Lipopolysaccharide escherichia coli serotype 0111 b4

Tamoxifen and lipopolysaccharide (Escherichia coli serotype 0111:B4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). TNF-α and IL-6 ELISA kits were obtained from Biolegend (San Diego, CA, USA). Blood urea nitrogen (BUN) assay kit was obtained from Arbor Assays (Ann Arbor, Michigan, USA). Kidney injury molecule-1 (KIM-1) assay kit was purchased from R&D (Minneapolis, MN, USA). Anti-Mouse Ly-6G (Gr-1)-FITC was purchased from eBioscience (San Diego, CA, USA). Goat anti-mouse ICAM-1 and VCAM-1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Phospho-STAT3 (Thr705), Stat3 (124H6), phospho-p44/42MAPK (ERK1/2) (Thr202/Tyr204), p44/42MAPK (ERK1/2), phosphorylated IκBα, IκBα, and β-actin antibodies were obtained from Cell Signal Technology (Boston, MA, USA).

Male C57BL/6 mice, 6–8 weeks old, were purchased from Nanjing Biomedical Research Institute of Nanjing University and maintained in the Experimental Animal Center of Zhejiang University. Mice were treated intratracheally with 20 μg/g of lipopolysaccharide (Escherichia coli serotype 0111:B4; Sigma-Aldrich, St. Louis, MO). After 4 h, mice were treated intratracheally with 0.1 mL of PBS containing 2% C57BL/6 serum with or without 5 × 10⁵ MSCs. As a vehicle control group, an equal volume of PBS containing 2% C57BL/6 serum was administered (PBS group). The PBS group consisted of 5 mice, and the LPS and LPS/MSC groups each consisted of 10 mice. The mice were euthanized on days 3 or 7 after MSC or PBS administration, and lung tissues were collected for histological analysis and prepared for lung immune cell separation.

Prior to the induction of ALI, the stress-related phenotype of WKY rats was confirmed by exposing SD and WKY rats to the open field followed by the elevated plus maze and behaviour was recorded. Rats were habituated to handling and received intraperitoneal (i.p.) injection of 0.89% NaCl sterile saline one day prior to systemic administration of LPS/GalN (20 μg/kg Lipopolysaccharide (Escherichia coli, serotype 0111:B4; Sigma-Aldrich, Dublin, Ireland, Ltd.) and 200 mg/kg d-galactosamine hydrochloride (Sigma-Aldrich, Dublin, Ireland, Ltd.) dissolved in 0.89% sterile saline) or saline vehicle (n = 8/group). Rats were returned to their home cage and sacrificed 6 and 24 h post injection. The dose and time of administration of LPS/GalN was chosen based on an in-house pilot study and published data [43 (link)] which demonstrate that this combination of doses induces liver injury with minimal mortality. Blood samples were taken by cardiac puncture into heparinised tubes and plasma separated and stored at −80 °C, until liver enzyme analysis. Livers were excised quickly, weighed and sections were snap frozen and stored at −80 °C or fixed for histological analysis.