Ethical statement
All animal experiments were conducted in strict accordance with the Guide to the Management and Use of Laboratory Animals issued by the National Institutes of Health. Animal Ethics Committee of Affiliated Hospital of Shandong University of Traditional Chinese Medicine approved all the experimental procedures used in this study (Approval number: 20,220,316).
Animal models and treatment
Sixty C57/BL6 female mice aged 36 weeks were obtained from JinanPengyYue-laboratory animal breeding Co., Ltd. (SYXK20180015, Jinan, China). All mice were housed in standard conditions with a 12-h light/dark cycle at 22 ± 2 °C and a humidity of 55 ± 5% with free access to food and water. We calculated the dose of EZTG to be taken by mice in the ratio of 9:1 According to the medication instructions, the therapeutic dose of EZTG for a woman weighing 60 kg is 18 g/day. The 60 mice aged 36 weeks were randomly distributed into three groups: an aged control group (old group), a low-dose EZTG group (EZTG-L, 2.7 g/kg/day by gavage), and a high-dose EZTG group (EZTG-H, 8.1 g/kg/day by gavage). The 20 mice aged 8 weeks were used as a young control group. The EZTG Decoction are produced by the Drug Manufacturing Unit of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, batch number 01-FZ032-03. The composition traditional Chinese herbs of EZTG Decoction include: Cuscuta chinensis (Tu Si Zi), Ligustrum lucidum (Nv Zhen Zi), Herba Ecliptae (Mo Han Lian), Lycium chinense Mill (Gou Qi Zi), Angelica sinensis (Dang Gui), Radix Rehmanniae Preparata (Shu Di Huang), Ligusticum wallichii (Chuan Xiong), Paeonia lactiflora Pall (Bai Shao), Rhizoma cyperi (Xiang Fu), Radix Glycyrrhizae Preparata (Zhi Gan Cao). The EZTG group was gavaged for 12 weeks, while the control group was gavaged with equivalent doses of normal saline. All mice were anesthetized and euthanized, and their blood and ovaries were collected for subsequent use, and the weight of mice and wet weight of ovaries were measured (ovarian index = ovarian mass/body weight, mg/g).
Histopathology
Ovaries were harvested and fixed with paraformaldehyde at a concentration of 4% for more than 24 h, and dehydration, wax leaching, and embedding were performed. Ovaries were cut into 4 μm sections., which were dewaxed with ethanol and stained with Haematoxylin and eosin dyes. The number of Primordial follicles, Primary follicles, Secondary follicles, and Mature follicles is recorded based on morphological characteristics.
Serum hormone levels
Serum samples obtained before the mice were euthanized were centrifuged at room temperature for 20 min at 3000×g. Serum hormone levels, including anti-Müllerian hormone (AMH), and follicle-stimulating hormone(FSH), were measured using ELISA kits (F9402-A, F2555-A, Shanghai Fanke Industrial Co., Ltd, Shanghai, China), according to the manufacturer’s instructions.
Mitochondrial membrane potential flow cytometric assay
Ovarian tissue was cut into small pieces, digesting it completely with trypsin, adding complete culture medium, and collecting the cell suspension. MMP levels were detected using the JC-1 assay kit (E-CK-A301, Elabscience). The cell suspension were removed in each group, and 1 mL JC-1 working solution was added after washing with PBS. Negative and positive control groups were set according to the instructions. The cell suspension stained with JC-1 staining buffer and detected by flow cytometry.
Mitochondrial ROS assay
ROS assay kits (AO036-100T/96S, Yishijiu Biotechnology Co., Ltd, Jiangsu, China) were utilised to quantify ROS levels in ovary tissue, according to the manufacturer’s instructions. In short, extract mitochondria from ovarian tissue to prepare homogenate. Upon centrifugation, 50 μl Mitochondrial homogenate was mixed with 50 μl catalyst solution and 100 μl(DCFH) solution for 30 min. The 2′,7′-dichlorodihydrofluorescein (DCF) levels were quantified using fluorescence spectrometry with excitation/emission (Ex/Em) at 480/530 nm.
Enzyme-linked immunosorbent assay
In short, weigh 0.1 g of ovarian tissue, add cold phosphate buffered saline at a ratio of 1:9, and prepare 10% ovarian tissue homogenate, centrifuged at 14,000 × g at 4 °C for 5 min, and collected supernatants. MDA, GSH,8-OHdG was determined by a colorimetric assay kit (F2221-A, F30753-A, F2658-A, Shanghai Fanke Industrial Co., Ltd, Shanghai, China) according to the manufacturer’s guidelines.
Transmission electron microscope
The ovaries were collected and fixed in Transmission Electron Microscope (TEM) (Servicebio, China) at 4 ℃, and dehydrated with gradient concentrations of ethanol. Then, the ovaries were penetrated and embedded with resin and sliced. Then, the ovary section was stained with uranium acetate and lead citrate. Finally, the ovary section was observed under TEM (Hitachi, Japan), and images were captured.
Immunofluorescence staining
In brief, Paraformaldehyde-fixed ovaries were dehydrated, embedded, and sliced. Primary antibodies against PINK1(proteintech,23274-1-AP,1: 200) and Parkin(proteintech,66674-1-Ig,1:50) were added at 4 °C overnight, while the secondary antibodies were added to the sample at 37 °C for 30 min (Parkin:Servicebio,GB22301,1:100; Pink1:Servicebio,GB21303,1:200). DAPI was added at room temperature for 10 min. Images were collected by section using a fluorescence microscope camera system and analyzed by Image J software, randomly select areas of the same size from each slice to measure fluorescence intensity, with three replicates per group.
Prussian blue staining
Prussian blue staining kit (Solarbio, China) was used to detect the deposition of iron-containing heme in ovarian tissue. The paraffin sections were prepared with a thickness of 4 μ m, subjected to routine dehydration and transparency, and then stained with Perls staining solution for 30 min. The slices were thoroughly washed with distilled water for 30–50 s, and the background was lightly stained with eosin staining solution for 15–30 s, followed by rinsing with distilled water for 2–3 s. Finally, the slices were dehydrated with anhydrous ethanol, transparent with xylene, sealed with neutral glue, and placed under a light microscope for observation.
Iron assay
In short, ovarian tissue was weighed and homogenized in 1 ml ice-cold phosphate-buffered saline, centrifuged at 14,000 × g at 4 °C for 5 min, and collected supernatants. The total iron content in the ovary were detected by the Total Iron Colorimetric Assay Kit (Elabscience, E-BC-K772-M). Spectrophotometry was finally adopted to detect the absorbance at a wavelength of 593 nm.
Western blot analysis
The expression levels of mitophagy-related proteins PINK1, Parkin and ferroptosis-related proteins GPX4 and ACSL4 were detected by Western blot. After adding radioimmunoprecipitation assay lysis buffer (Beyotime), tissues were ground in a homogenizer and then lysed on ice for 1 h. The Protein concentration was determined using the BCA kit (Solarbio, Beijing, China). The protein loading buffer was added to each sample which was then boiled for 10 min, centrifuged at 4 °C for 10 min and stored. Following protein gel electrophoresis, membrane transfer for 1 h at a constant current of 250 mA in a 4 ℃ transfer solution, blocking, and incubation of primary antibody and secondary antibody, the photographic developer was added to the membrane and the band was developed in ECL chemiluminescence instrument with glyceraldehyde-3-phosphate dehydrogenase as internal reference. The protein bands were analyzed by Image J software (version: v1.8.0). The antibodies as follows: anti-GPX4(Abcam; ab125066; 1:5000), anti-ACSL4(Abcam; ab155282; 1:10000), anti-Parkin(Abcam; ab77924; 1:1000), anti-PINK1(Abcam; ab300623; 1:1000), anti-GAPDH(Proteintech; 60004-1-Ig; 1:5000).
Statistical analysis
Continuous variables are expressed as mean ± SD throughout this study. Differences between the two groups were analyzed using one-way ANOVA followed by Duncan’s post hoc test. All statistical analyses were performed with the SPSS 25.0 and Image-J statistical software. A P-value < 0.05 was considered statistically significant.
Add Comment