Ningxin-Tongyu-Zishen formula alleviates the senescence of granulosa cells on D-galactose-induced premature ovarian insufficiency mice

Chemical composition analysis of NTZF

NTZF is composed of ten kinds of medicinal materials (Table 1), and its chemical composition is complex. Figure 1A shows total ion chromatography (TIC) of NTZF, and the TIC of single medicinal material is shown in Figure 1B–1K. The compounds contained in NTZF and single medicinal material are marked in the figure, including Adenosine (No. 1), Gallic acid (No. 2), 5-Hydroxymethyl-2-furaldehyde+ (No. 3), Magnoflorine (No. 4), Caffeic acid (No. 5), (+)-Catechin (No. 6), Chlorogenic acid (No. 7), Paeoniflorin (No. 8), Ferulic acid (No. 9), Quercetin (No. 10), Isorhamnetin (No. 11), and Linoleic acid (No. 12). The content of different compounds in NTZF was determined by single point external standard method, which were calculated according to the peak area ratio of standard substance to target substance in TIC (Table 2). The 2D structure of each compound was found in https://pubchem.ncbi.nlm.nih.gov/. The contents of different compounds in the formula of medicinal materials are shown in Supplementary Tables 1-1 and 1-2.

Figure 1. The experimental scheme and LC-MS/MSTIC of NTZF. (A) TIC of NTZF; (B–K) TIC of each Chinese medicinals in NTZF. Other labels: (No. 1) Adenosine, (No. 2) Gallic acid, (No. 3) 5-Hydroxymethyl-2-furaldehyde+, (No. 4) Magnoflorine, (No. 5) Caffeic acid, (No. 6) (+)-Catechin, (No. 7) Chlorogenic acid, (No. 8) Paeoniflorin, (No. 9) Ferulic acid, (No. 10) Quercetin, (No. 11) Isorhamnetin, and (No. 12) Linoleic acid.

Peak No.	Compound	Molecular formula	CAS	Content (μg/mL)	Chemical structure
1	Adenosine	C10H13N5O4	58-61-7	4.84
2	Gallic acid	C7H6O5	149-91-7	9.55
3	5-Hydroxymethyl-2-furaldehyde+	C6H6O3	67-47-0	7.68
4	Magnoflorine	C20H24NO4+	2141-09-5	4.16
5	Caffeic acid	C9H8O4	331-39-5	3.18
6	(+)-Catechin	C15H14O6	154-23-4	0.76
7	Chlorogenic acid	C16H18O9	327-97-9	0.52
8	Paeoniflorin	C23H28O11	23180-57-6	150.86
9	Ferulic acid	C10H10O5	1135-24-6	0.93
10	Quercetin	C15H10O7	117-39-5	0.35
11	Isorhamnetin	C16H12O7	480-19-3	0.07
12	Linoleic acid	C18H32O2	60-33-3	2.11

NTZF effectively improved the ovarian function of POI mice

Vaginal smears were used to evaluate the estrous cycle of experimental animals. The estrous cycle is divided into four stages [20], including proestrus, estrus, metestrus, and diestrus. Proestrus is dominated by nucleated epithelial cells (NEC); cornified epithelial cells (CEC) are the main components of estrus; epithelial cells, cornified epithelial cells, and leukocytes (L) can be observed in metestrus; the diestrus is marked by leukocytes (Figure 2A). Compared with control group, the estrous cycle of mice in model group was significantly prolonged (^*p < 0.05, Figure 2B), while M-NTZF could effectively restore the abnormal estrous cycle of mice (^#p < 0.05, Figure 2B). The body weight of the mice in each group was compared before and at the end of the experiment. The body weight of the model mice increased slowly, which was significantly different from control group (^**p < 0.01, Figure 2C). However, NTZF could effectively restore the weight gain of mice (^##p < 0.01, Figure 2C). We speculated that this result may be due to the overall conditioning characteristics of TCM formula.

Figure 2. NTZF can improve the estrous cycle, body weight, serum sex hormone levels and ovarian primordial follicle differentiation. (A) Representative pictures of different estrous cycles (×200 magnification). NEC indicates nucleated epithelial cells, CEC indicates cornified epithelial cells, L indicates leukocytes. (B) Statistical figure of estrous cycle, n = 8. (C) Statistical chart of the difference in body weight of mice before and at the end of the experiment, n = 8. (D) The concentration changes of FSH, LH, E2 and AMH in serum of mice, n = 4. (E) Representative images of HE staining (×400 magnification). Blue arrows indicate primordial follicles, yellow arrows indicate growing follicles, orange arrow indicates mature follicles, and black arrow indicates atretic follicles. (F) Statistics of the ratio of primordial follicles in the ovary of mice. The ratio of primordial follicles = the number of primordial follicles/the total number of follicles ×100%, n = 3. (^*p < 0.05, ^**p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01, versus model group).

The levels of FSH, LH, E₂, and AMH in the serum of mice were detected by ELISA (Figure 2D). Compared with control group, the levels of FSH (^*p < 0.05) and LH (^**p < 0.01) in model group were significantly increased, and E₂ was significantly inhibited (^**p < 0.01). Compared with model group, E₂ was significantly increased after NTZF or EV treatment (^##p < 0.01). At the same time, FSH of mice in H-NTZF group was significantly decreased (^##p < 0.01), and LH of mice in estradiol valerate (EV) group was relatively decreased (^#p < 0.05). In addition, AMH of mice in NTZF group was significantly higher than that in model group (^##p < 0.01) and showed a gradient upward trend with the increase of NTZF treatment concentration. The above results suggested that NTZF improves the disordered serum sex hormone levels in POI mice.

HE staining was used to observe the morphological changes of follicles in the ovaries of mice. The typical HE staining images of follicles in different treatment groups were shown in Figure 2E. It was known that the main defect caused by POI is the decrease in the ratio of primordial follicle differentiation [21]. To study the improvement effect of NTZF on this situation, we compared the ratio of primordial follicles in ovarian sections (Figure 2F). We found that the ratio of primordial follicles in model group was significantly lower than that in control group (^**p < 0.01). M-NTZF significantly increased the ratio of primordial follicles in POI mice (^#p < 0.05), but EV and H-NTZF had a more obvious effect on increasing the ratio of primordial follicles in POI mice (^##p < 0.01). The above results indicated that NTZF can improve the abnormal differentiation of primordial follicles caused by POI.

NTZF increased the antioxidant capacity of ovaries in POI mice

To evaluate the effect of NTZF on OS in the ovarian tissue of POI mice, we measured the levels of GPX and SOD (Figure 3A, 3B). Compared with control group, GPX (^*p < 0.05) and SOD (^**p < 0.01) in model group were significantly decreased. After EV and NTZF treatment, the decrease of GPX can be effectively reversed (^##p < 0.01). After NTZF treatment, the level of GPX in mice was significantly higher than that in control group (^*p < 0.05). At the same time, EV, M-NTZF, and H-NTZF treatment could up-regulate the level of SOD in mouse ovarian tissue (^#p < 0.05), and the ability of M-NTZF and H-NTZF to increase the level of SOD was particularly significant (^##p < 0.01). These results suggest that NTZF can play a therapeutic effect on POI by improving the antioxidant capacity of mouse ovaries.

Figure 3. NTZF alleviated oxidative stress and abnormal Sirt1/p53 signaling pathway in POI mice. (A) The level of GPX in mouse ovary. (B) The level of SOD in mouse ovary. (C, D) The levels of Sirt1 in the ovaries of mice were detected by IHC (×400 magnification). (E, F) The levels of p53 in the ovaries of mice were detected by IHC (×400 magnification). (G–I) The protein expression levels of Sirt1 and p53 in mouse ovaries were detected by western blot. (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, versus control group, n = 3; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, versus model group, n =3).

Because Sirt1/p53 signaling pathway plays an important role in regulating OS [22], we used IHC and western blot to evaluate the expression of Sirt1 and p53 in mouse ovarian tissue (Figure 3C, 3E, 3G). Compared with control group, there was no difference in the expression of Sirt1 in the ovary of model group, but H-NTZF treatment could significantly increase the expression of Sirt1 (^##p < 0.01, Figure 3D, 3H). In the ovaries of mice in model group, both IHC and western blot showed that the expression of p53 was significantly up-regulated compared with control group (^*p < 0.05, Figure 3F, 3I). Although IHC results suggested that all concentrations of NTZF could significantly reverse the up-regulation of p53 (^##p < 0.01, Figure 3F), western blot showed that only M-NTZF significantly inhibited the protein expression of p53 in mouse ovaries (^#p < 0.05, Figure 3I). These results indicate that NTZF can restore the proliferation of OGCs in the ovaries of POI mice by influencing Sirt1/p53 signaling pathway.

NTZF alleviated OGCs senescence and improved antioxidant capacity

To further explore whether NTZF could alleviate the senescence of OGCs through antioxidant effect to protect the ovaries of POI patients, we selected KGN cells for following research. In the preliminary experiment, we screened the optimum concentration of H₂O₂ (200 μM) and NTZF (1.06 mg/mL) to treat cells with CCK-8 and crystal violet staining (Supplementary Figure 1). According to the results of CCK-8 (Figure 4A) and EdU cell proliferation staining (Figure 4B, 4C), the cell proliferation rate of H₂O₂ group was significantly lower than that of control group (^***p < 0.001). Compared with H₂O₂ group, the proliferation rate of cells treated with p53 inhibitor—PFT-α was significantly increased (^##p < 0.01), while the proliferation rate of NTZF group and NTZF+PFT-α group was significantly increased (^###p < 0.001). At the same time, western blot results also suggested that the proliferation-related index PCNA also had higher protein expression in NTZF group and NTZF+PFT-α group (^#p < 0.05, Figure 4D, 4E). For in vivo experiments, IHC results of PCNA also showed that compared with control group, the expression of PCNA in the ovary of POI model group was significantly decreased (^***p < 0.001, Figure 4G), while EV and NTZF treatment promoted the expression of PCNA in the ovary (^##p < 0.01). The above results suggest that NTZF can increase the level of PCNA. In addition, we quantified the protein expression of γ-H2AX, a marker of DNA damage. The results showed that the cells in each group treated with H₂O₂ showed obvious DNA damage (Figure 4D, 4F). However, the expression of γ-H2AX protein in NTZF group and NTZF+PFT-α group was significantly lower than that in H₂O₂ group (^##p < 0.01), indicating that NTZF has a protective effect on H₂O₂-induced DNA damage in KGN cells. It was known that proliferation inhibition and DNA damage were typical characteristics of cell senescence. To further clarify the cell senescence of KGN cells, we also stained the biomarker SA-β-gal of cell senescence (Figure 4H). Compared with H₂O₂ group, the ratio of positive signals in PFT-α group, NTZF group, and NTZF+PFT-α group was significantly decreased (^###p < 0.001). In summary, NTZF had a significant protective effect on H₂O₂-induced KGN cells senescence.

Figure 4. NTZF alleviated OGCs senescence and improved antioxidant capacity. (A) The cell proliferation rate was detected by CCK-8. (B, C) Cell proliferation was detected by EdU cell proliferation staining (×200 magnification). The ratio of EdU positive cells = the number of EdU stained cells/the number of DAPI stained cells ×100%. (D–F) The protein expression of PCNA and γ-H2AX was detected by western blot. (G) The levels of PCNA in the ovaries of mice were detected by IHC (×400 magnification). (H) SA-β-gal staining was used to detect cell senescence (×400 magnification). Blue staining indicated cell senescence. (I) The intracellular ROS level was detected by DCFH-DA probe. Green fluorescence indicated ROS expression. (J) The level of intracellular GPX. (K) The level of intracellular SOD content (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, versus control group, n = 3; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, versus H₂O₂ group, n = 3).

To clarify the effect of NTZF on OS in OGCs, the levels of ROS, GPX, and SOD in KGN cells were detected again (Figure 4I–4K). The cells were labeled by DCFH-DA probe, and the results showed that the ROS level in KGN cells in H₂O₂ group was significantly increased (^***p < 0.001), while the ROS level in KGN cells treated with NTZF or NTZF combined with PFT-α in advance was significantly lower than that in H₂O₂ group (^###p < 0.001). At the same time, KGN cells pretreated with NTZF also showed a significant increase in GPX (^#p < 0.05) and SOD (^##p < 0.01). Therefore, NTZF improved the antioxidant capacity of ovarian granulosa cells. Therefore, NTZF can improve the antioxidant capacity of OGCs and correct the OS state of H₂O₂-induced imbalance between intracellular ROS and antioxidant levels.

NTZF protects OGCs by regulating Sirt1/p53 signaling pathway

In order to further clarify the mechanism of NTZF protecting H₂O₂-induced OGCs through Sirt1/p53 signaling pathway, we detected the mRNA and protein expression of Sirt1, p53 and p21 in cells. The expression of Sirt1 and p53 in each group of cells was semi-quantitatively determined by IF (Figure 5A–5D). The results showed that compared with control group, the expression of Sirt1 in H₂O₂ group was significantly decreased (^*p < 0.05), and the expression of p53 was significantly increased (^***p < 0.001). Compared with H₂O₂ group, the expression of Sirt1 in NTZF group and NTZF + PFT-α group was significantly increased (^###p < 0.001), and the expression of p53 was significantly decreased (^###p < 0.001). The expression levels of Sirt1, p53 and p21 mRNA were detected by q-PCR (Figure 5E–5G). It was found that there was no significant difference in the level of Sirt1 mRNA in each group (p > 0.05), but NTZF treatment could significantly inhibit the transcription of p53 mRNA (^#p < 0.05) and p21 mRNA (^##p < 0.01). In western blot detection (Figure 5H), the expression trend of Sirt1 protein in NTZF group and NTZF + PFT-α group was the same as that of IF (Figure 5I). The protein expression of p53 and p21 was significantly lower than that of H₂O₂ group (^#p < 0.05, Figure 5J, 5K), and the inhibitory effect of NTZF + PFT-α group on p53 protein expression was more significant (^##p < 0.01). In summary, NTZF can promote the expression of Sirt1 protein level, inhibit the transcription and protein expression of p53 and p21, and thus play a role in protecting OGCs.

Figure 5. NTZF protecting OGCs in vitro was associated with Sirt1/p53 signaling pathway. (A–D) The expression of Sirt1 and p53 in each group was detected by IF (×400 magnification). (E–G) The relative mRNA levels of Sirt1, p53, and p21 were analyzed by q-PCR. (H–K) Western blot was used to analyze the protein expression of Sirt1, p53, and p21 in each group (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, versus control group, n = 3; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, versus H₂O₂ group, n = 3).

Preparation of drugs

NTZF is composed of ten kinds of medicinal materials, including Rehmanniae Radix Praeparata, Testudinis Carapax et Plastrum, Codonopsis Radix, Cuscutae Semen, Angelicae Sinensis Radix, Ziziphi Spinosae Semen, Moutan Cortex, Paeoniae Radix Alba, Dioscoreae Rhizoma, and Bupleuri Radix (in a weight ratio of 10:4:4:4:5:4:3:4:5:2, Table 1). All medicinal materials were purchased from the Third Affiliated Hospital of Zhejiang Chinese Medicine University. NTZF was prepared as follows [38]: the medicinal materials were soaked in distilled H₂O (dH₂O, 1:8 w/v) for 30 min, and then boiled with strong fire until the water boiled. Subsequently, the medicinal materials were decocted with soft fire for 40 min, and then the water decoction was filtered out. The medicinal materials were decocted again in the same way. After mixing, the decoction was concentrated (2.7 g/mL). The liquid was stored at 4°C.

In this experiment, we also chose EV tablets (commonly used drugs for POI in clinic, J20171038, Bayer, Germany) as a positive control, and EV suspension (0.01 mg/mL) was diluted with dH₂O.

Chemical composition analysis of NTZF

We used Q-Orbitrap liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to qualitatively analyze NTZF, and a total of 71 chemical components were detected in previous studies [38]. In this study, we further quantified 12 of the 71 chemical components contained in NTZF. In addition, the contents of these 12 chemical components in single medicinal materials were also detected. The decoction was extracted using anhydrous methanol in a ratio of 5:1, and the resulting extract was utilized for subsequent analysis. The MS conditions were as follows: Ion source: electrospray ionization source (ESI); Scanning mode: positive and negative ion switching scanning; Detection methods: parallel reaction monitoring (PRM); Resolution: 17500; Scan range: 100-1000 m/z; Spary Voltage: 3.2 kV (Pos, Neg); Capillary Temperature: 300°C; Collision gas: high purity argon (purity≥99.999%); Sheath gas: nitrogen (purity≥99.999%), 40 Arb; Auxiliary gas: nitrogen (purity≥99.999%), 15 Arb, 350°C; Data acquisition time: 10.0 min. The chromatographic conditions were as follows: Chromatographic column: AQ-C18 150 × 2.1 mm, 1.8 μm, Welch; Flow rate: 0.3 mL/min; Needle washing liquid: methanol; Column oven temperature: 35°C; Automatic sampler temperature: 10°C; Automatic sampler injection volume: 5 μL; Water phase: 0.1% formic acid/water; Organic phase: 0.1% formic acid/acetonitrile. The gradient elution process of mobile phase is shown in Table 3. Reference standards, including Adenosine (B21356), Gallic acid (B20851), 5-Hydroxymethyl-2-furaldehyde+ (B21832), Magnoflorine (B20882), Caffeic acid (B20660), (+) -Catechin (B21722), Chlorogenic acid (B20782), Paeoniflorin (B21148). Ferulic acid (B20007), Quercetin (B20527), Isorhamnetin (B21554), Linoleic acid (B21421), were purchased from Shanghai Yuanye Bio-Technology Co., Ltd., (China).

Animals and treatment

Female C57BL/6 mice aged 6–8 weeks weighing 16–18 g, were purchased from Zhejiang Academy of Medical Sciences (China). The mice were routinely raised in Zhejiang Chinese Medical University Laboratory Animal Research Center. This is a specific pathogen-free (SPF) environment with 12 h light-dark cycles. Its ambient temperature is maintained at 20~25°C, and relative humidity is maintained at 40~70%.

Before the experiment, mice were acclimated for one week. These mice were randomly divided into six groups, including Control group, Model group, EV group (0.15 mg/kg), L-NTZF group (10.14 g/kg), M-NTZF group (20.27 g/kg) and H-NTZF group (40.54 g/kg). Except for the Control group, the other groups were subcutaneously injected with D-gal (200 mg/kg) to establish a POI mouse model [39]. The mice in control group were injected with an equal volume of normal saline (NS). Subcutaneous injection lasted from the first day of the experiment to the day of sacrifice for 42 days, and the day of subcutaneous injection was defined as Day1 (Figure 6). The clinical therapeutic dose of NTZF for women weighing 60 kg was 135 g/day, and the clinical therapeutic dose of EV tablets was 1 mg/day. The dose used in mice was calculated by the recommended human-mice conversion ratio (the ratio of human to mice was 1:9). In the study, the mice in the L-NTZF group were given 1/2 times the clinical equivalent dose, the mice in the M-NTZF group were given the same dose as the clinical equivalent dose, and the mice in the H-NTZF group were given 2 times the clinical equivalent dose. From Day15, each group began to give medicine intervention, and the dosage and duration of each group were shown in Table 4. On Day42, mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium solution (50 mg/kg), and blood and ovaries were collected. At the end of sampling, the mice were sacrificed by excessive anesthesia.

Figure 6. Overview of experimental scheme.

Observation of estrous cycle

From Day28, the estrous cycle of mice was examined daily for 15 days, and the body weight of each mouse was monitored before and at the end of the experiment. Vaginal smear cytology was performed at 15 p.m. every day. The vaginal cavity was repeatedly washed with 10 μL of NS. Vaginal secretions were smeared on slides and fixed with 95% ethanol to obtain smears. The smears were stained with 0.23% Alkaline methylene blue staining solution (R24063, Yuanye, China). Finally, the morphology of vaginal exfoliated cells was observed by optical microscope with ×100 magnification. According to the observed ratio of different cells, the stage of estrous cycle in mice was determined.

ELISA

ELISA was performed according to the manufacturer’s guidelines. The concentrations of FSH (MM-45654M1, Meimian, China), LH (MM-44039M1, Meimian, China), E₂ (MM-0566M1, Meimian, China), and AMH (MM-44204M1, Meimian, China) in mice serum were determined using mice-specific ELISA kits.

Ovarian follicle count and morphological observation

Mice ovarian tissues were fixed with 4% paraformaldehyde for at least 24 h. Then the fixed tissues were dehydrated with gradient concentration of alcohol, and embedded in paraffin. Mice ovarian wax blocks were made into 4 μm sections, baked at 60°C for 2 h, and then stained with HE. The digital pathological scanning system (Hamamatsu, Japan) was used to scan the pathological sections of mice ovarian tissue, and the number of follicles in ovarian sections was counted. Follicles can be divided into four stages [40] (Figure 2E): primordial follicles, growing follicles, mature follicles, and atresia follicles. Primordial follicles (blue arrows) were characterized by oogonia, which was surrounded by flat granulosa cells; growing follicles (yellow arrows) were characterized by one or more layers of cubic granulosa cells around the oocytes, and even follicular cavities; mature follicles (orange arrows) were characterized by a large follicular cavity with obvious cumulus; atresia follicles (black arrows) were characterized by unclear egg cell structure, shrunken zona pellucida, and collapsed follicle walls.

Cell culture and grouping

The KGN human granulosa cell line (cat. no. iCell-h298, China) conforming to short tandem repeats identification was purchased from iCell Biotechnology Co., Ltd. (China). KGN cells were cultured in DMEM/F-12 containing 10% FBS. The incubator conditions were 37°C and 5% CO₂. KGN cell in logarithmic growth phase was divided into control group, H₂O₂ group (200 μM H₂O₂) [41], PFT-α group (200 μM H₂O₂+1 μM PFT-α), NTZF group (200 μM H₂O₂ + 1.06 mg/mL NTZF), PFT-α + NTZF group (200 μM H₂O₂+1 μM PFT-α+1.06 mg/mL NTZF). NTZF diluted with PBS was added to NTZF group and PFT-α+NTZF group, and the same volume of PBS was added to the other groups. At the same time, PFT-α group and PFT-α+NTZF group were added with PFT-α dissolved in DMSO, and the other groups were added with equal volume of DMSO. After 24 h, H₂O₂ group, PFT-α group, NTZF group and PFT-α+NTZF group were added with 200 μM H₂O₂, and control group was added with the same volume of PBS. After 2 h of H₂O₂ intervention, cells in each group were subjected to subsequent detection.

Cell proliferation analysis

Cell Counting Kit (CCK-8, C0039, Beyotime, China) was used to evaluate the proliferation of KGN cells. KGN cells were inoculated into 96-well plates at 8 × 10³ cells/well. After the intervention, 10 μL CCK-8 was added to each well and continued to incubate for 2.5 h. Finally, the OD value was measured at 450 nm using a multifunctional microplate reader (M200 pro, Tecan, Switzerland).

According to the manufacturer’s guidelines in BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (C0075S, Beyotime, China), KGN cells were inoculated into 24-well plates and cultured. After the intervention, the proliferating cells were labeled with EdU. Click reaction solution was prepared according to the instructions. After 30 min incubation in the dark, the nucleus was labeled with Antifade Mounting Medium with DAPI (MA0222, Meilunbio, China). Finally, three random field images were taken with a fluorescence inverted microscope (Axio Observer A1, Zeiss, Germany). ImageJ V.1.8.0 software was used to calculate the number of EdU-stained cells and DAPI-stained cells.

SA-β-gal staining

The experiment was performed according to the guidelines in SA-β-Gal Staining Kit (C0602, Beyotime, China). To put it simply, we cultured KGN cells in 24-well plates. The cells were fixed with staining fixative at room temperature for 15 min, and then the staining solution was added and incubated at 37°C overnight. The next day, the staining results were observed under a microscope, and the percentage of senescent cells (blue stained cells) was counted using ImageJ V.1.8.0 software.

Biochemical estimations of OS markers

According to the manufacturer’s guidelines in the Reactive Oxygen Species Assay Kit (E004-1-1, Njjcbio, China), KGN cells were inoculated into 6-well plates and cultured. After the intervention, the cells were labeled with 5 μM DCFH-DA probe. Images were taken using a fluorescence inverted microscope (Axio Observer A1, Zeiss, Germany). The mean fluorescence intensity (MFI) of ROS in the image was analyzed by ImageJ V.1.8.0 software, and the data were normalized.

Proteins in mouse ovarian tissue or KGN cells were extracted with sample preparation solution, and the protein concentration was detected by BCA Protein Assay Kit (P0012, Beyotime, China). According to the guidelines of Total Glutathione Peroxidase Assay Kit (S0059S, Beyotime, China), various reagents were sequentially added to 96-well plates and incubated at room temperature for 30 min. Then DTNB solution was added and incubated at room temperature for 10 min. A multifunctional microplate reader (M200 pro, Tecan, Switzerland) was used to determine the OD value at a wavelength of 412 nm, which was used to calculate the GPX content of the sample. According to the guidelines of Total Superoxide Dismutase Assay Kit (S0101M, Beyotime, China), various reagents were added to 96-well plates in order. After incubation in 96-well plates at 37°C for 30 min, the OD value was measured at 450 nm using a multifunctional microplate reader (M200 pro, Tecan, Switzerland) to calculate the SOD content of the sample.

IHC staining

After dewaxing 4 μM mouse ovarian tissue sections, they were immersed in antigen repair solution and heated by microwave oven for antigen repair. Tissue sections were permeabilized with Enhanced Immunostaining Permeabilization Buffer (P0097, Beyotime, China) for 5 min, and then incubated with Enhanced Endogenous Peroxidase Blocking Buffer (PV-9000, ZSGB-BIO, China) for 10 min. At 37°C, the tissues were blocked with 5% goat serum for 30 min. The primary antibody was dropped and incubated overnight at 4°C. On the second day, the enhancer solution (PV-9000, ZSGB-BIO, China) was added dropwise and incubated at 37°C for 20 min. Then the secondary antibody was added dropwise and incubated at 37°C for 30 min. After dyeing with DAB, hematoxylin was used for re-dyeing. IHC sections of mouse ovarian tissue were scanned using a digital pathological scanning system (Hamamatsu, Japan). ImageJ V.1.8.0 software was used to analyze the positive expression area of the image, and the data were normalized. Detailed antibody information is listed in Supplementary Table 2.

IF staining

KGN cells were fixed with 4% paraformaldehyde for 15 min, and then washed with Immunol Staining Wash Buffer (P0106, Beyotime, China). Next, KGN cells were blocked with 10% goat serum at room temperature for 30 min, and incubated with primary antibody at 4°C overnight. The next day, cells were incubated with fluorescently labeled secondary antibodies at 37°C for 1 h, followed by sealing with Antifade Mounting Medium with DAPI (MA0222, Meilunbio, China). Images were taken with a fluorescence inverted microscope (Axio Observer A1, Zeiss, Germany). ImageJ V.1.8.0 software was used to analyze MFI of images and normalize the data. The detailed antibody information is listed in Supplementary Table 2.

q-PCR

Total RNA was extracted with Trizol, and the concentration and integrity of RNA were determined by spectrophotometry at 260 nm and 280 nm. It was reverse transcribed into cDNA using Evo M-MLV RT Premix for qPCR Kit (AG11706, AG, China) according to the manufacturer’s instructions. The relative mRNA expression levels of Sirt1, p53, and p21 were detected by SYBR Green Premix Pro Taq HS qPCR Kit (AG11718, AG, China). All samples were detected by QuantStudio3 Real-Time PCR System (ABI, USA). The relative expression of RNA was calculated using the 2^−ΔΔCt method. Target gene normalization was performed using GAPDH mRNA levels. The sequence of primers is listed in Table 5.

Western blot analysis

Proteins were extracted from mouse ovarian tissue and KGN cells using RIPA lysis buffer (G2002, Servicebio, China) containing protein phosphatase inhibitor mix (FD1002, Fdbio Science, China). The total protein concentration was measured using BCA protein assay kits (P0012, Beyotime, China). Samples (both cells and tissues) were denatured with 5× loading buffer (FD002, Fdbio Science, China) at 100°C for 5 min. Proteins were separated by SDS-PAGE and transferred to 0.22 μM PVDF membranes (Millipore, USA). It was blocked with 5% nonfat-dried milk for an hour and then incubated with primary antibody at 4°C overnight. The next day, the PVDF membrane was incubated with HRP-conjugated secondary antibody at room temperature for 1 hour. Finally, electrochemiluminescence (ECL) method was used for detection, and imaging was performed using a high-sensitivity imaging analysis system (Bio-Rad, USA). ImageJ V.1.8.0 software was used to analyze the gray value of western blot. According to the protein expression of GAPDH, the protein expression of other genes was normalized. Detailed antibody information is included in Supplementary Table 2.

Statistical analysis

Experiments were performed at least three times. The data of this study were expressed as mean ± SEM and analyzed using GraphPad Prism 8.0 software. One-way ANOVA was used for comparison between different groups. p < 0.05 was considered a statistically significant difference.