Application of vitexin in preparation of medicine for treating asthenospermia

Abstract

The invention discloses application of vitexin in preparation of a medicine for treating asthenospermia, and particularly relates to HFn (at) VI nanoparticles formed by loading vitexin VI on a human heavy chain ferritin HFn nanocage and used for preparing the medicine for treating asthenospermia. According to the invention, the pharmacological and diagnostic potentials of vitexin are unified, and a new platform is provided for precise medicine of male infertility.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to application of vitexin in preparing a medicine for asthenospermia. Background Art

[0002] Asthenozoospermia, a common form of male infertility characterized by reduced sperm motility, is increasingly being linked to oxidative stress and dysregulated iron metabolism, which together drive ferroptosis—a form of cell death driven by iron overload and lipid peroxidation. This pathological cascade begins with excessive production of reactive oxygen species (ROS), which disrupts antioxidant systems (particularly glutathione peroxidase 4 (GPX4)), promotes iron-dependent lipid peroxidation (a hallmark of ferroptosis), and ultimately impairs sperm motility. Current treatments targeting oxidative stress or iron chelation often have limitations in specificity and biocompatibility, necessitating innovative strategies that integrate dual-functional platforms capable of simultaneous imaging and targeted intervention.

[0003] Aggregation-induced emission (AIE) luminescent materials, a class of fluorescent materials that exhibit enhanced brightness upon molecular aggregation, have become transformative tools for biomedical imaging and therapeutics. Unlike conventional fluorescent probes that suffer from aggregation-induced quenching (ACQ), AIEgens retain or amplify emission in the aggregated state, enabling high-resolution imaging with minimal background noise. While synthetic AIEgens dominate research, their clinical translation is hampered by complex synthesis, environmental concerns, and biocompatibility issues. Notably, natural products with intrinsic AIE properties remain underexplored, despite their potential for synergistic pharmacological activity with imaging guidance—a paradigm shift termed "AIE pharmacology."

[0004] Vitexin (VI), a bioactive flavonoid glycoside extracted from a medicinal plant, exhibits potent antioxidant and antiferroptotic effects by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) pathway and upregulating glutathione peroxidase 4 (GPX4). Its ability to chelate iron and scavenge reactive oxygen species makes it a promising candidate for alleviating sperm cell ferroptosis. However, a key gap remains: whether vitexin exhibits AIE characteristics, enabling real-time tracking of its intracellular dynamics and therapeutic effects. Addressing this gap could unify its pharmacological and diagnostic potential, providing a new platform for precision medicine in male infertility. Summary of the Invention

[0005] The technical solution of the present invention is the application of vitexin in the preparation of asthenospermia drugs.

[0006] A better technical solution is to load vitexin VI with human heavy chain ferritin HFn nanocages to form HFn@VI nanoparticles for the preparation of asthenospermia drugs.

[0007] Vitexin (apigenin-8-C-glucoside) is a bioactive flavonoid monomer widely used in traditional Chinese medicine for health and medicinal purposes. It is extracted from plants such as Vitex flower, Passiflora incarnata, bamboo leaves, and cranberry. A growing number of studies have shown that vitexin is a bioactive flavonoid with broad-spectrum activities, including anti-tumor effects, modulation of inflammatory pathways, reduction of pain sensitivity, and protection of neuronal function. We discovered that vitexin (VI) exhibits unique photophysical characteristics characterized by aggregation-induced emission (AIE). BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 : Photophysical properties of vitexin (VI) and characterization of HFn@VI nanoparticles. (A) Photoluminescence (PL) spectra of VI in water / DMSO mixtures of different fractions. (B) Relationship between the maximum fluorescence intensity of VI and the water fraction. (C) Time-resolved emission decay curve of VI in the aggregated state. (D) Molecular orbital amplitude diagram of the HOMO and LUMO energy levels of VI. (E) TEM image of human ferritin nanoparticles (HFn NPs; scale bar = 50 nm). (F) Size distribution histogram of HFn NPs analyzed by transmission electron microscopy. (J) Vitexin-loaded HFn nanoparticles (HFn@VI NPs; scale bar = 50 nm). (H) Size distribution histogram of HFn@VI NPs analyzed by transmission electron microscopy. (I) UV-visible absorption spectra of HFn, free VI, and HFn@VI. (J) Fluorescence emission spectra of HFn, free VI, and HFn@VI. (K) Zeta potential measurement of HFn, free VI, and HFn@VI in PBS.

[0009] Figure 2 Subcellular localization of vitexin (VI) and testicular biodistribution of ferritin@vitexin (HFn@VI). (A). VI uptake behavior at different concentrations and time points. Scale bar = 20 μm. (B). Confocal images of GC-1 spg cells co-stained with organelle trackers targeting mitochondria (Mito-Tracker), endoplasmic reticulum (ER-Tracker), lysosomes (Lyso-Tracker), and lipid droplets (LDs-Tracker) for VI localization. Scale bar = 20 μm. (C). Fluorescence imaging of HFn@VI localization in testicular tissue sections after administration. Scale bar = 20 μm. (D). Quantification of HFn@VI fluorescence intensity within testicular tissue shown in panel C.

[0010] Figure 3In vitro inhibitory effect of HFn@VI on ferroptosis in GC-1-spg cells. (AC) Intracellular Fe2+, malondialdehyde (MDA), and glutathione peroxidase 4 (GPX4) levels were quantified using commercial assay kits. (D) Representative fluorescence images of intracellular reactive oxygen species (ROS); scale bar = 20 μm. (E) Measured ROS levels. (FH) Reduced glutathione (GSH), oxidized glutathione (GSSG) levels, and the GSH / GSSG ratio were measured using assay kits. (I) Fluorescence images of lipid peroxidation detected using the BODIPY™ C11 probe (scale bar = 20 μm). (J) Quantification of lipid peroxidation levels. Data: mean ± SEM; compared with the BU group: *P < 0.05, **P < 0.01, ***P < 0.001.

[0011] Figure 4 In vivo therapeutic effects of HFn@VI on a mouse model of asthenozoospermia induced by busulfan (BU). (A) Experimental timeline. (B) Mouse body and testis weights after drug administration. (C) Sperm parameters of BU-treated mice: total number (TC; M / mL), motile cells (MC; %), progressive cells (PC; %), static cells (SC; %), average path velocity (VAP; μm / s), curvilinear velocity (VCL; μm / s), linear velocity (VSL; μm / s), amplitude of lateral head displacement (ALH; μm), beat-crossing frequency (BCF; Hz), linearity (LIN; %), straightness (STR; %), and wobble (WOB; %). (D-I) Main sperm parameters after treatment: (D) TC, (E) MC, (F) PC, (G) SC, (H) VAP, (I) VCL, (J) VSL, (K) ALH, and (L) LIN. Data: mean ± SEM, n = 5; significance: * P < 0.05, ** P < 0.01, P < 0.001.

[0012] Figure 5 Effects of experimental treatments on testicular and epididymal morphology and organ indices. (A) Representative periodic acid-Schiff (PAS)-stained sections of testicular tissue from each experimental group. Scale bar = 20 μm. (B) Testicular weight ratio (testicular index) for each treatment group (n = 5). (C) Representative periodic acid-Schiff (PAS)-stained sections of epididymal tissue from each experimental group. Scale bar = 50 μm. (D) Epididymal to body weight ratio (epididymal index) for each treatment group (n = 5).

[0013] Figure 6HFn@VI activates the ferroptotic pathway in testicular tissue. (A) Representative immunofluorescence staining of ferritin heavy chain 1 (FTH1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and GPX4 (scale bar = 20 μm). (B, E) Quantitative fluorescence intensity of (B) FTH1, (C) NRF2, (D) HO-1, and (E) GPX4. Data: mean ± SEM; compared with the BU group: *P < 0.05, **P < 0.01, ***P < 0.001.

[0014] Figure 7 (A) Schematic diagram of the in vitro blood-testis barrier model using Sertoli cells (SC). (B) Quantification of fluorescence intensity in the basal chamber culture medium after addition of HFn@VI. T-HFn@VI: translocated HFn@VI.

[0015] Figure 8 : Sperm beating crossover frequency (Hz).

[0016] Figure 9 : Sperm straightness (%) of treated mice.

[0017] Figure 10 : Sperm motility (%) of treated mice.

[0018] Figure 11 H&E-stained sections of major organ systems: cardiovascular (heart), liver (liver), spleen (spleen), lung (lung), and kidney (kidney). (A) PBS control; (B) HFn@VI-treated mice. Scale bar = 50 μm.

[0019] Figure 12 HFn@VI: A nanotherapeutic system for the treatment of asthenozoospermia. The image shows plant-derived vitexin (VI) forming HFn@VI, which is transported across the blood-testis barrier and rescues sperm dysfunction by inhibiting iron sinking in spermatogenic cells through FTH1-mediated iron chelation and activation of the Nrf2 / GPX4 antioxidant pathway. DETAILED DESCRIPTION

[0020] Culture medium and penicillin-streptomycin were from Gibco. Vitexin (VI) and the glutathione peroxidase detection kit were from Beyotime, Jiangsu, China. MilliQ purified water was used in all experiments.

[0021] The following instruments were used for characterization: TEM (FEI Tecnai G2 S-Twin), UV-Vis spectrophotometry (Hitachi U-3100), DLS (Malvern Zetasizer Nano ZS), and PL spectroscopy (Edinburghinstruments F900).

[0022] Cell culture

[0023] Mouse spermatogonia GC-1 spg cells and mouse testicular somatic cells TM3 (Leydig cells) and TM4 (Sertoli cells) were cultured in high-glucose DMEM and stored in a humidified incubator at 37°C with 5% CO2.

[0024] Preparation of HFn@VI

[0025] Human heavy chain ferritin (HFn) protein was biosynthesized and purified according to existing protocols. HFn@VI was prepared via a pH-dependent disassembly / reassembly method. Briefly, an aqueous solution of HFn (1000 μg / mL) was mixed with varying concentrations of VI (0.01-2 mg / mL) in phosphate-buffered saline (PBS). HFn was decomposed with 1 M HCl, the pH was lowered, and the mixture was incubated for 15 minutes. The pH was then gradually adjusted to 7.4 with 0.2 M NaOH. The solution was stirred for 3 hours to allow VI encapsulation and HFn reassembly, forming HFn@VI. Unencapsulated VI was removed by filtration through a 0.22 µm membrane. VI loading was quantified by measuring UV-Vis absorbance at 340 nm (HFn background subtracted), resulting in a VI:HFn loading ratio of 1:10 (w / w).

[0026] Intracellular assay (GC-1 SPG cells)

[0027] Cells were treated with PBS, 1.0 mM BU, 20 µM VI, 1.0 µM HFn, 2 µM of the ferroptosis inhibitor Fer-1, or HFn@VI (20 µM VI) for 24 hours. GSH and GSSG levels were measured using an assay kit. After 12 hours of treatment, cells were stained with BODIPY581 / 591-C11 (10 µM) for 1 hour at 37°C and observed using a confocal microscope. Cells were treated for 0–12 hours in 6-well plates. GPX4 activity was measured using a peroxidase assay kit. After 12 hours of treatment in 6-well plates, intracellular iron levels were measured using an iron colorimetric assay kit.

[0028] Cross-well assay (blood-testis barrier)

[0029] Sertoli cells (1.0 × 10 6Cells were seeded on cell culture plates (100 cells / cm²). Barrier integrity was confirmed by monitoring transepithelial electrical resistance (TER) until TER ≥ 50 Ω·cm². To assess endocytosis, HFn@VI was added to the upper chamber. After 12 hours, the culture medium in the lower chamber was collected, and translocated HFn@VI was quantified by fluorescence intensity using a microplate reader.

[0030] Animal studies

[0031] Male ICR mice (3 weeks old) were obtained from Vital River (Beijing, China). All animal experiments were approved by the Zhejiang University Animal Care and Use Committee (ZJU20230447) and adhered to institutional guidelines. Mice (n = 5 / group) were randomly assigned to: 1) PBS control group; 2-6) busulfan model (single intraperitoneal injection of 20 mg / kg busulfan). After 3 weeks, each group received treatment every 2 days for 1 week: 2) BU (no treatment); 3) BU + VI (20 mg / kg VI, injection route unknown); 4) BU + HFn (20 mg / kg HFn, tail vein); 5) BU + Fe-1 (2 mg / kg Fe-1, intraperitoneal injection); and 6) BU + HFn@VI (20 mg / kg HFn@VI, tail vein). Body, testicular, and epididymal weights were recorded before treatment. The cauda epididymis was minced in DMEM / F12 at 37°C to release sperm. Sperm parameters were analyzed using an IVOS sperm analyzer. Testicular and epididymal tissue specimens were fixed, paraffin-embedded, sectioned, and stained with H&E or PAS for histological evaluation. For immunofluorescence detection of GPX4, HO-1, Nrf2, and FTH1, sections were incubated with labeled antibodies. Image data were analyzed using ImageJ.

[0032] Statistical analysis

[0033] Results are presented as mean ± standard deviation. All measurements were performed in triplicate unless otherwise noted. Statistical comparisons were performed using a two-tailed Student's t-test, with *p < 0.05, p < 0.01, and *p < 0.001 considered significant.

[0034] In water / DMSO mixtures, when the water content exceeds 90%, its fluorescence intensity increases sharply ( Figure 1A, B). In pure DMSO, VI exhibits negligible emission at ~580 nm, which is attributed to the flexible monomeric state, where unhindered intramolecular vibrations contribute to non-radiative energy dissipation. However, in a water-rich environment, molecular aggregation occurs due to the bipolar-nonpolar nature of VI (hydrophilic glucoside vs. hydrophobic flavonoid core). This aggregation restricts intramolecular motion (RIM), suppresses non-radiative decay, and enhances radiative recombination, thereby activating fluorescence. Time-resolved emission decay measurements further support this aggregation-induced emission (AIE) mechanism, revealing that the aggregated state has a significantly longer fluorescence lifetime (τ ≈ 12 ns) compared to the dissolved state, which is almost luminescent and therefore impossible to measure the fluorescence lifetime ( Figure 1 C). This behavior distinguishes VI from conventional aggregation-induced quenching (ACQ) molecules, making it a promising candidate for luminescent probes in biological systems. Structural analysis revealed the AIE mechanism of apigenin-8-C-glucoside. In the monomeric state, the conformational flexibility of the C8-glycosylated flavonoid backbone, particularly the rotation / vibration of the C-chain glucuronide moiety and hydroxyl group, promotes non-radiative energy dissipation ( Figure 1 D). Upon aggregation, the restriction of intramolecular motion (RIM) suppresses these dynamics, reducing nonradiative decay and enhancing fluorescence. Vitexin's donor-acceptor structure features an electron-rich 4′-hydroxyl group (donor) and an electron-deficient pyrrolidone carbonyl group (acceptor) on the b-ring, resulting in a spatially separated, weakly emissive charge transfer (CT) state. Density functional theory (DFT) calculations confirm this mechanism: the basal electron density is primarily localized on the b-ring donor, while the excited state resides on the pyrrolidone carbonyl acceptor, promoting π-π* transitions ( Figure 1 D). The calculated HOMO / LUMO energy levels (e.g., −6.01 eV / −1.86 eV) yield a band gap of 4.45 eV, consistent with typical flavonoid AIE emitters and their emission behavior in aggregates.

[0035] To improve the water solubility and bioavailability of vitexin (VI) and enable it to cross the blood-testis barrier (BTB)—a key requirement for the treatment of asthenozoospermia

[41] —we exploited the unique properties of human heavy chain ferritin (HFn) nanocages, building on previous findings demonstrating the ability of HFn to cross the BTB

[42] . HFn nanocages were successfully loaded with VI to form HFn@VI nanoparticles (NPs). Transmission electron microscopy (TEM) characterization confirmed the uniform spherical morphology of bare HFn (average size: 12.10 ± 0.83 nm; Figure 1 Importantly, the HFn@VI NPs maintained good monodispersity with a slightly reduced average size of 11.58 ± 0.67 nm ( Figure 1GH), indicating effective VI encapsulation without inducing aggregation. Optical characterization showed that HFn@VI has broad absorption and emission bands, reaching peaks at 340 nm and 580 nm, respectively ( Figure 1 - j), the fluorescence was significantly enhanced after VI encapsulation ( Figure 1 - j). The zeta potential of HFn@VI in PBS is -17.83 mV ( Figure 1 K), confirming that encapsulation did not significantly alter the surface charge, thereby balancing biocompatibility (minimizing RES clearance) and colloidal stability (via electrostatic repulsion), which is required for in vivo efficacy.

[0036] Figure 2 A shows the changes in cellular uptake of VI over time and concentration. Uptake increased significantly with longer incubation time and higher concentrations. Figure 2 B shows that the AIE property of VI makes it very suitable for cell imaging, effectively labeling GC-1 spg cells with different subcellular localization. Merged confocal microscopy images confirmed its distribution in multiple compartments, including mitochondria, endoplasmic reticulum, lysosomes and lipid droplets. Verification confirmed that HFn@VI was able to penetrate the blood-testis barrier. Using the known mechanism, HFn nanocarriers crossed the BTB through transferrin receptor 1 (TfR1) on Sertoli cells. Using an in vitro BTB model (supporting cells forming tight junctions on Transwell plates), introduction of HFn@VI into the apical compartment showed obvious translocation. Fluorescence measurements ( Figure 7 A) showed that the signal intensity in the basal chamber was significantly higher than that in the control group, but significantly lower than that in the apical chamber ( Figure 7 B), confirming tfr1-mediated cytosis. To investigate in vivo localization, we injected HFn@VI intravenously and performed confocal microscopy on testicular sections. Notably, HFn@VI was widely distributed in the seminiferous tubules, with evidence indicating its presence in various germ cell types ( Figure 2 CD). This confirms the ability of HFn@VI to cross the BTB in vivo and deliver cargo directly to target cells within the male reproductive system.

[0037] Collectively, these results provide compelling evidence for the BTB-crossing ability and testicular accumulation of HFn@VI. The combination of in vitro mechanistic studies and in vivo distribution analysis provides a comprehensive understanding of HFn@VI's behavior in male reproductive biology, laying a solid foundation for the development of HFn@VI-based drug delivery systems to treat male reproductive disorders and overcome BTB challenges. The molecular design of VI and the nanocarrier properties of HFn exhibit a strong synergistic effect. Encapsulation enables the AIE activity of VI by restricting intramolecular motion (RIM) within the nanocage, overcoming the limitations of traditional aggregation-induced quenching dyes in aqueous environments. Simultaneously, HFn@VI NPs offer optimal targeted delivery properties: compact size (<20 nm; favorable for biodistribution), stable surface charge, inherent biocompatibility, and transferrin receptor-mediated BTB penetration. This functional integration makes HFn@VI a versatile therapeutic platform, combining AI-based bioimaging with targeted therapeutic delivery to the testis. This work exemplifies how rational nanocarrier design (HFn encapsulation) can exploit specific molecular photophysics (AIE) to overcome key biological barriers, paving the way for advanced fluorescence-guided therapeutics and precision medicine in reproductive health.

[0038] In GC-1 spg cells, exposure to BU (Busulfan) triggered key iron-induced apoptotic events. Compared with the PBS control group, intracellular Fe 2 + Levels of ferroptosis (the main driver of ferroptosis) were significantly increased ( Figure 3 A). This iron overload was effectively reversed by HFn@VI treatment, with performance comparable to that of the established ferroptosis inhibitor ferrostatin-1 (fer1), demonstrating the ability of HFn@VI to mitigate key initiators of ferroptosis. Concomitantly, BU exposure significantly increased malondialdehyde (MDA), a marker of lipid peroxidation, indicating oxidative membrane damage ( Figure 3 B). Both HFn@VI and Fer-1 inhibited MDA elevation, indicating reduced membrane degradation. In addition, BU reduced GPX4, a central regulator that inhibits iron sinking, while HFn@VI and Fer-1 restored GPX4 to near basal levels ( Figure 3 C), highlighting the conservation of this key defense mechanism.

[0039] In GC-1 spg cells, exposure to BU (Busulfan) triggered key iron-induced apoptotic events. Compared with the PBS control group, intracellular Fe 2 + Levels of ferroptosis (the main driver of ferroptosis) were significantly increased ( Figure 3A). This iron overload was effectively reversed by HFn@VI treatment, with performance comparable to that of the established ferroptosis inhibitor ferrostatin-1 (fer1), demonstrating the ability of HFn@VI to mitigate key initiators of ferroptosis. Concomitantly, BU exposure significantly increased malondialdehyde (MDA), a marker of lipid peroxidation, indicating oxidative membrane damage ( Figure 3 B). Both HFn@VI and Fer-1 inhibited MDA elevation, indicating reduced membrane degradation. In addition, BU reduced GPX4, a central regulator that inhibits iron sinking, while HFn@VI and Fer-1 restored GPX4 to near basal levels ( Figure 3 C), highlighting the conservation of this key defense mechanism.

[0040] Oxidative stress was further confirmed by the significant increase in intracellular reactive oxygen species (ROS) induced by BU, as visualized by fluorescence imaging ( Figure 3 D) and quantify ( Figure 3 E). HFn@VI and Fer-1 significantly reduced ROS production. This reduction was consistent with the restoration of glutathione homeostasis: BU decreased reduced glutathione (GSH) and the GSH / GSH ratio, while increasing oxidized glutathione (GSSG), a deleterious shift effectively reversed by HFn@VI, normalizing GSH levels and GSH / GSH balance ( Figure 3 Elevated GSH supports GPX4 activity, strengthening the link between HFn@VI treatment and enhanced cellular antioxidant capacity. Direct assessment of lipid peroxidation using the BODIPY-C11 probe confirmed these findings. Bu-treated cells exhibited strong fluorescence, indicating robust lipid peroxidation ( Figure 3 I), which was significantly quenched by HFn@VI and Fer-1 ( Figure 3 J). Taken together, these results suggest that HFn@VI effectively inhibits ferroptosis in spermatogenic cells through a multi-pronged mechanism: chelating excess labile iron (Fe 2 + ), scavenging ROS, maintaining glutathione redox balance (GSH / GSSG), restoring GPX4 activity, and preventing lipid membrane peroxidation. The consistent similarity of HFn@VI's effects with those of the specific ferroptosis inhibitor Fer-1 strongly suggests that HFn@VI is a potent ferroptosis modulator. This makes HFn@VI a promising therapeutic candidate for diseases in which pathogenic iron dysregulation and oxidative stress aggregate to drive iron-induced cell death.

[0041] To evaluate the therapeutic effect of HFn@VI on the asthenozoospermia mouse model, we used busulfan, a commonly used chemotherapy drug, to induce long-term azoospermia in the experimental model

[43] . Due to its strong cytotoxicity, BU mainly targets testicular cells, inducing apoptosis, autophagy, and ferroptosis of mitotic spermatogonia, ultimately leading to infertility. Utilizing this mechanism, we established a reversible infertility model in male mice by a single intraperitoneal injection of 20 mg / kg BU on day 0. We hypothesized that in this model, HFn@VI treatment could alleviate sperm ferroptosis and improve sperm quality ( Figure 4 A). As asthenozoospermia is a major cause of male infertility, our primary aim was to evaluate whether HFn@VI could alleviate this condition.

[0042] The successful establishment of the asthenozoospermia model was confirmed by the absence of significant changes in body or testicular weight and a significant decrease in sperm parameters: total number (TC), percentage of motile cells (MC), percentage of progressing cells (PC), mean path velocity (VAP), and curvilinear velocity (VCL), which together indicated severe sperm dysfunction ( Figure 4 To evaluate the therapeutic effects, mice were intravenously injected with PBS, VI, HFn, Fer-1, or HFn@VI on days 21, 22, 24, and 26 (three injections in total). Post-treatment analysis showed that sperm motility indicators were significantly improved in all treatment groups. MC, PC, and VAP were significantly enhanced in the HFn@VI cohort compared with the control group ( Figure 4 DL). While individual treatments (VI, HFn, and Fer-1) partially restored TC, MC, PC, VAP, and VCL, the most robust restoration was achieved with HFn@VI, demonstrating the synergistic benefit of combined ferroptosis inhibition. Treatments, particularly HFn@VI, significantly alleviated the deficits induced by busufen. For example, TC in the HFn@VI group was nearly double that of the BU group, approaching 83% of PBS levels. MC and PC were also significantly higher with HFn@VI than with BU, and quiescent cells (SC) were reduced to 20% ± 2%, similar to PBS values. The velocity parameter of HFn@VI was significantly better than that of BU but still slightly lower than that of PBS. Notably, while the individual agents showed modest benefits, HFn@VI consistently outperformed the individual treatments, suggesting a synergistic or enhanced nanoparticle delivery effect. Indicators such as amplitude of lateral head movement (ALH) and linearity (LIN) showed less significant differences; HFn@VI only partially restored ALH in BU, while LIN, BCF, STR, and WOB remained comparable between groups, suggesting that certain sperm motility dynamics may be less responsive to treatment or less important for overall motility improvement ( Figure 8-10 ).

[0043] Collectively, these results demonstrate that Buruli ulcer causes severe testicular damage and sperm dysfunction, characterized by decreased counts, impaired motility, and altered velocity profiles. Therapeutic intervention, particularly HFn@VI, effectively reversed these defects, with HFn@VI showing the most robust effects across multiple sperm metrics. The superior recovery of HFn@VI suggests that its design (e.g., enhanced drug delivery) could better target testicular damage compared to single agents, offering hope for alleviating chemotherapy-induced male infertility. Further studies will explore the mechanistic basis, such as protection of spermatogonial stem cells or reduction of oxidative stress, to validate these findings and guide clinical translation.

[0044] To further elucidate the therapeutic effects, periodic acid-Schiff (PAS) staining of mouse testicles showed significant histological differences ( Figure 5 A). Control testes showed tightly organized seminiferous tubules, densely packed spermatogenic cells of various stages, and abundant sperm in the tubular lumen. In stark contrast, busulfan (BU)-treated groups exhibited severe atrophy: germ cell organization was disrupted, mature cells were depleted, spermatozoa were sparse, tubules were irregular, interstitial vacuolation was observed, and vascular degeneration was observed. Treatment with VI, HFn, Fer-1, and particularly HFn@VI, significantly alleviated these pathologies, restoring tubular structure and spermatogenic cell density. Notably, the testis-to-body weight ratio remained consistent across all groups ( Figure 5 B), indicating that structural restoration was independent of total body weight change. Epididymal histology confirmed these findings ( Figure 5 C). The control group showed fallopian tubes filled with live sperm, whereas Buruli ulcer treatment resulted in almost complete depletion of sperm in the fallopian tubes, leaving hollow cavities. Intervention with VI, HFn, Fer-1, and especially HFn@VI, preserved some sperm, indicating functional restoration of spermatogenesis and post-testicular maturation. Stable epididymal index ( Figure 5 D) Further emphasizes that although local treatment is effective, there is no systemic toxicity.

[0045] To elucidate the molecular basis of testicular protection, we evaluated key biomarkers associated with iron poisoning using immunofluorescence staining and quantitative analysis of testicular tissues from different groups (PBS, BU, VI, HFn, Fer-1, HFn@VI). Compared with the PBS control group, Busulfan (BU) treatment significantly reduced the fluorescence intensity / expression of key proteins involved in iron homeostasis (FTH1), antioxidant response (Nrf2, HO-1), and iron poisoning protection (GPX4). Figure 6 AE). Specifically, FTH1 intensity was significantly reduced, indicating impaired iron sequestration and risk of toxic iron accumulation. Nrf2 showed reduced nuclear localization and intensity, indicating weakened activation of antioxidant responses. Consistent with impaired Nrf2 signaling, HO -1GPX4 expression was dramatically reduced, and it showed weak fluorescence, indicating increased sensitivity to ferroptosis.

[0046] Therapeutic intervention reversed these deficits. Treatment with VI (vehicle) or HFn alone induced only partial recovery of all biomarkers. In contrast, HFn@VI induced significant increases: FTH1 levels returned to levels comparable to those of PBS, suggesting enhanced iron sequestration, possibly through its iron chelating or transport properties; Nrf2 and its downstream effector HO -1 Significantly upregulated, HO -1 The expression of GPX4 increased significantly, reaching a level comparable to that of PBS. -1 The effects of HFn@VI on ferroptosis and GPX4 were the same as those of the known iron deposition inhibitor Fer-1, which strongly suggests that HFn@VI acts through an inhibitory iron deposition mechanism, such as maintaining GPX4 activity to prevent lipid peroxidation. Hematoxylin-eosin (HE) staining of major organs revealed no treatment-related histopathological abnormalities, confirming systemic biocompatibility ( Figure 11 ).

[0047] Effects of HFn@VI on FTH1, Nrf2, and HO -1 The synergistic upregulation of HFn@VI and GPX4 highlights a multi-pronged protective strategy: enhanced iron chelation (FTH1) reduces free iron toxicity; Nrf2 activation transcriptionally enhances antioxidant defenses and effectively induces HO-1; and direct stabilization of GPX4 blocks ferroptosis. These actions together alleviate oxidative stress and ferroptosis associated with bu-induced testicular damage and germ cell loss. The similar efficacy of HFn@VI and Fer-1 supports the hypothesis that HFn@VI exerts its protective effects through iron chelation and activation of endogenous antioxidant pathways, synergistically maintaining testicular homeostasis. In summary, HFn@VI potently activates protective pathways (Nrf2 / HO -1 , GPX4) and enhance iron sequestration (FTH1), providing a promising therapeutic approach to combat oxidative and iron-induced damage, with potential implications for protecting fertility from chemotherapy- or oxidative stress-induced impairment.

Claims

1. Application of vitexin in the preparation of asthenospermia drugs.

2. The use according to claim 1, characterized in that Human heavy chain ferritin HFn nanocages were loaded with vitexin VI to form HFn@VI nanoparticles, which were used to prepare asthenospermia drugs.

Images