A pharmaceutical composition for treating asthenospermia by activating the Nrf2 / HO-1 / GPx4 pathway and its application.

Abstract

This invention discloses a pharmaceutical composition for treating asthenospermia by activating the Nrf2 / HO-1 / GPx4 pathway and its application. The pharmaceutical composition comprises taurine and geranium as active ingredients, wherein the mass ratio of taurine to geranium is 3-5:1. The pharmaceutical composition of this invention can synergistically activate the Nrf2 / HO-1 / GPx4 antioxidant signaling pathway, upregulate the expression of downstream antioxidant enzymes GPx4 and HO-1, inhibit lipid peroxidation, protect sperm mitochondrial function, and simultaneously improve the microenvironmental metabolic profile of the epididymis—the site of sperm maturation—thereby enhancing sperm motility. The pharmaceutical composition provided by this invention can be used to prepare a treatment for asthenospermia, particularly oxidative stress-induced asthenospermia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition and its application in the preparation of drugs for treating asthenospermia. In particular, it relates to a pharmaceutical composition containing taurine and geranium, which is used to treat oxidative stress-induced asthenospermia by activating the nuclear factor E2-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) / glutathione peroxidase 4 (GPx4) antioxidant signaling pathway. Background Technology

[0002] Asthenospermia is one of the main causes of male infertility, clinically characterized by low sperm motility and a lower proportion of progressively motile sperm than the reference value. The World Health Organization's diagnostic criteria are: sperm concentration greater than 15 × 10⁻⁶. 6 The percentage of forward-motile sperm is less than 32%, or the total sperm motility is less than 40%. Although patients with asthenospermia have normal sperm concentration and morphology, their sperm motility is insufficient, making it difficult for them to penetrate cervical mucus and complete the fertilization process, directly leading to decreased fertility or even infertility.

[0003] Oxidative stress is one of the core pathological mechanisms in the development of asthenospermia. When the level of reactive oxygen species (ROS) is too high or the antioxidant defense capacity is reduced, the oxidative and antioxidant systems become unbalanced, resulting in oxidative stress. Sperm cell membranes are rich in polyunsaturated fatty acids, which give them good fluidity but also make them sensitive to lipid peroxidation. Excessive ROS attack the polyunsaturated fatty acids in the sperm membrane, causing lipid peroxidation, which leads to membrane structure damage, increased permeability, and affects the normal function of ion channels and receptors on the sperm membrane. Oxidative stress can also directly damage sperm mitochondria, leading to a decrease in mitochondrial membrane potential and a reduction in adenosine triphosphate (ATP) production, directly resulting in decreased sperm motility.

[0004] Currently, the main drugs used clinically to treat asthenospermia include antioxidants (such as vitamin E, vitamin C, coenzyme Q10, and L-carnitine), androgen drugs, and some traditional Chinese medicine preparations. However, these existing therapies have significant limitations: their efficacy is uncertain, and the clinical studies of most antioxidants have small sample sizes and inconsistent quality; their mechanisms of action are singular, with most antioxidants targeting a single site, making it difficult to comprehensively correct the complex pathological network of asthenospermia; there are side effects, as long-term or high-dose use of certain antioxidants may interfere with the normal regulation of endogenous redox signals in the body; and patient compliance is poor, with some drugs requiring long-term, repeated administration.

[0005] Taurine is a sulfur-containing amino acid widely found in mammalian tissues, especially abundant in the male reproductive system. Taurine possesses strong antioxidant capabilities, directly scavenging reactive oxygen species (ROS), stabilizing cell membranes, regulating intracellular calcium homeostasis, and exerting indirect antioxidant effects by upregulating the activity of antioxidant enzymes. Geraniol is a naturally hydrolyzable ellagitannin compound with multiple biological activities, including anti-inflammatory, antioxidant, antitumor, and anti-hyperglycemic effects.

[0006] Although the antioxidant effects of taurine and geranium have been reported individually, there are currently no reports in published literature and patents regarding the combined use of taurine and geranium for the treatment of asthenospermia. In particular, whether the combination of the two can exert a therapeutic effect on asthenospermia by synergistically activating the Nrf2 / HO-1 / GPx4 antioxidant signaling pathway, inhibiting lipid peroxidation, and improving the metabolic profile of the epididymal microenvironment remains a technological gap. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a pharmaceutical composition that can effectively treat asthenospermia, especially asthenospermia induced by oxidative stress.

[0008] To address the aforementioned technical problems, this invention provides a pharmaceutical composition for treating asthenospermia by activating the Nrf2 / HO-1 / GPx4 pathway and its application.

[0009] The pharmaceutical composition of the present invention comprises taurine and geranium as active ingredients, and exerts its effect by activating the Nrf2 / HO-1 / GPx4 pathway. The taurine has CAS number 107-35-7, molecular formula C2H7NO3S, and molecular weight approximately 125.15 Da; the geranium is a naturally hydrolyzable ellagitannin compound with CAS number 60976-49-0 and molecular formula C... 41 H 28 O 27 Its molecular weight is approximately 952.64 Da.

[0010] Preferably, the mass ratio of taurine to geranium is 3-5:1.

[0011] The pharmaceutical composition of this invention exerts the following effects by synergistically activating the Nrf2 / HO-1 / GPx4 antioxidant signaling pathway: upregulating the expression of downstream antioxidant enzymes GPx4 and HO-1; inhibiting sperm membrane lipid peroxidation, reducing the production of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE); protecting sperm mitochondrial function and restoring ATP synthesis; and improving the metabolic profile of the epididymal microenvironment, providing a favorable microenvironment for sperm maturation. Ultimately, it significantly improves sperm motility and effectively treats asthenospermia.

[0012] The mechanism of action of the pharmaceutical composition of the present invention is as follows:

[0013] The two work synergistically to promote the translocation of Nrf2 into the cell nucleus, thereby strongly upregulating the expression of downstream key antioxidant enzymes—heme oxygenase-1 (HO-1) and glutathione peroxidase 4 (GPx4)—forming a multi-layered antioxidant defense network. HO-1 exerts broad-spectrum antioxidant and anti-inflammatory effects by degrading heme to produce biliverdin (a direct antioxidant) and carbon monoxide (an anti-inflammatory mediator); GPx4 specifically reduces phospholipid hydroperoxides, directly protecting sperm cell membrane lipids from oxidative damage.

[0014] This antioxidant network effectively inhibits lipid peroxidation, restores sperm mitochondrial membrane potential, and improves the metabolic profile of the epididymal microenvironment. Thus, it achieves systemic protection at the molecular, cellular, tissue, and overall functional levels, ultimately significantly enhancing sperm motility.

[0015] The pharmaceutical composition described in this invention can be prepared into any pharmaceutical formulation suitable for use, including but not limited to: tablets, capsules, granules, powders, oral liquids, suspensions, emulsions, etc.

[0016] In preparing the above formulations, pharmaceutically acceptable excipients may be added as needed, including but not limited to: fillers (such as lactose, starch, microcrystalline cellulose), binders (such as povidone, hydroxypropyl methylcellulose), disintegrants (such as crospovidone, sodium carboxymethyl starch), lubricants (such as magnesium stearate, talc), flow aids (such as micronized silica gel), flavoring agents (such as steviol glycosides, fruit flavorings), preservatives (such as sodium benzoate, parabens), and solvents (such as water, ethanol, propylene glycol), etc.

[0017] Compared with the use of geranium or taurine alone, the pharmaceutical composition of the present invention has the following beneficial effects: synergistic effect, significantly improving sperm motility and concentration in asthenospermia model animals; clear mechanism, by activating the Nrf2 / HO-1 / GPx4 antioxidant signaling pathway, inhibiting lipid peroxidation (4-HNE), and reducing oxidative stress damage; improved metabolism, correcting the metabolic disorder of the epididymal microenvironment (confirmed by metabolomics), providing a favorable environment for sperm maturation; dual protection, directly protecting spermatocytes (GC-2spd) from oxidative damage at the cellular level. Attached Figure Description

[0018] Figure 1 The effect of the taurine and geranium compound in Example 1 on the viability of mouse spermatocyte GC-2spd cells. ### p<0.001, compared with the blank control group; *p<0.05, **p<0.01, compared with the model group.

[0019] Figure 2The effect of the taurine and geranium combination in Example 1 on the mitochondrial membrane potential of mouse spermatocyte GC-2spd cells was investigated. a. Control group; b. Model group (H2O2, 200 μM); c. Taurine group (Tau, 10 mM); d. Geranium group (Ge, 10 μM); e. Taurine + Geranium group (Tau + Ge, 10 mM + 10 μM).

[0020] Figure 3 The effect of the combination of taurine and geranium in Example 1 on the expression of Nrf2 signaling pathway proteins in mouse spermatocyte GC-2spd cells.

[0021] Figure 4 The effects of the taurine and geranium compound in Example 2 on the gonadal index and biochemical indicators of rats were shown. a. Testicular index, b. Epididymal index, c. SOD level in testicular tissue, d. MDA level in testicular tissue; # p<0.05, ## p<0.01, ### p<0.001, compared with the blank control group; * p<0.05, ** p<0.01, *** p<0.001, compared with the model group.

[0022] Figure 5 The effects of the taurine and geranium compound combination in Example 2 on sperm density and sperm motility. a. Changes in rat sperm density, b. Changes in rat sperm motility; ## p<0.01, compared with the blank control group; * p<0.05, ** p<0.01, compared with the model group.

[0023] Figure 6 The effect of the taurine and geranium combination in Example 2 on the mitochondrial membrane potential of rat sperm. a. Control group; b. Model group; c. Taurine group (Tau, 200 mg / kg / d); d. Geranium group (Ge, 50 mg / kg / d); e. Taurine + geranium group (Tau + Ge, 200 + 50 mg / kg / d).

[0024] Figure 7 The effect of the taurine and geranium combination in Example 2 on the morphological changes of rat testicular tissue. a. Control group; b. Model group; c. Taurine group; d. Geranium group; e. Taurine + geranium group (Tau + Ge).

[0025] Figure 8The effect of the combination of taurine and geranium in Example 2 on the expression of Nrf2 signaling pathway proteins in rat testicular tissue.

[0026] Figure 9 The effect of the taurine and geranium compound combination in Example 2 on the metabolic profile of rat epididymal fluid. a. PCA score plot; b. PLS-DA score plot. Detailed Implementation

[0027] The present invention will be described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited to the following. Unless otherwise specified, the scientific and technical terms used herein are based on the understanding of those skilled in the art. In the following embodiments, unless specific conditions are specified, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments used without specified manufacturers are all commercially available conventional products.

[0028] It should be noted that the mass ratio of taurine to geranium in the pharmaceutical composition of this invention is based on the mass of the active pharmaceutical ingredients and is suitable for preparing solid dosage forms or as a pharmaceutical raw material. In in vitro cell experiments, because the molecular weight of geranium (approximately 952.64 Da) is significantly greater than that of taurine (approximately 125.15 Da), the optimal molar concentrations of the two in the culture medium differ (geranium is in the micromolar range, while taurine is in the millimolar range). When the composition is prepared according to a mass ratio (3~5:1), it exhibits a synergistic effect in both in vivo animal experiments and in vitro cell experiments. In other words, the mass ratio range defined by this invention ensures that the two active ingredients achieve an effective synergistic concentration relationship in vivo after administration.

[0029] Regarding the difference in concentration representation between in vitro cell experiments and in vivo animal experiments, the following explanation is necessary: ​​In the in vitro cell experiments of Example 1, taurine (molecular weight 125.15 Da) and geranium (molecular weight 952.64 Da) had significantly different effective concentration ranges in cell culture medium due to their significant molecular weight difference (approximately 7.6 times). Taurine, as a small molecule amino acid, is effective at millimolecular (mM) concentrations; while geranium, as a large molecule polyphenol compound, exhibits biological activity at micromolar (μM) concentrations. Table 1 shows that the various dose groups (low, medium, and high) employed different molar concentration ratios to explore the optimal synergistic window at the cellular level. All dose groups showed a synergistic protective effect superior to the single-drug groups. Figure 1-3 In the in vivo animal experiment of Example 2, to simulate the clinical administration scenario and simplify dosage control, a mass ratio of 4:1 (i.e., taurine: geranium extract = 200 mg / kg / d : 50 mg / kg / d) was used for intervention. At this ratio, the synergistic effect was significant. Figure 4-9The differences between in vitro and in vivo experimental systems stem from variations in cellular microenvironment and overall animal metabolism, but both confirm that the pharmaceutical compositions of the present invention synergistically activate the Nrf2 / HO-1 / GPx4 pathway within the stated ratio range.

[0030] Example 1: In vitro cell experiments

[0031] 1.1 Experimental Materials

[0032] Mouse spermatocyte GC-2spd and DMEM (high glucose) medium were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; fetal bovine serum was purchased from Sangon Biotech (Shanghai) Co., Ltd.; flow cytometry reagents, nucleoprotein and plasma protein extraction kits were purchased from Jiangsu Kaiji Biotechnology Co., Ltd.; primary antibody was purchased from Beijing Bio-Sen Biotechnology Co., Ltd., and secondary antibody was purchased from Affinity Biosciences.

[0033] 1.2 Experimental Methods

[0034] (1) Effect of the drug composition on the viability of GC-2spd cells as determined by MTT assay

[0035] To verify the protective effect of the pharmaceutical composition of this invention on oxidative stress-induced GC-2spd cell damage, a "pretreatment-damage" model was used. The specific steps are as follows: Mouse spermatocytes (GC-2spd) in the logarithmic growth phase were digested with trypsin to prepare a cell suspension, which was then added to 96-well plates at 100 μl per well. The plates were incubated at 37°C and 5% CO2 for 24 h. Afterward, complete culture medium containing different concentrations of the drug (taurine, geranium extract, and their combination) was added for pretreatment for 12 h. Subsequently, the culture medium was discarded, and serum-free culture medium containing 200 μmol / L H2O2 was added for further incubation for 2 h to induce oxidative stress damage. The blank control group received no drug or H2O2, while the model group received only H2O2 and no drug.

[0036] The groupings and corresponding drug concentrations are shown in Table 1.

[0037]

[0038] After treatment, 10 μl of thiazolyl blue (MTT) solution was added to each well and incubated for another 4 h. The culture medium was then discarded, and 100 μl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the crystals. The absorbance of each well was measured at 492 nm using a microplate reader, and the cell viability of each group was calculated.

[0039] (2) Flow cytometry analysis of the effect of the drug composition on the mitochondrial membrane potential of GC-2spd cells GC-2spd cell suspension was cultured at 2 ml / well in 6-well plates for 24 hours. During the experiment, blank control group, model group (H2O2, 200 μM), taurine group (Tau, 10 mM), geranium group (Ge, 10 μM), and taurine + geranium group (Tau + Ge, 10 mM + 10 μM) were set up, with treatment methods as described above. After drug treatment, cells were collected with trypsin (without EDTA), washed three times with phosphate-buffered saline (PBS), and 2 μl of JC-1 (mitochondrial membrane potential fluorescent probe) was added to 1000 μl of Incubation Buffer and mixed well. The cells were then resuspended in 1000 μl of this solution and incubated at 37°C in the dark for 20 min. After centrifugation and removal of supernatant, the cells were resuspended in 1000 μl of Incubation Buffer, and mitochondrial membrane potential was detected by flow cytometry.

[0040] (3) Western blot analysis of the effect of the drug composition on the expression of oxidative stress proteins in GC-2spd cells

[0041] GC-2spd cells were cultured in 6-well plates and administered drugs in groups. Cells were collected and washed three times with PBS (as described above). Nuclear and plasma proteins were extracted according to the instructions of the KGI Nuclear-Plasma Separation Kit. Proteins were separated by SDS-PAGE with polyacrylamide gel electrophoresis. After electrophoresis, the target proteins were transferred to a PVDF membrane, blocked with 5% skim milk, and incubated overnight at 4°C with primary antibodies including anti-Nrf2, anti-HO-1, anti-GPx4, and anti-4-HNE. After washing with TBST, the membrane was incubated with secondary antibody at room temperature for 90 min, exposed in a dark chamber using an electrochemiluminescence system, and images were captured using ChemiCapture software.

[0042] 1.3 Experimental Results

[0043] (1) The drug composition enhances cell viability

[0044] Cellular experiments showed that the cell viability of the model group decreased significantly to approximately 43% (P<0.001), indicating the successful establishment of the oxidative stress injury model. Compared with the model group, the drug composition of this invention can dose-dependently increase the viability of GC-2spd cells, with the high-dose group showing a recovery of cell viability to approximately 88% (P<0.01), which is superior to each single-drug group, demonstrating a synergistic protective effect. Figure 1 ).

[0045] (2) The drug composition enhances the mitochondrial membrane potential of cells.

[0046] Mitochondrial membrane potential (MMP) is a core indicator for evaluating mitochondrial function, directly reflecting the cell's ATP synthesis capacity. The energy required for sperm motility mainly comes from mitochondrial oxidative phosphorylation; a decrease in MMP leads to ATP depletion and loss of sperm motility. Therefore, detecting MMP can elucidate the mechanism by which the composition of this invention improves sperm motility from an energy metabolism perspective. When the mitochondrial membrane potential is normal, the fluorescent probe JC-1 forms a polymer in the mitochondrial matrix, producing red fluorescence (FL1 channel, horizontal axis); while when the mitochondrial membrane potential is low, JC-1 exists in monomeric form in the cytoplasm, producing green fluorescence (FL2 channel, vertical axis). Therefore, the stronger the red fluorescence and the weaker the green fluorescence, the higher the mitochondrial membrane potential and the more normal the mitochondrial function (Q3 represents the proportion of cells with low MMP). Figure 2 As shown, the model group cells exhibited a significant decrease in MMP, indicating that oxidative stress led to the collapse of mitochondrial membrane potential. Both taurine and geranium extract alone improved MMP levels, while the composition of this invention restored MMP to levels close to the blank control group, superior to each of the single-use groups. These results demonstrate that the combined application of taurine and geranium extract can synergistically protect spermatocyte mitochondrial function, maintain mitochondrial membrane potential stability, and provide sufficient energy supply for sperm motility.

[0047] (3) The drug composition upregulates cellular Nrf2, HO-1, and GPx4, and downregulates 4-HNE protein expression.

[0048] To elucidate the protective mechanism of the pharmaceutical composition of this invention against oxidative stress damage in GC-2spd cells, Western blotting was used to detect the protein expression levels of Nrf2 (an upstream transcription factor, reflecting whether the drug activates the antioxidant defense initiation program), HO-1 and GPx4 (downstream effector molecules of Nrf2, representing broad-spectrum antioxidant capacity and specific membrane lipid peroxide scavenging capacity, respectively), and 4-HNE (lipid peroxidation end products, reflecting the degree of oxidative damage). β-actin (cytoplasmic protein) and Lamin B (nuclear protein) were used as internal controls for calibration. Results are as follows: Figure 3 As shown, in the model group, Nrf2 underwent compensatory nuclear translocation, downstream HO-1 and GPx4 expression was downregulated, and 4-HNE was significantly increased. Taurine and geranium extract alone could partially reverse these changes, while the combination restored Nrf2 to near-normal levels, significantly upregulated HO-1 and GPx4, and significantly downregulated 4-HNE. This indicates that the combined application of taurine and geranium extract can synergistically activate Nrf2 and its downstream HO-1 and GPx4, effectively inhibiting lipid peroxidation and protecting GC-2spd cells from oxidative stress damage.

[0049] Example 2: Animal Experiment

[0050] 2.1 Experimental Materials

[0051] Male SD rats were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd. (SCXK(Su)2024-0001), weighing 180-220 g. The rest of the procedure was the same as in Example 1.

[0052] 2.2 Experimental Methods

[0053] (1) Creation and treatment of animal models of asthenospermia

[0054] Forty-two male SD rats were randomly divided into seven groups, and the drug administration regimens were as follows:

[0055] Ornidazole induces oxidative stress by inhibiting antioxidant enzyme activity and disrupting mitochondrial function, damaging the epididymis and leading to decreased sperm motility. It is used to establish a model of asthenospermia. Ornidazole, taurine, and geranium suspension were dissolved in 0.5% CMC-Na at the concentrations shown in Table 2, and prepared fresh each time. Rats were administered the solution via gavage in groups, with the blank control group receiving 0.5% CMC-Na. Rats were weighed daily to adjust the dosage. The treatment lasted for 3 weeks, during which the condition of each group was observed.

[0056] (2) Effects of the drug composition on the gonadal index and biochemical indicators of rats

[0057] Three weeks after administration, rats in each group were weighed, sacrificed, and dissected. The wet weight of each gonadal tissue was quickly measured and recorded, and the gonadal index was calculated. The testicular tissue was prepared into a 5% tissue homogenate using a ball mill, and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in the testicular tissue were detected.

[0058] (3) Effects of the drug composition on sperm density, sperm motility, and testicular morphology in rats During rat dissection, the bilateral epididymal tails were harvested, minced, and incubated in physiological saline at 37°C. Sperm were then fully freed and rapidly counted under a microscope, with at least 200 sperm counts recorded. The number of motile sperm, the number of non-motile sperm, and the total sperm count were recorded. The formulas for calculating the total sperm count and sperm motility are as follows:

[0059]

[0060] The left testis of a rat was removed, fixed in 4% paraformaldehyde, embedded in paraffin, and tissue sections were prepared. After staining with hematoxylin and eosin (H&E), the testicular tissue sections were observed under an inverted microscope (×200 field of view).

[0061] (4) Effect of the drug composition on the mitochondrial membrane potential of rat sperm detected by fluorescence microscopy

[0062] Rat sperm were collected using the method described above. The JC-1 staining method was the same as in Example 1, "Flow Cytometry Detection of the Effect of Drug Composition on Mitochondrial Membrane Potential of GC-2spd Cells". One drop was spread on a glass slide and the fluorescence intensity was observed under a fluorescence microscope.

[0063] (5) Western blot analysis of the effect of the drug composition on the expression of oxidative stress proteins in rat testicular tissue

[0064] During rat dissection, testicular tissue was collected, proteins were extracted, and the expression level of the target protein was detected, using the same method as in Example 1, "Western Blot Detection of the Effect of Drug Composition on Oxidative Stress Protein Expression in GC-2spd Cells".

[0065] (6) Effect of metabolomics on the metabolic profile of drug composition on rat epididymal fluid

[0066] Epididymal fluid provides the necessary microenvironment for sperm maturation, and its metabolic composition directly affects sperm motility. Epididymal fluid metabolomics can comprehensively assess the effects of drugs on improving the sperm microenvironment, providing direct evidence of metabolic levels for enhancing sperm motility.

[0067] (6.1) Sample pretreatment: After sperm counting, the sperm and epididymal fluid suspensions were centrifuged to obtain the supernatant, which was then frozen at -70℃. 100 μL of epididymal fluid was taken, and cold methanol containing the internal standard was added to precipitate the protein. The supernatant was centrifuged and evaporated to dryness. Oxime treatment (methoxyamine pyridine, 16 h) and derivatization (MSTFA + 1% TMCS, 1 h) were performed sequentially. Finally, heptane containing the external standard was added, and the supernatant was centrifuged for GC-MS analysis.

[0068] (6.2) Data Acquisition and Processing: GC-MS (Agilent 7890A-5975C), full ion scan mode. Total ion chromatograms (TICs) were acquired, and deconvolution analysis was performed using AMDIS software. Peaks with a signal-to-noise ratio >30 were filtered out, compounds were identified, and peak areas were automatically integrated. An RSD <15% for both internal and external standard peak areas was considered acceptable, and all peak areas were corrected using the internal standard. Multivariate analysis, including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), was performed using MetaboAnalyst 6.0.

[0069] 2.3 Experimental Results

[0070] (1) The drug composition improves the gonadal index and biochemical indicators in rats ( Figure 4 )

[0071] like Figure 4As shown in a and b, the testicular index of rats in the treatment group was significantly higher than that in the model group. The epididymal index in the single-drug group was slightly higher than that in the model group, but the difference was not statistically significant. However, the drug combination significantly increased the epididymal index to near the control group, indicating that the synergistic effect of the drug combination significantly improved ornidazole-induced testicular and epididymal atrophy in rats to near-normal levels. MDA is the end product of lipid peroxidation, reflecting the degree of oxidative damage; SOD is a key antioxidant enzyme that scavenges superoxide anions in the body. The antioxidant effect of the drug combination on rat testicular tissue can be evaluated from both damage and defense dimensions. Figure 4 As shown in c and d, the drug groups can significantly improve the decrease in SOD level and increase in MDA level caused by ornidazole. The efficacy of the drug composition is better than that of the drug alone, indicating that the drug composition of the present invention can effectively reduce lipid peroxidation damage in testicular tissue and enhance antioxidant enzyme activity.

[0072] (2) The drug composition improves sperm density and sperm motility in rats. Figure 5 )

[0073] The sperm density in the model group rats was slightly lower than that in the control group, but the difference was not significant. The sperm density in the treatment group showed no significant change. Figure 5 a). The model group rats showed a highly significant decrease in sperm motility (P<0.01), while the drug treatment groups all significantly improved the decrease in sperm motility induced by ornidazole (P<0.05), and the efficacy of the drug combination was superior to that of the single-drug group (P<0.01). Figure 5 (b) indicates that the pharmaceutical composition of the present invention synergistically and effectively improves asthenospermia in rats.

[0074] (3) The drug composition improves the mitochondrial membrane potential of rat sperm ( Figure 6 )

[0075] The sperm of rats in the blank control group showed a state of high red fluorescence and low green fluorescence under a fluorescence microscope. Figure 6 a); The sperm of the model group rats showed the opposite trend of high green fluorescence and low red fluorescence, with significant differences ( Figure 6 b); Taurine ( Figure 6 c) Geranyl ketone ( Figure 6 d) In the single-drug group, green fluorescence was weakened and red fluorescence was enhanced; the drug combination showed this trend more significantly, with no significant difference compared to the control group. The results indicate that the combined use of taurine and geranium synergistically protects sperm mitochondrial function, maintains mitochondrial membrane potential stability, and provides sufficient energy supply for sperm motility.

[0076] (4) The drug composition improved the morphological changes of rat testicular tissue ( Figure 7 )

[0077] In the control group, the basement membrane of the seminiferous tubules in the testis was intact, and the spermatogenic cells at all levels within the lumen were arranged in an orderly and layered manner, with a large number of filamentous sperm visible in the center of the lumen. In the model group, the basement membrane was thickened, the interstitium was widened, a large number of spermatogenic cells died and sloughed off, and intercellular vacuoles formed, with only a very small number of sperm visible in the center of the lumen. In the treatment group, the basement membrane was intact, the spermatogenic cells were arranged in a more orderly manner, and the number of spermatogenic cells was slightly reduced compared to the control group, but significantly increased compared to the model group. The filamentous sperm in the center of the lumen were arranged radially. In particular, the drug composition could improve the overall structure to be close to that of the control group.

[0078] (5) The drug composition upregulated Nrf2, HO-1, and GPx4 in rat testicular tissue and downregulated 4-HNE protein expression. Figure 8 )

[0079] The trends in Nrf2, HO-1, and GPx4 protein expression in animal testicular tissue were consistent with the results of in vitro cell experiments (comparison). Figure 3 and Figure 8 The mutual corroboration of these in vivo and in vitro results indicates that the pharmaceutical composition of the present invention can synergistically activate Nrf2 and its downstream antioxidant enzyme network, thereby reducing lipid peroxidation damage and improving sperm motility.

[0080] (6) The drug composition improves the metabolic profile of the epididymal microenvironment in rats ( Figure 9 )

[0081] Results of metabolomics analysis ( Figure 9 The results showed that the metabolic profiles of the model group and the blank control group were significantly separated; the single-drug group approached the control group, while the combined drug group approached the control group. This indicates that combined drug therapy can improve the metabolic disorders of the epididymal microenvironment, provide a favorable microenvironment for sperm maturation and motility, thereby enhancing sperm motility and treating asthenospermia.

[0082] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and scope, and all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating asthenospermia by activating the Nrf2 / HO-1 / GPx4 pathway, characterized in that: It contains taurine and geraniol.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of taurine to geraniol is 3~5:

1.

3. The use of the pharmaceutical composition according to any one of claims 1-2 in the preparation of a medicament for treating asthenospermia.

4. The application according to claim 3, characterized in that, The asthenospermia mentioned above is induced by oxidative stress.

5. The application according to claim 3, characterized in that, The drug works by activating the Nrf2 / HO-1 / GPx4 antioxidant signaling pathway.

6. The application according to claim 5, characterized in that, The drug improves sperm motility by upregulating the expression of GPx4 and HO-1, inhibiting lipid peroxidation, protecting sperm mitochondrial function, and improving the metabolic profile of the epididymal microenvironment.

7. A pharmaceutical preparation for treating asthenospermia, characterized in that, It comprises the pharmaceutical composition according to any one of claims 1 to 2 and a pharmaceutically acceptable excipient.

8. The pharmaceutical preparation according to claim 7, characterized in that, The dosage form of the preparation is selected from tablets, capsules, granules, powders, oral liquids, suspensions, and emulsions.

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