress was assessed at the 2-cell (2C), 4-cell (4C), 8-cell (8C), morula, and 96-h blastocyst stages to verify whether furodixine could rescue the effects of inosine on mouse embryonic development and improve early embryonic developmental potential. The experiment was independently repeated three times. ROS was detected using mouse 2-cell embryos, and fluorescence intensity was quantified. 1.5‰ dimethyl sulfoxide (DMSO) was used as a blank control group, and 25 μM inosine alone was used as a treatment group.

[0095] 2. Experimental Results

[0096] As a key enzyme in the downstream metabolism of inosine, purine nucleoside phosphorylase (PNP) is crucial for inosine-induced proliferation and bioenergesis. This study aimed to assess whether inosine accumulation could mitigate its impact on early embryonic development by intervening in PNP. To eliminate the interference of furodixin, embryos from fertilized eggs to the blastocyst stage were treated with different concentrations of furodixin (5 μM, 12.5 μM, 25 μM, and 50 μM). Figure 8 As shown in Figure B), it was confirmed that different concentrations of furofisin did not affect early embryonic development in mice. Subsequently, 25 μM inosine was added in combination with different concentrations of furofisin.

[0097] The results showed that furosemide significantly rescued the inhibitory effect of inosine on early embryonic developmental potential. Figure 8 (A and C), improved blastocyst formation rate. qPCR experiments further confirmed that furoficil inhibited PNP activity, which is crucial for rescuing inosine-induced early embryonic developmental arrest in mice. Figure 8 (D). Furodexin inhibits PNP activity in the embryo, not only preventing the conversion of inosine to hypoxanthine, thus preventing xanthine oxidase from carrying out the oxidation reaction due to the lack of substrate (hypoxanthine), but also blocking the accompanying ROS burst ( Figure 8 (E and F in the middle).

[0098] Example 5: Testing the effect of PNP gene-targeted interfering RNA design on mouse embryonic development.

[0099] 1. Sample Source and Experimental Methods

[0100] The initial mouse embryo collection process in this experiment was the same as in Example 3. 25 μM inosine was added to the mouse embryo culture medium, and 20-50 μM small interfering RNA (siRNA) targeting PNP was microinjected into the mouse fertilized eggs. Figure 9(A in the diagram is a flowchart) Developmental progress was assessed at the 2-cell embryonic stage (2C), 4-cell embryonic stage (4C), 8-cell embryonic stage (8C), morula stage, and 96h blastocyst stage to verify whether interfering RNA molecules could rescue inosine from mouse embryonic development and improve early embryonic developmental potential. The experiment was independently repeated three times. A non-targeted blank control siRNA (siNC) with no significant homology to any known mouse gene was used as a control, and separately treated embryos (siNC) cultured in 25 μM inosine medium were used as controls.

[0101] The designed siRNA (siPNP) sequence targeting PNP is as follows:

[0102]

[0103] The same concentrations of siPNP_1, siPNP_2, and siPNP_3 were mixed in a volume ratio of 1:1:1 and then injected into the embryo at the fertilized egg stage.

[0104] 2. Experimental Results

[0105] Experimental results are as follows Figure 9 As shown, purine nucleoside phosphorylase (PNP), a key enzyme in the downstream metabolism of inosine, is crucial for inosine-induced proliferation and bioenergy support. This study aimed to determine whether inosine accumulation could mitigate its impact on early embryonic development by intervening in PNP. Therefore, the embryonic development rate under the combined action of inosine and siRNA was assessed. qPCR experiments further confirmed that siRNA targeting PNP significantly inhibited PNP expression, which was crucial for rescuing inosine-induced early embryonic developmental arrest in mice. Figure 9 (B) The results showed that siRNA significantly rescued the inhibitory effect of inosine on early embryonic developmental potential (B). Figure 9 (C and D) improved blastocyst formation rate.

[0106] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this application. It should be understood that the above descriptions are merely specific embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Use of purine nucleoside phosphorylase inhibitors in the preparation of medicaments for antagonizing hydrosalpinx toxicity in subjects or improving reproductive function in subjects.

2. The use according to claim 1, characterized in that, The hydrosalpinx toxicity is caused by purine metabolites; and / or The improvement in the subject's reproductive function includes one or more of the following: (1) Improve fallopian tube function; (2) Reduce miscarriage rate; (3) Inhibit abnormal activation of zygote genome in embryonic cell development; (4) Improve the stability of the embryonic cytoskeleton; (5) Enhance the developmental potential of fertilized eggs; (6) Improve the embryo's tolerance to metabolic stress; (7) Reduce embryo damage; (8) Enhance embryonic developmental potential; (9) Inhibits the burst of reactive oxygen species in embryonic cells; (10) Prevent or reverse embryonic developmental arrest; (11) Improve embryo formation rate; (12) Improve embryo quality; (13) Increase the natural conception rate; (14) Reduce infertility rate; (15) Improve embryo implantation rate; and (16) Increase pregnancy rate.

3. The use according to claim 1, characterized in that, The subjects were mammals.

4. The use according to claim 1, characterized in that, The subject suffered from hydrosalpinx.

5. The use according to claim 2, characterized in that, The embryo is either an embryo formed in vivo or an embryo produced by in vitro fertilization.

6. The use according to claim 3, characterized in that, The subject's hydrosalpinx led to decreased reproductive function.

7. The use according to claim 1, characterized in that, The dosage form of the drug is selected from intrauterine irrigation solution, suppository, injection, ointment, paste or gel.

8. Use of purine nucleoside phosphorylase inhibitors in the preparation of reagents for embryo culture and gamete treatment.

9. The use according to claim 8, characterized in that, The embryo is a mammalian embryo; and / or The gametes are those of mammals.

10. The use according to claim 1 or 8, characterized in that, The purine nucleoside phosphorylase inhibitor is selected from: furodioxin or a pharmaceutically acceptable salt thereof, urodioxin or a pharmaceutically acceptable salt thereof, or siRNA that inhibits the expression of the purine nucleoside phosphorylase gene.

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