Lycopene attenuates DEHP-induced oxidative stress, mitochondrial dysfunction, and apoptosis in human granulosa cells via SIRT1-dependent mechanisms

To characterize DEHP cytotoxicity, KGN and COV434 cells were exposed to DEHP (50–800 µM) for 24, 48, or 72 h. DEHP at 50 and 100 µM did not significantly affect cell viability in KGN or COV434 cells after 24, 48, or 72 h of exposure (Fig.  1 D–F). After 24 h, cell viability declined from 100% in the control group to 73.1 ± 6.1% at 400 µM and 43.4 ± 6.9% at 800 µM ( p  < 0.05; Fig.  1 A). At 48 h, significant reductions were observed at 200 µM (60.6 ± 8.1%), 400 µM (46.8 ± 9.1%), and 800 µM (23.4 ± 6.8%; p  < 0.05; Fig.  1 B). The inhibitory effect became more pronounced after 72 h, with viability decreasing to 47.8 ± 8.4%, 33.4 ± 7.6%, and 16.3 ± 12.7% at 200, 400, and 800 µM, respectively ( p  < 0.05; Fig.  1 C). A similar trend was observed in COV434 cells. After 24 h, DEHP exposure significantly decreased cell viability at 400 µM (54.8 ± 18.0%) and 800 µM (46.9 ± 20.9%; p  < 0.05; Fig.  1 D). At 48 h, viability further declined at 200 µM (60.4 ± 14.4%), 400 µM (47.2 ± 18.5%), and 800 µM (28.8 ± 13.2%; p  < 0.05; Fig.  1 E). After 72 h, COV434 cells exhibited heightened sensitivity, with viability reduced at 100 µM (65.5 ± 7.8%), 200 µM (52.8 ± 7.4%), 400 µM (33.0 ± 17.5%), and 800 µM (16.2 ± 16.1%; p  < 0.05; Fig.  1 F). Overall, DEHP treatment caused a progressive and concentration-dependent loss of viability in both granulosa cell lines, with more pronounced effects at longer exposure durations. Fig. 1 DEHP decreases cell viability and increases caspase-3/-7 activity in human granulosa cells in a concentration- and time-dependent manner. ( A – C ) KGN cells were treated with increasing concentrations of DEHP (50–800 µM) for 24 ( A ), 48 ( B ), and 72 h ( C ), cell viability was determined using the CCK-8 assay. ( D – F ) COV434 cells were treated with increasing concentrations of DEHP (50–800 µM) for 24 ( D ), 48 ( E ), and 72 h ( F ), cell viability was determined using the CCK-8 assay. ( G – I ) KGN and ( J – L ) COV434 cells were analyzed for caspase-3/-7 activity following the same DEHP treatments for 24 ( G ), 48 ( H ) and 72 h ( I ). ( J – L ) COV434 cells were analyzed for caspase-3/-7 activity following the same DEHP treatments for 24 ( J ), 48 ( K ) and 72 h ( L ). Data are represented as mean ± SD ( n  = 3). * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 compared to control group DEHP decreases cell viability and increases caspase-3/-7 activity in human granulosa cells in a concentration- and time-dependent manner. ( A – C ) KGN cells were treated with increasing concentrations of DEHP (50–800 µM) for 24 ( A ), 48 ( B ), and 72 h ( C ), cell viability was determined using the CCK-8 assay. ( D – F ) COV434 cells were treated with increasing concentrations of DEHP (50–800 µM) for 24 ( D ), 48 ( E ), and 72 h ( F ), cell viability was determined using the CCK-8 assay. ( G – I ) KGN and ( J – L ) COV434 cells were analyzed for caspase-3/-7 activity following the same DEHP treatments for 24 ( G ), 48 ( H ) and 72 h ( I ). ( J – L ) COV434 cells were analyzed for caspase-3/-7 activity following the same DEHP treatments for 24 ( J ), 48 ( K ) and 72 h ( L ). Data are represented as mean ± SD ( n  = 3). * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 compared to control group To assess apoptosis, caspase-3/-7 activity was quantified following DEHP exposure. DEHP at 50 and 100 µM did not significantly affect caspase-3/-7 activity in KGN or COV434 cells after 24, 48, or 72 h of exposure (Fig.  1 G–L). After 24 h, activity rose to 190.3% and 223.8% of control levels at 400 and 800 µM, respectively (Fig.  1 G). At 48 h, significant increases were detected at 200 µM (164.3%), 400 µM (215.2%), and 800 µM (259.5%; Fig.  1 H). By 72 h, activity remained elevated at 200 µM (203.4%), 400 µM (249.4%), and 800 µM (303.9%; Fig.  1 I). COV434 cells exhibited a comparable pattern. At 24 h, caspase-3/-7 activity increased to 209.2% and 245.2% at 400 and 800 µM, respectively (Fig.  1 J). After 48 h, activity rose further at 200 µM (199.3%), 400 µM (229.9%), and 800 µM (289.9%; Fig.  1 K). By 72 h, DEHP exposure sustained significant increases at 200 µM (227.2%), 400 µM (283.8%), and 800 µM (349.4%; Fig.  1 L). Because lycopene was used as a protective co-treatment, we first assessed its intrinsic cytotoxicity across the tested range (0.3–30 µM). Lycopene treatment led to a gradual, concentration-dependent decline in KGN cell viability at all examined time points. After 24 h, a significant reduction was observed only at 30 µM (60.37 ± 12.38% of control; p  < 0.05), while lower concentrations (≤ 10 µM) did not cause appreciable changes (Fig.  2 A). At 48 h, viability decreased significantly at 10 µM (71.57 ± 11.15%) and 30 µM (50.13 ± 11.45%; p  < 0.05), indicating a time-dependent increase in cytotoxicity (Fig.  2 B). Prolonged exposure for 72 h further intensified the inhibitory effect, with viability declining to 61.63 ± 13.63% at 10 µM and 41.40 ± 14.51% at 30 µM ( p  < 0.05; Fig.  2 C). These findings indicate that lycopene significantly reduces KGN cell viability at concentrations ≥ 10 µM, particularly following extended treatment durations. Fig. 2 Lycopene reduces cell viability in a concentration- and time-dependent manner and mitigates DEHP-induced cytotoxicity and apoptosis. ( A – C ) KGN cells were treated with increasing concentrations of lycopene (0.3–30 µM) for 24 ( A ), 48 ( B ), and 72 h ( C ), cell viability was determined using the CCK-8 assay. ( D – F ) COV434 cells were treated with increasing concentrations of lycopene (0.3–30 µM) for 24 ( D ), 48 ( E ), and 72 h ( F ), cell viability was determined using the CCK-8 assay. ( G – H ) Co-treatment with lycopene (1 µM or 3 µM) partially restored DEHP-suppressed viability in both cell lines. ( I – J ) Lycopene attenuated DEHP-induced caspase-3/-7 activation in a concentration-dependent fashion. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 Lycopene reduces cell viability in a concentration- and time-dependent manner and mitigates DEHP-induced cytotoxicity and apoptosis. ( A – C ) KGN cells were treated with increasing concentrations of lycopene (0.3–30 µM) for 24 ( A ), 48 ( B ), and 72 h ( C ), cell viability was determined using the CCK-8 assay. ( D – F ) COV434 cells were treated with increasing concentrations of lycopene (0.3–30 µM) for 24 ( D ), 48 ( E ), and 72 h ( F ), cell viability was determined using the CCK-8 assay. ( G – H ) Co-treatment with lycopene (1 µM or 3 µM) partially restored DEHP-suppressed viability in both cell lines. ( I – J ) Lycopene attenuated DEHP-induced caspase-3/-7 activation in a concentration-dependent fashion. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 A comparable concentration- and time-dependent cytotoxic response was observed in COV434 cells. After 24 h, lycopene significantly decreased cell viability at 30 µM (65.33 ± 6.43%; p  < 0.05), whereas lower concentrations exerted minimal effects (Fig.  2 D). At 48 h, viability declined significantly at 10 µM (75.30 ± 9.36%) and 30 µM (56.17 ± 7.94%; p  < 0.05; Fig.  2 E). By 72 h, sustained cytotoxicity was evident, with viability further reduced to 65.63 ± 12.35% at 10 µM and 46.53 ± 12.97% at 30 µM ( p  < 0.05; Fig.  2 F). Overall, lycopene induced a significant and consistent decline in viability in both KGN and COV434 granulosa cell lines in a concentration- and time-dependent manner. The similar sensitivity patterns observed across both cell types suggest that granulosa cells are comparably susceptible to lycopene-induced cytotoxicity under prolonged exposure. Next, we evaluated whether lycopene protects granulosa cells from DEHP injury. Co-treatment with lycopene partially restored DEHP-suppressed viability in both granulosa cell lines in a clear concentration-dependent manner. In COV434 and KGN cells, lycopene increased viability relative to DEHP alone at both tested concentrations, with the higher concentration producing a greater rescue (Fig.  2 G–H; significant vs. DEHP). DEHP-induced apoptosis was likewise alleviated by lycopene. Caspase-3/-7 activity elevated by DEHP was reduced in both cell lines, with a larger reduction at the higher lycopene concentration (Fig.  2 I–J; significant vs. DEHP). Collectively, these findings indicate that lycopene mitigates DEHP-induced cytotoxicity and apoptotic signaling in a concentration-dependent fashion across both KGN and COV434 cells. Given the central role of oxidative injury in phthalate toxicity, we next examined ROS and mitochondrial integrity. DEHP exposure markedly increased intracellular ROS accumulation in both granulosa cell lines, indicating the induction of pronounced oxidative stress. Co-treatment with lycopene significantly attenuated this response, restoring ROS levels closer to those of untreated controls (Fig.  3 A–B). The consistent reduction observed in both COV434 and KGN cells suggests that lycopene acts as a potent antioxidant, effectively neutralizing excessive reactive oxygen species generated by DEHP. In alignment with the reduced oxidative burden, lycopene co-treatment mitigated DEHP-induced mitochondrial dysfunction. DEHP exposure led to a marked dissipation of the mitochondrial membrane potential, signifying impaired electron transport chain activity and loss of mitochondrial integrity. Lycopene markedly restored mitochondrial membrane potential in both COV434 and KGN cells (Fig.  3 C–D). Furthermore, DEHP exposure significantly decreased mitochondrial DNA (mtDNA) copy number, reflecting compromised mitochondrial biogenesis and genomic stability. Lycopene partially reversed this decline, bringing mtDNA levels closer to normal in both cell lines (Fig.  3 E–F). Fig. 3 Lycopene alleviates DEHP-induced oxidative stress and mitochondrial dysfunction in human granulosa cells. ( A – B ) Intracellular ROS levels were measured by DCFH-DA fluorescence in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( C – D ) Mitochondrial membrane potential was assessed using JC-1 staining in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( E – F ) Mitochondrial DNA (mtDNA) copy number was quantified by qPCR in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( G - H ) SOD levels were determined by SOD assay kit in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( I - J ) CAT levels were determined by CAT assay kit in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( K - L ) MDA activities were determined by MDA assay kit in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 Lycopene alleviates DEHP-induced oxidative stress and mitochondrial dysfunction in human granulosa cells. ( A – B ) Intracellular ROS levels were measured by DCFH-DA fluorescence in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( C – D ) Mitochondrial membrane potential was assessed using JC-1 staining in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( E – F ) Mitochondrial DNA (mtDNA) copy number was quantified by qPCR in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( G - H ) SOD levels were determined by SOD assay kit in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( I - J ) CAT levels were determined by CAT assay kit in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( K - L ) MDA activities were determined by MDA assay kit in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 To determine whether lycopene reinforces endogenous antioxidant defenses, we measured SOD and CAT activities. DEHP exposure significantly impaired endogenous antioxidant defenses in both granulosa cell lines, as reflected by reduced superoxide dismutase (SOD) and catalase (CAT) activities. Lycopene co-treatment consistently restored these enzyme activities toward control levels in COV434 and KGN cells, with effects that were statistically significant relative to DEHP alone (Fig.  3 G–J). The concordant recovery of both SOD and CAT suggests that lycopene does not act on a single antioxidant pathway but broadly reinforces enzymatic detoxification of superoxide and hydrogen peroxide, thereby dampening propagation of oxidative injury. Notably, the rescue was observed across two distinct granulosa models, indicating that the protective action of lycopene is robust to cell-line–specific differences. In parallel, lipid peroxidation—indexed by malondialdehyde (MDA)—was markedly elevated by DEHP and significantly reduced by lycopene co-treatment in both lines (Fig.  3 K–L; vs. DEHP). The inverse relationship between restored SOD/CAT activities and lowered MDA supports a coherent mechanism in which lycopene limits ROS accumulation and downstream membrane damage. Because SIRT1 is a key regulator of cellular stress resistance and mitochondrial homeostasis [ 26 ], we next assessed whether it is modulated by DEHP and lycopene. DEHP exposure markedly suppressed SIRT1 transcription in both granulosa cell lines, whereas lycopene co-treatment significantly restored mRNA expression toward control levels (Fig. 4 A–B; vs. DEHP). The concordant responses in COV434 and KGN cells suggest a robust effect across distinct granulosa models, consistent with lycopene counteracting DEHP-induced transcriptional repression of SIRT1. Fig. 4 Lycopene restores SIRT1 expression suppressed by DEHP in human granulosa cells. ( A – B ) SIRT1 mRNA levels were measured by RT-qPCR in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( C – D ) qRT-PCR analysis of SIRT1 mRNA expression in COV434 and KGN cells following si-NC or si-SIRT1 transfections. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 Lycopene restores SIRT1 expression suppressed by DEHP in human granulosa cells. ( A – B ) SIRT1 mRNA levels were measured by RT-qPCR in COV434 and KGN cells exposed to control, DEHP, or DEHP + lycopene. ( C – D ) qRT-PCR analysis of SIRT1 mRNA expression in COV434 and KGN cells following si-NC or si-SIRT1 transfections. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 At the protein level, DEHP similarly reduced SIRT1 abundance, which was significantly rescued by lycopene in both lines (Fig.  4 C–D; vs. DEHP). Together with the parallel improvements in oxidative stress and mitochondrial readouts, these data support a model in which lycopene restores SIRT1 signaling at both transcript and protein levels, thereby contributing to its cytoprotective and antioxidative actions in human granulosa cells. To test whether SIRT1 is required for lycopene’s protective effects, we silenced SIRT1 using siRNA. As expected, DEHP significantly reduced cell viability, and lycopene co-treatment improved survival in both COV434 and KGN cells. However, SIRT1 knockdown partially abrogated this improvement, yielding viability levels significantly lower than the DEHP + lycopene condition (Fig.  5 A–B). These findings indicate that SIRT1 activation is required for the full cytoprotective effect of lycopene. Fig. 5 SIRT1 knockdown attenuates the protective effects of lycopene against DEHP-induced cytotoxicity and apoptosis. ( A – B ) Cell viability and ( C – D ) caspase-3/-7 activity were assessed in COV434 and KGN cells treated with DEHP alone, DEHP + lycopene, or DEHP + lycopene following SIRT1 siRNA transfection. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 SIRT1 knockdown attenuates the protective effects of lycopene against DEHP-induced cytotoxicity and apoptosis. ( A – B ) Cell viability and ( C – D ) caspase-3/-7 activity were assessed in COV434 and KGN cells treated with DEHP alone, DEHP + lycopene, or DEHP + lycopene following SIRT1 siRNA transfection. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 Consistently, caspase-3/-7 assays showed that DEHP increased apoptotic activity, which was significantly suppressed by lycopene in both lines. Silencing SIRT1 partially reversed this anti-apoptotic effect, resulting in higher caspase activity compared with DEHP + lycopene (Fig.  5 C–D). Together, these data support a SIRT1-dependent mechanism by which lycopene mitigates DEHP-induced cytotoxicity and apoptosis in human granulosa cells. We next asked whether the ROS-lowering and mitochondrial-protective actions of lycopene depend on SIRT1. DEHP robustly increased intracellular ROS in both granulosa cell lines, and lycopene co-treatment significantly suppressed this oxidative surge (Fig.  6 A–B). Silencing SIRT1 partially negated the antioxidant effect of lycopene, yielding ROS levels that were higher than the DEHP + lycopene condition yet still lower than DEHP alone (vs. DEHP + lycopene, p  < 0.05). The same pattern was observed in COV434 and KGN cells, indicating a consistent SIRT1 contribution to lycopene’s ROS-lowering action across distinct granulosa models. Mitochondrial functional readouts showed concordant changes. DEHP reduced mitochondrial membrane potential, whereas lycopene significantly restored it in both cell lines (Fig.  6 C–D). SIRT1 knockdown attenuated this recovery, resulting in membrane potential values intermediate between DEHP and DEHP + lycopene (vs. DEHP + lycopene, p  < 0.05). Fig. 6 SIRT1 silencing reverses lycopene-mediated antioxidant and mitochondrial protection in DEHP-exposed granulosa cells. ( A – B ) Intracellular ROS production, ( C – D ) MMP and ( E – F ) mtDNA copy number were measured in COV434 and KGN cells following DEHP, DEHP + lycopene, and DEHP + lycopene + si-SIRT1 treatments. ( G – J ) Superoxide dismutase (SOD) and catalase (CAT) activities and ( K – L ) malondialdehyde (MDA) content were quantified as markers of oxidative stress and lipid peroxidation in COV434 and KGN cells following DEHP, DEHP + lycopene, and DEHP + lycopene + si-SIRT1 treatments. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 SIRT1 silencing reverses lycopene-mediated antioxidant and mitochondrial protection in DEHP-exposed granulosa cells. ( A – B ) Intracellular ROS production, ( C – D ) MMP and ( E – F ) mtDNA copy number were measured in COV434 and KGN cells following DEHP, DEHP + lycopene, and DEHP + lycopene + si-SIRT1 treatments. ( G – J ) Superoxide dismutase (SOD) and catalase (CAT) activities and ( K – L ) malondialdehyde (MDA) content were quantified as markers of oxidative stress and lipid peroxidation in COV434 and KGN cells following DEHP, DEHP + lycopene, and DEHP + lycopene + si-SIRT1 treatments. Data are expressed as mean ± SD ( n  = 3). Significant differences between treatment groups were indicated as * P  < 0.05, ** P  < 0.01 and *** P  < 0.001 Measures of mitochondrial content followed a similar trajectory. DEHP decreased mtDNA copy number, while lycopene increased it toward control levels in both COV434 and KGN cells (Fig.  6 E–F). SIRT1 silencing partially reduced this increase (vs. DEHP + lycopene, p  < 0.05), suggesting that lycopene’s enhancement of mitochondrial biogenesis/maintenance is mediated in part by SIRT1. Collectively, these findings demonstrate that lycopene mitigates DEHP-induced oxidative stress and mitochondrial dysfunction through mechanisms that require intact SIRT1 signaling, with effects that are reproducible across both granulosa cell lines. Finally, we evaluated whether SIRT1 is required for lycopene’s effects on antioxidant defenses and lipid peroxidation. DEHP exposure markedly suppressed antioxidant enzyme activities in both COV434 and KGN granulosa cells, while lycopene co-treatment significantly enhanced SOD and CAT activities (Fig.  6 G–J; vs. DEHP). Silencing SIRT1 partially abrogated these improvements, yielding enzyme activities that were significantly lower than the DEHP + lycopene condition in both cell lines ( p  < 0.05). Conversely, malondialdehyde (MDA), a lipid-peroxidation marker, was elevated by DEHP and significantly reduced by lycopene; this reduction was partially reversed by SIRT1 knockdown in both models (Fig.  6 K–L; p  < 0.05 vs. DEHP + lycopene). Overall, these results indicate that SIRT1 activation is critical for lycopene’s antioxidative actions, as SIRT1 silencing compromises lycopene-mediated reinforcement of antioxidant defenses and attenuation of lipid peroxidation under DEHP challenge.

Human granulosa-like tumor cell lines KGN and COV434 were used as in vitro models of ovarian granulosa cells due to their stable steroidogenic and proliferative characteristics. Both cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 (DMEM/F12, Gibco, Thermo Fisher Scientific, Waltham, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific) and 1% penicillin–streptomycin solution (100 U/mL penicillin, 100 µg/mL streptomycin, Sigma-Aldrich, St. Louis, USA). Cells were maintained in a humidified incubator at 37 °C with 5% CO₂ and 95% relative humidity. Di(2-ethylhexyl) phthalate (DEHP) and lycopene were purchased from Sigma-Aldrich. The DEHP concentration range was selected based on a previous study [ 28 ]. DEHP was first dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 M stock solution and diluted in complete medium to final working concentrations of 50, 100, 200, 400, and 800 µM. Lycopene (Sigma-Aldrich), a highly hydrophobic carotenoid, was dissolved in tetrahydrofuran containing 0.01% butylated hydroxytoluene to prevent oxidative degradation. The stock was freshly diluted into the medium to obtain final concentrations of 0.3, 1, 3, 10, and 30 µM immediately before use. The concentration range selection of lycopene was based on a previous study [ 28 ]. For concentration–response experiments, KGN and COV434 cells were exposed to DEHP (50–800 µM) for 24, 48, or 72 h. For lycopene cytotoxicity testing, cells were treated with various concentrations of lycopene (0.3–30 µM) for identical durations. For co-treatment experiments, cells were pre-incubated with lycopene (1 or 3 µM) for 2 h and subsequently treated with DEHP (400 µM) for an additional 48 h. Control wells received medium containing the corresponding vehicle only. Each condition was assayed in triplicate wells and repeated in at least three independent experiments. To probe SIRT1’s role, we performed RNA interference using a SIRT1-targeting siRNA (si-SIRT1) and a non-targeting control (si-NC) from GenePharma (Shanghai, China). Cells were plated in six-well dishes at 2 × 10⁵ cells per well and, at 60–70% confluence, transfected with 50 nM siRNA via Lipofectamine 3000 (Invitrogen, Carlsbad, USA) per the manufacturer’s instructions. After 6 h, transfection medium was replaced with complete medium, and cells recovered for 24 h before drug exposure. Viability was measured via the CCK-8 assay kit (Beyotime, Beijing, China) [ 28 ]. Cells (5 × 10³/well, 96-well format) received the designated treatments, then 10 µL CCK-8 was added and plates were incubated 2 h at 37 °C. Absorbance at 450 nm was captured using a SpectraMax reader (Molecular Devices, San Jose, USA). Data are presented as a percentage of control after blank subtraction. Apoptosis was assessed with the Caspase-Glo ® 3/7 kit (Promega, Madison, USA) [ 29 ]. Following treatment, an equal volume of reagent was dispensed into each well and plates were incubated for 30 min at room temperature in the dark. Luminescence was recorded on a BioTek luminometer (USA). Data are reported as caspase activity relative to control (fold change). Intracellular ROS were assessed with 2’,7’-dichlorofluorescein diacetate (DCFH-DA; Beyotime) [ 30 ]. After treatment, cells were rinsed in PBS and loaded with 10 µM DCFH-DA in serum-free medium for 30 min at 37 °C in the dark. Following two PBS washes, fluorescence was read at 488/525 nm on a microplate fluorimeter. Signals were normalized to protein content and expressed as % of control. The JC-1 assay (Beyotime) was employed to measure MMP [ 31 ]. Post-exposure, cells received 5 µg/mL JC-1 for 30 min at 37 °C, were washed twice with PBS, and evaluated by fluorescence reader or microscope. A red-to-green fluorescence ratio served as the readout of mitochondrial integrity. Genomic DNA was isolated with DNeasy kit (Qiagen, Hilden, Germany). mtDNA abundance was determined by SYBR-based qPCR (Bio-Rad, Hercules, USA) with primers for ND1 (mtDNA) and β-globin (nuclear control) [ 32 ]. Data are reported as 2⁻ ΔΔCt . Following treatment, cells were rinsed with ice-cold PBS and lysed in RIPA buffer (Beyotime). Lysates were clarified by centrifugation at 12,000 × g for 10 min at 4 °C, and protein abundance was quantified using the BCA kit (Thermo Fisher Scientific). SOD and CAT activities were then assayed with commercial kits from Nanjing Jiancheng (Nanjing, China) per the supplier’s instructions, normalized to total protein, and reported as units per mg protein. Lipid peroxidation was quantified as MDA using thiobarbituric acid–reactive substances assay (Nanjing Jiancheng, China) [ 33 ]. Cell lysates were combined with TBA reagent, heated at 95 °C for 40 min, cooled, and centrifuged. Supernatant absorbance was read at 532 nm, and MDA was determined from a standard curve. Total RNA was extracted using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. RNA purity and concentration were verified by measuring the A260/A280 ratio using a Nanodrop spectrophotometer. Complementary DNA (cDNA) was synthesized using the PrimeScript RT Reagent Kit (Takara, Tokyo, Japan). Quantitative PCR was conducted using SYBR Green Master Mix (Bio-Rad, USA) on a CFX96 Real-Time PCR Detection System. Primer sequences were designed to specifically amplify SIRT1 and GAPDH as an internal control. All experiments were performed in triplicate and independently repeated at least three times. Data were presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, USA). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, while pairwise comparisons were analyzed using the Student’s t-test. A p-value < 0.05 was considered statistically significant.

Our work demonstrated that DEHP exposure induces pronounced cytotoxicity, oxidative stress, and mitochondrial dysfunction in human granulosa-like KGN and COV434 cells, and that lycopene effectively mitigates these detrimental effects through activation of the SIRT1 pathway. Specifically, DEHP significantly decreased cell viability and increased caspase-3/-7 activity in a concentration- and time-dependent manner, confirming its pro-apoptotic potential in granulosa cells. Concomitantly, DEHP elevated intracellular ROS levels, suppressed antioxidant enzyme activities, disrupted mitochondrial membrane potential, and reduced mitochondrial DNA (mtDNA) copy number—hallmarks of oxidative and mitochondrial injury. Lycopene co-treatment restored antioxidant capacity, preserved mitochondrial integrity, and attenuated apoptosis, while SIRT1 silencing partially reversed these protective effects. Together, the main unique contribution of this study is mechanistic and we demonstrate in human granulosa cell models that lycopene’s protection against DEHP is functionally SIRT1-dependent linking redox control and mitochondrial maintenance to reduced caspase activation. DEHP and its bioactive metabolite MEHP drive a multifaceted injury program in granulosa cells characterized by excessive ROS generation, mitochondrial depolarization, mtDNA loss, and caspase-dependent apoptosis—lesions that align with our findings in KGN and COV434 cells [ 28 ]. Granulosa-cell apoptosis is a biologically meaningful endpoint because it directly relates to follicular atresia and loss of follicular competence. In situ observations of granulosa apoptosis in mammalian ovaries further support the relevance of apoptosis readouts for ovarian physiology [ 34 ]. Importantly, our dataset is internally coherent across outcome domains: DEHP increased ROS while weakening enzymatic antioxidant defenses (SOD/CAT) and elevating lipid peroxidation (MDA), which is consistent with the observed decline in mitochondrial membrane potential and mtDNA copy number. This pattern supports a feed-forward model in which oxidative burden impairs mitochondrial function, and dysfunctional mitochondria further amplify ROS, thereby accelerating lipid peroxidation and caspase activation. Beyond classical oxidative damage, recent studies broaden this landscape: co-exposure models show polystyrene microplastics synergize with DEHP to intensify ROS production and trigger DNA damage, cell-cycle arrest, and necroptosis via CNR1/CRBN/YY1/CYP2E1 and Hippo signaling [ 35 ]; activation of the p62–Keap1–Nrf2 axis emerges as a compensatory antioxidant response that curbs MEHP-driven oxidative injury and excessive autophagy [ 36 ]; and Nrf2 signaling also intersects with ferroptosis pathways in mouse ovaries exposed to DEHP/MEHP [ 37 ]. These reports reinforce that DEHP/MEHP toxicity may converge on multiple stress programs (e.g., antioxidant compensation, autophagy changes, alternative cell-death routes). In this context, our data identify SIRT1 restoration as a key mechanistic checkpoint associated with improved redox and mitochondrial endpoints in granulosa cells under DEHP challenge. Parallel endocrine disruptions further implicate mitochondrial stress in impaired steroidogenesis: MEHP suppresses aromatase transcripts and estradiol output in granulosa cells [ 38 ], and DEHP dampens FSH-stimulated steroidogenic programs by inhibiting cAMP/CREB and ERK1/2 signaling—defects that can be partially rescued by EGFR activation [ 39 ]. While we did not quantify steroidogenic outputs, mitochondrial impairment is tightly linked to steroid hormone biosynthesis, suggesting that the oxidative–mitochondrial injury we observe could plausibly contribute to endocrine dysfunction in addition to reduced cell survival. Additional death programs appear relevant, including pyroptosis via the SLC39A5/NF-κB/NLRP3 axis [ 40 ], while mitochondrial-targeted protection with melatonin (via Drp1/AMPK-PGC-1α) [ 41 ] or phytochemicals such as Shatavarin-IV [ 42 ] can mitigate ROS accumulation, stabilize MMP, and restore steroidogenic gene expression. In addition, ultrastructural studies in granulosa cells exposed to toxicants show organelle-level damage and cytopathology [ 43 ], which is consistent with our functional evidence of mitochondrial depolarization and mtDNA loss. Together, the literature supports a central model in which DEHP/MEHP impose redox pressure that destabilizes mitochondrial homeostasis and can engage multiple death modalities; our data place caspase activation prominently in granulosa cells and show that lycopene blunts this trajectory. Lycopene, beyond directly scavenging ROS and restoring antioxidant enzyme capacity in our granulosa models, engages conserved stress-response programs that preserve mitochondrial quality control across organs exposed to DEHP. Our results demonstrated that lycopene not only lowered ROS but also restored antioxidant enzyme activity, reduced lipid peroxidation, and increased mtDNA copy number toward control levels. This aligns with the broader reproductive literature suggesting natural antioxidant compounds can ameliorate infertility-related oxidative injury through multi-target mechanisms rather than a single endpoint effect [ 44 ]. In vivo studies show that lycopene prevents DEHP-induced hepatic mitochondrial quality control disorder by coordinating the SIRT1–PGC-1α axis, restraining PINK1/Parkin-dependent mitophagy, and tempering the mitochondrial unfolded protein response, thereby stabilizing MMP and mitochondrial ultrastructure [ 45 ]. Lycopene also limits ER stress triggered by nuclear xenobiotic receptor activation (AHR/PXR/CAR) and restores CYP450 homeostasis in liver and testis, highlighting AHR–Nrf2 crosstalk and UPRER as complementary antioxidant nodes under DEHP challenge [ 46 , 47 ]. In kidney and heart, lycopene attenuates DEHP-evoked mitochondrial depolarization, aberrant mitophagy, and inflammatory signaling by engaging cGAS–STING, suppressing TLR/MyD88–JNK–driven cytokine induction, and rebalancing mitochondrial biogenesis/dynamics [ 48 – 50 ]. Parallel activation of Nrf2/Keap1 dampens oxidative injury and curbs Caspase-1/NLRP3-dependent pyroptosis in kidney and ferroptosis in hippocampus, extending lycopene’s cytoprotection to inflammasome and iron-lipid peroxidation axes [ 51 , 52 ]. Mechanistically, lycopene’s benefits also involve maintaining mitochondria–ER coupling (MAM) and restraining HIF-1α-driven metabolic reprogramming (PPARα/γ, FXR/LXR) that accompanies DEHP toxicity [ 47 , 53 ]. Taken together with our granulosa-cell data, this external evidence supports a unifying model in which lycopene reinforces mitochondrial homeostasis and redox signaling—via SIRT1-centered and innate stress pathways—to blunt DEHP-induced oxidative injury, organellar stress responses, and downstream cell-death programs. Beyond our granulosa-cell data, multiple independent models reinforce SIRT1 as the hub through which lycopene confers cytoprotection; in hepatocytes, lycopene counters toxin-triggered mitochondrial damage and coordinated cell-death programs, and critically, SIRT1 knockdown diminishes lycopene’s benefit (PANoptosis model in FB1-injured LMH cells) [ 54 ]. In vascular and metabolic contexts, lycopene reactivates SIRT1 to improve insulin resistance, angiogenic capacity, and microvascular integrity in aging models [ 55 ], and combined flavonoid–carotenoid strategies (e.g., quercetin + lycopene) stabilize and activate SIRT1 while suppressing the Nox4–ROS axis and inflammatory transcriptional programs [ 56 ]. Consistently, lycopene attenuates oxidative injury in endothelial cells via SIRT1/Nrf2/HO-1, and in endothelial progenitor cells via AMPK/SIRT1 [ 57 ], indicating convergent SIRT1-centered signaling across cell types. Notably, in a DEHP in vivo setting, lycopene preserves mitochondrial quality control through a SIRT1–PGC-1α/PINK1–mitophagy–UPRmt axis, aligning with our observation that SIRT1 integrity is required for lycopene to restore mitochondria and blunt oxidative stress [ 45 ].Together, these studies substantiate a unifying mechanism in which lycopene engages SIRT1-anchored oxidative and mitochondrial governance, and loss of SIRT1 function curtails lycopene’s efficacy, thereby providing external validation for the SIRT1 dependence we demonstrate in human granulosa cells. Given the widespread environmental exposure to DEHP, understanding potential protective interventions is of substantial clinical and public health interest. The ability of lycopene to counteract DEHP-induced oxidative and mitochondrial dysfunction highlights its potential as a dietary or nutraceutical strategy for reducing reproductive toxicity associated with phthalate exposure. Since lycopene is naturally abundant in the human diet and possesses a favorable safety profile, it may serve as a practical preventive antioxidant for populations at risk of chronic environmental exposure. Furthermore, because oxidative stress and mitochondrial damage are common pathogenic mechanisms underlying various ovarian disorders—such as polycystic ovary syndrome (PCOS), diminished ovarian reserve, and premature ovarian insufficiency—lycopene supplementation might also have broader therapeutic benefits in reproductive health beyond toxicant exposure. However, while our findings provide strong mechanistic support in vitro, the translational application of lycopene requires further validation in animal models and clinical settings to determine optimal dosing, bioavailability, and long-term safety. This study provides compelling mechanistic evidence but is not without limitations. First, the experiments were conducted in immortalized granulosa-like cell lines, which, while useful for mechanistic exploration, may not fully replicate the complex hormonal and paracrine interactions present in vivo. Second, only a limited concentration range of DEHP and lycopene was evaluated, and concentration optimization under physiological conditions remains necessary. Third, we observed that higher lycopene concentrations reduced granulosa cell viability, raising the possibility of off-target or context-dependent cytotoxic effects at supraphysiological levels. Therefore, the protective effects reported here should be interpreted within an optimized, non-cytotoxic concentration range, and careful dose selection and formulation will be critical for translational relevance. Additionally, although SIRT1 was identified as a major mediator of lycopene’s effects, other signaling pathways, such as Nrf2, AMPK, and PI3K/Akt, may also contribute to its antioxidative properties and warrant further investigation. Future studies employing animal models or ex vivo human follicle systems could provide more comprehensive insights into lycopene’s pharmacodynamics, tissue distribution, and reproductive outcomes. Moreover, exploring the potential synergistic effects of lycopene with other antioxidants or SIRT1 activators could enhance its protective efficacy. In conclusion, DEHP induced marked cytotoxicity in human granulosa-like KGN and COV434 cells, accompanied by oxidative stress, impaired antioxidant defenses, mitochondrial dysfunction, and increased caspase-3/-7 activity. Lycopene significantly counteracted these effects by restoring redox balance and mitochondrial integrity and suppressing apoptotic signaling. DEHP downregulated SIRT1, whereas lycopene restored SIRT1 expression, and SIRT1 silencing partially weakened lycopene’s protection, supporting a SIRT1-dependent mechanism. These findings identify lycopene as a potential dietary strategy to reduce phthalate-related granulosa cell injury and highlight SIRT1 as a candidate target for mitigating environmental reproductive toxicity. Future work should validate these effects in primary granulosa cells and in vivo models and refine physiologically relevant exposure and dosing conditions.

Female reproductive capacity relies heavily on the proper function of ovarian granulosa cells, which nurture oocyte maturation, synthesize steroid hormones, and regulate follicular development [ 1 ]. Damage to these cells can impair folliculogenesis, disrupt endocrine balance, and ultimately lead to infertility [ 2 ]. In recent years, reproductive toxicology has increasingly highlighted how environmental and consumer-product chemicals perturb ovarian physiology by targeting granulosa cell survival programs, mitochondrial homeostasis, and stress-adaptation pathways, thereby accelerating follicular loss and reducing ovarian reserve [ 3 ]. In addition to oxidative injury and apoptosis, emerging mechanisms, such as dysregulated autophagy and impaired maintenance of the primordial follicle pool, are now recognized as important contributors to toxicant-induced ovarian dysfunction and female infertility [ 4 , 5 ]. Among ubiquitous environmental contaminants, di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer in polyvinyl chloride materials, is one of the most prevalent endocrine-disrupting chemicals in daily life [ 6 ]. DEHP is extensively utilized in food packaging, cosmetics, and medical devices, and is continuously released into the environment, where it readily accumulates in biological systems [ 7 , 8 ]. Recent biomonitoring studies have detected DEHP and its metabolites in human serum, urine, and even follicular fluid, indicating chronic exposure among women of reproductive age [ 9 , 10 ]. Growing epidemiological and experimental evidence implicates DEHP exposure in diminished ovarian reserve, anovulation, and poor oocyte quality, underscoring its detrimental impact on ovarian function [ 11 , 12 ]. At the cellular level, granulosa cells appear particularly susceptible: toxicant-induced ultrastructural and functional alterations in granulosa cells are closely associated with follicular atresia and reduced fertility, consistent with observations that granulosa cell degeneration is a pivotal event in follicular demise [ 13 ]. DEHP, together with its major metabolite MEHP, damages cells through convergent mechanisms that provoke oxidative stress, impair mitochondrial function, and induce programmed cell death [ 14 , 15 ]. Following cellular uptake, DEHP metabolites promote reactive oxygen species (ROS) production, disrupt redox equilibrium, and impair mitochondrial oxidative phosphorylation [ 16 ]. The overaccumulation of ROS initiates lipid peroxidation, DNA oxidation, and protein modification, triggering structural and functional mitochondrial damage. Consequent loss of mitochondrial membrane potential (MMP) and release of pro-apoptotic factors such as cytochrome c activate downstream caspase-3/-7 cascades, leading to programmed cell death [ 17 ]. Furthermore, oxidative stress induced by DEHP could suppress activities of key antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), while elevating malondialdehyde (MDA) levels, a biomarker of lipid peroxidation [ 17 ]. The resulting imbalance between ROS generation and antioxidant defense contributes to mitochondrial genome instability and reduced mitochondrial DNA (mtDNA) copy number [ 18 ]. These alterations ultimately compromise granulosa cell viability and hormone synthesis, contributing to follicular atresia and reduced fertility. Although these mechanistic insights are well-documented, the cellular defense pathways that mitigate DEHP-induced oxidative and mitochondrial injury in granulosa cells remain poorly understood. Lycopene, a red carotenoid pigment abundant in tomatoes, watermelon, and other fruits, is widely recognized as one of the most potent naturally occurring antioxidants [ 19 , 20 ]. Owing to its long chain of conjugated double bonds, it can effectively deactivate singlet oxygen and eliminate reactive free radicals, reducing oxidative injury to cellular lipids and membranes [ 19 , 20 ]. In addition to its direct radical-scavenging capacity, lycopene can modulate redox-sensitive transcription factors and upregulate endogenous antioxidant enzymes through Nrf2 and other signaling pathways [ 21 ]. Experimental evidence has demonstrated that lycopene supplementation mitigates oxidative damage and inflammation in cardiovascular, hepatic, and neural tissues, highlighting its broad cytoprotective potential. In reproductive systems, lycopene has shown promise in improving ovarian morphology, preserving follicular integrity, and restoring hormonal balance in animal models exposed to oxidative or chemical stressors [ 22 – 24 ]. However, despite these promising attributes, the molecular mechanisms by which lycopene confers protection against phthalate-induced oxidative stress, particularly in the context of human granulosa cells, remain largely unexplored. The Sirtuin 1 (SIRT1) pathway represents a crucial regulator of oxidative metabolism, mitochondrial biogenesis, and apoptosis [ 25 ]. As a NAD + -dependent deacetylase, SIRT1 modulates the activity of multiple downstream effectors, thereby enhancing antioxidant defenses and promoting mitochondrial resilience under stress conditions [ 25 ]. Activation of SIRT1 facilitates cellular adaptation to oxidative challenges by increasing antioxidant gene transcription, improving mitochondrial membrane potential, and suppressing caspase-mediated apoptosis [ 26 ]. Dysregulation of SIRT1 has been implicated in various reproductive disorders, such as ovarian aging, PCOS, and endometriosis, highlighting its central role in ovarian homeostasis [ 27 ]. Given its ability to regulate oxidative metabolism, SIRT1 may represent a critical mediator through which lycopene exerts its antioxidative and mitochondrial protective functions. Based on this background, we hypothesized that lycopene alleviates DEHP-induced oxidative stress, mitochondrial dysfunction, and apoptosis in human granulosa cells through SIRT1-dependent mechanisms. To test this hypothesis, we employed KGN and COV434 granulosa-like cell lines to model DEHP-induced cytotoxicity and examined the protective efficacy of lycopene across multiple cellular endpoints. Specifically, we evaluated changes in cell viability, caspase-3/-7 activity, ROS generation, MMP, mtDNA copy number, and activities of key antioxidant enzymes (SOD and CAT), along with lipid peroxidation markers (MDA). Furthermore, to delineate the mechanistic involvement of SIRT1, we used siRNA-mediated SIRT1 silencing to determine whether inhibition of this pathway compromises lycopene’s protective actions. This integrative approach provides a comprehensive understanding of how lycopene modulates oxidative and mitochondrial pathways to preserve granulosa cell function under phthalate-induced stress, offering new insights into potential nutritional or pharmacological interventions against environmental reproductive toxicants.