FSHR-targeting tetrahedral DNA frameworks ameliorate ovarian aging through oxidative stress elimination

Abstract

Ovarian aging, characterized by declining ovarian reserve, is a pacemaker of aging in the female body. Oxidative stress leads to apoptosis, mitochondrial dysfunction, inflammation, and telomere shortening, accelerating ovarian aging. Scavenging reactive oxygen species (ROS) has been shown to delay ovarian aging; however, there remains a significant lack of antioxidants with both proven efficacy and minimal side effects. DNA tetrahedral nanostructure (DTN) is a promising nucleic acid framework with antioxidant and anti-apoptotic properties. We developed FSH-DTN, a modified nanoparticle equipped with a follicle-stimulating hormone receptor-targeting peptide (FSH33-53) to enhance ovarian accumulation. Compared to native DTN, FSH-DTN showed superior ovarian targeting efficiency as confirmed by in vivo imaging. In both in vivo and in vitro models of acute, subacute, and chronic ovarian aging, FSH-DTN demonstrated superior antioxidant, anti-apoptotic, and anti-aging effects. Further investigation revealed that FSH-DTN can directly eliminate ROS in the ovaries while enhancing ovarian antioxidant capacity by activating the NRF2 signaling pathway, thereby protecting ovarian function. In this study, we offer a new strategy for neutralizing oxidative stress to delay ovarian aging.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04111-z.

Introductionction

In recent years, the global fertility rate has been well below the replacement level, accelerating population aging and putting pressure on public finances and social development. Delayed childbearing has also led to an increase in infertility rates, further exacerbating population imbalance [1]. Ovarian aging is one of the primary causes of declining fertility, characterized by a reduction in both the quantity and quality of oocytes, leading to loss of reproductive capacity and endocrine dysfunction, accompanied by menstrual irregularities [2, 3]. At around the age of 50, women experience ovarian aging and enter menopause, with about one-third of their life span being postmenopausal. During this stage, ovarian aging contributes to multi-system health problems such as osteoporosis, cardiovascular diseases, obesity, and cognitive decline [4]. Although ovarian aging is a natural process, many women experience primary ovarian insufficiency (POI) before the age of 40, which not only shortens their reproductive lifespan but also increases the risk of miscarriage, pregnancy complications, and birth defects [5]. Therefore, delaying ovarian aging and preventing subsequent systemic organ aging are crucial for maintaining women’s health.

Ovarian aging is a multifaceted process affected by numerous internal and environmental variables, with oxidative stress playing a key role. The free radical theory, a classic theory of aging, posits that aging is the result of accumulated oxidative stress within cells, with oxidative stress as the main culprit of ovarian aging [6, 7]. Reactive oxygen species (ROS), mainly produced in mitochondria, serve a significant function in the ovary’s physiological processes. However, when ROS production exceeds the redox homeostasis, oxidative stress occurs, leading to cell structure damage, including lipids, proteins, and DNA [8]. Oxidative stress within the ovary has been demonstrated to induce aberrant functionality in oocytes and granulosa cells (GCs), thereby impacting follicle development and hormone synthesis, ultimately accelerating the aging of ovary [9, 10]. ROS are known to impair ovarian cell function by inducing mitochondrial DNA damage, which consequently diminishes ATP production and augments apoptotic rates [11]. These abnormalities ultimately lead to the depletion of ovarian follicle, reduced oocyte quality, and disrupted hormone production. Therefore, targeting ROS constitutes a pivotal strategy for mitigating the progression of ovarian aging.

In recent years, antioxidant drugs have gained attention for their potential in combating oxidative stress. Antioxidants against ovarian aging, including natural and synthetic ones, such as resveratrol, melatonin, vitamin C, ubiquinone, etc [12]. However, current research shows controversy regarding the efficacy and applicability of antioxidants in treating aging, especially some antioxidants may exert opposite effects. For instance, experimental evidence has demonstrated that resveratrol exhibits a dose- and time-dependent inhibition of self-renewal capacity while promoting cellular aging [13]. Moreover, at higher concentrations, this compound induces replication stress, leading to the aggregation of DNA strand breaks and the subsequent induction of cellular senescence [14]. Furthermore, antioxidant supplementation has been shown to modulate the activity of various transcriptional regulators and cellular enzymes, which in turn complicates the hierarchical signaling cascade of upstream and downstream molecules [15]. Consequently, this complexity poses significant challenges in precisely controlling the therapeutic efficacy of antioxidants. Traditional antioxidants face issues such as poor stability, low bioavailability, and non-specific distribution. Many of these antioxidants are prone to degradation or loss of activity during storage and use. For example, antioxidants such as vitamin C and vitamin E are quickly oxidized when exposed to air, light, or high temperatures, leading to loss of their effectiveness [16, 17]. Additionally, many water-soluble antioxidants would be rapidly excreted after passing through the gastrointestinal tract, making it difficult to reach sufficient concentrations in the body. Hence, more research is urgently needed to explore effective antioxidant treatment strategies to delay ovarian aging.

Nano-antioxidants offer significant advantages over traditional antioxidants, including enhanced stability and increased bioavailability due to small sizes and high surface area of nanoparticles, targeted delivery through specific surface modifications or carrier designs, and controlled release capabilities [18]. These features provide nano-antioxidants with distinct benefits compared to traditional antioxidants, especially in the domains of pharmaceutical distribution and therapeutic intervention, where they hold great potential for future applications. The DNA tetrahedral nanostructure (DTN) possesses intrinsic antioxidant properties, primarily attributed to the redox activity of DNA bases. Among these, guanine (G) exhibits the strongest reducing capacity, enabling direct redox reactions with ROS to achieve antioxidant effects [19]. It is composed of four DNA single strands and has excellent cell absorption and tissue penetration capabilities [20]. DTN are characterized by excellent biocompatibility, precise addressability, structural stability, controllable self-assembly, and customizable functionalities [21]. DTNs can also serve as drug delivery carriers to enhance drug uptake efficiency, including the delivery of small molecules, nucleic acids, short peptides, and aptamers. Previous research has demonstrated capacity of DNA nanostructures to mitigate oxidative stress damage in diseases such as acute kidney injury [22], rheumatoid arthritis [23], and ischemic stroke [24]. Given its demonstrated efficacy and safety profile, DTN represents a promising candidate for ROS scavenging to mitigate ovarian aging.

After being introduced into the body, DTNs are rapidly distributed to various organs through the bloodstream, primarily accumulating in the liver and kidneys before being metabolized or cleared [22]. However, the therapeutic application of DTN lacks effective targeting, particularly for organs with limited blood flow, such as the ovaries, where drug accumulation is minimal. To enhance treatment specificity, we propose incorporating an ovarian-targeting peptide into the DTN. Follicle-stimulating hormone (FSH), secreted by the pituitary gland, exerts effects on the ovaries via the FSH receptor (FSHR). The FSHR is a highly tissue-specific receptor, primarily located on the surface of sertoli cells and ovarian granulosa cells, with minimal presence in non-gonadal tissues [Sertoli cells and ovarian granulosa cells, with minimal presence in non-gonadal tissues [25]. The FSHβ (33–53) peptide (YTRDLVYKDPARPKIQKTCTF) is a classic FSHR-targeting peptide known for its precise ovarian localization [FSHR-targeting peptide known for its precise ovarian localization [26], which potentially functions as a partial FSH agonist, demonstrating significant stimulation of basal estradiol synthesis. We hypothesize that FSH33 peptide-modified DTNs could serve as a potential target therapeutic agent for ovarian aging.

In this study, we synthesized FSH33 peptide-modified DTNs (FSH-DTNs) and explored their therapeutic effects in delaying ovarian aging (Fig. 1). Experiments were performed across diverse cellular and animal models of ovarian aging, during their acute, subacute, and chronic phases. The FSH-DTNs demonstrated a range of beneficial properties, including the mitigation of oxidative stress, inhibition of cell apoptosis, and delay of cell senescence. Additionally, they improved mitochondrial function in ovarian granulosa cells, underscoring their potential as a therapeutic intervention for ovarian aging. FSH-DTNs can significantly delay ovarian aging in mice, as evidenced by the improvement in ovarian hormone synthesis function and fertility. Furthermore, FSH-DTNs demonstrated favorable safety profiles, with no significant detrimental effects observed on the health of both the parental generation and their progeny. The data we present suggest that FSH-DTNs possess the potential to mitigate diverse manifestations of ovarian aging, thereby holding significant promise for translational research and clinical application.

Fig. 1.

Schematic diagram of FSH-DTN synthesis and its application in ovarian aging

Methods

Synthesis and characterization of DTN/FSH-DTN

DTNs were prepared using a DNA self-assembly procedure, involving heating at 95 °C for 10 min followed by cooling at 4 °C for 20 min in a 1x TAE-Mg²⁺ (TM) buffer (10 mM Tris-HCl, 50 mM MgCl₂, pH 8.0). The azide-modified FSH33-53 peptide (FSH-N3) was synthesized and purified to > 95% purity by Qiangyao Biological Technology (Wuhan, China). To construct the FSH-DTN, the FSH-N3 peptide was first reacted with a DBCO-functionalized oligonucleotide (DBCO-A20) to form FSH-A20 at 37 °C, 400 rpm for 3 h. This reaction mixture was then mixed with the pre-synthesized DTNs at room temperature to form the FSH-DTN complex via DNA hybridization. After the complementary base pairing between A20 and T20 bases, four FSH33-53 peptides will bind to each DTN. Following incubation, the samples were purified by ultrafiltration (Millipore Amicon Ultra 3 K device, 4000 rpm, 10 min, repeated three times) and centrifugation to remove unreacted FSH-N3. Agarose gel electrophoresis was used to characterize the self-assembly of DTN and FSH-DTN. DNA samples were separated on a 2% agarose gel in a 1×TAE running buffer containing 12.5 mM MgAc2, and the electrophoresis was performed at 110v for 25 min at room temperature.el electrophoresis was used to characterize the self-assembly of DTN and FSH-DTN. DNA samples were separated on a 2% agarose gel in a 1×TAE running buffer containing 12.5 mM MgAc₂, and the electrophoresis was performed at 110V for 25 min at room temperature.

The morphology of FSH-DTN was examined via Transmission Electron Microscopy (TEM). 10 µL sample was adsorbed onto the carbon-coated copper grids (400 mesh) surface for 3 min. The excess solution was wicked away with filter paper, and the grid was immediately washed deionized water to remove residual buffer salts. The sample was then stained with 2% uranyl acetate solution for 1 min. The grid was wicked dry and completely air-dried before imaging.Imaging was performed using a FEI TECNAI G2 20 TWIN microscope operated at 200 kV.ple was adsorbed onto the carbon-coated copper grids (400 mesh) surface for 3 min. The excess solution was wicked away with filter paper, and the grid was immediately washed deionized water to remove residual buffer salts. The sample was then stained with 2% uranyl acetate solution for 1 min. The grid was wicked dry and completely air-dried before imaging.Imaging was performed using a FEI TECNAI G2 20 TWIN microscope operated at 200 kV.

AFM samples of 12.5 µM FSH-DTN were prepared on freshly cleaved muscovite mica. A small aliquot of the sample was deposited and allowed to adsorb briefly. Subsequently, the liquid cell was filled with 1x TAE-Mgnd allowed to adsorb briefly. Subsequently, the liquid cell was filled with 1x TAE-Mg² buffer to fully immerse the sample and tip. The DTNs were scanned in tapping mode under fluid conditions using a Pico-Plus AFM (Agilent Technologies). Imaging utilized NP-S silicon nitride tips.

The particle sizes of DTN and FSH-DTN were characterized using Dynamic Light Scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, United Kingdom). Solutions of DTN and FSH-DTN were prepared at a concentration of 50 µM in 1.0 mL of phosphate buffer (pH 7.4). The suspensions were homogenized by filtration using a 0.22 μm syringe filter. DTN/FSH-DTN samples were combined with PBS and 10% fetal bovine serum (FBS) and incubated for durations of 0, 2, 4, 8, 12, and 24 h at 37 °C. Subsequently, the stability of DTN/FSH-DTN was assessed using agarose gel electrophoresis.

Scavenging radicals with DTNs

The hydroxyl radical scavenging efficiency was tested using a Hydroxyl Free Radical assay kit (Catalog number: A018-1-1. Nanjing Jiancheng, Inc., China). The superoxide radical scavenging efficiency was assessed with the Inhibition and produce superoxide anion assay kit (Catalog number: A052-1-1. Nanjing Jiancheng, Inc., China). The DPPH radical scavenging efficiency was tested using a DPPH Free Radical Scavenging Capacity Assay Kit (Catalog number: A018-1-1. Nanjing Jiancheng, Inc., China). All tests were performed following the protocol provided by the kits.

COV434 cells culture and treatment

COV434 cells, a human ovarian granulosa cell line, were procured from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The cells were maintained in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and incubated at 37 °C under 5% CO₂. COV434 cells were treated with D-gal (200mmol L^− 1, Catalog number: G5388. Sigma-Aldrich, USA) for 48 h, or H₂O₂ (500 µmol L^− 1, Catalog number: 88597. Sigma-Aldrich, USA) for 1 h, NaAsO₂ (10 µmol L^− 1, Catalog number: S7400. Sigma-Aldrich, USA) for 6 h.

Animals

Eight-week-old female C57BL/6 mice were acquired from Beiente Bio-Technology Co., Ltd. (Hubei, China). They were provided with food ad libitum under a 12-h light and 12-h dark cycle. All mice were kept under regulated temperature (22 ± 2 °C) and humidity (55 ± 10%). All procedures received approval and oversight from the ethical committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China (Approval Number: TJH-202212019). Sixty female mice were randomly divided into four groups: control group (treated with saline), Dgal group (injected subcutaneously in the neck for 42 consecutive days with Dgal), Dgal + DTN group (injected Dgal subcutaneously, tail vein injected DTN 0.1 nmol daily), Dgal + FSH-DTN (injected Dgal subcutaneously, tail vein injected FSH-DTN 0.1 nmol daily) group. Following a 7-week exposure period, the female mice were permitted to mate with male mice for reproduction. Pups and dams live together until animals are sacrificed.

Cellular uptake

The DTNs were labeled with Cy3 fluorescence. After the cells were treated with Cy3-DTN and Cy3-FSH-DTN for 1.5, 3, 6, 12, 24, 48 h separately, the cells were rinsed with PBS for 3 times and harvested for fluorescence microscopy observation.

Cell viability assay

Cells were seeded into 96-well plates at a density of 5000 cells per well. Subsequently, 100 µL of CCK8 solution was added, and the plate was incubated for 2 h at 37 °C. The absorbance of each well was quantified at a wavelength of 450 nm with a microplate spectrophotometer.

Living animal image

Cy5-labeled DTN/FSH-DTN (1 µmol L^− 1, 100 µL) was injected into the mice through the tail vein. Fluorescence images at several time points (0.5, 1, 3, 6, and 12 h) were collected and analyzed. Mice were sacrificed, and their organs were dissociated and rinsed with PBS, subsequently positioned into a bioluminescence imaging system to examine the distribution of DTN/FSH-DTN.

Tunel staining

Ovarian cell apoptosis in mice was evaluated by TUNEL staining using a commercial kit (Beyotime, C1088). Frozen sections were subjected to a PBS wash, permeabilized with 0.3% Triton X-100, and incubated with the reaction mixture at 37 °C for 1 h. After staining the nuclei with DAPI, the samples were mounted and imaged under a fluorescence microscope for apoptosis assessment.

Western blot

Ovarian tissues and COV434 cells were lysed in RIPA buffer for protein extraction. The extracted proteins were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking, the membranes were incubated overnight at 4 °C with the following primary antibodies: GAPDH, SOD2, GPX1, NRF2, CDKN1A, CDKN2A, KEAP1, P-H2AX (all at 1:1000, NFKB: AP1528, SOD2: A19576, GPX1: A1110, KEAP1: A25951, P-H2AX: A11412 (ABclonal); GAPDH: 10494-1-AP, NRF2: 16396-1-AP, CDKN1A: 10355-1-AP, CDKN2A: 10355-1-AP (Proteintech)). This was followed by a 1 h incubation with a HRP conjugated Goat anti-Rabbit IgG (H + L) (1:3000) secondary antibody at room temperature. Signal quantification was performed using Image Lab software (Bio-Rad), normalizing all data to the GAPDH loading control.

ROS detection

ROS was evaluated using ROS Assay Kit (Beyotime, S0033S). For DCFH staining, COV434 cells were incubated with the oxidation-sensitive fluorescent probe [dichlorofluorescein (DCFH)] (10 mM) for 30 min at 37 °C in PBS containing 0.5% BSA. After being washed three times with PBS containing 0.5% BSA, the COV434 cells were transferred to glass slides for examination with a fluorescence microscope.

Quantitative real-time polymerase chain reactions (qRT-PCR)

Following RNA extraction with TRIzol reagent (Invitrogen, 10296010) and reverse transcription of 1 µg RNA into cDNA (Vazyme, R223-01), quantitative PCR was conducted using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) on a Bio-Rad CFX96 system. Target gene expression was quantified via the 2^(-ΔΔCt) method.

JC-1 staining

The mitochondrial membrane potential of oocytes was evaluated with the JC-1 Assay Kit (Beyotime, C2003S) according to the manufacturer’s instructions. Briefly, oocytes were loaded with JC-1 dye in M2 medium for 30 min at 37 °C, washed, and then immediately visualized under a fluorescence microscope.

GSH detection

GSH was evaluated using GSH Assay Kit (Beyotime, S0053). After grinding the tissue, protein removal reagent was added, and the upper layer solution was collected after centrifugation at 4 °C. GSH reductase, DNTB reserve solution and total glutathione detection buffer were prepared as glutathione detection working solution in proportion. After adding the samples to the 96-well plate, the GSH detection working solution was added, thoroughly mixed, and incubated at room temperature for 5 min. This was followed by the addition of NADPH solution, mixing, and immediate measurement of the absorbance at 412 nm.

CAT detection

Catalase (CAT) activity was assessed using a CAT Assay Kit (Beyotime, S0051). Cell lysates were diluted to an appropriate protein concentration. A reaction mixture was prepared by combining CAT (100 µL) assay buffer with the diluted lysate (30 µL). The reaction was initiated by adding H₂O₂ (20 µL) substrate, followed by incubation at 25 °C for 5 min. To terminate the reaction, 100 µL of stop solution was added, and the absorbance was subsequently measured at 520 nm with a microplate reader.

GPX detection

Glutathione peroxidase (GPX) activity was determined using a GPX Assay Kit (Beyotime, S0056). Cell lysates were diluted to an appropriate protein concentration. A reaction mixture was prepared by mixing GPX (100 µL) assay buffer, 10 µL of the diluted lysate, 10 µL of GSH, and 10 µL of NADPH. The reaction was initiated by adding 10 µL of cumene hydroperoxide. Absorbance at 340 nm was measured every minute for 5 min using a microplate reader.

MDA detection

Using an MDA Assay Kit (Beyotime, S0131), 0.1 mL of sample was combined with 0.2 mL of detection working solution, heated at 100 °C for 15 min, and cooled. The mixture was then centrifuged at 1000 xg for 10 min, and the absorbance of the supernatant (200 µL) was determined at 532 nm.

SOD detection

Superoxide dismutase (SOD) activity was determined using a commercial assay kit (Beyotime, S0101) according to the manufacturer’s instructions. Briefly, the samples, SOD detection buffer, WST-8/enzyme working solution, and reaction initiation solution were added sequentially in a 96-well plate. After thorough mixing and incubation at 37 °C for 30 min, the absorbance was measured at 450 nm.

Serum hormone testing

Following serum separation from whole blood by centrifugation, estradiol and follicle-stimulating hormone levels were quantified with commercial ELISA kits (CUSABIO) as per the manufacturer’s protocols.

HE staining and follicle counting

Ovaries were fixed in 4% paraformaldehyde and processed for H&E staining. Follicles at all developmental stages were then enumerated and classified according to standard criteria, enabling the calculation of the rates of atresia, primordial follicles, and growing follicles.

β-Galactosidase staining

β-Galactosidase Staining Kit (Beyotime, Shanghai, China) was used to detect the senescent cells in different groups according to the standard protocol. After overnight incubation at 37℃ in the staining solution, the samples were detected via microscopy observation.

Immunohistochemistry staining

Immunohistochemical staining for SOD2, GPX1, NRF2, and CAT was performed on ovarian sections. Following deparaffinization and rehydration, sections were incubated overnight at 4 °C with specific primary antibodies (all from ABclonal; CAT: A11220, SOD2: A19576, GPX1: A1110, NRF2: A1244; all at 1:200). Subsequent to PBS washing, sections were treated with a secondary antibody for 1 h at 37 °C, and the signal was developed with DAB (G1212, Servicebio) for visualization.

Dihydroethidine (DHE) staining

Frozen ovarian sections were incubated with DHE (2 µmol L^− 1) at 37℃ for 30 min and then stained with DAPI for 5 min, and visualized by fluorescence microscopy.

RNA-sequencing

Total RNA of ovary treated with or without FSH-DTN for 24 h were extracted using TRIzol reagent (10296010, Invitrogen, USA). RNA sequencing and subsequent bioinformatic analysis were conducted by APExBIO Technology LLC (Shanghai, China). Briefly, 2 µg of total RNA was reverse-transcribed into cDNA using oligo-dT primers, and the resulting cDNA was then linearly amplified for second-strand synthesis. The synthesized cDNA products were purified with the AMPure XP system (Beckman Coulter, Beverly, USA) and used for library preparation. The final library quality was assessed on an Agilent 2100 Bioanalyzer (Agilent, USA), and qualified libraries were sequenced on an Illumina NovaSeq 6000 platform to generate raw reads (paired-end 150).

Statistical analysis

All data are presented as the mean±SEM. Prism software (GraphPad, La Jolla, CA, USA) was used to display all graphics. One-way analysis was used to compare differences among four groups. P < 0.05 is considered significant.

Results

Synthesis and characterization of DTN/FSH-DTN

Equimolar amounts of the four single-stranded DNAs were mixed in TM buffer, denatured at 95 °C, and then gradually cooled to 4 °C to promote self-assembly through hybridization. DTN is assembled through base pairing of unmodified staple strands (TableS1), as illustrated in Fig. 1. To verify whether DTN was successfully synthesized, agarose gel electrophoresis (AGE) and high performance liquid chromatography (HPLC) were performed. As shown in Fig. 2A and B, DTN migrated slowly in the gel, matching the 200 bp band on the DNA ladder, the molecular weight of FSH-DTN was slightly larger than that of DTN. Based on the molecular weight of the electrophoresis bands, we successfully synthesized the expected nucleic acid nanostructure. The morphology of the FSH-DTN was characterized using TEM and AFM (Fig. 2C and D). From the TEM and AFM images, we observed triangular nanoparticles and some polymers, indicating that FSH-DTN was well dispersed, uniform in size (≈ 40 nm), and triangular in structure. Dynamic light scattering (DLS) measurements showed that the zeta potential of the DTN was −4.56 ± 0.25 mV, with an average size of 27.26 ± 0.60 nm. After FSH conjugation, the zeta potential was −5.53 ± 0.63 mV, and the average size of the FSH-DTN was about 32.11 ± 2.40 nm (Fig. 2E). FSH-DTN exhibits a more negative zeta potential and a larger size. These findings suggest that FSH-DTN has enhanced structural stability, as predicted by the Derjaguin, Landau, Verwey, and Overbeek (DLVO) colloidal stability theory. Additionally, the interaction between FSH33 and DTN appears to contribute to the observed increase in particle size. To further investigate stability, AGE was used to measure the degradation of DTN and FSH-DTN in PBS and 10% FBS at 37 °C for 2, 4, 8, 12, and 24 h (Fig. 2F). FSH-DTN and DTN were not degraded in PBS buffer at 37 °C for 24 h, showing good stability. As time increases, there is no significant difference in the degradation of DTN and FSH-DTN in 10% serum. The degradation of DTN and FSH-DTN in 10% serum is not obvious within 8 h and is still present at 12 h. These results indicate that FSH-DTN has similar biological stability to DTN. Based on the above results, we have successfully synthesized stable DTN/FSH-DTN, which can be used for subsequent cellular and animal experiments.

Fig. 2.

Characteristics of DTN/FSH-DTN. (A) HPLC and (B) AGE validation of DTN/FSH-DTN synthesis. M, Marker; lane 1, single-strand mixtures; lane 2, double-strand mixtures; lane 3, triple-strand mixtures; lane 4, quadruple-strand mixtures (DTN); lane 5, FSH-DTN. (C) Representative TEM image of FSH-DTN. Scale bar: 200 nm, 50 nm. (D) Representative AFM image of FSH-DTN. (E) Average particle size and zeta potential tested via DLS. n = 3. (F) Stability of DTN/FSH-DTN in PBS and 10% serum. (G, H, I) Analysis of the scavenging efficiencies of DPPH/O₂·/OH· radicals. n = 5. Data are represented as mean±SEM. *p < 0.05; ****p < 0.0001

Next, to investigate the antioxidant properties of DTN and FSH-DTN, we conducted comprehensive free radical scavenging assays targeting three predominant reactive oxygen species: DPPH (2,2-Diphenyl-1-picrylhydrazyl) (Fig. 2G), hydroxyl radicals (•OH) (Fig. 2H), and superoxide radicals (•O₂) (Fig. 2I). Quantitative analysis revealed that all DNA materials, including single stranded DNA (ssDNA), DTN, and FSH-DTN, exhibited significant reactivity with these ROS species, demonstrating potent in vitro antioxidant activity through efficient ROS neutralization (Fig. 2G and I). Notably, comparative assessment showed that both DTN and FSH-DTN achieved superior ROS scavenging efficiency compared to ssDNA, highlighting the enhanced antioxidant capacity of nanostructured DNA assemblies.

FSH-DTN demonstrates superior ovarian targeting capabilities

To investigate the targeting specificity of FSH-DTN, we first examined the expression of FSHR. The mRNA expression of FSHR in the mouse ovary was significantly higher than that in other organs of the body (Fig.S1A). Immunofluorescence results suggested that FSHR was highly expressed in the ovaries, while it was almost not expressed in other organs, including the heart, liver, spleen, lungs, kidneys, and uterus (Fig. S1B). At the cellular level, mouse granulosa cells expressed FSHR positively (Fig. S1C). The concentrated expression of FSHR in the ovary indicated that FSHR was feasible as a target for nanomedicine.

To track DNA nanostructure in ovarian granulosa cells (COV434), we loaded the fluorescent dye Cy3 into one strand of ssDNA and synthesized Cy3-DTN and Cy3-FSH-DTN. After incubating COV434 cells with Cy3-DTN and Cy3-FSH-DTN, we observed the uptake of DNA nanostructure using fluorescence microscopy (Fig. 3 A). The images revealed that a significant amount of Cy3-DTN and Cy3-FSH-DTN were stably maintained in the cytoplasm. As shown in Fig. 3 A, the Cy3 fluorescence in COV434 cells increased significantly over time, with notable uptake achieved by 12 h. The uptake of DNA nanostructure was quantitatively analyzed at different time points (Fig. 3B), the cellular uptake rate of Cy3-FSH-DTN reached 8.4 times that of Cy3-DTN at 12 h. These findings demonstrate that the targeting peptide FSH33-53 substantially enhances the transcytosis of DTN in ovarian granulosa cells.

Fig. 3.

Cellular uptake and in vivo distribution of DTN/FSH-DTN. (A) Fluorescence microscopy images of the cellular internalization at different time points. Scale bar: 20 μm. (B) Statistical analysis of fluorescence microscopy images of the cellular internalization (n = 4). (C) IVIS imaging of organs in mice after tail vein injection of cy5-labelled DTN and FSH-DTN. (D) Live fluorescence images of ovary at 0.5, 1, 3, 6, and 12 h after tail vein injection of DTN and FSH-DTN. (nd FSH-DTN. (E) Statistics of the mean radiant efficiency of Cy5-labelled DTN and FSH-DTN in the ovary at 1 h. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001

Next, we explored the distribution of DTN/FSH-DTN in mice. Cy5-labeled DTN and FSH-DTN was administered intravenously to mice via the tail vein, respectively. Subsequent ex vivo imaging showed that the signal of DNA nanostructure in all organs began to decrease immediately after injection, with a short circulating half-life, predominantly accumulating in the liver and kidneys, and mostly cleared by the kidneys (Fig. 3C). However, imaging of the isolated ovaries revealed that DTN was completely metabolized within 1 h, while FSH-DTN fluorescence remained in the ovaries for 6–12 h (Fig. 3D). The accumulation of FSH-DTN in the ovaries at 1 h was significantly 8.3 times higher than that of DTN (p = 0.0028) (Fig. 3E). The FSH peptide-modified tetrahedral DNA nanostructure demonstrated excellent targeting capabilities, as evidenced by greater accumulation in the ovaries at the same time point and longer retention within the ovaries.

DTN/FSH-DTN reduces ROS level in ovarian granulosa cells under conditions of acute, subacute, and chronic oxidative stress

First, the safe concentration of DTN and FSH-DTN was determined using the Cell Counting Kit-8 (CCK8) in vitro. When the concentration of DTN reached 100 nmol L^− 1, the cell activity decreased significantly (p < 0.05). While the concentration of FSH-DTN reached 50 nmol L^− 1, the cell activity decreased significantly (p < 0.05). The results indicated that the safe dosage of DTN should not exceed 50 nmol L^− 1 (Fig. S2A).

Dgal is frequently employed to establish a model of chronic oxidative stress leading to ovarian aging [odel of chronic oxidative stress leading to ovarian aging [27], whereas NaAsO₂ is used to induce subacute oxidative stress [28], and H₂O₂ is applied to create acute oxidative stress models [29]. The CCK8 results indicate that both DTN and FSH-DTN can significantly enhance cell viability in the treatment with Dgal, NaAsO₂, and H₂O₂ (Fig. S2B-D). Then, we evaluated the protective effect of DTN/FSH-DTN against oxidative stress at different intervention time points. Specifically, DTN/FSH-DTN was administered before, simultaneously with, or after the addition of Dgal, NaAsO₂, and H₂O₂ to assess its protective effects. To explore whether FSH-DTN can mitigate ROS induced by Dgal, NaAsO₂, and H₂O₂ in human ovarian granulosa cells (COV434), we employed the DCFH-DA probe to measure ROS levels. In the Dgal model, both pre-treatment (p < 0.01) and delayed treatment (p < 0.05) with FSH-DTN significantly reduced ROS levels. In the NaAsO₂ model, pre-treatment (p < 0.01) and simultaneous intervention (p < 0.05) with FSH-DTN also exhibited significant ROS-lowering effects. Meanwhile, in the H₂O₂ model, protective strategies at different time points all demonstrated good effects (Fig. S2E). As shown in Fig. 4A and B, both FSH-DTN and DTN significantly reduced ROS generation induced by Dgal, NaAsO₂ and H₂O₂. In the Dgal-induced oxidative stress cell model, FSH-DTN exhibited superior ROS-scavenging capacity compared to DTN (p < 0.05). The ROS overproduction induced significant cellular apoptosis, as quantitatively assessed by TUNEL staining. Notably, treatment with DTN/FSH-DTN dramatically inhibited apoptosis in COV434 cells induced by Dgal, NaAsO₂ and H₂O₂ (p < 0.0001, Fig. 4C). These findings collectively indicate that DTN-based nanostructures, particularly FSH-DTN, can effectively mitigate oxidative damage across acute, subacute, and chronic stress paradigms in ovarian granulosa cells through robust ROS reduction.

Fig. 4.

DTN/FSH-DTN reduces ROS level in ovarian granulosa cells under conditions of acute, subacute, and chronic oxidative stress. Intracellular ROS were detected by fluorescence microscopy images (A) and flow cytometric analysis (B). (C) Tunel fluorescence staining for cell apoptosis detection. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

DTN/FSH-DTN improves the antioxidant levels in ovarian granulosa cells

The cellular antioxidant defense system, primarily mediated through enzymatic regulators such as SOD, CAT and GPX, plays a crucial role in ROS homeostasis. To investigate the molecular mechanisms underlying DTN/FSH-DTN-mediated antioxidant effects, we systematically analyzed the expression and functional activity of these key antioxidant enzymes. Quantitative RT-PCR analysis revealed that DTN/FSH-DTN treatment significantly upregulated the mRNA levels of key antioxidant enzymes, including SOD2, GPX1, GPX3, CAT, and the master regulator Nuclear factor erythroid 2-related factor (NRF2), Kelch-like ECH-associated protein 1 (KEAP1) in Dgal-treated cells, with FSH-DTN demonstrating superior efficacy compared to DTN alone (Fig. 5A). Western blot analysis confirmed parallel increases in protein expression levels of SOD2, GPX1, CAT, NRF2 and KEAP1 (Fig. 5B, S2F). Functional enzymatic assays demonstrated that both DTN and FSH-DTN effectively restored the Dgal-induced impairment of SOD, GPX, and glutathione (GSH) activities in COV434 cells. While no significant intergroup differences were observed in CAT activity and malondialdehyde (MDA) levels (Fig. S2G), FSH-DTN treatment exhibited particularly robust enhancement of GSH activity compared to DTN (Fig. 5C), suggesting its superior capacity in maintaining cellular redox homeostasis.

Fig. 5.

The protective effect of FSH-DTN on COV434 cells from Dgal induced aging model. (A) The relative mRNA expression levels of CAT, GPX1, GPX3, SOD2, NRF2 and KEAP1 in COV434 cells. n = 3. (B) Western blot analysis results for GPX1, SOD2, CAT, NRF2 and KEAP1 expression. (C) Detection of SOD, GPX, GSH in vitro. (D) Mitochondrial membrane potential was detected by JC-1 staining in different groups. (E) Statistical analysis of the results of JC-1 staining in COV434. n = 4. (F) β-Galactosidase staining for cell senescence detection. (G) Statistical analysis of the number of β‐Galactosidase-positive cells in COV434. n = 4. (H) The relative mRNA expression levels of CDKN1A, CDKN2A, NFKB and γH2AX cells. n = 3. (I) Western blot results for CDKN1A, CDKN2A, NFKB, and γH2AX in COV434 cells. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Excessive oxidative stress can lead to mitochondrial dysfunction. Subsequently, we assessed mitochondrial membrane potential using JC-1 staining, a critical indicator of mitochondrial function. A hallmark of functional mitochondria is their high membrane potential, which drives ATP synthesis and is indicated by red JC-1 fluorescence. Conversely, green fluorescence signifies a loss of this potential, a feature of depolarized mitochondria. Our results demonstrated that Dgal-treated oocytes showed a significantly reduced red/green fluorescence ratio compared to control COV434 cells, while DTN and FSH-DTN-treated oocytes exhibited restored red/green fluorescence ratio (Fig. 5D, F). Excessive oxidative stress can also induce cellular senescence, as assessed by β-Gal staining. In COV434 cells, Dgal-induced senescence was significantly mitigated by DTN/FSH-DTN treatments, with FSH-DTN demonstrating superior efficacy (Fig. 5E, G). Furthermore, DTN/FSH-DTN treatments effectively downregulated the expression of senescence-associated markers, including CDKN1A, CDKN2A, NFKB, and γH2AX (Fig. 5H and I, S2H).

In models of oxidative stress induced by NaAsO₂ and H₂O₂, we similarly examined the protective effects of DTN/FSH-DTN on COV434 cells. In the NaAsO₂ model, DTN/FSH-DTN treatment upregulated the mRNA expression of key antioxidant enzymes, including SOD1, GPX1, GPX3, CAT, and NRF2 (Fig. S3A). Similarly, in the H₂O₂ model, DTN/FSH-DTN increased the mRNA expression of SOD1, CAT, and NRF2 (Fig. S3B). However, in both models, DTN/FSH-DTN showed a trend towards improving antioxidant enzyme levels, but no significant differences were observed (Fig. S3C, D). Western blot analysis further confirmed that, despite the upregulation of mRNA, the expression levels of antioxidant-related proteins showed no significant differences after DTN/FSH-DTN treatment, except for a notable increase in NRF2 protein in the NaAsO₂ model group (Fig. S3E-H). Importantly, both models demonstrated significant improvements in mitochondrial membrane potential and a reduction in β-galactosidase accumulation (Fig. S4). These results suggest that DTN/FSH-DTN exhibits antioxidant protective effects in both acute and sub-acute oxidative stress cell models; however, the effectiveness is weaker compared to the chronic oxidative stress model.

FSH-DTN shows good biosafety in vivo

Age-related ovarian aging is mainly caused by chronic oxidative stress, mirroring the mechanistic pathways observed in Dgal-induced oxidative stress models. To evaluate the therapeutic potential of DTN and FSH-DTN, we established a mice model of ovarian aging through daily subcutaneous injections of Dgal in the neck region for 42 consecutive days, with concurrent intravenous delivery of DTN or FSH-DTN via the tail vein (Fig. 6 A). Body weight and organ indices showed that FSH-DTN and DTN did not affect the normal growth of the mice (Fig. 6B, C). H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) at the end of the treatment showed no significant histological damage, supporting the biosafety of these nanostructures (Fig. 6D). Comprehensive biochemical analysis, including assessment of liver function markers (aspartate transaminase (AST) and alanine aminotransferase (ALT)), renal function markers (creatinine (CREA) and urea (UREA)), revealed no significant differences among treatment groups in parental mice (Fig. 6E). Interestingly, in the offspring of Dgal-induced ovarian aging mice, DTN and FSH-DTN treatments improved the elevation of ALT levels caused by Dgal, while showed no differences in AST and renal function between the groups (Fig. 6 F). These comprehensive findings establish that both FSH-DTN and DTN exhibit exceptional biosafety profiles across generations, demonstrating no detectable adverse effects on vital organ systems from parental to offspring mice.

Fig. 6.

In vivo administration and safety of DTN/FSH-DTN in mice. (A) Experiment plan for the treatment of DTN/FSH-DTN in Dgal-induced ovarian aging mice. (B) Organ index of Dgal-induced ovarian aging mice. n = 15. (C) Changes in body weight during the treatment period. Data are presented as mean (n = 15 for each group). (D) H&E staining of major organs. Scale bar (250 μm). (E) The level of serum UREA, CREA, ALT, AST in Dgal-induced ovarian aging mice. n = 7. (F) The level of serum UREA, CREA, ALT, AST in the offspring of Dgal-induced ovarian aging mice. n = 4. *p < 0.05, **p < 0.01

FSH-DTN restores ovarian endocrine and reproductive function in mice of ovarian aging

To investigate the protective effects of DTN and FSH-DTN on mouse ovaries, we euthanized the mice after the treatment and recorded the morphology and weight of their uteri and ovaries (Fig. 7A-C). The results showed significant improvement in ovarian volume and ovarian index in the DTN and FSH-DTN treatment groups compared to the Dgal group (p < 0.05). While the Dgal model group exhibited a significant disruption in regularity compared to the control group (p < 0.05), FSH-DTN treatment effectively improved the regularity of the estrous cycle, while DTN treatment did not (p > 0.05, Fig. 7D). For histological analysis, we performed H&E staining on the ovaries. Representative histological images of the ovaries in each group showed some healthy follicles (primordial follicles, PMF; primary follicles, PF; secondary follicles, SF; antral follicles, AF) and atretic follicles (ATF) (Fig. 7E). Ovarian function is determined by the number of follicles, and we counted follicles at different stages. Compared to the control group, exposure to Dgal notably decreased the ratio of secondary follicles and concurrently raised the ratio of atretic follicles (Fig. 7F). Conversely, FSH-DTN treatment significantly increased the proportion of secondary follicles and decreased the proportion of atretic follicles (Fig. 7F). To further characterize the ovarian endocrine function, we measured serum hormone levels. Compared to the Dgal group, FSH-DTN treatment significantly elevated serum E2 levels and reduced FSH levels (Fig. 7G, H). These results indicate that FSH-DTN treatment can significantly improve the ovarian endocrine function in mice.

Fig. 7.

The protective effects of DTN/FSH-DTN on ovarian function. (A) Ovary and uterus size, morphology. (B, C) Ovary and uterus index. (D) The proportion of regular or irregular estrous cycles (n = 15 for each group). (E) The representative H&E staining images of mouse ovaries. (F) The proportion of follicle according to ovary serial sections (n = 3 for each group). (G, H) Serum E2 and FSH level. n = 5. (I) The proportion of successful pregnant mice. n = 4. (J) Average days for successful gestation. (K) The litter size of total mice. (L) The sex ratio and (M) average birth weight of the offspring mice. Data are represented as mean±SEM. *p < 0.05, **p < 0.01, ***p < 0.001

To evaluate the effect of FSH-DTN on reproductive function, a mating test was carried out following the treatment. We found that the Dgal group had a lower rate of successful pregnancies compared to the control group, while FSH-DTN treatment improved it (Fig. 7I). Notably, the time to successful pregnancy did not differ significantly between the Dgal group and the FSH-DTN-treated group (Fig. 7J). Additionally, the Dgal group showed reduced litter size and an abnormal sex ratio, both of which were restored to normal levels in the FSH-DTN-treated group, matching the control group (Fig. 7K, L). Additionally, there were no statistically significant differences in birth weight across all groups (p > 0.05) (Fig. 7M). These results suggest that FSH-DTN treatment can ameliorate the reduction in fertility caused by Dgal. These findings collectively suggest that FSH-DTN treatment effectively alleviates the reduction in fertility caused by Dgal.

In summary, FSH-DTN treatment demonstrates the potential to enhance ovarian function in Dgal-induced ovarian aging mice, offering a promising approach for delaying ovarian aging.

FSH-DTN treatment improves ovarian resistance to oxidative stress

We next explored the protective effects of DTN-based nanostructures against oxidative stress in ovarian tissue. DHE fluorescence staining revealed significantly elevated ROS levels in Dgal-treated ovarian tissue, which were markedly attenuated by both DTN and FSH-DTN treatments, with FSH-DTN demonstrating superior ROS-scavenging capacity (p < 0.0001, Fig. 8A, B). Quantitative PCR analysis showed that Dgal exposure downregulated key antioxidant genes (GPX1, GPX3, SOD2, CAT, and NRF2), while DTN/FSH-DTN effectively restored their expression, particularly in the FSH-DTN group (Fig. S5A). Western blot results showed corresponding increases in antioxidant protein levels (CAT, GPX1, SOD2, and NRF2) following DTN/FSH-DTN treatment (Fig. S5B, C). Immunohistochemical evaluation further validated the enhanced expression and tissue distribution of these antioxidant markers in DTN/FSH-DTN-treated groups (p < 0.0001, Fig. 8C, D). Biochemical analysis of serum samples revealed that FSH-DTN significantly elevated the content of SOD (p < 0.05) and GPX (p < 0.01) activities while reducing MDA levels (p < 0.05), indicating improved systemic antioxidant status (Fig. S5D).

Fig. 8.

FSH-DTN improves ovarian anti-oxidative stress capacity. (A) Representative DHE staining of ovary sections. Scale bar, 50 μm. (B) Statistical analysis of the results of DHE staining (n = 3). (C) The immunohistochemical of CAT, SOD, GPX, and NRF2 in ovary tissues. (D) Statistical analysis of the results of IHC staining (n = 3). Scale bar, 50 μm. (E) Heat map of differential expression genes (DEGs) between Dgal and FSH-DTN-treated groups. (F, G) GO and KEGG analysis of DEGs between Dgal and FSH-DTN-treated groups. **p < 0.01, ***p < 0.001, and ****p < 0.0001

The enhanced ovarian antioxidant capacity translated into significant improvements in cellular senescence and apoptosis profiles. Dgal treatment induced pronounced ovarian granulosa cellular senescence, which was effectively mitigated by DTN/FSH-DTN treatment (Fig. S5E). TUNEL results indicated that both DTN and FSH-DTN significantly inhibited Dgal-induced granulosa cells apoptosis (pronounced ovarian granulosa cellular senescence, which was effectively mitigated by DTN/FSH-DTN treatment (Fig. S5E). TUNEL results indicated that both DTN and FSH-DTN significantly inhibited Dgal-induced granulosa cells apoptosis (p < 0.01), with FSH-DTN showing particularly robust protective effects (Fig. S5F). These in vivo findings collectively demonstrate that FSH-DTN effectively attenuates oxidative stress-mediated ovarian cellular senescence and apoptosis.

Building upon these findings, we conducted RNA sequencing to further elucidate the underlying mechanism underlying FSH-DTN-mediated ovarian antioxidant protection. Transcriptomic profiling revealed that substantial alterations in gene expression patterns following FSH-DTN treatment, as shown in the hierarchical clustering heatmap (Fig. 8E). To systematically characterize the key biological processes and signaling pathways modulated by FSH-DTN during antioxidation, we conducted integrated bioinformatics analysis incorporating gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment (Fig. 8 F, G). GO analysis demonstrated significant upregulation of antioxidant activity processes in the FSH-DTN-treated ovaries (Fig. antioxidant activity processes in the FSH-DTN-treated ovaries (Fig. 8 F). Furthermore, KEGG pathway analysis further identified substantial modulation of key signaling cascades, including adehesion molecules, cytokine-cytokine receptor interaction, and critical inflammatory pathways such as TNF, PPAR, and IL-17 signaling networks (Fig. hesion molecules, cytokine-cytokine receptor interaction, and critical inflammatory pathways such as TNF, PPAR, and IL-17 signaling networks (Fig. 8G). These results suggest that FSH-DTN exerts its protective effects through coordinated regulation of antioxidant defense systems and inflammatory signaling pathways in ovarian tissue.

Discussion and conclusions

Oxidative stress represents a pivotal mechanistic driver of age-related ovarian aging, yet the development of precise and effective therapeutic interventions remains an ongoing challenge. In this study, we engineered a novel targeted delivery system by conjugating FSH33 peptide to DNA tetrahedral nanostructures (FSH-DTN), designed to enhance radical scavenging capacity and mitigate ovarian aging. The FSH33 modification significantly improved the nanostructures’ ovarian targeting specificity and cellular internalization efficiency. Our experimental results demonstrate that FSH-DTN effectively attenuates oxidative stress-induced granulosa cell senescence and apoptosis across acute, subacute, and chronic stress paradigms, consequently restoring ovarian endocrine function and fertility in a murine model of ovarian aging. Beyond conventional ROS neutralization approaches, FSH-DTN was shown to fundamentally enhance the ovarian antioxidant defense system through multiple molecular mechanisms. These findings provide novel insights into targeted therapeutic strategies for ovarian aging and establish a foundation for nanotherapy-based interventions. The demonstrated biosafety and therapeutic efficacy of FSH-DTN highlight its potential for clinical translation in delaying ovarian aging.

DTN has gained significant attention as promising therapeutic tools due to its excellent biocompatibility and potential for targeted delivery. Previous studies have shown that DTN prevents oxidative stress-induced retinal ischemia-reperfusion injury through activation of the Akt/NRF2 pathway [30]. Furthermore, it has shown efficacy in mitigating retinal neovascular diseases via the same mechanism [31]. It also promotes antioxidant activity and enhances angiogenesis, thereby improving wound healing in diabetic conditions [32]. Additionally, DTN exerts its therapeutic effects by activating autophagy through upregulation LC3-II, Beclin1, and Atg7. It also exhibits anti-apoptotic effects by modulating of the BCL2/BAX/caspase-3 pathway. Additionally, DTN inhibits oxidative stress by modulating the NRF2/HO-1 signaling pathway [33]. These findings collectively highlight the multifaceted therapeutic potential of DTN in various pathological conditions. In our study, we demonstrated that FSH-DTN significantly reduced oxidative stress and enhanced antioxidant enzyme activity in both aging granulosa cells and ovaries of aged mice. To the best of our knowledge, this is the first application of DTN in the treatment of ovarian aging, presenting a novel approach for selective ROS-scavenging therapy for this condition.

In this study, we discovered that FSH-DTN not only neutralizes ROS, but also enhances the antioxidant capacity of the ovaries by activating NRF2/KEAP1 pathway. This pathway is central to cellular response to oxidative stress, where NRF2, a transcription factor with seven NRF2-ECH homology domains, plays a pivotal role in maintaining cellular redox homeostasis and regulating the expression of antioxidant genes [34]. Under normal conditions, NRF2 forms a complex with the protein Keap1, leading to its ubiquitination and subsequent degradation in the cytosol, thereby keeping NRF2 levels low [35]. However, when ROS levels increase, NRF2 dissociates from Keap1, becomes activated, translocates to the nucleus, and binds to Antioxidant Response Element (ARE) in the DNA. This binding triggers the upregulation of antioxidant genes, thereby enhancing the cell’s defense mechanisms against oxidative stress. NRF2 may play a role in the decline of ovarian reserve function, while the deletion of NRF2 accelerates ovarian aging. NRF2 has a protective effect on granulosa cells, but its depletion adversely affects oocyte maturation and spindle assembly. Overexpression of NRF2 mitigates age-related meiotic defects in oocytes [36]. FSH-DTN’s dual action—neutralizing ROS directly and enhancing the NRF2/Keap1 pathway—makes it a potent protector against oxidative damage in the ovaries, potentially offering therapeutic benefits in conditions associated with oxidative stress, such as ovarian aging or reproductive health issues.

Oxidative stress-mediated ovarian injury encompasses acute, subacute, and chronic [37]. Acute oxidative stress arises from sudden and intense bursts of ROS, such as during ischemia-reperfusion or radiation exposure, leading to immediate ovarian cell damage. Subacute oxidative stress involves prolonged exposure to substances like chemotherapy drugs or environmental endocrine disruptors, causing gradual ovarian dysfunction. Chronic oxidative stress, often linked to aging, results from long-term ROS accumulation, leading to ovarian degeneration. In this study, H₂O₂, NaAsO₂, and Dgal were employed to establish in vitro models of acute, subacute, and chronic oxidative stress in granulosa cells. The findings reveal that FSH-DTN effectively protects granulosa cells across all models, with enhanced efficacy in chronic conditions. In vivo, D-gal exposure was used to mimic age-related ovarian aging. The study demonstrates that FSH-DTN reduces ROS levels, inhibits granulosa cell senescence, and prevents apoptosis, all critical for preserving ovarian function. These results underscore FSH-DTN’s ability to mitigate oxidative damage, particularly in chronic settings, offering promising avenues for treating age-related ovarian decline and conditions like chemotherapy-induced ovarian damage.

While FSH-DTN therapy has shown promising results in the mouse model, there are important considerations for its potential application in humans. Prior to clinical adoption, it is crucial to comprehensively evaluate the immunostimulatory properties of DNA nanostructures, as their introduction into the body may elicit immune responses. Furthermore, the potential risks of genetic disturbances arising from the use of artificially designed DNA nanostructures remain underexplored. Additionally, the pharmacokinetics, circulation, metabolism, and excretion of DNA nanostructures within the body need further investigation. The main challenges for the future application of FSH-DTN therapy include determining the optimal intervention scenarios, timing, dosage, and delivery methods, all of which may influence patient compliance and individual variability. The long-term stability and efficacy of these nanostructures across different physiological contexts must also be thoroughly assessed. Although significant challenges remain before the clinical translation of FSH-DTN, these findings underscore the potential of FSH-DTN as an alternative and targeted approach to ROS-scavenging therapies for ovarian aging.

Author contributions

Yun Dai and Yican Guo contributed equally to this work. Experimental design involved Yun Dai, Yican Guo, Dawei Jiang, Jun Dai, Meng Wu and Shixuan Wang. Data collection and analysis were conducted by Yun Dai, Yican Guo, Dan Chen, Pinghui Li, Liru Xue, Yourong Feng, Weicheng Tang, Chuqing Wu, Simin Wei. Manuscript drafting were performed by Yun Dai, Yican Guo, Dawei Jiang, Jun Dai, Meng Wu and Shixuan Wang. Project management was overseen by Dawei Jiang, Jun Dai, Meng Wu and Shixuan Wang. Lastly, review and revisions were carried out by Yun Dai, Yican Guo, Dawei Jiang, Jun Dai, Meng Wu and Shixuan Wang. Final approval was passed through all authors.

Funding

This work was financially supported by the National Key Research and Development Program of China (no. 2022YFC2704100, 2024YFC2707404), National Natural Science Foundation of China (no. 82471678, 82403482, 22277031, 8240062640), the China Postdoctoral Science Foundation (no. 2024M752492).

Data availability

The data that support the findings of this study are available in the supplementary material of this article.

Declarations

Ethics approval and consent to participate

All animal procedures were approved and supervised by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China (Approval Number: TJH-202212019).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.