Ethylene diamine tetraacetic acid (edta)

Pure AST was provided by Shanghai Yuanye Biotechnology Co., Ltd. For experiments, AST was dissolved in phosphate buffered saline (PBS) and diluted with normal saline. Pentobarbital sodium, papain and cysteine were purchased from Guangzhou Chemical General Factory, and hematoxylin, water-soluble eosin Y, toluidine blue O, paraformaldehyde and EDTA were acquired from Takara Bio, Inc. Anti-TNF (cat. no. ab1793), IL-1 (cat. no. ab9722), β-actin (cat. no. ab8227), AKT (cat. no. ab8805), NF-κB (cat. no. ab28849), PI3K (cat. no. ab140307) and IL-6 (cat. no. ab6672) antibodies were all purchased from Abcam. The quantitative PCR detection and Prime Script RT kits were purchased from Bao Biological Engineering Co. Ltd., and Takara Biotechnology Co. Ltd., respectively.

Blood was collected from elbow veins of fasted pregnant women into anticoagulant tubes with ethylenediaminetetraacetic acid (EDTA, TaKaRa, China) before delivery. Mononuclear cells were extracted over 30 min in 1 mL Total RNA Extraction Reagent (TRIzol, TaKaRa, China), and the cells were homogenized, mixed, and stored at −80 °C. Placental specimens were cut from the maternal surface near the umbilical cord, avoiding obvious fibrosis and calcified areas. One piece was quickly stored in liquid nitrogen, and the other was fully rinsed, fixed in 10% formaldehyde solution for at least 24 h, placed in an embedding box, dehydrated, and embedded in paraffin. Placental tissues (50–100 mg) were homogenized adequately, 1 mL TRIzol was added, and the tissues were incubated for 5 min at room temperature to ensure complete dissociation of the nucleic acid protein complex.