Antibodies.
Fig 1.
Identification of mexenone as a permeability blocker.
A. BAECs were grown to a monolayer on biotin-labeled fibronectin for two days. After 90 min of drug (1 uM each) pretreatment, LPS (2 ug/mL) was added to the EC monolayer for 3 h to induce endothelial permeability. FITC-streptavidin was added and allowed to bind to exposed biotin-fibronectin for 3 min. After washing, the cells were fixed and observed by fluorescence microscopy. Fluorescent areas were imaged and quantified using ImageJ. B. Mexenone induced the blockade of endothelial permeability. BAEC monolayers were incubated with the indicated concentrations of mexenone for 90 min before stimulation with LPS for 3 h. The FITC-avidin bound area was calculated and normalized to the non-treated control group. *p < 0.05 (n = 3, two-way ANOVA,Bonferroni posttests). Permeability indicates the relative fold-increase of the FITC-stained area compared to non-treated controls. C. Chemical structure of mexenone (MW: 242.27), benzophenone-10, which has been used as a UV-absorbing agent in sunscreen cosmetics. D. BAEC monolayers were treated with clinical library compounds, including mexenone, stimulated with LPS for 3 h, and probed for the indicated antibodies by immunoblotting. BAEC:Bovine Endothelial Cells, LPS:Lipopolysaccharides.
Fig 2.
Effect of mexenone on VE-cadherin phosphorylation and localization.
A. ECs were incubated with LPS after mexenone pretreatment and fixed and stained for VE-cadherin. B. Confluent BAECs were stimulated with LPS for 3 h with or without mexenone pretreatment. Cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. C. The phosphorylation of VE-cadherin Y658 was measured using phospho-specific antibodies and normalized to total VE-cadherin expression (n = 3, two-way ANOVA, Bonferroni posttests, *p < 0.05). EC:Endothelial Cells.
Fig 3.
Effect of mexenone on LPS-induced lung permeability in vivo.
A. In vivo permeability study design. Mexenone (10 mg/kg) was intraperitoneally injected into mice 1 h before LPS injection. Seventeen and a half hours after LPS injection, the mice were tail vein-injected with Evans blue dye (4 mg/kg) and perfused after 30 min of circulation. The lungs were isolated, and the dye was extracted and quantified. B. Evans blue dye was extracted from the lungs and quantified spectrophotometrically by measuring absorbance at 611 nm. One-way ANOVA, Tukey’s multiple comparison test, *p < 0.05.
Fig 4.
Mexenone prevents LPS-induced lung injury.
A. Mice were intraperitoneally injected with vehicle or mexenone (10 mg/kg) for 1 h before the intraperitoneal injection of LPS (18 mg/kg). BAL fluid was collected by PBS instillation, and total cell numbers were counted. One-way ANOVA, Tukey’s multiple comparison test, *p < 0.05. B. Protein concentrations in BAL fluid were measured by the Bradford assay (n = 6–10, Student’s t-test). C. Lung tissue morphology changes were demonstrated by H&E staining of the control group, LPS group, and mexenone/LPS group. D. Histological lung injury scores were analyzed (n = 14–22, one-way ANOVA, Tukey’s multiple comparison test, *p < 0.05). BAL:Bronchoalveolar lavage.