Patients and Sample Collection

All samples were obtained from Changzhou Maternity and Child Health Care Hospital between January 2023 and June 2023. Placental villous samples were collected from 20 women who experienced SA at 6–12 weeks. Women with immune disorders, endocrine imbalances, genetic diseases, genital malformation, infection and other factors were excluded. Tissues in the normal control (NA) group were obtained from 20 age-matched women who underwent elective abortion. Women in the NA group had no history of diabetes, hypertension or adverse pregnancy. Both the SA and NA groups underwent vacuum aspiration to collect intrauterine tissue containing villi, which was then washed by PBS, stored at -80℃ or fixed in formalin for further study. The present study was approved by the Ethics Committee of Changzhou Maternity and Child Health Care Hospital. All patients signed informed consent before sample collection.

Immunohistochemical Staining

Tissue specimens were fixed in formalin immediately, followed by standard paraffin embedding. The sections were boiled in citrate buffer (pH 6.0) for 10 min for antigen retrieval. Hydrogen peroxide (3%) in methanol was used to eliminate endogenous peroxidase activity. Then the sections were blocked using 5% BSA (Beijing Solarbio Science & Technology Co., Ltd), labeled at 4℃ with anti-HMGB1 antibody (1:100, Proteintech) or anti-ACSL4 antibody (1:100, Proteintech) overnight, then biotinylated with horseradish peroxidase (HRP)-conjugated rabbit secondary antibody at room temperature for 1 h. The sections were placed in the solution for DAB reaction and examined with a microscope.

Western Blot

Total protein was extracted from villous tissues or HTR-8/SVneo cells with RIPA lysis buffer (Beyotime, China) containing protease inhibitors (Beyotime, China). Every 20 µg of protein was separated from each sample by 10% SDS-PAGE, transferred to PVDF membranes and blocked with 5% skimmed milk in TBST for 1 h at room temperature. The membranes were incubated with specific primary antibodies, including anti-HMGB1 (1:1000, Proteintech), anti-ACSL4 (1:1000, Proteintech) and anti-β-actin (1:5000, Bioworld) overnight at 4℃. Then, the PVDF membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, and visualized by chemiluminescence. The intensity of each band was quantified by Image J.

Cell Culture

Human trophoblast cell line HTR-8/SVneo was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd., cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco BRL, Grand Island, NY, USA) at 37℃ in a 5% CO2 incubator. When the confluence reached 80% or above, the cells were treated with 100 ng/ml LPS (Sigma, St. Louis, MO, USA) for 24 h.

Cell Viability

Cell viability was measured using Cell Counting Kit-8 (CCK-8, PF00004, Proteintech Group, Inc). HTR-8/SVneo cells were seeded into 96-well plates at a density of 5000 cells/well and treated as indicated for 24 h. Then, 10 µl of incubation reagent and 90 µl of medium were added to each well and incubated at 37 ℃ for 1 h. Absorbance was measured at 450 nm by a microplate reader (BioTek Instruments, Inc.).

Malondialdehyde (MDA) Assessment

The relative concentration of lipid peroxidation was calculated by MDA level measured using an MDA assay kit (Beyotime, S0131S) according to the manufacturer’s instructions. The MDA in each sample was reacted with thiobarbituric acid (TBA) to form an MDA-TBA mixture that was then measured colorimetrically at 532 nm using a microplate reader (BioTek Instruments, Inc.).

Glutathione (GSH) Measurement

The relative GSH level was measured by a GSH assay kit (Beyotime, S0053) according to the manufacturer’s instructions. Briefly, the sample was mixed with the GSH working solution, and the absorbance was read at 405 nm by a microplate reader (BioTek Instruments).

Iron Assay

The relative ferrous iron (Fe2+) level was tested using an iron assay kit (Abcam, ab83366, Cambridge, UK) according to the manufacturer’s manuals. Briefly, 5 µl of iron reducer was added to each sample and the reaction was incubated at 25℃ for 30 min. Then, 100 µl of the iron probe was added to the mixture for 60 min in the dark. Finally, the absorbance was measured at 593 nm.

Reactive Oxygen Species (ROS) Measurement

The intracellular level of ROS was assessed using a cellular ROS assay (Beyotime, S0033S). The cell culture medium was removed and incubated with 10 µM DCFH-DA at 37℃ for 20 min. After removing excessive DCFH-DA by washing thrice with PBS, the cells were visualized under a fluorescence microscope (Leica).

Cell Transfections

Overexpression plasmids pCMV3-HMGB1-Flag and pCMV3-HA-ACSL4 were bought from SinoBiological (Beijing, China). siRNAs targeting HMGB1 or ACSL4 (RiboBio, Guangzhou, China) were used to knock down the HMGB1 or ACSL4 gene in HTR-8/SVneo cells. The HMGB1 and ACSL4 overexpression plasmids were transiently transfected by Lipofectamine 3000 (Invitrogen). si-HMGB1 and si-ACSL4 were transfected using riboFECT™ CP (RiboBio) according to the manufacturer’s instructions.

CHX Chase Assay

After transfection with plasmid pCMV3-HMGB1-Flag and the vector control for 48 h, HTR-8/SVneo cells were treated with CHX (50µM) for 0, 1.5, 3, and 6 h, respectively. Total proteins of the HTR-8/SVneo cells with different treatment times were obtained for subsequent detection of ACSL4 protein levels via Western blot.

Co-Immunoprecipitation (Co-IP)

Co-IP assay was used to verify the binding between HMGB1 and ACSL4 in HTR-8/SVneo cells. The Co-IP complexes were purified by the PierceTM Crosslink Magnetic IP/Co-IP Kit (Thermo Fisher, 88805). The dosage of the IP antibody was 4 µg of anti-HMGB1 (Proteintech). The whole lysates and the antigen eluate were measured by Western blot.

Statistical Analysis

Statistical analysis was performed using SPSS 22.0, GraphPad Prism 8 and Image J. Continuous variables in a normal distribution were expressed as mean ± SEM. Student’s t-test or Chi-square-test was used for comparison between two groups, ANOVA for comparison among three or more groups. Correlation was tested by Pearson’s correlation coefficient. Significant results were indicated by *P < 0.05 and **P < 0.01.