PM2.5 sample collection

PM2.5 samples were collected in Gulou District, Fuzhou City, using a Thermo Scientific GUV-15-H-1 large flow sampler equipped with a G1200-41 PM2.5 sampling head and a quartz fiber membrane (Whatman, USA) at a flow rate of 1.13 m³/min. Before sampling, the fiber membrane was calcined at 450 ℃ for 5 h in a Maboiler furnace to remove any residual organic matter. The fiber membrane was replaced every 24 h. The PM2.5-collecting fiber membrane was cut into 1 cm×1 cm pieces, immersed in ultrapure water, placed in an ultrasonic oscillator, shaken at 4 °C for 2 h and then filtered through eight layers of sterile gauze. The filtered liquid was added to a sterile centrifuge tube, centrifuged at low temperature and high speed (4 °C, 12,000 rpm, 30 min), and the supernatant was removed. The precipitates were then dried in a vacuum freeze-dryer for 12 h and stored at 4 °C for later use. The PM2.5 exposure concentration standard was based on the 24-hour average annual concentration limit (35.75 µg/m³) from the People’s Republic of China Ambient Air Quality Standard (GB3095-2012) and the Ambient Air Quality Index (AQI) Technical Regulations (Trial). Values exceeding 150 µg/m³ were classified as heavy pollution. PM2.5 powder was weighed and exposed to ultraviolet irradiation for 1 h on an ultra-clean bench. Phosphate-buffered saline (PBS) was added to achieve the optimal concentration and then sterilized for later use.

Mouse model for PM2.5 exposure

A total of 30 healthy SPF female SD mice, each weighing approximately 50 g, were obtained from the Animal Medical Center of Comparative Medicine Laboratory Department of the Ninth Hospital with the production license No: SCXK (Shanghai) 2017-0001. After a seven-day adaptation period, the 30 SD mice were randomly divided into three groups of 10: the control group, the PM2.5 low-dose group, and the PM2.5 high-dose group. The mice received PM2.5 suspension via airway infusion once every 3 days. The exposure doses for the mouse model were based on the 24-hour average PM2.5 concentration standards in China. The low-dose group was exposed to 35 µg/m³, while the high-dose group was exposed to 150 µg/m³. The exposure dose was calculated based on the ventilation and lung deposition rates of healthy adult mice, with the low dose being 3.9 µL and the high dose 16.65 µL. The concentration of the suspension was 2 µg/µL. After five months of PM2.5 airway infusion, castration surgery was performed, and the ovaries were carefully sectioned for subsequent hematoxylin and eosin (H&E) staining.

Histology

Ovarian tissue of mice was fixed in 4% paraformaldehyde overnight, dehydrated using ethanol gradient, and embedded in paraffin. H&E staining (Boster, Wuhan, China) was performed on Serial 5-µm ovary sections mounted on glass slides. The tissue sections were examined under a microscope.

Cell culture and treatment

The ovarian granulosa cell line (KGN cells) was purchased from Procell (Wuhan, China) and confirmed as ovarian granulosa cells. KGN cells were cultured in DMEM/F12 (Procell, China), while HEK293T cells were cultured in DMEM (BasalMedia, China). The culture medium was supplemented with 1% Penicillin-Streptomycin solution (100 U/mL; Bioss, China) and 10% fetal bovine serum (FBS; Gemini, USA). All cells were incubated at 37℃ in a 5% CO2 atmosphere.

Cell viability analysis

A 6-well white plate was cultured KGN with PM2.5 concentrations of 0, 50, 100, 150, 200 and 300 µg/mL ( The selection of 100 µg/ml was based on the reference to China’s Ambient Air Quality Standards (GB3095-2012) for Class I and II 24-hour average annual concentration limits (35 µg/m³ and 75 µg/m³), as well as the annual average PM2.5 concentration of 150 µg/m³ reported in Greenpeace’s “2015 Annual PM2.5 Concentration Rankings of 336 Cities in China ), and the cell state under each concentration was observed under a microscope. All steps were repeated three times to ensure high repeatability of the experiment.

Cell viability was analyzed by adding PBS supplemented with Cell Titer-Glo (Promega, USA) to the affected cells in 96-well white plates at 0, 12, 24, 36, 48, and 60 h after the addition of PM2.5 (100 µg/mL).

Apoptosis analysis

KGN were immersed in PM2.5(100 µg/mL) suspension for 24 and 36 h, then directly collected into centrifuge tubes and centrifuged. The precipitated cells were washed once with incubation binding buffer, then 500 µL of buffer was added for resuspension. The suspended cells were incubated with fluorescent Annexin V-FITC (Biolegend, USA) solution for 15 min at 4 °C, avoiding any exposure to light. Fluorescent Propidium Iodide (PI, Sangon Biotech, China) was then added and incubated for 30 min before flow cytometry analysis. The flow cytometer was set with an excitation wavelength of 488 nm to detect Fluorescein isothiocyanate (FITC) fluorescence signals using a 515 nm passband filter, while a filter with a wavelength greater than 560 nm was used to detect PI.

Cell cycle analysis

The KGN were co-cultured with PM2.5 (100 µg/mL) for 24 h, then directly collected into a centrifugal tube. The cells were washed twice and then mixed with anhydrous ethanol and PBS in a 7:3 ratio. The mixture was slowly resuspended and fixed overnight at 4 °C. The cells were stained with PI solution and incubated at room temperature. The cell cycle was identified using flow cytometry and the data were processed with FlowJo-10 software.

Western blot analysis

KGN cells were washed twice with pre-cooled PBS, followed by the addition of RIPA buffer in the appropriate ratio. Proteins were separated by SDS-PAGE based on their molecular weight and then transferred to a PVDF membrane, which was subsequently blocked with 5% skimmed milk. The membrane was incubated overnight at 4 °C with various primary antibodies. Afterward, the membrane was incubated with the corresponding secondary antibody. The antibodies used were as follows: anti-ATF3 (Abcam, USA, 1:1000), anti-BAX (Cell Signaling, USA, 1:1000), anti-BCL2 (Cell Signaling, USA, 1:1000), anti-GAPDH (SAB, China, 1:10000), and anti-β-ACTIN (SAB, China, 1:10000).

Inflammatory cytokines analysis by ELISA

Inflammatory cytokines TNF-α, IL-6, and IL-1β were measured in cell supernatants using Human IL-1β and IL-6 ELISA kits (Multi Science, China) and a Human TNF-α ELISA kit (Boster, China) following the manufacturers’ instructions. The preparation of standards and samples followed the instructions provided with each kit. Standard curves for TNF-α, IL-6, and IL-1β were generated, and their concentrations in the samples, assayed in duplicate, were calculated.

ROS assay

Reactive oxygen species (ROS) were measured after 36 h incubations with PM2.5 (100 µg/mL). The cells were incubated with a MitoSOX working solution containing the DCFH-DA probe (Beyotime, China) for 20 min at 37℃. After incubation, the MitoSOX working solution was removed, and the cells were collected. Finally, resuspended with 500 µL of the serum-free medium and measured using FACS. After adding the MitoSOX working solution, it is necessary to avoid light exposure.

Cell transfection and lentiviral infection

For lentivirus production, lentiviral vectors were co-transfected into HEK293T cells along with packaging vectors psPAX2 (#12260, Addgene) and pMD2.G (#12259, Addgene). The medium was changed 6 h post-transfection. Infectious lentivirus particles were harvested at 48 h after transfection, filtered through 0.45 μm and 0.22 µM PVDF filters, and subsequently transduced into cells. The knockdown ATF3 primer sequence was designed (Table 1).

Table 1 Primer sequence of ATF3RNA sequencing (RNA-seq)

Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA), and index codes were added to assign sequences to each sample according to the manufacturer’s recommendations. Total RNA was isolated from KGN cells treated with PM2.5 (100 µg/mL) or from control cells using Trizol reagent. Poly(A) RNA was purified using the PolyTtract mRNA Isolation System, and cDNA libraries were then generated. All samples were sequenced using the Novogene HiSeq 4000 platform. Sequence reads were mapped to the human genome (hg38) using the Novogene sequence analysis pipeline. Average gene expression values (FPKM > 30) from three independent studies were used for the subsequent analysis.