Reagents and chemicals

Zearalenone (ZEA) powder was acquired from Sigma‒Aldrich (St. Louis, MO, USA). Asiaticoside (AS) and LY294002 were purchased from Glpbio Technology Inc. (Montclair, CA, USA). The assay kits for malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The primary antibodies used in this study were obtained from Abcam (Cambridge, UK) or Cell Signaling Technology (Boston, MA, USA). All other chemicals were obtained from Sigma‒Aldrich unless otherwise indicated.

Animals and experimental design

Female C57BL/6 mice (7 weeks old, 20–21 g) were procured from Liaoning Changsheng Biotechnology Co. Ltd. C57BL/6 Nrf2 knockout (Nrf2−/−, strain Nrf2tm1Ywk/J) mice were purchased from Jackson Laboratory (Bar Harbor, ME). All the mice were maintained on a 12-h light/dark cycle at a temperature between 22 °C and 24 °C. All the animals were given a normal chow diet and drinking water. After seven days of adaptive feeding, they were randomly allocated to four treatment groups (n = 8 per group) (Fig. 1B): the healthy control (Ctrl), AS treatment (AS), ZEA treatment (ZEA), and AS + ZEA treatment (AS_ZEA) groups. ZEA was dissolved in 5% ethanol, and AS was dissolved in 5% CMC-Na. The Ctrl and AS groups were intragastrically administered 5% ethanol within 1–7 days, whereas the other groups were orally administered ZEA (20 mg/kg BW) once a day. The AS and AS + ZEA groups were treated with AS (i.e., 40 mg/kg) within 1–14 days, whereas the other groups were given 5% CMC-Na orally once a day. To explore the connection between the PI3K/Akt signaling pathway and apoptosis, an inhibitor of PI3K, LY294002 (30 mg/kg), was injected intraperitoneally 2 h before AS treatment once a day for 14 days (Fig. 6A). To investigate the effect of the Nrf2 pathway on the MAPK pathway and oxidative stress, an Nrf2 inhibitor (ML385) was used in subsequent experiments. ML385 (30 mg/kg) pretreatment was administered intraperitoneally 1 h before the administration of AS (Fig. 4F). To explore the role of the Nrf2 pathway in protecting against ZEA-induced uterine injury, we conducted Experiment 3 with Nrf2−/− mice (four groups, n = 8 per group) (Fig. 5A). Both wild-type (WT) and Nrf2−/− mice were treated with ZEA to induce uterine damage. During this time, half of the WT and Nrf2−/− mice received AS treatment. The dose and method of administration were the same as those described above. On the 15th day of the experiment, the body weights of the mice were measured. Blood and uterine tissue samples were subsequently collected after euthanasia, and the uterus was weighed. The uterine index was calculated according to the following formula: uterine index (%) = uterine weight/body weight × 100% [7]. All animal experiments in this study strictly abided by the principles of animal welfare and scientific ethics and were carried out under the approval and guidance of the Animal Protection and Use Committee of Jilin University (Permit number: SY202309056).

Assessment of serum reproductive hormones

The mice were euthanized, and the serum was collected by centrifugation. The contents of P4, LH, and E2 in the serum were detected via enzyme-linked immunosorbent assay (ELISA) kits (Elabscience, Wuhan, China).

Histological analysis

After being fixed with 4% formaldehyde for 24 h, the uterine samples were embedded in wax blocks following dehydration. The samples were subsequently cut into 5 μm slices via a microtome. The uterine sections were subsequently stained with hematoxylin and eosin (H&E). Finally, the histopathology of these slices was observed via a light microscope (Olympus, Tokyo, Japan), and images were acquired. Histological scores were estimated as previously described with minor modifications [23]: (i) Hyperaemia/edema: 0, normal; 2, mild; 4, severe. (ii) The number of neutrophils in the visual field: 0, 0–1; 1, 2–5; 2, 6–10; 3, 11–15; 4, 16–20; 5, > 20. The endometrial thickness of each slide was measured from each cross-section under high magnification. In brief, a total of eight high-power fields with 45° intervals in each cross-section were selected, measured, and averaged for each HE-stained section.

Detection of oxidative stress

The appropriate amounts of extraction buffer were added to the uterine tissues, which were then homogenized via a homogenizer. The uterine tissue homogenates were centrifuged, and the protein concentration of the supernatants was determined via a BCA protein concentration assay kit. The activities of SOD and CAT, as well as the content of MDA in the uterine tissues, were measured via commercial kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute).

Determination of uterine apoptosis via TUNEL staining

Apoptosis in uterine tissue was detected via a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit according to the manufacturer’s instructions (Yeasen Biotechnology, Shanghai, China). The sections were observed under a fluorescence microscope, and cells displaying green fluorescence were identified as TUNEL-positive apoptotic cells.

Immunohistochemistry (IHC)

The slicers of uterine tissues were heated in 0.01 M citric acid buffer for antigen retrieval. After incubation with 3% H2O2 at room temperature in the dark to remove endogenous peroxidase activity, the sections were blocked with 3% BSA. The tissue slides were incubated with primary antibodies at 4 °C overnight and then with a secondary antibody (HRP) for 1 h at room temperature. Finally, the sections were treated with a DAB chromogenic reagent kit (Solarbio, Beijing, China) and counterstained with hematoxylin. The average optical density (AOD) of the IHCs was calculated via ImageJ software (National Institutes of Health).

Western blot analysis

The uterine tissues were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Roche AG, Mannheim, Germany). The nuclear proteins of the uterine tissues were extracted via a nuclear and cytoplasmic protein extraction kit (Yeasen Biotechnology). Protein samples were subjected to sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE). Next, immunoblotting was performed as previously described [24]. Finally, the intensity of the protein bands was measured with ImageJ software, and normalization was conducted with reference to GAPDH.

Statistical analysis

All the data are expressed as the means ± SEMs and were analyzed with GraphPad Prism version 8.0 (GraphPad software, San Diego, CA, USA). To compare normally distributed variables, an independent sample t test was performed. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used to compare data between three or more groups. Unless otherwise stated, the data shown in this study represent at least three independent experiments, and a p value < 0.05 was considered statistically significant.