Lipopolysaccharides lps from escherichia coli o55 b5

Six linear oligopeptides (>95% pure) acetylated and amidated at the N- and C-termini, respectively (Table 1), the linear (Vi45-51), and the cyclic-retro-inverse-vasoinhibin-(45-51)-peptide (CRIVi45–51) were synthesized by GenScript (Piscataway, NJ). Recombinant vasoinhibin isoforms of 123 (Vi1-123) (16 (link)) or 48 residues (Vi1-48) (15 (link)) were produced as reported. Recombinant human prolactin (PRL) was provided by Michael E. Hodsdon (17 (link)) (Yale University, New Haven, CT). Human recombinant plasminogen activator inhibitor 1 (PAI-1) was from Thermo Fisher Scientific (Waltham, MA), and human tissue plasminogen activator (tPA) from Sigma Aldrich (St. Louis, MO). Rabbit monoclonal anti-PAI-1 [EPR17796] (ab187263, RRID:AB_2943367) and rabbit polyclonal anti-β-tubulin antibodies (Cat# ab6046, RRID:AB_2210370) were purchased from Abcam (Cambridge, UK), and mouse monoclonal anti-uPA receptor (anti-uPAR) from R&D systems (Minneapolis, MN, Cat# MAB807, RRID:AB_2165463). The NF-κB activation inhibitor BAY 11-7085 and lipopolysaccharides (LPS) from Escherichia coli O55:B5 were from Sigma Aldrich. Recombinant human vascular endothelial growth factor-165 (VEGF) was from GenScript, and basic fibroblast growth factor (bFGF) was donated by Scios, Inc. (Mountain View, CA).

Oxygen consumption rate (OCR) was assessed in the extracellular analyzer XFp (Seahorse Agilent Technologies). Mitochondrial respiration assays were performed following the described protocol [14 (link)] with minor modifications in reagents concentrations: 2.6 μM oligomycin (#75351, Sigma-Aldrich), 1.0 μM carbonyl cyanide 4-(trifluoromethoxy)-phenyl-hydrazone (FCCP) (C2920, Sigma-Aldrich) and 1.0 μM rotenone/antimycin A (R8875 and A8674, respectively, both from Sigma-Aldrich). 40.000 cells/well were seeded for approximately 24 hours on XFp plates prior to performing the test. Data was obtained using Agilent Seahorse Wave 2.6.3.5 software (Seahorse Agilent Technologies). When indicated, cells were preincubated during 4 hours with 200 ng/mL Lipopolysaccharides (LPS) from Escherichia coli O55:B5 (L6529, Sigma-Aldrich) before testing.

Nine to twelve weeks old C57BL/6 male mice were given intraperitoneal (i.p.) injection with 14 μg/kg mouse anti-CALR antibody (aCALR) (Abcam, Cambridge, MA, catalog number: ab223614) and intratracheal (i.t.) injection with 5 mg/kg Lipopolysaccharides (LPS) from Escherichia coli O55:B5 (Sigma, St. Louis, Missouri, USA). The mice were injected with the same doses of goat IgG isotype and LPS were used as untreated control group. The mice were treated with PBS as naïve controls. Two days after treatment, bronchoalveolar lavage (BAL) and lung tissues were collected for analysis. All animals were housed and treated according to the guidelines of the Institutional Animal Care and Use Committee of the Fudan University, Zhongshan Hospital in China. All experiments were approved by the committee.

Plant extracts: rosemary extract from Rosmarinus officinalis (standardized to 6% carnosol; 15% carnosic acid) was obtained from Flavex (Rehlingen, Germany), ashwagandha extract from Withania somnifera (standardized to 2% withaferin A) was obtained from Verdure Sciences (Noblesville, IN, USA), and luteolin (standardized to 98% luteolin, from Sophora japonica) was obtained from Jiaherb (Pine Brook, NJ, USA). For making PB125 solutions, the rosemary, ashwagandha, and luteolin powders were mixed at a 15:5:2 ratio by mass, and then extracted at 50 mg of mixed powder per mL in ethanol overnight and the supernatant was isolated [24 (link)]. Cell culture: media and antibiotics were purchased from Thermo Fisher Scientific (Waltham, MA, USA). LPS (lipopolysaccharides from Escherichia coli O55:B5) was from Sigma-Aldrich (St. Louis, MO, USA).