L3755

Human adipose‐derived stem cells (hADSC, PT‐5006, Clonetics, Lonza) were cultured in keratinocyte‐SFM (17005‐042, GIBCO‐Invitrogen), supplemented with 2 mmol/L N‐acetyl‐L‐cysteine (NAC, A8199, SIGMA), L‐ascorbic acid 2‐phosphate (Asc 2P, A8960, SIGMA) and 5% foetal bovine serum (16000044, GIBCO‐Invitrogen).
Human umbilical vein endothelial cells (HUVECs, BCRC No. H‐UV001) were cultured in medium 199, supplemented with 10% foetal bovine serum, 25 U/mL heparin (H‐3149, SIGMA), 30 µg/mL endothelial cell growth supplement (ECGS, 02‐102, Millipore), 2 mmol/L L‐glutamine, 1.5 g/L sodium bicarbonate and 1X penicillin/streptomycin.
For culture media collection, cell culture was limited to eight passages. 1 × 10⁶ hADSC cells were cultured in 10 mL serum‐free media, with/without 1 μg/mL lipopolysaccharides (LPS, L3755, SIGMA), for 24 hours. The culture media (500 mL) were harvested and centrifuged at 300 g for 5 minutes to remove cells and cell debris. The supernatants were concentrated using the Amicon^® Ultra‐15 (UFC900324, Merck‐Millipore) and transferred into new tubes for further use.

The mice used for the sepsis models were 8–10-week-old females. The CLP sepsis model was established following a previously reported method^{62 (link)} with slight modification. Briefly, mice were anesthetized using avertin, which was formulated by dissolving 0.5 g 2,2,2-tribromoethanol (Sigma-Aldrich, T48402) in 1 mL of 2-methyl-2-butanol (Sigma-Aldrich, 240486). PBS was added to prepare a working solution of 1.25% avertin, which was filtered using a 0.22-μm syringe filter (Merck, SLGP033RS) prior to use. After disinfecting the abdomen with 70% ethanol, a small midline incision was made, and the cecum was exposed. The cecum was ligated below the ileocecal valve (about 75% of total cecum length) with Black Silk (Ailee, SK447), punctured twice with an 18-gauge needle, and the abdomen was closed. The wound was sutured with 5-0 Surgifit (Ailee, AV521). The sham control mice were operated on without performing the ligation and puncture process. The LPS sepsis model was established by injecting 30 mg/kg of PBS-diluted LPS (Sigma-Aldrich, L3755) intraperitoneally. Rg6 (20 mg/kg) was dissolved in PBS and IP injected into each mouse once (2 h prior to LPS) for the pre-treat, and twice (1 and 2 h post-LPS injection) for the post-treat system.

Mice were anesthetized (ketamine 100 mg/kg and xylazine 10 mg/kg) and received one intratracheal instillation of 10 μg per mouse of Escherichia coli LPS (026:B6; L3755, Sigma Aldrich, St. Louis, MO, United States) suspended in phosphate-buffered saline (PBS) to induce acute lung injury and ARDS. Briefly, anesthetized mice were positioned on an inclined platform (approximately 60–70°) with fixation at the incisors to gain free excess to the oral cavity and were hold the tongue with forceps to straighten the throat for instilling LPS into the trachea (proximal to the bifurcation) using a pipette (the injected volume depended on mouse bodyweight). The upper body of the mouse was kept in an upright position for 30 s to avoid leakage of the fluid from the trachea. The control groups received sterile PBS intratracheally instead of LPS. Once the mice recovered from anesthesia, they were returned into an individually ventilated cage and allowed free access to food and water.

C57BL6/j mice were purchased from Charles River Laboratories, bred and maintained in the animal facilities of the Department of Cellular, Computational and Integrative Biology (CIBIO) under pathogen-free conditions and according to the authorization received from the Italian Health Ministry ethical committee for animal experimentation (#629-2018). To measure inflammatory factor secretion, 8-week-old C57BL6/j mice were injected i.p. with LPS (Sigma-Aldrich, L3755) at 150 μg/25 g body weight. Dexamethasone (10 mg/kg) and TMs (40 mg/kg) were co-administrated with LPS via i.p. injection in a solution containing 20% Kolliphor EL and 5% DMSO in PBS. Blood samples were collected 90 min later by cardiac puncture, and two serial 10 min centrifugations, at 850 g at 4°C and at 3500 g at 4°C, were performed to collect sera.