Application of aspirin in prevention and / or treatment of male reproductive injury caused by rare earth elements

Abstract

The invention relates to the technical field of environmental medicine and reproductive toxicology, in particular to application of aspirin in prevention and / or treatment of male reproductive injury caused by rare earth elements. The invention also relates to a method for improving sperm quality reduction caused by rare earth element exposure. The method comprises the step of administering an effective dose of aspirin or a pharmaceutically acceptable salt thereof to an individual in need thereof. The invention also relates to a pharmaceutical composition for preventing and / or treating male reproductive injury caused by rare earth elements. The cognitive limitation of a traditional oxidative stress mechanism is broken through, a new mechanism of rare earth reproductive toxicity is disclosed from a new perspective of testis immune microenvironment for the first time, and an intervention scheme aiming at the new mechanism is provided. According to the invention, the aspirin is creatively used for solving the novel public health problem of low-dose long-term exposure of rare earth elements for the first time, and a new application field is developed for non-steroidal anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the fields of environmental medicine and reproductive toxicology, specifically to new pharmaceutical uses of aspirin, and more particularly to its use in the preparation of medicaments for the prevention and / or treatment of male reproductive damage caused by subchronic exposure to environmentally relevant concentrations of rare earth elements. Background Technology

[0002] Aspirin (acetylsalicylic acid) is a long-established nonsteroidal anti-inflammatory drug (NSAID). Its clinical applications mainly include antipyretic analgesia, antirheumatic effects, and low-dose antiplatelet aggregation to prevent cardiovascular and cerebrovascular thrombotic diseases. In recent years, its role in tumor chemoprevention has also received widespread attention. However, to date, neither the classic nor newly discovered uses of aspirin have involved the field of environmental toxicology, especially its use in the prevention or treatment of specific organ damage induced by exposure to environmental pollutants.

[0003] With industrial development, the emission of rare earth elements into the environment is increasing, and yttrium nitrate, as a common rare earth compound, has attracted attention for its environmental toxicity. Previous studies on yttrium nitrate toxicity have largely focused on high-dose acute exposure, characterized by short-term exposure (30 days) at 100 mg / kg / day, with rapid and severe toxic effects, significantly different from long-term, low-dose exposure in the natural environment. However, long-term human exposure to environmentally relevant concentrations of yttrium nitrate through air, water, and food exhibits cumulative, latent, and complex toxic effects. While acute exposure studies exist, limitations in research technology, such as the long and costly duration of chronic exposure experiments, the insensitivity of early detection technologies to subtle toxic effects, disciplinary cognitive inertia, the "acute-first" research paradigm, the locked-in oxidative stress mechanism pathway, biases in understanding testicular immune privilege, and the disconnect between environmental exposure realities and research design (e.g., lack of environmental monitoring data, and misconceptions about dose-response relationships), have prevented researchers in this field from prioritizing chronic yttrium nitrate exposure studies.

[0004] However, clarifying the effects of long-term, low-dose exposure to yttrium nitrate on the male reproductive system is crucial, and there is currently a gap in this field that urgently needs to be filled. Therefore, there is an urgent need in this field for a novel intervention program that can target male reproductive damage caused by subchronic exposure to environmentally relevant concentrations of rare earth elements.

[0005] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a new pharmaceutical use for aspirin in the prevention and / or treatment of male reproductive damage caused by rare earth elements. This invention provides a method for the application and therapeutic effects of aspirin in the prevention or treatment of male reproductive damage caused by subchronic exposure to rare earth elements, offering a new approach for the clinical treatment or prevention of male reproductive damage caused by subchronic exposure to rare earth elements.

[0007] Existing technologies attribute the mechanism of yttrium nitrate exposure-induced sperm quality damage in mouse testes to "oxidative stress." This introduces a strong technical bias in those skilled in the art: to address the toxicity of yttrium nitrate, antioxidants (such as vitamin C and NAC) should be used. This invention is the first to discover that under specific conditions of environmentally relevant concentrations and subchronic exposure, the primary mechanism undergoes a fundamental shift—from "oxidative stress" to a specific mechanism of "blood-testis barrier disruption → immune microenvironment imbalance → immune inflammation."

[0008] The testes have long been considered "immune privileged" organs, believed to be tightly protected by the blood-testis barrier and relatively isolated from the systemic immune system. Traditional thinking held that internal inflammatory responses were atypical or secondary. This invention breaks with this understanding, demonstrating for the first time that environmental pollutants can disrupt this "privilege," triggering local immune inflammation and becoming a core element of damage.

[0009] The technical solution of this invention is as follows: This invention is the first to discover that aspirin or a pharmaceutically acceptable salt thereof can be used to prepare a medicament for the prevention and / or treatment of male reproductive damage caused by rare earth elements, wherein the reproductive damage is characterized by an imbalance of the testicular immune microenvironment and an immune inflammatory response. Preferably, the rare earth element includes yttrium, more preferably yttrium nitrate.

[0010] Preferably, the damage is caused by subchronic exposure to environmentally relevant concentrations (0.01-10 mg / kg) of yttrium nitrate.

[0011] Preferably, the damage is caused by subchronic exposure to yttrium nitrate at environmentally relevant concentrations of 0.01 mg / kg, 1 mg / kg, or 10 mg / kg.

[0012] The uses described in this invention are achieved through the anti-inflammatory effect of aspirin, which is effective in: Reduce the expression of pro-inflammatory factors in the testes (such as IL-1β, IL-6, Ccl2, Cxcl10); Suppress testicular immune inflammatory response; Reduce sperm apoptosis rate; Increase sperm mitochondrial membrane potential; Reduce oxidative damage to sperm DNA (e.g., reduce 8-OHdG levels); Reduce sperm abnormality rate.

[0013] The present invention also provides a method for improving sperm quality decline caused by rare earth element exposure, the method comprising administering an effective dose of aspirin or a pharmaceutically acceptable salt thereof to an individual in need. Preferably, the effective dose is 5-20 mg / kg / day.

[0014] Preferably, the effective dose is 10 mg / kg / day.

[0015] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of male reproductive damage caused by rare earth elements, comprising an effective dose of aspirin or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers. The pharmaceutical composition is formulated to relieve testicular immune inflammation.

[0016] The beneficial effects achieved by this invention are as follows: 1. This invention breaks through the limitations of traditional understanding of oxidative stress mechanisms, and for the first time reveals a new mechanism of rare earth reproductive toxicity from the new perspective of "testicular immune microenvironment", and provides an intervention plan for this new mechanism; 2. This invention is the first to creatively repurpose aspirin, addressing the novel public health issue of long-term low-dose exposure to rare earth elements, thus opening up new application areas for nonsteroidal anti-inflammatory drugs (NSAIDs). Furthermore, aspirin is inexpensive, safe, and easy to use, making it readily adaptable to preventative or therapeutic applications for specific high-risk groups (such as residents of rare earth mining areas and those with occupational exposure), demonstrating significant public health value and market potential. 3. Animal experiments have shown that aspirin intervention can reverse deep sperm damage caused by rare earth elements in a multi-target and comprehensive manner, with clear and significant effects. Attached Figure Description

[0017] Figure 1 This is a graph showing the effect of different doses of yttrium nitrate on sperm count.

[0018] Figure 2 This is a graph showing the effect of different doses of yttrium nitrate on sperm abnormality rate.

[0019] Figure 3 These are representative images of 2-cell embryos formed after in vitro fertilization of sperm from the control group and sperm treated with yttrium nitrate.

[0020] Figure 4 The in vitro fertilization rate was statistically analyzed in the control group and the groups treated with different concentrations of yttrium nitrate.

[0021] Figure 5 These are representative images of blastocyst development in the control group and the yttrium nitrate treatment group.

[0022] Figure 6 The study investigated the blastocyst formation rate in the control group and the groups treated with different concentrations of yttrium nitrate.

[0023] Figure 7 These are representative images of newborn mice produced after female mice mate with male mice treated with different concentrations of yttrium nitrate.

[0024] Figure 8 The number of offspring was counted in the control group and the groups treated with different concentrations of yttrium nitrate.

[0025] Figure 9 This represents the birth weight of offspring produced from female mice mating with male mice treated with different concentrations of yttrium nitrate. Scale bar = 100 µm.

[0026] Figure 10 These are representative images of sperm DNA damage in each group, and the DNA damage intensity in the control group and the yttrium nitrate treatment group is statistically analyzed.

[0027] Figure 11 The percentage of sperm with high mitochondrial membrane potential (hMMP) in the control group and the yttrium nitrate treatment group was detected and statistically analyzed by flow cytometry.

[0028] Figure 12 The protein expression levels of apoptosis-related molecules in each group of sperm were detected by Western blotting.

[0029] Figure 13 These are representative histopathological images of mouse testis tissue. Red arrows: disordered cell arrangement; black arrows: vasodilation. Scale bar = 50 µm.

[0030] Figure 14 The ultrastructural changes of the blood-testis barrier induced by yttrium nitrate were observed using transmission electron microscopy. Red arrows indicate the blood-testis barrier, triangles indicate its disintegration, and circles indicate edematous Support cells. Scale bar = 2 µm.

[0031] Figure 15 The mRNA expression levels of blood-testis barrier-related genes in mouse testes were detected by real-time quantitative PCR.

[0032] Figure 16 The infiltration of CD45+ cells in mouse testes was detected by flow cytometry.

[0033] Figure 17 The detection of [something] in mouse testes was performed using real-time quantitative PCR. IL-1β , IL-6 , Ccl2 and Cxcl10 The relative expression level of mRNA.

[0034] Figure 18 The detection of TM3 and TM4 cells in the testes was performed using real-time quantitative PCR. IL-1β , IL-6 , Ccl2 and Cxcl10 The relative expression level of mRNA.

[0035] Figure 19 The method used was RT-qPCR to detect the levels of yttrium nitrate and / or aspirin in the testes of mice treated with 10 mg / kg yttrium nitrate and / or aspirin. IL-1β , IL-6 , Ccl2 and Cxcl10 The relative expression level of mRNA.

[0036] Figure 20 The sperm apoptosis status after treatment with 10 mg / kg yttrium nitrate and / or aspirin was assessed by Annexin V-PI flow cytometry.

[0037] Figure 21 The percentage of sperm with high mitochondrial membrane potential (hMMP) in the 10 mg / kg yttrium nitrate and / or aspirin treatment groups was detected and statistically analyzed using flow cytometry.

[0038] Figure 22 This presents representative images of sperm DNA damage in the 10 mg / kg yttrium nitrate and / or aspirin treatment groups, along with quantitative analysis results of 8-hydroxydeoxyguanosine (8-OHdG) fluorescence intensity.

[0039] Figure 23 The percentage of sperm abnormalities in mice treated with 10 mg / kg yttrium nitrate and / or aspirin is the percentage of sperm abnormalities in mice treated with these drugs.

[0040] Among them, compared with the control group, * p <0.05,** p <0.01, *** p <0.001. Detailed Implementation

[0041] Unless otherwise specified, the experimental materials used in the following experimental methods can be readily obtained from commercial companies. Without departing from the spirit of this invention, those skilled in the art can make numerous modifications to this invention using publicly available technology, and such modifications also fall within the protection scope of this invention.

[0042] Example 1: Assessment of sperm quality and fertility after subchronic exposure to yttrium nitrate 1.1 Animal modeling of sub-chronic exposure to yttrium nitrate Mice in the three yttrium nitrate exposure groups were administered Y(NO3)3 solution orally via gavage at daily doses of 0.01 mg / kg, 1 mg / kg, and 10 mg / kg, respectively. Eighty male BALB / c mice were randomly assigned to four experimental groups (n=20) based on body weight: (1) Control group: The same volume of physiological saline was administered by gavage daily; (2) Low-dose Y(NO3)3 group: 0.01 mg / kg by gavage daily; (3) Medium-dose Y(NO3)3 group: 1 mg / kg administered by gavage daily; (4) High-dose Y(NO3)3 group: 10 mg / kg by gavage daily.

[0043] All groups were continuously intervened for 180 days, during which the weight of mice was recorded daily.

[0044] 2.1 Sperm morphological analysis Sperm were collected from the epididymal tails of male mice in the control group (same as 1.1) and those treated with different doses (0.01 mg / kg / d, 1 mg / kg / d, 10 mg / kg / d) of Y(NO3)3. A sterile gauze pad was placed in a centrifuge tube using forceps, the supernatant was collected, and any fragments were discarded to prepare a suspension. 10 µL of the sperm suspension was dropped onto a erythrocyte counting plate, and the number of sperm in five squares was counted under an optical microscope. This counting was repeated three times, and the average value was taken as the final count.

[0045] Sperm count = N / 5 × 25 × 10 4 × Sperm suspension volume (mL).

[0046] Adjust the sperm concentration of the test suspension to 1×10⁻⁶ 6 For each sperm cell count, 20 µL of the treated specimen was taken and smeared. The smear was allowed to air dry naturally, then fixed with methanol for 10 minutes, followed by staining with 2% eosin aqueous solution for 20 minutes. The smear was then rinsed gently with water and dried. Manual counting analysis was performed, with 200 sperm cells continuously counted from multiple regions on each smear. Normal and abnormal sperm cells were distinguished under an optical microscope (×400), and the corresponding numbers were recorded. The sperm abnormality rate was also calculated.

[0047] The specific criteria for identifying sperm abnormalities are as follows: Normal sperm morphology: The head is pear-shaped or slightly flattened, and the tail is relatively long and naturally curved; Abnormal sperm morphology: The head may exhibit abnormalities such as large head, small head, pointed head, double head, no hook, or irregular shape; the neck may show obvious twisting or bending; the body may show abnormalities such as double body, wavy body, body fold, or rolled body; the tail may show abnormalities such as double tail, folded tail, broken tail, or wavy tail. If any of the four parts of the sperm shows an abnormality, it is considered an abnormal sperm.

[0048] 2.2 Experimental Results and Analysis like Figure 1 and Figure 2 As shown, compared with the control group, the sperm count was significantly reduced and the sperm abnormality rate was significantly increased in the 10 mg / kg yttrium nitrate treatment group; while no statistically significant changes were observed in the 0.01 mg / kg and 1 mg / kg yttrium nitrate treatment groups.

[0049] 3.1 In vitro fertilization experiment Sperm were collected from the epididymal tails of male mice in the control group (same as 1.1) and those treated with different doses (0.01 mg / kg / d, 1 mg / kg / d, 10 mg / kg / d) of Y(NO3)3. The specific method was as follows: the epididymal tail was excised, cut open, and its contents released into G-IVF medium (Vitrolife, Gothenburg, Sweden). The sperm suspension was incubated at 37°C and 5% CO2 for 1 hour. Then, the capacitated fresh sperm were co-incubated with mature oocytes in G-IVF medium (covered with paraffin oil) for 4 hours to complete fertilization. Double pronucleated zygotes were transferred to KSOM medium (Caisson, Smithfield, USA), covered with mineral oil, and cultured in a humidified incubator at 37°C. After 4 days of culture, the blastocyst formation rate was calculated to assess embryonic development.

[0050] 3.2 Experimental Results and Analysis like Figure 3 and Figure 4 As shown, daily administration of 10 mg / kg yttrium nitrate significantly reduced the conception rate, while the 0.01 mg / kg and 1 mg / kg dose groups showed no significant difference compared to the control group. Figure 5 and Figure 6 As shown, the blastocyst formation rate in the 10 mg / kg yttrium nitrate group was also significantly lower than that in the control group.

[0051] 4.1 Reproduction Experiment Male mice were treated with saline (control group) and different doses (0.01 mg / kg / d, 1 mg / kg / d, 10 mg / kg / d) of Y(NO3)3 by gavage for 180 days, and then mated with female mice of normal reproductive capacity. The vaginal plugs were checked and recorded the following morning to confirm successful mating. The number of pups per female and the birth weight of the offspring were recorded as indicators of reproductive capacity.

[0052] 4.2 Experimental Results and Analysis like Figure 7 and Figure 8 As shown, the male reproductive capacity of the 10 mg / kg yttrium nitrate treatment group was significantly reduced compared to the control group. Furthermore, compared to the control group, as... Figure 9 As shown, the birth weight of male rats significantly decreased after being given 10 mg / kg yttrium nitrate.

[0053] All of the above results indicate that yttrium exposure reduces sperm quality, fertility, and birth weight of offspring in mice.

[0054] Example 2 Assessment of sperm DNA damage, mitochondrial membrane potential and apoptosis after subchronic exposure to yttrium nitrate To clarify the specific damage to sperm caused by yttrium nitrate exposure, this application evaluated sperm DNA oxidative damage.

[0055] 1.1 Detection of DNA oxidative damage (8-OHdG immunofluorescence assay) After anesthetizing and euthanizing mice in the control group (same as Example 1.1) and the three experimental groups (0.01 mg / kg / d, 1 mg / kg / d, 10 mg / kg / d Y(NO3)3), the epididymal tails were quickly separated, cut into small pieces, and incubated in preheated PBS at 37°C for 15 minutes to allow sperm to fully swim out. The sperm suspension was collected and fixed with 4% paraformaldehyde at room temperature for 15 minutes.

[0056] Fixed sperm cells were washed three times with PBS, followed by treatment with 0.1% Triton X-100 solution for 10 minutes to increase cell membrane permeability. The permeabilization solution was discarded, and PBS solution containing 1% bovine serum albumin (BSA) was added. The cells were then blocked at room temperature for 1 hour to inhibit nonspecific binding.

[0057] Remove the blocking solution and add mouse anti-8-OHdG monoclonal antibody prepared with antibody dilution buffer (1:200 dilution, Santa Cruz, USA), and incubate overnight at 4°C.

[0058] After washing thoroughly with PBS three times, add FITC-labeled goat anti-mouse IgG secondary antibody (1:500 dilution, SABBiological Services, USA) and incubate at room temperature in the dark for 1 hour.

[0059] After washing again with PBS in the dark, sperm were dropped onto a glass slide and mounted using a DAPI-containing anti-fluorescence quenching mounting medium. Finally, images were observed and acquired under a confocal laser scanning microscope. The DAPI channel shows the cell nucleus (blue), and the FITC channel shows an 8-OHdG positive signal (green).

[0060] At least five fields of view were randomly selected from each group, and the mean fluorescence intensity was calculated using ImageJ software to perform a semi-quantitative analysis of 8-OHdG expression levels. Data are expressed as mean ± standard deviation, and one-way ANOVA was used for inter-group comparisons. p A value <0.05 is considered statistically significant.

[0061] 1.2 Experimental Results and Analysis like Figure 10 As shown, high-dose (10 mg / kg) yttrium nitrate exposure can induce DNA oxidative damage in sperm compared to the control group.

[0062] 2.1 Detection of sperm mitochondrial membrane potential Mice in the control group (same as in Example 1.1) and three experimental groups (0.01 mg / kg / d, 1 mg / kg / d, and 10 mg / kg / d Y(NO3)3) were anesthetized and euthanized. The epididymal tail was rapidly isolated. The epididymal tail was placed in PBS solution pre-warmed at 37°C with 1% BSA, slightly minced, and incubated for 15 minutes to allow sperm to fully swim out. The obtained sperm suspension was filtered through a 200-mesh sieve to remove tissue debris, and then centrifuged at 500 ×g for 5 minutes to collect the sperm cell pellet.

[0063] Strictly follow the instructions for the JC-1 Mitochondrial Membrane Potential Detection Kit (Beyotime Biotechnology Research Institute, China). Gently resuspend the sperm cell pellet in PBS and adjust the cell density to 1×10⁻⁶. 6 cells / mL. Take 1 mL of cell suspension, add an equal volume of JC-1 staining working solution, gently vortex to mix, and incubate in a 37°C cell culture incubator in the dark for 20 minutes.

[0064] After incubation, centrifuge at 500 ×g for 5 minutes, carefully discard the supernatant to remove unbound JC-1 dye. Wash the cell pellet once with pre-chilled PBS, and finally resuspend the cells in 500 μL PBS for immediate analysis.

[0065] Fluorescence intensity analysis was performed using flow cytometry. The excitation wavelength for JC-1 monomers (green light) was 488 nm, and the emission wavelength was 530 nm; the emission wavelength for JC-1 aggregates (red light) was 590 nm. The dual-color fluorescence signal for each cell was detected and calculated using flow cytometry software.

[0066] In sperm cells with normal mitochondrial membrane potential, JC-1 forms JC-1- aggregates, exhibiting red fluorescence; while in cells with decreased membrane potential, JC-1 exists as monomers, exhibiting green fluorescence. The number of sperm with high mitochondrial membrane potential was quantitatively assessed by comparing the proportion of positive cells in the FL2 (red fluorescence) channel. Results are expressed as mean ± standard deviation, and one-way ANOVA was used for inter-group comparisons.* p A value <0.05 is considered statistically significant.

[0067] 2.2 Experimental Results and Analysis like Figure 11 As shown, compared with the control group, the proportion of sperm with high mitochondrial membrane potential decreased significantly after high-dose (10 mg / kg) yttrium nitrate treatment. This observation suggests that the mitochondrial-derived superoxide anion production may be excessive in the 10 mg / kg yttrium nitrate treatment group.

[0068] 3.1 Western Blot Analysis of Sperm Apoptosis-Related Protein Expression Take appropriate amounts of testicular tissue or collected sperm cells from control group (same as Example 1 1.1) and three experimental groups (0.01 mg / kg / d, 1 mg / kg / d, 10 mg / kg / d Y(NO3)3) mice, add RIPA high-efficiency lysis buffer containing PMSF, and homogenize thoroughly on ice. Then, centrifuge at 12000 ×g for 15 minutes at 4°C, and carefully aspirate the supernatant, which is the total protein solution.

[0069] Using the BCA protein concentration assay kit, strictly follow the instructions to determine the total protein concentration of each sample. This ensures consistent sample loading rates in subsequent tests.

[0070] Take 20 μg of total protein, mix it with 5× SDS-PAGE protein loading buffer, and boil at 100°C for 5 minutes to denature the protein. Then, perform electrophoresis using a 10% SDS-polyacrylamide gel. After electrophoresis, transfer the protein to a PVDF membrane using a wet transfer method under ice bath conditions.

[0071] After transfer, the PVDF membrane was blocked with TBST solution containing 5% skim milk at room temperature for 1 hour. Following blocking, the membrane was incubated overnight at 4°C with the following specific primary antibody: The primary antibody dilution ratios were Bcl-2 (1:1000, Affinity Biosciences, USA), Bax (1:1000, Affinity Biosciences, USA), Caspase-3 (1:2000, Affinity Biosciences, USA), cleaved Caspase-3 (c-Caspase3) (1:2000, Affinity Biosciences, USA), and β-actin (1:1000, CST Biosciences, USA), with β-actin used as an internal control.

[0072] The following day, the membrane was washed three times with TBST for 10 minutes each time, and then incubated with the corresponding HRP-labeled secondary antibody (1:5000) at room temperature for 1 hour.

[0073] PVDF membranes were treated with an ECL chemiluminescence kit, and then exposed, developed, and fixed under a chemiluminescence imaging system or X-ray film to obtain protein band images.

[0074] ImageJ software was used to quantify the grayscale values ​​of the target band. The calculations were as follows: Divide the gray value of Bcl-2 by the gray value of Bax (both are β-actin normalized).

[0075] Divide the gray value of c-Caspase3 by the gray value of Caspase-3 (both are β-actin normalized).

[0076] The experiment was repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA was used for between-group comparisons. p A value <0.05 is considered statistically significant.

[0077] 3.2 Experimental Results and Analysis like Figure 12 As shown, compared with the control group, the Bcl-2 / Bax ratio was significantly reduced and the c-Caspase3 / Caspase3 ratio was increased in the high-dose (10 mg / kg) yttrium nitrate treatment group, indicating that the sperm apoptosis pathway was activated.

[0078] These results indicate that yttrium exposure exacerbates sperm apoptosis and mitochondrial dysfunction, leading to sperm DNA damage.

[0079] Example 3: Morphological changes and inflammatory response of testicular tissue induced by subchronic exposure to yttrium nitrate 1.1 Histopathological analysis of testicular tissue (hematoxylin-eosin staining method) After anesthesia and euthanasia, mice in the control group and the three experimental groups (0.01 mg / kg / d, 1 mg / kg / d, and 10 mg / kg / d Y(NO3)3) were rapidly dissected and their bilateral testicular tissues were completely removed. The tissues were gently rinsed with pre-cooled physiological saline to remove surface blood. Subsequently, the testicular tissues were immediately immersed in 4% paraformaldehyde phosphate buffer and fixed at 4°C for 24 hours.

[0080] The fixed tissue blocks were dehydrated using a gradient of ethanol (70%, 80%, 90%, 95%, 100%), with each concentration dehydrating for 1 hour. Subsequently, the tissue blocks were placed in xylene for clearing treatment until the tissue became transparent. Finally, the tissue blocks were immersed in molten paraffin and embedded to form paraffin blocks.

[0081] The paraffin block was continuously sectioned using a paraffin microtome to a thickness of 5 μm. The cut tissue sections were flattened in 40°C warm water and then mounted onto poly-L-lysine-treated glass slides. The slides were then placed in a 60°C oven for 2 hours to ensure the paraffin sections adhered tightly to the slides.

[0082] The slides were immersed in xylene I and xylene II for 10 minutes each to dewax them, then hydrated with a gradient of ethanol (100%, 95%, 90%, 80%, 70%) for 3 minutes each, and finally rinsed with distilled water.

[0083] Immerse the slide in hematoxylin staining solution for 5-10 minutes, then gently rinse with running water to restore the blue color.

[0084] After brief differentiation with 70% and 90% ethanol, the slide was counterstained in eosin staining solution for 3 minutes.

[0085] The stained slides were dehydrated successively with 95% ethanol and 100% ethanol, cleared with xylene, and finally sealed with neutral resin.

[0086] Sections were observed and images acquired using an optical microscope (Nexcope, China) at magnifications of 100x, 200x, and 400x. Two pathology researchers, whose experimental groups were unknown, performed a semi-quantitative assessment of the following indicators according to relevant literature standards: Count the number and quantity of spermatogenic cell layers within a unit area or a single seminiferous tubule cross section.

[0087] Observe whether the arrangement of spermatogenic cells in the seminiferous tubules is clear and whether there are any phenomena such as cell shedding or vacuolization.

[0088] Observe whether the diameter of the blood vessels in the interstitial region of the testis is abnormally increased.

[0089] 1.2 Experimental Results and Analysis like Figure 13As shown, compared with the control group, high-dose (10 mg / kg) yttrium nitrate exposure can lead to significant changes in the structure of seminiferous tubules, including a significant decrease in spermatogenic cell density, cell structure disorder, and significant vasodilation in the testicular interstitium.

[0090] 2.1 Ultrastructural observation of the blood-testis barrier (transmission electron microscopy) After anesthesia and euthanasia, testicular tissue was rapidly removed from mice in the control group and the three experimental groups (0.01 mg / kg / d, 1 mg / kg / d, and 10 mg / kg / d Y(NO3)3). The tissue was then gently rinsed with pre-cooled 0.1 M phosphate-buffered saline (PBS, pH 7.4). Subsequently, the tissue was trimmed into small pieces with a volume of less than 1 mm³ and immediately immersed in pre-cooled 0.1 M PBS fixative containing 2.5% glutaraldehyde. The fixation was carried out at 4°C for at least 4 hours to ensure good fixation of deep tissues.

[0091] The tissue block was rinsed three times with 0.1 M PBS buffer for 15 minutes each time to completely remove residual glutaraldehyde. Then, the tissue block was transferred to 1% osmium tetroxide (OsO4) solution and fixed at 4°C in the dark for 2 hours.

[0092] The fixed tissue blocks were dehydrated using a gradient of ethanol (50%, 70%, 80%, 90%, 95%, 100%) for 15 minutes at each stage. Subsequently, they were impregnated with a mixture of ethanol and epoxy resin, and finally embedded in pure epoxy resin in an oven.

[0093] The impregnated tissue block was placed into an embedding plate pre-injected with fresh epoxy resin and precisely positioned. The embedding plate was then placed in a constant temperature oven and heated to polymerize according to the program (37°C overnight, 45°C for 12 hours, 60°C for 24 hours) to harden the resin into a solid embedding block.

[0094] The embedded blocks were sectioned using an ultramicrotome to a thickness of approximately 60 nm. The cut ultrathin sections were then transferred onto a copper grid. Subsequently, they were stained with a dual electron stain of uranyl acetate and lead citrate to enhance the electronic contrast of the cell structures.

[0095] The prepared copper mesh was placed under a transmission electron microscope (e.g., Hitachi HT-7800, Japan) and observed at an accelerating voltage of 80 kV. The focus was on observing the tight junctions formed by the lateral membranes of adjacent supporting cells in the seminiferous epithelium, assessing their continuity, density, and the presence of breaks or vacuolation. The morphology of the basement membrane and organelles within the cytoplasm of supporting cells was also observed. Samples from at least five mice were randomly selected from each group, and at least ten different sections of the seminiferous tubules were observed from each sample, with digital images acquired.

[0096] The images were interpreted by two researchers whose groupings were unknown. The assessment criteria for blood-testis barrier integrity included: clear and continuous tight junction structures, high electron density, and no significant separation or interruption. The proportion of cross-sections showing tight junction structural anomalies (such as blurring, breakage, or dissociation) was recorded and semi-quantitative analysis was performed.

[0097] 2.2 Experimental Results and Analysis like Figure 14 As shown, in the control group, the cell junctions that constitute the blood-testis barrier (including tight junctions, basal ectoplasmic specialization, gap junctions, and desmosome junctions) remained intact between adjacent supporting cells; while the high-dose (10 mg / kg) yttrium nitrate treatment group showed significant disintegration of the blood-testis barrier.

[0098] 3.1 RT-qPCR analysis of mRNA expression of genes related to blood-testis barrier function Approximately 30 mg of testicular tissue from the control group and three experimental groups (0.01 mg / kg / d, 1 mg / kg / d, and 10 mg / kg / d Y(NO3)3) mice was taken and thoroughly ground under liquid nitrogen conditions. Subsequently, 1 mL of TRIzol reagent (TransGold, China) was added to the tissue powder, and total RNA was extracted according to the manufacturer's instructions. The extracted RNA was dissolved in RNase-free water.

[0099] The absorbance values ​​of RNA at 260 nm and 280 nm were measured using a micro spectrophotometer to assess the concentration and purity of RNA (an A260 / A280 ratio between 1.8 and 2.0 was considered acceptable).

[0100] Take 1 μg of total RNA and perform reverse transcription using the HiScript III first-strand cDNA synthesis kit (Novizan, China). The reaction volume is 20 μL, including 4 μL of 5× HiScript III qRT SuperMix, 1 μg of RNA template, and RNase-free water to a final volume of 20 μL. The reaction conditions are: 37°C for 15 minutes (reverse transcription), 85°C for 5 seconds (enzyme inactivation). The resulting cDNA is stored at -20°C for later use.

[0101] All qPCR primers for all genes were designed to span exon adapters to avoid amplification of genomic DNA. The primers used in this example... Primer sequences are shown in Table 1.

[0102] Table 1 Primer sequences for reverse transcription polymerase chain reaction (RT-PCR)

[0103] Amplification was performed using ChamQ Universal SYBR qPCR Master Mix (Novizan, China). The 10 μL reaction mixture consisted of: 5 μL 2× ChamQ SYBR Master Mix, 0.4 μL forward and reverse primer mixture (10 μM), 1 μL cDNA template (appropriately diluted), and 3.6 μL RNase-free water.

[0104] Run the following program on a real-time quantitative PCR instrument (such as Bio-Rad CFX96): pre-denaturation at 95°C for 30 seconds; then perform 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60°C for 30 seconds; finally plot the melting curve to verify the specificity of the amplified product.

[0105] Using the comparison Ct method (2^ -ΔΔCt The relative mRNA expression level of the target gene was calculated using a method (method). Gapdh Genes were standardized as internal reference genes. Downregulation of gene expression level was defined as a 2:2 reduction in expression level between the experimental group and the control group. -ΔΔCt Values ​​less than 1. Results are expressed as mean ± standard deviation. One-way ANOVA was used for inter-group comparisons. p A value <0.05 is considered statistically significant.

[0106] 3.2 Experimental Results and Analysis like Figure 15 As shown, compared with the control group, the mRNA expression levels of blood-testis barrier-related genes (including tight junction genes Claudin-11, Occludin, and basal exoplasmic differentiation gene N-cadherin) were downregulated in the high-dose (10 mg / kg) yttrium nitrate treatment group, indicating that the integrity of the blood-testis barrier was disrupted.

[0107] 4.1 Flow cytometry analysis of immune cell infiltration in testicular tissue After anesthetizing and euthanizing mice in the control group and the three experimental groups (0.01 mg / kg / d, 1 mg / kg / d, 10 mg / kg / d Y(NO3)3), testicular tissue was quickly removed and weighed in pre-cooled PBS.

[0108] Use ophthalmic scissors to thoroughly mince the tissue, transfer it to a digestion solution containing appropriate amounts of type IV collagenase (1 mg / mL) and DNase I (20 μg / mL), and digest it in a constant temperature shaker at 37°C for 45 minutes.

[0109] Blow on the tissue every 10 minutes during digestion until the tissue block is basically gone.

[0110] Filter the digestion solution through a 200-mesh cytometer to remove undigested tissue clumps. Collect the filtrate, centrifuge at 500 × g for 5 minutes, and discard the supernatant.

[0111] Add 2-3 mL of erythrocyte lysis buffer (ACK Lysis Buffer) to the cell pellet, gently pipette to mix, and incubate at room temperature in the dark for 3 minutes to lyse the erythrocytes. Add excess PBS to terminate lysis, centrifuge at 500 ×g for 5 minutes, and discard the supernatant. This step can be repeated once to ensure complete removal of erythrocytes.

[0112] Wash the cell pellet once with pre-chilled PBS, centrifuge, and discard the supernatant. Resuspend the cells in PBS containing 1% BSA, and adjust the cell density to 1 × 10⁶ cells / mL using a cell counting chamber or automated cell counter. 7 cells / mL.

[0113] Take 100 μL of cell suspension (approximately 1×10⁻⁶ cells / mL) 6 (1 cell) was added to the flow cytometry tube.

[0114] Add 0.5 μg / test of anti-mouse CD45 antibody (PerCP-Cy5.5 labeled, catalog number: 45-0451-82, eBioscience).

[0115] At the same time, an unstained control group (cells only) and an isotype control group (using the same dose of non-specific immunoglobulin of the same species, subtype and label) were set up.

[0116] After gently vortexing to mix, incubate at 4°C in the dark for 30 minutes.

[0117] After incubation, add 2 mL of flow cytometry staining buffer to each tube, centrifuge at 500 × g for 5 minutes, and discard the supernatant to remove unbound antibodies. Resuspend the cells in 300–500 μL of flow cytometry staining buffer and immediately perform flow cytometry analysis.

[0118] Detection was performed using a flow cytometer such as the Beckman Coulter CytoFLEX.

[0119] First, use FSC-A / SSC-A scatter plots to delineate lymphocyte and monocyte populations, excluding cell debris.

[0120] Within the delineated target cell population, the percentage of CD45-positive cells was analyzed.

[0121] CD45 is a universal marker of leukocytes. This study quantitatively assessed the infiltration of immune cells in testicular tissue by comparing the percentage of CD45-positive cells in the experimental and control groups. Results are expressed as mean ± standard deviation. One-way ANOVA was used for inter-group comparisons.* pA value <0.05 is considered statistically significant.

[0122] 4.2 Experimental Results and Analysis like Figure 16 As shown, compared with the control group, daily administration of 10 mg / kg yttrium nitrate significantly increased the infiltration level of CD45+ cells in testicular tissue, while the low-dose groups (0.01 mg / kg and 1 mg / kg) had no such effect.

[0123] 5.1 Effects of subchronic exposure to yttrium nitrate on the expression of pro-inflammatory factor mRNA in mouse testicular tissue At the animal level, we investigated the effects of subchronic exposure to yttrium nitrate on the transcriptional levels of key pro-inflammatory cytokines and chemokines in the testicular immune microenvironment.

[0124] After anesthetizing and euthanizing control mice and three experimental groups (0.01 mg / kg / d, 1 mg / kg / d, 10 mg / kg / d Y(NO3)3), bilateral testicular tissue was rapidly dissected and removed. The tissue was rinsed thoroughly with pre-cooled PBS and then blotted dry with filter paper. One testicular tissue was immediately flash-frozen in liquid nitrogen and then transferred to a -80°C cryogenic freezer for long-term storage for RNA extraction.

[0125] Total RNA extraction: Approximately 30 mg of frozen testicular tissue was thoroughly ground into powder using a homogenizer under liquid nitrogen conditions. 1 mL of TRIzol reagent was added to the powder, and subsequent steps were performed strictly according to the manufacturer's instructions for total RNA extraction. The extracted RNA was dissolved in RNase-free water.

[0126] RNA quality control and cDNA synthesis: RNA concentration and purity (A260 / A280 ratio) were determined using a micro-spectrophotometer. 1 μg of total RNA was used for reverse transcription using the HiScript III first-strand cDNA synthesis kit, with a reaction volume of 20 μL. The reaction conditions were: 37°C for 15 minutes, followed by 85°C for 5 seconds.

[0127] Real-time quantitative PCR: Reaction system: ChamQ Universal SYBR qPCR Master Mix was used in a 96-well plate. The 10 μL reaction mixture consisted of: 5 μL Master Mix, 0.4 μL forward and reverse primer mixture (10 μM), 1 μL cDNA template (appropriately diluted), and 3.6 μL RNase-free water.

[0128] Amplification program: Run on a Bio-Rad CFX96 real-time quantitative PCR instrument: 95°C for 30 seconds; followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds; finally plot the melting curve.

[0129] Target gene: Il-1β , IL-6 , Ccl2 and Cxcl10 .by Gapdh This gene serves as an internal reference. Primer sequences were validated using BLAST to ensure specificity. The primer sequences used in this example are shown in Table 2.

[0130] Table 2 Primer sequences for reverse transcription polymerase chain reaction (RT-PCR)

[0131] Data analysis: using 2^ -ΔΔCt The relative mRNA expression levels of each target gene were calculated using the method described above. Results are expressed as mean ± standard deviation. Independent samples t-tests were used for comparisons between groups. p A value <0.05 is considered statistically significant.

[0132] 5.2 Experimental Results and Analysis like Figure 17 As shown, treatment with 10 mg / kg yttrium nitrate significantly upregulated the mRNA expression of multiple pro-inflammatory cytokines / chemokines in the testes, such as interleukin (…). IL-1β , IL-6 ), chemokine (CC motif) ligand 2 ( Ccl2 ) and chemokine (CXC motif) ligand 10 ( Cxcl10 ).

[0133] 6.1 Effects of Yttrium Nitrate on the Expression of Pro-inflammatory Factors mRNA in Testicular Cells (TM3 / TM4) At the cellular level, we will verify whether yttrium nitrate can directly induce pro-inflammatory responses in testicular interstitial cells (TM3) and supporting cells (TM4), thereby confirming its direct toxic effects.

[0134] The specific experimental steps are as follows: Cell culture: Mouse testicular interstitial cells (TM3) and mouse Sertoli cells (TM4) were purchased from Pronoss. Cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin antibiotics in an incubator at 37°C and 5% CO2.

[0135] Cell treatment and grouping: Experiments were conducted when the cells reached 70%-80% confluence.

[0136] TM3 cell experimental groups: ① Control group (complete culture medium); ② Yttrium nitrate treatment group (50, 100, 150 μg / mL Y(NO3)3).

[0137] TM4 cell experimental group: grouping is the same as TM3 cell.

[0138] Each group had 3 replicates. Cells were cultured in drug-containing medium for another 48 hours.

[0139] Total RNA extraction from cells: After treatment, discard the culture medium and gently wash the cells twice with pre-cooled PBS. Add 1 mL of TRIzol reagent directly to each well and incubate at room temperature for 5 minutes to allow for complete cell lysis. Transfer the lysis buffer to RNase-free centrifuge tubes, and follow the same RNA extraction steps as in in vivo experiments.

[0140] cDNA synthesis and qPCR detection: This part of the process, reaction system, amplification procedure, target gene and internal reference gene are completely consistent with the in vivo experimental part (steps 4 and 5 above) to ensure methodological comparability.

[0141] Data analysis: using 2^ -ΔΔCt Relative gene expression levels were calculated using [method / method / method]. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and LSD-t tests were used for further pairwise comparisons. p A value <0.05 is considered statistically significant.

[0142] 6.2 Experimental Results and Analysis like Figure 18 As shown, similar results were obtained after in vitro treatment of mouse testicular interstitial cells (TM3) and supporting cells (TM4) with yttrium nitrate.

[0143] In summary, these results indicate that yttrium nitrate can induce an inflammatory response in the testes.

[0144] Example 4: Effect of Aspirin on Sperm Damage Caused by Yttrium-Induced Testicular Inflammation To elucidate the role of yttrium-induced testicular inflammation in sperm damage, we administered aspirin to yttrium-treated mice.

[0145] 1. Animal grouping and treatment Forty 4-week-old clean-grade male BALB / c mice were randomly divided into the following 4 groups (n=10): Control group: administered an equal volume of physiological saline by gavage daily.

[0146] Aspirin control group: 10 mg / kg aspirin solution was administered by gavage daily.

[0147] Yttrium nitrate model group: 10 mg / kg yttrium nitrate solution was administered by gavage daily.

[0148] Yttrium nitrate + aspirin intervention group: 10 mg / kg yttrium nitrate solution and 10 mg / kg aspirin solution were administered by gavage simultaneously every day.

[0149] All treatments lasted 180 days. Aspirin was administered 1 hour before yttrium nitrate gavage.

[0150] 2. Sample Collection After treatment, the mice were weighed, anesthetized, and euthanized. The following samples were collected for subsequent analysis: Testicular tissue: a portion was flash-frozen at -80°C for qPCR analysis; another portion was fixed with 4% paraformaldehyde for histological analysis.

[0151] Epididymal tail: used to collect sperm for testing various functional indicators.

[0152] 3. Detection Indicators and Methods 3.1 Expression of testicular inflammatory factor mRNA Method: Real-time quantitative PCR.

[0153] Procedure: Frozen testicular tissue was collected, total RNA was extracted and reverse transcribed into cDNA. Pro-inflammatory cytokines were detected. IL-1β , IL-6 , Ccl2 and Cxcl10 The mRNA expression level of ). Gapdh As an internal reference, 2^ -ΔΔCt The method is used for relative quantification.

[0154] 3.2 Sperm apoptosis rate detection Method: Annexin V-FITC / PI double staining flow cytometry.

[0155] Procedure: Epididymal sperm were collected, resuspended in PBS, and cell density was adjusted. Annexin V-FITC and PI staining solution were added according to the kit instructions, and the cells were incubated in the dark before flow cytometry analysis. The sum of early and late apoptotic cells was calculated as the total apoptosis rate.

[0156] 3.3 Detection of sperm mitochondrial membrane potential Method: Flow cytometry using JC-1 fluorescent probe.

[0157] Procedure: Sperm samples were collected and incubated with JC-1 staining working solution in the dark. The ratio of red to green fluorescence intensity was detected by flow cytometry. A decrease in membrane potential was manifested as weakened red fluorescence and enhanced green fluorescence (increased JC-1 monomer ratio).

[0158] 3.4 Detection of oxidative DNA damage in sperm Method: Immunofluorescence staining with 8-hydroxydeoxyguanosine.

[0159] Procedure: Sperm smears are fixed and cleared, incubated with primary antibody against 8-OHdG, and then incubated with FITC-labeled secondary antibody. After mounting, the smears are observed under a fluorescence microscope or confocal microscope, and the 8-OHdG level is semi-quantitatively determined by calculating the average fluorescence intensity.

[0160] 3.5 Sperm morphological analysis Methods: Sperm smears were stained with eosin.

[0161] Procedure: Sperm were collected from the tail of the mouse epididymis. A sterile gauze pad was placed in a centrifuge tube using forceps. The supernatant was collected, and any fragments in the tube were discarded to prepare a suspension. 10 µL of the sperm suspension was dropped onto a red blood cell counting plate, and the number of sperm in 5 squares was counted under an optical microscope. This counting was repeated 3 times, and the average value was taken as the final count result.

[0162] Sperm count = N / 5 × 25 × 10 4 × Sperm suspension volume (mL).

[0163] Adjust the sperm concentration of the test suspension to 1×10⁻⁶ 6 For each sperm cell count, 20 µL of the treated specimen was taken and smeared. The smear was allowed to air dry naturally, then fixed with methanol for 10 minutes, followed by staining with 2% eosin aqueous solution for 20 minutes. The smear was then gently rinsed with water and dried. Manual counting analysis was performed, with 200 sperm cells continuously counted from multiple regions on each smear. Normal and abnormal sperm cells were distinguished under an optical microscope (×400), and the corresponding numbers were recorded. The sperm abnormality rate was also calculated.

[0164] The above method is the same as the method in Examples 1-3, but the target animals are the animals in the animal group in Example 1 above.

[0165] 2. Experimental Results and Analysis All data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups; if significant differences were found, LSD-t tests were used for pairwise comparisons. p <0.05, ** p <0.01, *** p A difference of <0.001 is considered statistically significant.

[0166] like Figure 19 As shown, aspirin significantly alleviated the increase in testicular inflammatory factors induced by yttrium exposure. It is noteworthy that, as... Figure 20 As shown, aspirin treatment effectively reduced the yttrium-mediated increase in sperm apoptosis rate. Furthermore, as... Figure 21As shown, aspirin also reversed yttrium-induced damage to sperm mitochondrial membrane potential (MMP). Furthermore, as... Figure 22 As shown, aspirin can effectively protect sperm from yttrium-mediated oxidative DNA damage and significantly reduce yttrium-induced sperm toxicity by decreasing sperm abnormality rates. Figure 23 As shown.

[0167] In summary, these data suggest that testicular inflammation plays a central role in yttrium-mediated sperm damage, and aspirin can be used as a key drug to alleviate the damage.

[0168] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Use of aspirin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of male reproductive damage caused by rare earth elements.

2. The use of aspirin according to claim 1 in the prevention and / or treatment of male reproductive damage caused by rare earth elements, characterized in that: The reproductive damage is characterized by an imbalance in the testicular immune microenvironment and an immune inflammatory response.

3. The use of aspirin according to claim 1 in the prevention and / or treatment of male reproductive damage caused by rare earth elements, characterized in that: The male reproductive damage caused by rare earth elements is due to subchronic exposure to environmentally relevant concentrations.

4. The use of aspirin according to claim 3 in the prevention and / or treatment of male reproductive damage caused by rare earth elements, characterized in that: The environmentally relevant concentration is 0.01-10 mg / kg.

5. The use of aspirin according to any one of claims 1-4 in the prevention and / or treatment of male reproductive damage caused by rare earth elements, characterized in that: The rare earth element includes yttrium.

6. The use of aspirin according to claim 5 in the prevention and / or treatment of male reproductive damage caused by rare earth elements, characterized in that: The rare earth element is yttrium nitrate.

7. The use according to any one of claims 1-4, characterized in that, The drug is used for one or more of the following purposes: (a) Reduces the expression levels of pro-inflammatory cytokines IL-1β, IL-6, Ccl2 and Cxcl10 in testicular tissue; (b) Reduce sperm apoptosis rate; (c) Increase sperm mitochondrial membrane potential; (d) Reduce oxidative damage to sperm DNA; (e) Reduce sperm abnormality rate.

8. A method for improving sperm quality decline caused by rare earth element exposure, characterized in that: The method involves administering an effective dose of aspirin or a pharmaceutically acceptable salt thereof to an individual in need.

9. The method for improving sperm quality decline caused by rare earth element exposure according to claim 8, characterized in that: The effective dose is 5-20 mg / kg / day.

10. A pharmaceutical composition for preventing and / or treating male reproductive damage caused by rare earth elements, characterized in that: It contains an effective dose of aspirin or a pharmaceutically acceptable salt thereof as the active ingredient, and one or more pharmaceutically acceptable carriers.

Images