Peptide C248 from PRDX4 may improve testicular hormone cells by protecting their mitochondria

Human PRDX4 and murine Prdx4 protein sequences were retrieved from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/↗, accessed on 8 April 2022). Peptides were synthesized via Fmoc chemistry using a Shimadzu PSSM-8 automated solid-phase peptide synthesizer and analyzed by Agilent 110 automated HPLC (Table 1).

A Fam fluorescent tag was conjugated to the target peptide for tracing. Logarithmic phase cells were seeded in confocal dishes; after adhesion, the medium was replaced with one containing fluorescently labeled peptides and incubated at 37 °C with 5% CO₂ for 4 h. The medium was discarded, and the cells were fixed with 4% paraformaldehyde for 10 min and washed 3× with PBS (5 min each). The cells were stained with Hoechst 33342 (Invitrogen, Carlsbad, CA, USA, Cat. No. 2747528) in the dark for 30 min, then washed 3× with PBS (5 min each). Antifluorescence quenching mounting medium was added for sealing, and the samples were observed/imaged under a confocal microscope (Leica STELLARIS, Wetzlar, Germany; Fam: 488 nm excitation/520 nm emission; Hoechst 33342: 350 nm excitation/460 nm emission).

Mouse Leydig tumor cells (MLTC-1) were purchased from the American Type Culture Collection (ATCC). The MLTC-1 cells were cultured in DMEM/1640 medium (Gibco, New York, NY, USA, Cat. No. 11885084) supplemented with 10% fetal bovine serum (Gibco, Cat. No. A5256701) and 1% penicillin–streptomycin solution (HCM, Cat. No. C100C5). The cells were maintained in a humidified incubator at 37 °C with 5% CO₂. When the cell confluence reached 80%, they were subcultured using 0.25% trypsin–EDTA solution (HCM, Cat. No. C100C1).

To establish a model of oxidative stress-induced cellular senescence, the cells were treated with 100 μM hydrogen peroxide (H₂O₂, Sigma-Aldrich, St. Louis, MO, USA, Cat. No. H1009) for 24 h. Both C248 and NMN were originally stored as dry powders. Double-distilled water (ddH₂O) was used as the solvent to prepare stock solutions, with concentrations of 1 mM for C248 and 10 mM for NMN. In the intervention experiments, the cells were treated as follows: (1) Pep-C248 group. The cells were cotreated with C248 at a concentration of 50 nM and H₂O₂ for 24 h. (2) NMN group. The cells were pretreated with 500 μM NMN (MedChemExpress, Monmouth Junction, NJ, USA, Cat. No. HY—12941) for 2 h and then treated with H₂O₂.

Equal volumes of peptide solutions (0–992 μM) were mixed with 0.1 mM 2,2-Diphenyl-1-picrylhydrazyl (DPPH) ethanol solution. After a 30 min reaction at room temperature, 200 μL of the reaction mixture was added to a microplate, and absorbance at 517 nm was measured (A_sample). Controls were set as follows: 200 μL absolute ethanol for 517 nm absorbance (A_blank) and 200 μL ddH₂O mixed with 200 μL 0.1 mM DPPH ethanol solution, reacted at room temperature for 30 min. Then, a 200 μL mixture was measured at 517 nm (A_control).radical scavenging rate%=(1−Asample− AblankAcontral) × 100%

The three-dimensional structure of the C248 peptide (sequence: CYGRKKRRQRRRALEKEVSVLEKEVSALEKEPGLFYTDKHGEVCPAG) was predicted using the AlphaFold3 online server (https://alphafoldserver.com/↗, accessed on 4 June 2025). Quantum chemical calculations were carried out with Gaussian 16 software. Density Functional Theory (DFT) in conjunction with the SMD implicit solvation model (scrf = (SMD, solvent = Water)) was employed to simulate an aqueous environment. Geometry optimization was conducted separately for the reactant and product structures. Electronic energies were computed, and thermodynamic corrections were applied to derive the enthalpy change (ΔH) and Gibbs free energy change (ΔG). The prediction of the active pocket in Prdx4 decamers was performed via CASTpFold.

Hydrogen peroxide levels were measured using a Hydrogen Peroxide Assay Kit (Beyotime, Shanghai, China, S0038). For 1 × 10⁶ cells, the kit-provided lysis buffer was added; after ultrasonic disruption, supernatants were collected by centrifugation. Then, 50 μL samples or standards were added to assay wells, followed by 100 μL of detection reagent. After a 30 min incubation at room temperature, the absorbance at 560 nm was measured immediately.

Cells were seeded in confocal dishes and treated for 24 h when confluency reached 60%. After removing the medium, 500 μL of 4% paraformaldehyde was added for 25 min fixation at room temperature. Following 3 PBS rinses, the cells were permeabilized with Beyotime immunostaining permeabilization solution for 10 min and then washed 3 times with PBS (5 min each). After blocking with Biyuntian immunostaining blocking solution for 10 min, primary antibodies (3β-HSD, 1:100, ProteinTech, Rosemont, IL, USA, Cat. No. 15516-1-AP; P21, 1:100, ProteinTech, Cat. No. 28248-1-AP; 4-HNE, 1:25, Abcam, Waltham, MA, USA, Cat. No. ab48506; Mouse IgG, same concentration as the primary antibody, ProteinTech, Cat. No. B900620; Rabbit IgG, same concentration as the primary antibody, ProteinTech, Cat. No. 98136-1-RR) were added and incubated overnight at 4 °C. After removing the primary antibodies and PBS washing, the secondary antibodies were incubated for 1 h (Anti-Mouse secondary antibody, 1:500, Invitrogen, Cat. No. A21202; Anti-Rabbit secondary antibody, 1:500, Invitrogen, Cat. No. A21207). The nuclei were counterstained with Hoechst before mounting (Hoechst 33342, 1:500, Invitrogen, Cat. No. H3570). Fluorescence quantitative analysis was performed using ImageJ version 1.54q (National Institutes of Health, USA), with identical threshold parameters set to measure the fluorescence signal intensity. Cell counting was conducted by enumerating Hoechst-positive cells with intact morphology using ImageJ. At least 150 cells were counted per image. Data were expressed as the total fluorescence intensity of each image divided by the number of cells in the same image.

After 24 h of drug treatment, the cell pellets in a 6-well plate were collected. The samples were lysed with protein lysis buffer and stored on ice. GSH, catalase, and MDA levels were detected using a reduced glutathione (GSH) assay kit (Nanjing Jiancheng, Nanjing, China, Cat. No. A006-2-1), a Catalase assay kit (Beyotime, Cat. No. S0051), and a Lipid Peroxidation MDA assay kit (Beyotime, Cat. No. S0131S).

For the GSH assay, cell pellets were resuspended in 0.2 mL PBS and ultrasonically disrupted at 4 °C. A 0.1 mL aliquot was mixed with Reagent 1 (precipitant), followed by centrifugation to collect the supernatant. The supernatant was used for BCA protein quantification. For GSH measurement, a blank group (PBS) and a standard group (20 μmol/L GSH standard solution) were included. Briefly, 100 μL of supernatant/PBS/standard was added to each reaction well, followed by 25 μL of buffer and 100 μL of chromogenic solution. The plate was incubated at room temperature for 5 min, and the absorbance of each well was measured at 405 nm using a microplate reader. For the Catalase assay kit, the lysis buffer was used for the test samples.

For CAT detection, 5 μL of a test sample was mixed with 35 μL of catalase assay buffer, followed by the addition of 10 μL of 250 mM hydrogen peroxide solution. The mixture was rapidly blended with a pipette and incubated at room temperature for 3 min; then, the reaction was terminated by adding 450 μL of stop solution. A 10 μL aliquot of the terminated reaction mixture was mixed with 40 μL of buffer, and 10 μL of this dilution was combined with 200 μL of chromogenic working solution. After incubation for 15 min, the absorbance was measured at 520 nm (A520). For the standard curve, serially diluted standard solutions were prepared, and 4 μL of each dilution was mixed with 200 μL of chromogenic working solution for detection under the same conditions.

For the MDA assay, cell pellets were lysed with 200 μL of lysis buffer to prepare test samples, and aliquots were used for BCA protein quantification. For MDA measurement, 0.1 mL of a test sample or serial dilutions of the standard were added to 0.2 mL of the MDA assay working solution. After thorough mixing, the mixture was heated at 100 °C for 15 min, cooled to room temperature, and centrifuged to collect the supernatant. The absorbance of the supernatant was measured at 532 nm (A532) using a microplate reader.

All assays were performed in accordance with the manufacturers' instructions, and the final concentrations of GSH, CAT, and MDA were normalized to the total protein content (determined by the BCA assay) for quantitative analysis. Three independent biological replicates were conducted for each group.

After 24 h of drug treatment, cells were harvested and counted. Approximately 5 × 10⁶ cells were resuspended in 200 μL PBS and subsequently subjected to ultrasonic cell lysis to release intracellular contents. The lysate was then centrifuged at 1500× g for 10 min at 4 °C to remove cell debris. Thereafter, the supernatant was collected and used for the detection of 8-hydroxydeoxyguanosine (8-OHDG) following the manufacturer's instructions for the 8-OHDG ELISA kit (Elabscience, Houston, TX, USA, Cat. No. E-EL-0028).

Cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and treated according to different experimental purposes. After the treatment period, 10 μL of CCK8 reagent (BIOSHARP, Beijing, China, Cat. No. BL502A) was added to each well, and the plates were incubated at 37 °C for 1 h. The absorbance at 450 nm was measured using a microplate reader to evaluate cell viability.

The cells were fixed with staining fixative for 10 min and then rinsed twice with PBS to remove residual fixative. Subsequent staining was performed strictly following the manufacturer's instructions for the SA-β-gal Staining Kit (Beyotime, Cat. No. C0602). After staining, positive cells (characterized by blue staining) were counted under a light microscope, with 5–10 random fields counted per sample to ensure statistical reliability.

Total RNA was extracted from the cells using Trizol reagent (Invitrogen, Cat. No. 15596018). The RNA was reverse-transcribed into cDNA using the HiScript III RT SuperMix reverse transcription kit (Vazyme, Nanjing, China, Cat. No. R323—01). qPCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Cat. No. Q311—02) on an Applied Biosystems StepOnePlus Real-Time PCR System. The relative expression levels of target genes were calculated using the 2^−ΔΔCt method and normalized to β-actin. The primer sequences for Trp53, p21, CYP11A1, 3β-HSD, ND1, and Cox1 are shown in Table 2.

Cell culture supernatants were collected 24 h after treatment and immediately centrifuged at 1000× g for 5 to remove any floating cells or debris. The resulting supernatants were either assayed immediately or stored at −80 °C until analysis to prevent testosterone degradation. Testosterone levels were measured strictly following the manufacturer's instructions for the testosterone ELISA kit (Elabscience, Cat. No. E-EL-M0033c), with all reagents equilibrated to room temperature (25 ± 2 °C) prior to use. After the reaction was completed, absorbance was measured at 450 nm using a microplate reader. Testosterone concentrations were calculated based on a standard curve generated from serial dilutions of testosterone standards, with each sample assayed in duplicate to ensure measurement accuracy.

Cell apoptosis was detected using the TUNEL Brightgreen Apoptosis Detection Kit (Vazyme, Cat. No. A112—01). Briefly, cells were fixed with PFA at room temperature for 20 min and then rinsed three times with PBS. Subsequently, the cells were permeabilized with 0.1% Triton X-100 for 10 min, followed by another three washes with PBS. For the positive control group, the cells were first equilibrated with DNase I Buffer for 5 min and then incubated with 20 U/mL DNase I working solution at room temperature for 10 min. All groups were then treated with Equilibration Buffer for 30 min. The cells were incubated with the freshly prepared TUNEL reaction mixture (negative group without TdT enzyme in the reaction) in a humidified chamber at 37 °C for 60 min, protected from light to prevent fluorophore degradation. Fluorescence signals were visualized using a Leica confocal microscope (STELLARIS) with appropriate excitation/emission filters. For quantification, apoptotic cells (exhibiting positive fluorescent staining) were counted using ImageJ software. Three independent biological replicates were performed, with at least 150 cells counted per replicate.

Intracellular ROS levels were measured using DCFH-DA (Beyotime, Cat. No. S0033), and mitochondrial membrane potential was evaluated using TMRE (Beyotime, Cat. No. C2006). The cells were incubated with 10μM DCFH-DA or 200 nM TMRE at 37 °C for 30 min. After incubation, the cells were washed and analyzed by confocal microscopy using a Leica SP8 STED microscope (Leica Microsystems, Wetzlar, Germany), and the fluorescence intensity was quantitatively measured.

Total protein was extracted from the cells using RIPA lysis buffer (Beyotime, Cat. No. P0013B), and the protein concentration was determined by a BCA protein quantification kit (Beyotime, Cat. No. P0012S). Thirty micrograms of protein was separated by SDS-PAGE and then transferred onto a PVDF membrane (Millipore, Molsheim, France, Cat. No. ISEQ00010). The membrane was incubated overnight at 4 °C with primary antibodies against p53 (1:10000, proteintech, Cat. No. 80077—1—RR), p21 (1:1000, proteintech, Cat. No. 28248—1—AP), Hsd3b2 (1:5000, proteintech, Cat. No. 15516—1—AP), CYP11A1(1:4000, proteintech, 13363-1-AP), and Vinculin (1:10,000, Abcam, Cat. No. AB129002). After incubation with HRP-conjugated secondary antibodies (1:10,000, proteintech), the protein bands were detected using Super ECL chemiluminescence detection reagent (YEASEN, Shanghai, China, Cat. No. 36208ES76).

The mitochondria were labeled with 100 nM MitoTracker Deep Red FM (Beyotime, Cat. No. C1046) for 30 min at 37 °C with 5% CO₂. Meanwhile, the cell nuclei were stained with Hoechst 33342. The cells were imaged using an HIS-SIM Live-Cell Super-Resolution Microscope System (ChaoShiJi Technology, Guangzhou, China). The mean mitochondrial branch length was quantified using the Mitochondria Analyzer plugin in Fiji (ImageJ, National Institutes of Health, USA). Three biological replicates were included per group, with 10 cells analyzed per replicate, resulting in a total of 30 cells for statistical analysis in each group.

The ATP content in the cells was measured according to the instructions for the ATP Assay Kit (Beyotime, Cat. No. S0026). Briefly, cells were lysed using the lysis buffer, and the supernatant was collected after centrifugation (12,000× g, 4 °C, 5 min) for subsequent detection. First, 100 μL of ATP assay working solution was added to each test well of a 96-well plate, followed by 20 μL of sample supernatant or ATP standard solution. Luminescence intensity was measured using a multifunctional microplate reader. The ATP concentration of each sample was calculated based on the standard curve, and the results were normalized to the total protein content.

Experimental data were presented as the mean ± standard deviation (mean ± SD) from at least three independent experiments. First, the premise assumptions of one-way analysis of variance (ANOVA) were verified. The data in each group followed a normal distribution, and homogeneity of variances was assessed among groups. If the data were normally distributed and variances were homogeneous, one-way ANOVA followed by Tukey's post hoc test was performed for multiple comparisons. If variances were heterogeneous, Brown–Forsythe and Welch ANOVA tests were employed instead. Statistical analyses were conducted using GraphPad Prism 10 software (Version 10.1.2). Differences with p < 0.05 were considered statistically significant.

To investigate the antioxidant active sites of the Prdx4 protein, we predicted the three-dimensional (3D) structure of the Prdx4 monomer using AlphaFold (Figure 1a). Subsequently, we employed the CASTp tool to predict active pockets in the 3D structure of the Prdx4 multimeric assembly (Figure 1b,c), and screened the top 10 potential active pockets based on parameters, including pocket volume and accessible surface area (Table 3). Combined with UniProt annotation analysis, the results revealed that the active pockets harboring amino acid residues at positions 127 and 248 of Prdx4 accounted for half of these top 10 key pockets. This distribution pattern suggested that residues 127 and 248 may represent core regions through which Prdx4 mediates substrate binding, catalytic reactions, or dynamic conformational regulation. Furthermore, the comparative analysis demonstrated that human Prdx4 and mouse Prdx4 share identical sequences in the fragment region containing the antioxidant active sites (Figure 1d).

Guided by the identified active sites, we engineered peptides C127 and C248 with an N-terminal TAT cell-penetrating sequence for membrane translocation, alongside a negative control peptide E1-ctrl (Figure 1e). To evaluate their antioxidant capacity, a DPPH radical scavenging assay was conducted. Among the three peptides, C248 showed a significantly higher scavenging activity against DPPH radicals (Figure 1f,g), with an EC50 value of 268.4 μM (p < 0.05) (Table 4). To further elucidate the ROS-scavenging capacity of C248, a detailed analysis of its electronic properties was conducted. The three-dimensional structure of C248 was predicted using AlphaFold3 (Figure 1h). The per-residue confidence score (pLDDT) indicated that 87% of the residues were of high confidence (pLDDT ≥ 70). This finding indicates that C248 possesses excellent structural stability and high predictive reliability, which supports the feasibility of subsequent functional investigations. To characterize the reactivity of C248 toward H₂O₂, we performed quantum chemical calculations using highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) analyses. The HOMO electron density distribution highlighted electron-donating regions (Figure 1i), whereas LUMO indicated electron-accepting sites (Figure 1j). Single-point energy calculations revealed a standard molar Gibbs free energy of formation (ΔG) of −4926.208196 Hartree for the active fragment of C248 and −151.527532 Hartree for H₂O₂. Following thermodynamic correction, the reaction enthalpy (ΔH) and Gibbs free energy (ΔG) were determined to be −282.0968475 kJ/mol and −249.2623445 kJ/mol, respectively (Table 5). These results confirm that the reaction is both exothermic and spontaneous.

Low concentrations of H₂O₂ can induce the senescence of LCs. Previous studies have confirmed that H₂O₂ successfully induces the senescence of embryonic fibroblasts [28]. Our experiments showed that H₂O₂ stimulation can dose-dependently decrease LC viability (Figure S1a–d). As the H₂O₂ concentration increased in the medium, the proliferation rate of LCs slowed, and the cells exhibited an elongated morphological change. When the H₂O₂ concentration reached 400 μM or higher in the medium, the LCs displayed additional morphological features of excessive damage, such as shrinkage and fragmentation (Figure S1e). Concurrently, the proportion of senescence-associated β-galactosidase (SA-β-gal)-positive cells increased in a dose-dependent manner (Figure S1f,g), and the mRNA and protein levels of the senescence-related factors Trp53 and p21 were significantly upregulated (Figure S1h–j).

To investigate whether C248 could alleviate the H₂O₂-induced senescence of LCs, we first assessed the cytotoxicity of C248 on LCs (NMN as the control). The results showed that within the concentration range tested in this experiment, NMN had no significant effect on cell viability. Similarly, no obvious cytotoxicity was observed for C248 when its concentration was ≤100 nM (Figure 2a–d). Subsequently, the senescent cells were treated with 50 nM C248 or 500 μM NMN, respectively, and both C248 and NMN significantly improved the viability of senescent cells (Figure 2e). To evaluate the effect of C248 on apoptosis in the senescent cells, TUNEL staining was performed to label DNA fragmentation. The senescent cells treated with H₂O₂ exhibited a significantly higher percentage of TUNEL-positive cells (8.8 ± 2.0%, p ≤ 0.05) compared with the non-senescent control cells (1.0 ± 0.6%). Notably, the treatment with C248 or NMN markedly reduced the proportion of apoptotic cells (Figure 2f,g). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the supplementation with C248 or NMN significantly downregulated the mRNA expression levels of the senescence markers p21 and Trp53 (Figure 2h,i). Further immunofluorescence staining analysis showed that the C248 and NMN treatments significantly decreased the protein expression level of p21 in the senescent cells (Figure 2j,k and Figure S4a). Western blot quantification analysis confirmed that C248 or NMN significantly reduced the expression levels of the p21 and Trp53 proteins (Figure 2l). Collectively, these results demonstrate that both C248 and NMN effectively alleviate the senescence of LCs.

Cellular senescence is often accompanied by a decline in core cellular functions. For LCs, their key physiological function is androgen biosynthesis. In the present study, we found that in the H₂O₂-induced senescence model of LCs, the expression levels of two key enzymes in androgen biosynthesis, CYP11A1 and 3β-HSD, were significantly decreased at both the transcriptional level and the protein level (Figure S2b–d). Concurrently, the amount of testosterone secreted also decreased (Figure S2a). These findings further confirm that H₂O₂-induced senescence impairs the androgen biosynthetic function of LCs.

To determine whether C248 can reverse the aforementioned functional impairment, we investigated the effects of C248 on androgen biosynthesis and secretion in the senescent LCs. First, the senescent cells were treated with C248 for 24 h (NMN as the control). Subsequently, the cell culture medium was collected, and the testosterone concentration in the medium was measured using an enzyme-linked immunosorbent assay (ELISA). The results showed that both C248 and NMN effectively restored the androgen-secreting capacity of senescent cells (Figure 3a). Given that androgen biosynthesis involves multiple enzymatic reactions, and CYP11A1 and 3β-HSD are two key rate-limiting enzymes in the steroidogenesis pathway, we further detected the expression levels of CYP11A1 and 3β-HSD. The results demonstrated that the supplementation with C248 or NMN significantly upregulated the mRNA and protein expression levels of CYP11A1 and 3β-HSD in the senescent cells (Figure 3b–f). Furthermore, the protein expression level of 3β-HSD was further verified by immunofluorescence staining. The result was consistent with those of the Western blot and qRT-PCR, confirming that C248 or NMN could effectively restore the protein expression of 3β-HSD in senescent cells (Figure 3g,h and Figure S4b).

Given that C248 can directly scavenge ROS, we further investigated its role in consuming intracellular ROS in LCs. Firstly, to confirm whether C248 can enter into LCs, we conjugated a FAM fluorescent tag to the C-terminal of the C248 molecule. After co-incubating the tagged C248 with LCs for 4 h, fluorescent signals were detected in the cells. The results showed a dose-dependent increase in intracellular fluorescence intensity (Figure 4a,b and Figure S5a,b). This indicated that C248 can smoothly penetrate the cell membrane to enter the cytoplasm and thereby exert its synergistic effects, both inside and outside of the cell. Subsequently, we examined the effects of C248 on the intracellular H₂O₂ level (NMN as the control). The results demonstrated that both the C248 and NMN treatments significantly reduced the intracellular H₂O₂ content in the senescent cells (Figure 4c). Further investigation into the short-term effect of C248 revealed that C248 rapidly and effectively decreased the total intracellular ROS levels in the LCs (Figure 4d,e). Treatment with C248 led to a dose-dependent decrease in intracellular ROS levels, suggesting that C248 plays a key role in mediating the reduction in intracellular ROS (Figure S3a,b). To comprehensively evaluate the ameliorative effect of C248 on oxidative damage, we further detected the levels of key peroxidation products in the senescent cells, including the lipid peroxidation products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), as well as the DNA oxidative damage marker 8-hydroxy-2′-deoxyguanosine (8-OHDG). The results showed that the C248 and NMN treatments significantly downregulated the levels of these three peroxidation products (Figure 4f–i and Figure S4c). The antioxidant enzyme system is a core defense mechanism against oxidative stress. We further examined the effect of C248 on key indicators of this system. The results showed that the C248 treatment effectively maintained the stable level of glutathione (GSH) in the senescent cells and significantly increased the activity of catalase (CAT) (Figure 4j,k).

Given that oxidative stress triggers mitochondrial functional impairment, we further investigated the effects of peptide C248 on the restoration of mitochondrial function in LCs (NMN as the control). First, a TMRE fluorescent probe was used to detect mitochondrial membrane potential. The results showed that both the C248 and NMN treatments significantly reversed the oxidative stress-induced decrease in mitochondrial membrane potential (Figure 5a,b), suggesting that both molecules can maintain the homeostasis of basic mitochondrial functions. ATP is a key indicator of mitochondrial energy metabolism, and its level is directly related to the mitochondrial functional status. In the present study, our results demonstrated that the C248 and NMN treatments effectively increased the ATP level in the senescent cells (Figure 5c), indicating that both molecules can enhance mitochondrial energy production capacity.

MtDNA copy number decreases with the progression of cellular senescence and oxidative damage. Notably, the ND1 and COX1 gene fragments in mtDNA are highly conserved, and their copy numbers can indirectly reflect the quantity and functional status of mitochondria. In the present study, we further detected the copy numbers of these two genes. The results showed that both peptide C248 and NMN could effectively maintain the mtDNA copy number in the senescent LCs (Figure 5d), demonstrating that both molecules can protect mtDNA from oxidative damage.

Finally, to evaluate changes in mitochondrial morphology, we used a Mitotracker fluorescent probe to specifically label the mitochondrial membrane of living cells. Combined with high-resolution structured illumination microscopy (HIS-SIM), we observed the proportion of cells with abnormal mitochondrial morphology. Our results showed that C248 alleviated mitochondrial fragmentation and preserved the tubular structure of mitochondria (Figure 5e–g). Collectively, these results demonstrate that C248 can improve mitochondrial function in Leydig cells.