Preventive Effect of Fisetin on Follicular Granulosa Cells Senescence via Attenuating Oxidative Stress and Upregulating the Wnt/β-Catenin Signaling Pathway

Hy-line white chickens (Gallus domesticus) were purchased from a local commercial farm and raised with free access to feed and water. Ovaries were isolated from hens approximately 280 days old (high-laying period) and 580 (low-laying period) days old, respectively, and stored in the ice-cold sterile phosphate-buffered saline (PBS). The prehierarchical follicles (SYFs, 6–8 mm), ovarian cortex, and atretic small yellow follicles (ASYFs) were separated from the collected ovaries, and fixed in 4% paraformaldehyde (PFA) for morphological examination. Specifically, the ASYFs were collected from the 580-day-old hens. A portion of the SYFs from both age groups were used for granulosa layer isolation. Briefly, the SYFs were washed with cold-PBS, punctured with the tweezers, and the yolk was removed as clean as possible. The remaining granulosa layers were gently stripped from the theca layers, washed three times using PBS, and used for subsequent Western blot or quantitative real-time polymerase chain reaction (qRT-PCR) analysis. All procedures were approved by the Committee on the Ethics of Animals Experiments of Zhejiang University and conducted in accordance with guidelines the Guiding Principles for the Care and Use of Laboratory Animals of Zhejiang University (ZJU20220085).

The granulosa layers stripped from the SYFs of D280 hens were digested with collagenase 2 (Gibco, Grand Island, NY, USA), filtered through a 200-mesh steel sieve (75 μm) and centrifuged at 1500 rpm for 5 min. After washed with PBS twice, the cell mass was resuspended, counted, and cultured with DMEM/F12 medium (Hyclone, Tauranga, New Zealand) containing with 5% fetal calf serum (FCS, Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin. The cells were incubated at 38 °C and in 5% CO₂ to allow cell attachment overnight. After attachment, GCs were treated with different concentrations of fisetin (0, 2.5, 5, 10, 20, 40, 80 µM, MB5836, Meilunbio, Dalian, China) for 24 h to test the effect of fisetin on cell viability. To examine the anti-aging effect of fisetin on granulosa cells, the cells were pretreated with fisetin (0, 5, 10, 20, 40 µM) for 24 h, followed by incubation with the D-gal (200 mM, MB1853-2, Meilunbio, Dalian, China) for another 24 h. For inhibitor experiments, GCs were pretreated with each specific inhibitor for 2 h before fisetin exposure. ML385 (10 µM, HY-100523, MedChemExpress, Nanjing, China), the Nrf2 inhibitor and IWR-1 (10 µM, HY-12238, MedChemExpress), the β-catenin inhibitor, were used in these experiments.

SYFs from late-laying (aged 580 days) hens were transferred into DMEM/F12 complete medium (Hyclone) supplemented with 5% FCS (Hyclone) and 1 x ITS mixture (10 mg/mL insulin, 5 mg/mL transferrin, 30 nM selenite, and 2 mM glutamine). The follicles were cultured in 48-well plates (Corning Inc., Corning, NY, USA) at 38 °C and in a 5% CO₂ atmosphere for 72 h. The treatment groups were designed as follows: fisetin (20 µM), fisetin + ML385 (10 µM), fisetin + IWR-1 (10 µM), ML385 (10 µM), and IWR-1 (10 µM). After 48 h of culture, the medium was supplemented with bromodeoxyuridine (BrdU, Sigma Aldrich, Saint Louis, MO, USA) at a final concentration of 10 µg/mL, followed by a further 24 h incubation.

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8, FD3788, Fudebio, Hangzhou, China) according to the manufacturer's instructions. Briefly, GCs were grown in 96-well plates and cultured for 24 h until reaching approximately 90% confluency, then treated with experimental conditions. For the assay, 10 µL of CCK-8 solution was added into each well containing 100 µL medium, and incubated for 2 h. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

Cellular senescence of D-gal-induced GCs was detected using a β-galactosidase staining kit (G1580, Solarbio, Beijing, China) according to the kit's protocols. Briefly, after being incubated, the GCs were washed twice with PBS and fixed in β-galactosidase fixative solution for 15 min at room temperature. Following three washes with PBS, the cells were incubated in 1 mL dyeing liquid (10 µL β-galactosidase staining fluid A, 10 µL fluid B, 930 µL fluid C, and 50 µL X-Gal solution) overnight at 37 °C. The cells were observed and imaged using an Eclipse 80i microscope (Nikon, Tokyo, Japan). The cytoplasm of SA-β-gal-positive cells appeared as blue uniform particles. For each experimental condition, at least five randomly selected microscopic fields were analyzed, and the number of positive-stained cells per 200 cells was quantified using ImageJ v2.3.0 software (NIH, Bethesda, MD, USA) to distinguish blue cells (positive) from unstained cells (negative) by setting a color threshold. All experiments were performed in triplicate.

The effects of different treatments on cell cycle progression were analyzed by flow cytometry. Briefly, GCs were plated at 1 × 10⁶ cells per 60 mm dish. After indicated treatments, the cells were trypsinized, washed with PBS, and fixed with 75% ethanol on ice followed by resuspension in 500 µL of propidium iodide solution (480 µL staining buffer, 10 µL PI, and 10 µL RNase A) and incubated at 37 °C for 30 min. Cell cycle distribution were assayed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with an extraction wavelength of 488 nm. Cell cycle distribution was analyzed using FlowJo v10.8.1 software (FlowJo, LLC, Ashland, OR, USA).

Cell proliferation was assessed using an EdU assay kit (C0071S, Beyotime, Hangzhou, China) according to the manufacturer's protocol. GCs were incubated with 10 µM EdU for 2 h at 38 °C. The treated GCs subsequently underwent fixation with 4% PFA for 15 min at room temperature and permeabilized with into 0.5% Triton X-100 for 15 min. After three washes with PBS, the GCs were incubated with 0.5 mL click reaction solution (430 µL click reaction buffer, 20 µL CuSO₄, 1 µL Azide 594, and 50 µL click additive solution) for 30 min in the dark. Nucleic acids were marked using DAPI (C1006, Beyotime, Hangzhou, China). Images were captured by a fluorescence microscope (Olympus IX70, Tokyo, Japan). All experiments were performed in triplicate.

Mitochondrial membrane potential (MMP), an indicator of mitochondrial inner and outer membrane integrity, was estimated using an enhanced mitochondrial membrane potential assay kit with the fluorescent probe JC-1 (C2003S, Beyotime) following the instruction. Cells were plated in 6-well culture plates at a density of 1 × 10⁶ cells per well and allowed to adhere for 24 h. After the indicated treatment, GCs were subjected to two PBS rinses and then incubated with 1 mL of JC-1 working solution per well at 38 °C for 20 min. Subsequently, cells were rinsed twice with premade JC-1 staining buffer and analyzed for red and green fluorescence signals using either laser confocal microscopy (Olympus IX81-FV1000, Tokyo, Japan) or flow cytometry. Quantification of red and green fluorescence was performed using ImageJ (v2.3.0, NIH, Bethesda, MD, USA), and the ratio was calculated from the mean optical density of each. FlowJo v10.8.1 software was performed to analyze the flow cytometry.

Intracellular ROS was measured using a ROS Assay Kit (S0033S, Beyotime) according to the manufacturer's instructions. Briefly, after exposure to fisetin and D-gal, GCs were collected and incubated with 10 µM fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 20 min at 38 °C. Following two washes with pre-cooled PBS twice, fluorescence was measured by flow cytometry with excitation at 488 nm and emission fluorescence at 525 nm.

After treatments, cells or SYFs were harvested to assess the intracellular levels of CAT, T-SOD, MDA, GSH, and ATP. For the measurement of oxidative parameters in the cultured ovarian follicles, tissues were homogenized in PBS and then centrifuged at 2500 rpm for 10 min at 4 °C to obtain a 10% tissue homogenate. The supernatants were used for the determination of total protein concentration and subsequent measurements of oxidative parameters. Total protein concentration, CAT, T-SOD activities, and the levels of MDA, GSH, and ATP were measured according to the manufacturer's instruction using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The SYFs, ASYFs, ovarian cortex, and the cultured follicles were washed with PBS three times and fixed in 4% paraformaldehyde for 24 h at 4 °C. Samples were then dehydrated, embedded in paraffin, and sectioned at a thickness of 5 µm. Hematoxylin and eosin (H&E) staining was performed following standard procedures. For IHC staining, paraffin sections were deparaffinized in xylene, rehydrated through a graded alcohol series, and treated with 3% hydrogen peroxide to block endogenous peroxidase activity. Antigen retrieval was conducted according to standard protocols. The sections were washed three times using PBS and then blocked by 10% normal goat serum for 20 min. Subsequently, the slides were incubated overnight at 4 °C with primary antibody of rabbit anti-p53 (1:50, ET7107-33, Huabio, Hangzhou, China). After washing with PBS three times, sections were incubated with goat anti-rabbit IgG-HRP (1:500, HA1001, Huabio) at 37 °C for 1 h. Immunoreactivity was visualized using DAB substrate, followed by hematoxylin counterstaining for nuclear visualization. Images were captured using an Eclipse 80i microscope (Nikon).

The chicken follicular slides were deparaffinized, hydrated, and subjected to antigen retrieval following established laboratory procedures. GCs were seeded onto sterilized glass coverslips placed in 24-well plates and cultured according to a standard protocol. After the indicated treatments, the cell-covered coverslips were washed with PBS and fixed in 4% paraformaldehyde for 10 min. Samples were then washed three times with PBS, permeabilized with 0.5% Triton X-100 for 10 min, and then blocked with 10% normal goat serum for 20 min at room temperature. The follicular sections and cell coverslips were incubated overnight at 4 °C with anti-β-catenin (1:50, Huabio), anti-BrdU antibody (1:200, G3G4; DSHB, Iowa City, IA, USA), or anti-Lamin B1 (1:100, ET1606-27, Huabio). After washing three times with PBS, samples were incubated with goat anti-rabbit Alexa Fluor 594 (1:500, A11037, Abclonal, Wuhan, China) for 1 h at 37 °C in the dark. Nuclei was stained with DAPI (Beyotime) for 5 min at room temperature. Images were captured using laser confocal microscopy (Olympus IX81-FV1000).

Western blotting was performed using the conventional protocol. Briefly, total protein from tissues and cells was extracted on ice using RIPA buffer (P0013B, Beyotime) supplemented with phosphatase inhibitor cocktail. Protein concentration was determined with a BCA protein assay kit (P0009, Beyotime). Cytoplasmic and nuclear extracts were prepared using the Nuclear Protein Extraction Kit (R0050, Solarbio, Beijing, China) following the manufacturer's instructions. Protein samples were diluted to 2 µg/µL using loading buffer and denatured at 100 °C for 10 min. Equal amounts (10 µL) of protein were loaded onto a 10% SDS-polyacrylamide gel (8.6 cm × 6.8 cm × 1.0 mm) prepared using the SDS-PAGE Gel Preparation Kit (FD2190, Fudebio) and separated at a constant voltage of 200 V for 1 h. The separated proteins were electrotransferred onto nitrocellulose membranes (Millipore, Darmstadt, Germany) at a constant current of 200 mA for 45 min at room temperature. Membranes were blocked with 5% non-fat milk for 1 h at room temperature, then incubated overnight at 4 °C with the following primary antibodies: CCND1 (1:500, ER0722, Huabio), CDK2 (1:500, R1309-3, Huabio), CDK1 (1:500, ER31213, Huabio), Cyclin B1 (1:500, R1308-13, Huabio), Caspase-3 (1:500, ER1802-42, Huabio), cleaved Caspase-3 (Asp175) (1:500, 9664, Cell Signaling Technology, Beverly, MA, USA), TOMM20 (1:1000, ET1609-25, Huabio), HO-1 (1:500, ER1802-73, Huabio), NQO1 (1:500, ER1802-85, Huabio), Nrf2 (1:500, ER1706-41, Huabio), phospho-Nrf2 (1:1000, ET1608-28, Huabio), Lamin B1 (1: 1000, Huabio), p53 (1:500, Huabio), CDKN1A/p21 (1:500, HA500156, Huabio), GSK3 beta (1:500, ET1607-7, Huabio), phospho-GSK3 beta (1:500, ET1607-60, Huabio), β-catenin (1:500, Huabio), Histone H3 (1:500, R1105-1, Huabio), and β-actin (1:5000, EM2001-07, Huabio). Following three washes with TBST, the membranes were probed with an HRP-conjugated secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized using clarity ECL Western blot substrate kits (Bio-Rad, #1705061, Hercules, CA, USA) and imaged with a ChemiScope 3400 Mini machine (Clinx, Shanghai, China). Band intensities were quantified using ImageJ v2.3.0 software (NIH), and target protein expression levels were normalized to β-actin as control.

Total RNA was extracted from tissues using Trizol reagent. Reverse transcription was conducted using the HiScript II 1st Strand cDNA Synthesis Kit (R211-01, Vazyme, Nanjing, China) with 1 µg of total RNA in accordance with the manufacturer's instructions. The qRT-PCR was performed to assess the expression of gene which involved in glycolysis by using a HiScript II One Step qRT-PCR SYBR Green Kit (Q221-01, Vazyme, Nanjing, China). The primers used in the PCR are listed in Table S1. The relative mRNA expression levels were calculated using the 2^−∆∆Ct method, with β-actin serving as the internal reference gene.

The cultured SYFs were collected and fixed in 2.5% glutaraldehyde overnight at 4 °C, followed by post-fixed in buffered 1% osmium tetroxide for 1.5 h. Sample were then dehydrated through a grade series of ethyl alcohol or acetone, and eventually wrapped in epoxypropane resin according to the standard TEM procedures. Ultrathin sections (70–90 nm) were obtained using an ultramicrotome (Leica EM UC7, Wetzlar, Germany), stained with 8% aqueous uranyl acetate and Reynold's lead citrate, and observed under a Tecnai G2 Spirit (FEI Company, Hillsboro, OR, USA) with an acceleration voltage of 120 kV at various magnifications.

All data are presented as the mean ± SEM. T-test was used to assess the difference between two samples. Statistical differences between groups were analyzed and determined by one-way analysis of variance (ANOVA) followed by Tukey's test or Dunnett's test. The results were regarded as significant when p < 0.05.

As shown in Figure 1A, compared with the control group, GCs exposed to 10 µM, 20 µM and 40 µM fisetin for 24 h exhibited a significant increase in cell viability, with 20 µM concentration displaying the highest effect. As expected, treatment with 20 µM fisetin significantly restored the viability of GCs following D-gal exposure (Figure 1B). β-galactosidase staining, a widely used approach for detecting cellular senescence, further confirmed these findings. is the most commonly used approach for detecting senescent cells. As presented in Figure 1C,D, pretreatment with 20 µM fisetin markedly reduced the proportion of β-galactosidase-positive cells. These results demonstrated that fisetin reversed the D-gal-induced senescence in SYF-GCs.

Both replicative and premature senescent cells exhibit significant proliferation inhibition. As shown in Figure 2A–E, D-gal markedly decreased the expression of the proliferation-related proteins CCND1, CDK2, CDK1, and Cyclin B1, whereas fisetin pretreatment effectively reversed these alterations. Specifically, CCND1 expression was elevated in the groups co-treated with D-gal and fisetin at doses of 5, 10, 20, and 40 µM compared with the D-gal group, with the 5 and 10 µM combination groups showing no significant difference from the control. The D-gal-induced downregulation of CDK2 was significantly reversed by fisetin at 10, 20, 40 µM, although expression in the 40 µM fisetin and D-gal combination group did not reach control levels. Similarly, CDK1 expression was elevated in the 5, 10, 20, and 40 µM fisetin-pretreated groups relative to the D-gal alone, except in the 5 and 40 µM groups, were comparable to the control. Consistently, Cyclin B1 levels exhibited a dose-dependent upregulation in D-gal-induced SYF-GCs with increasing concentrations of fisetin from 10 to 40 µM. Our previous findings further suggested cellular senescence by demonstrating a marked G1 phase arrest in D-gal-treated SYF-GCs, aligning with the key feature of cell cycle arrest [1]. However, fisetin pretreatment effectively alleviated this cell-cycle arrest triggered by D-gal -induced damage (Figure 2F,G). Furthermore, fisetin mitigated the reduction in EdU-labeling cells caused by D-gal treatment, thereby restoring SYF-GCs proliferation capacity (Figure 2H,I). The mRNA levels of follicle-stimulating hormone (FSHR), steroidogenic acute regulatory (StAR), cytochrome p450 family 11 subfamily A member1 (CYP11A1), leukemia inhibitory factor (LIF), leukemia inhibitory factor receptor (LIFR), cyclooxygenase 2 (COX2), specificity protein 1 (SP1), GATA binding 6 (GATA6), insulin-like growth factor 1 receptor (IGFR1), and inhibin activin subunit beta A (INHBA) in D-gal-induced senescent GCs were significantly upregulated after the addition of fisetin (Figure 2J,K).

The results of JC-1 staining revealed that compared with the control, D-gal-treated GCs showed reduced red fluorescence, enhanced green fluorescence and a decreased MMP, indicating mitochondrial dysfunction. The effect was effectively reversed in GCs pretreated with fisetin (Figure 3A,B). Similarly, Western blot analysis showed that TOMM20, a mitochondrial mass marker, was significantly upregulated in D-gal-induced senescent GCs following fisetin treatment. GCs treated with fisetin alone also exhibited higher TOMM20 expression compared to the control group (Figure 3C,D). Notably, GCs co-exposed to D-gal and fisetin exhibited a low cleaved-caspase-3 (C-Caspase-3)/Caspase-3 ratio, indicating that the resulting mitochondrial damage occurred independently of apoptosis in GCs (Figure 3E,F). Pretreatment of fisetin further enhanced the mRNA levels of mitochondrial biogenesis-related genes, including mitochondrial transcription factor B2 (TFB2M), mitochondrial transcription factor A (TFAM), RNA polymerase, mitochondrial transcriptional (POLRMT), and ATP synthase membrane subunit 8 (ATPase8) in D-gal-induced senescent GCs (Figure 3G). In addition, the decreased ATP content in D-gal-induced senescent GCs was reversed by fisetin pretreatment, while fisetin administration alone significantly elevated ATP levels relative to the control (Figure 3H).

Flow cytometry analysis following DCF-DA staining showed that ROS levels were markedly enhanced in D-gal-induced senescent GCs, whereas fisetin pretreatment significantly attenuated this increase (Figure 4A,B). Similarly, the D-gal-induced reduction in antioxidant enzyme activities, including T-SOD and CAT, was effectively restored by fisetin pretreatment (Figure 4C,D). Consistent with these findings, MDA levels in the D-gal group were significantly higher than the other two groups (Figure 4E). WB assay showed that the protein levels of p-Nrf2/Nrf2, HO-1, and NQO1 were significantly decreased in D-gal-induced senescent GCs relative to control, and fisetin pretreatment prevented this decline effectively. To further elucidate the role of Nrf2 in mediating fisetin's protective effects against D-gal-induced oxidative stress, Nrf2/HO-1 signaling was inhibited using the specific Nrf2 inhibitor, ML385. Pretreatment with ML385 blocked the fisetin-induced activation of Nrf2/HO-1 signaling and attenuated GSH activity in D-gal-induced senescent GCs (Figure 4F–H). At the transcriptional level, qRT-PCR analysis revealed that inhibition of Nrf2/HO-1 signaling reversed the fisetin-induced upregulation of antioxidant genes, including SOD, CAT, glutathione S-transferase alpha (Gsta), glutathione reductase (Gsr), and microsomal glutathione S-transferase (Mgst), in D-gal-induced senescent GCs (Figure 4I). In addition, the improvements in mitochondrial dysfunction observed in fisetin-treated senescent GCs were abolished by Nrf2/HO-1 inhibition (Figure 4J,K).

As shown in Figure 5A, p53 expression in the granulosa layers of ovarian follicles from D580 hens markedly higher than in D280 hens, except in the vitelline membrane, and was strongly expressed in the ASYFs. IF staining showed β-catenin was predominantly localized in the cytoplasm of GCs in SYFs and ovarian follicles from D280 hens. In contrast, β-catenin expression was markedly reduced in ovarian follicles and SYFs from D580 hens and was almost undetectable in ASYFs (Figure 5B). WB analysis indicated that Lamin B1, β-catenin, and the proliferation-related protein CCND1, were all significantly down-regulated in SYF-GCs at D580 hens compared to D280 hens, whereas p53 expression was markedly increased with aging. (Figure 5C,D). In addition, the senescence-related genes p53, p21, and p15 mRNA levels showed a significant increase in SYF-GCs from D580 hens, while the gene of SIRT1 showed a down-regulation expression in SYF-GCs from D580 hens compared to that from D280 hens (Figure 5E).

Compared to the control, D-gal treatment significantly decreased β-catenin while increasing Axin. In contrast, fisetin partly restored this phenomenon (Figure 6A). Additionally, qRT-PCR analysis revealed that fisetin reduced the D-gal-induced upregulation of key senescence-related genes, namely p53, p21, and p16, in SYF-GCs. Fisetin treatment robustly promoted β-catenin accumulation in the nucleus, as evidenced by IF staining and confirmed through separate Western blot analysis of nuclear and cytoplasmic protein extracts (Figure 6B,C). To investigate the involvement of β-catenin in fisetin-mediated effects on GC proliferation and senescence, we employed the specific inhibitor IWR-1. Inhibition of β-catenin completely abolished the ability of fisetin to downregulate p53 and p21 expression, thereby also preventing the fisetin-induced increase in CDK1 activity in senescent GCs (Figure 6D,E). Correspondingly, IWR-1 treatment prevented fisetin from alleviating D-gal-induced cell cycle arrest (Figure 6F,G). Furthermore, the upregulation of Lamin B1 by fisetin was suppressed upon co-treatment with the β-catenin inhibitor (Figure 6H).

In naturally aged SYFs from D580 hens, fisetin treatment activated Nrf2 signaling and increased β-catenin expression, whereas the addition of the inhibitors ML385 (Nrf2 inhibitor) and IWR-1 (β-catenin inhibitor) reversed these effects (Figure 7A,B). Morphology observation indicated a notable promotion of follicular development and maintenance of structural integrity in SYFs by fisetin, as evidenced by tightly arranged granulosa and theca layers relative to the control group. Conversely, inhibition of Nrf2 and β-catenin expression concurrently disrupted GC integrity and altered follicle morphology with loosely and irregularly arranged granulosa layers (Figure 7C). TEM analysis confirmed that the inhibitor abolished the protective effect of fisetin on mitochondrial ultrastructure in GCs from D580-SYFs (Figure 7D–G). Biochemical analysis showed that fisetin increased the activities of T-SOD and CAT, as well as GSH content, and reduced MDA levels in D580 SYFs; these effects were abrogated by co-treatment with ML385 or IWR-1 (Figure 7H–K). Western blotting further confirmed the upregulation of TOMM20 by fisetin, which was effectively suppressed by the inhibitor (Figure 7L,M). Fisetin treatment increased ATP content in D580-SYFs, which was blocked by inhibition of Nrf2 and β-catenin (Figure 7N). BrdU incorporation assays revealed that fisetin significantly increased the proliferation of D580 SYFs compared to control, whereas inhibition of Nrf2 and β-catenin signaling attenuated this effect (Figure 8A,B). Correspondingly, fisetin-induced upregulation of CDK1, CDK2, and Cyclin B1 was markedly reduced by Nrf2 or β-catenin inhibition (Figure 8C–F).