Suppressive effects of polyozellin on TGFBIp-mediated septic responses in human endothelial cells and mice  Byeongjin Jung, Eun-Ju Yang, Jong-Sup Bae  Nutrition Research  Volume 36, Issue 4, Pages 380-389 (April 2016) DOI: 10.1016/j.nutres.2015.12.009 Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 1 Effects of POZ on release of TGFBIp and expression of receptors. A, HUVECs were treated with the indicated concentrations of POZ for 6 hours after stimulation with LPS (100 ng/mL, 1 hour), and TGFBIp release was measured by ELISA. B, Male C57BL/6 mice that underwent CLP were administered POZ at 10 or 20 μM/mouse each intravenously 12 hours after CLP (n = 5). Mice were euthanized 24 hours after CLP. Serum TGFBIp levels were measured by ELISA. C, The same as panel A, except that real-time qRT-PCR analysis was performed using specific primers for TGFBIp and actin, as described in the Materials and Methods section. D, Confluent HUVECs were activated with LPS (100 ng/mL, 3 hours), followed by incubation with POZ for 6 hours. Expression of αvβ3 (white bar) and αvβ5 (black bar) was determined by cell-based ELISA. E, Effect of POZ on cellular viability was measured by MTT assay. D = 0.2% DMSO is the vehicle control. Values are expressed as means ± SEM (triplicate, n = 5/group). *P < .05 vs LPS alone (A, C, D) or CLP alone (B). DMSO indicates dimethyl sulfoxide; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction. Nutrition Research 2016 36, 380-389DOI: (10.1016/j.nutres.2015.12.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 2 Effects of POZ on TGFBIp-mediated permeability in vitro and in vivo. A, The effects of posttreatment with different concentrations of POZ for 6 hours on the barrier disruptions caused by TGFBIp (5 μg/mL, 6 hours) were monitored by measuring the flux of Evans blue bound albumin across HUVECs. B, The effects of POZ at 10 or 20 μM/mouse on TGFBIp-induced (0.1 mg/kg, intravenously) vascular permeability in mice were examined by measuring the amount of Evans blue in peritoneal washings (expressed μg/mouse, n = 5). C, Staining for F-actin. HUVEC monolayers grown on glass coverslips were stimulated with TGFBIp for 1 hour, treated with POZ for 6 hours, and stained for F-actin. Arrows indicate intercellular gaps. D, HUVECs were activated with TGFBIp (5 μg/mL, 6 hours), followed by treatment with different concentrations of POZ for 6 hours. The effects of POZ on TGFBIp-mediated expression of phospho p38 were determined by ELISA. Results are expressed as the means ± SEM of at least 3 independent experiments (triplicate, n = 5/group). *P < .05 vs TGFBIp alone. Nutrition Research 2016 36, 380-389DOI: (10.1016/j.nutres.2015.12.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 3 Effects of POZ on TGFBIp-mediated proinflammatory responses. A-C, HUVECs were stimulated with TGFBIp (5 μg/mL) for 6 hours, followed by treatment with POZ for 6 hours. TGFBIp-mediated (A) expression of VCAM-1 (white bar), ICAM-1 (gray bar), and E-selectin (black bar) in HUVECs; (B) adherence of human neutrophils to HUVEC monolayers; and (C) migration of human neutrophils through HUVEC monolayers were analyzed. D, The effects of posttreatment with POZ at 10 or 20 μM/mouse on leukocyte migration into the peritoneal cavities of mice caused by TGFBIp (0.1 mg/kg, intravenously) were analyzed. Values are expressed as means ± SEM (triplicate, n = 5/group). *P < .05 vs TGFBIp. Nutrition Research 2016 36, 380-389DOI: (10.1016/j.nutres.2015.12.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 4 Effects of POZ on lethality or pulmonary injury after CLP. A, Male C57BL/6 mice (n = 20) were administered POZ (10 μM/mouse, □; 20 μM/mouse, ■) at 12 hours and 50 hours after CLP. Animal survival was monitored every 6 hours after CLP for 132 hours. Control CLP mice (●) and sham-operated mice (○) were administered sterile saline (n = 20). The Kaplan-Meier survival analysis was used for determination of overall survival rates vs CLP-treated mice. (B) The same as panel A, except that mice were euthanized 96 hours after CLP. Histopathologic scores of the lung tissue were recorded as described in the Materials and Methods section. *P < .05 vs CLP. C, Photomicrographs of lung tissues (hematoxylin and eosin staining, ×200). Sham group (grade 1); CLP group (grade 3); right, CLP + POZ (10 or 20 μM/mouse; grade 2). Illustrations indicate representative images from 3 independent experiments. D-G, The same as panels B and C, except that mice were bled and euthanized. Levels of AST (D), ALT (D), creatinine (E), BUN (F), and lactate dehydrogenase (G) in plasma were measured. Values are expressed as means ± SEM (triplicate, n = 20/group). *P < .05 vs CLP. LDH indicates lactate dehydrogenase. Nutrition Research 2016 36, 380-389DOI: (10.1016/j.nutres.2015.12.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Nutrition Research 2016 36, 380-389DOI: (10.1016/j.nutres.2015.12.009) Copyright © 2016 Elsevier Inc. Terms and Conditions