Trypsin

Peptides of at least 95% purity were synthesized by Bootech (Shanghai, China). Bovine serum albumin (BSA) was purchased from Tbdscicece (Tianjin, China). Trypsin was purchased from Amresco (US). Ovalbumin (OVA) was purchased from Sigma (US). Oligonucleotide primers were synthesized by Invitrogen (Shanghai, China). Streptavidin-coated magnetic beads were purchased from Promega (US). Affimag UF magnetic microspheres (7−8 um) were purchased from BaseLine ChromTech (Tianjin, China).

The extraction of CP-PGN is based on the TCA method^{13 (link)}. The harvested bacterial sludge was washed in 0.9% saline solution until it turned milk white. The clean sludge was dissolved in 10% TCA (w/v = 1:10), incubated in a boiling bath for 20 min, then centrifuged at 4,000 rpm for 10 min. The sediment were washed with sterile water for 3 times and centrifuged at 4,000 rpm for 10 min. Collecting the sediment and treated with 8% SDS (Beyotime Biotechnology, Jiangsu, China), incubated in a boiling bath for 30 min, then centrifuged at 4,000 rpm for 30 min. After centrifugation, the sediment was washed in sterile water for 3 times. Adding the 1 mg/ml trypsin (Amresco, USA) buffer to the above sediment at 37 °C in a shaking bath (240 rpm) for 3 hours, centrifuged at 3,000 rpm in 4 °C for 5 min, then collected the supernatant. The collecting supernatant was centrifuged at 12,000 rpm for 15 min. The sediment was added to aether and rested for 30 min, then centrifuged for 15 min to collect sediment. The harvested sediment were finally added to absolute alcohol and drying at 70 °C to get the hazel target product, then stored in an airtight container at −20 °C for further analysis.

The uterus from CD1 mice was split longitudinally on d 4 of pregnancy. Luminal epithelial cells were removed by digestion in HBSS containing 1% trypsin (AMRESCO Inc., Solon, OH) and 6 mg/ml dispase (Roche Diagnostics GmbH, Mannheim, Germany). The remaining tissues were incubated with 0.15 mg/ml collagenase I (Invitrogen). The supernatants were filtrated through 70 μm wire gauze, and centrifuged to collect the stromal cells. The cell pellets were washed twice with HBSS, and resuspended in complete DMEM/F-12 medium (Sigma-Aldrich) with 10% charcoal-treated fetal bovine serum (cFBS, Invitrogen, Carlsbad, CA). Cells were seeded onto 24-well culture plates at a concentration of 2 × 10⁵ cells/well. After an initial 30-min culture, cells were further cultured in fresh medium with 2% cFBS.

The whole research was carried out by human normal hepatocyte cell line L02, which was purchased from Kunming Institute of Zoology, Chinese Academy of Sciences. The cell was maintained in an incubator (MCO-20AIC CO₂ Incubator, Japan's Sanyo Electric Co., Ltd.) with 5% carbon dioxide, 95% air, at 37°C in RPMI-1640 medium (Gibco Invitrogen Corporation, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone). 0.25% trypsin (1~2 mL) (Amresco, USA) was used to passage cells at 80~90% confluence.
L02 cells were inoculated into 6-well plates at a cell density of 3 × 10⁵ and then incubated for 48 h till 80~90% confluences with 10% FBS-RPMI 1640 medium. Cells were incubated in G₀ medium (0.2% FBS-RPMI 1640 medium) for 24 h to induce the cell cycle synchronous before the test. In the subsequent experiments, the control group was incubated with 10% FBS-RPMI 1640 medium alone, while the model group was incubated with 5% fat emulsion-10% FBS-RPMI 1640 medium for 48 h, respectively. After 48 h, cell morphological changes were investigated by inversion fluorescence microscope (ECLIPSE TS100, Nikon Corporation, Japan).