Inflammation ( # 2015) DOI: 10.1007/s10753-015-0199-9

Ameliorative Effect of Vicenin-2 and Scolymoside on TGFBIpInduced Septic Responses Wonhwa Lee,1,2 Sae-Kwang Ku,3 and Jong-Sup Bae1,4

Abstract—Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein whose expression in several cell types is greatly increased by TGF-β. TGFBIp is released by the human umbilical vein endothelial cells (HUVECs) and functions as a mediator of experimental sepsis. Cyclopia subternata is a medicinal plant commonly used in traditional medicine to relieve pain in biological processes. In this study, we investigated the antiseptic effects and underlying mechanisms of vicenin-2 and scolymoside, two active compounds in C. subternata against TGFBIp-mediated septic responses in HUVECs and mice. The anti-inflammatory activities of vicenin-2 or scolymoside were determined by measuring permeability, human neutrophils adhesion and migration, and activation of pro-inflammatory proteins in TGFBIp-activated HUVECs and mice. According to the results, vicenin-2 or scolymoside effectively inhibited lipopolysaccharide-induced release of TGFBIp and suppressed TGFBIp-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. In addition, vicenin-2 or scolymoside suppressed the production of tumor necrosis factor-α and interleukin 6 and activation of nuclear factor-κB and extracellular regulated kinases 1/2 by TGFBIp. Vicenin-2 or scolymoside reduced cecal ligation and puncture (CLP)-induced septic mortality and pulmonary injury. Collectively, these results indicate that vicenin-2 and scolymoside could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the TGFBIp signaling pathway. KEY WORDS: vicenin-2; scolymoside; TGFBIp; sepsis; inflammation; HUVEC.

INTRODUCTION Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein that can be highly expressed in various cell types [1–4]. TGFBIp contains an N-terminal secretory signal peptide, followed by a cysteine-rich domain, four internal homologous repeats Wonhwa Lee and Sae-Kwang Ku contributed equally to this work. 1

College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Dahak-ro, Buk-gu, Daegu, 702-701, Republic of Korea 2 Department of Biochemistry and Cell Biology, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu, 702-701, Republic of Korea 3 Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan, 712-715, Republic of Korea 4 To whom correspondence should be addressed at College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Dahak-ro, Buk-gu, Daegu, 702-701, Republic of Korea. E-mail: [email protected]

(FAS1 domain), and a C-terminal tripeptide Arg-Gly-Asp (RGD) motif [1]. Several studies suggest TGFBIp is involved in cell growth, cell differentiation, wound healing, tumorigenesis, wound healing, and apoptosis [2–5]. Very recently, we reported that TGFBIp is a promising therapeutic target for the treatment of severe vascular inflammatory diseases, such as sepsis and septic shock [4, 6]. In fact, the blockade of TGFBIp, even at later times after the onset of infection, has been shown to rescue mice from lethal sepsis [6]. TGFBIp also acts as a lethal mediator in conditions such as sepsis, in which serum TGFBIp levels are substantially increased [4, 6]. Once released into the extracellular milieu, TGFBIp can bind to cell surface receptors, such as integrins αvβ3 and αvβ5 in human endothelial cells [7]. Teas and herbal infusions are natural beverages which contain compounds that are of particular interest to the health sciences due to their potential in vivo biological properties [8, 9]. One such source is Cyclopia subternata Vogel (Family: Fabaceae; Tribe: Podalrieae), an endemic South African fynbos plant that traditionally has been used

0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York

Lee, Ku, and Bae as a herbal tea called honeybush after Bfermentation,^ a high temperature oxidative process required to produce its characteristic sweet aroma and flavor [10]. Mostly ignored in terms of its commercial potential in the previous century, commercial production of C. subternata commenced in the 1990s to meet the demand for honeybush by the local South African and international markets [10]. The healthpromoting properties of active compounds in C. subternata have been documented such as antioxidant, antiinflammatory, and enhancement of recognition memory [11–13]. It is known that abundant flavonoids are contained in C. subternata, particularly, vicenin-2 and scolymoside [14]. Although some biological activities and pharmacological functions of vicenin-2 and scolymoside have been reported, antiseptic effects of vicenin-2 and scolymoside in TGFBIp- or CLP-induced septic responses in human endothelial cells and mice are not known. Together with our previous reports, which demonstrated the potential effects of TGFBIp on vascular inflammatory responses [4, 6], this study was conducted in an effort to understand the mechanism of the antiseptic action of vicenin-2 and scolymoside by investigating their antiseptic effect on TGFBIp- or CLP-induced septic responses in human endothelial cells and mice.

Cecal Ligation and Puncture For induction of sepsis, male mice were anesthetized with 2 % isoflurane (Forane, JW pharmaceutical, South Korea) in oxygen delivered via a small rodent gas anesthesia machine (RC2, Vetequip, Pleasanton, CA), first in a breathing chamber and then via a facemask. They were allowed to breath spontaneously during the procedure. The cecal ligation and puncture (CLP)-induced sepsis model was prepared as previously described [7]. This protocol was approved by the Animal Care Committee at Kyungpook National University prior to conduct of the study (IRB No. KNU 2012–13). Cell Culture Primary human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex Bio Science (Charles City, IA) and maintained as described previously [7, 15–21]. Briefly, the cells were cultured to confluency at 37 °C and 5 % CO2 in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science). All experiments were carried out with HUVEC at passages 3– 5. Human neutrophils were freshly isolated from whole blood (15 mL) obtained by venipuncture from five healthy volunteers and maintained as previously described [22, 23].

MATERIALS AND METHODS

ELISA for TGFBIp

Reagents

TGFBIp concentrations in cell culture media or mouse serum were determined by competitive ELISA, as described previously [4, 7].

Vicenin-2 and scolymoside, lipopolysaccharide (LPS, serotype: 0111:B4, L5293, used at 100 ng/mL), Evans blue, and crystal violet were obtained from Sigma (St. Louis, MO). Vybrant DiD (used at 5 μM) was obtained from Invitrogen (Carlsbad, CA). TGFBIp protein was purified as described previously [7]. Animals and Husbandry Male C57BL/6 mice (6–7 weeks old, weighing 18– 20 g), purchased from Orient Bio Co. (Sungnam, Republic of Korea), were used after a 12-day acclimatization period. Animals were housed five per polycarbonate cage under controlled temperature (20–25 °C) and humidity (40– 45 %) and a 12:12 h light:dark cycle. Animals received a normal rodent pellet diet and water ad libitum during acclimatization. All animals were treated in accordance with the ‘Guidelines for the Care and Use of Laboratory Animals’ issued by Kyungpook National University (IRB No. KNU 2012–13).

RNA Preparation and Real-Time qRT-PCR HUVEC monolayers were treated with LPS (100 ng/ mL) for 16 h, followed by each compound for 6 h. RNA was isolated using TRI-Reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s suggested protocol. An aliquot (5 μg) of extract RNA was reverse transcribed into first-strand cDNA using a PX2 Thermal Cycler (Thermo Scientific), using 200 U/μL M-MLV reversetranscriptase (Invitrogen, Grand Island, NY) and 0.5 mg/ μL of oligo (dT)-adapter primer (Invitrogen, Grand Island, NY) in a 20-μL reaction mixture. Real-time PCR for HMGB1 and α-actin was performed using a MiniOpticon real-time PCR system (Bio-Rad, Hercules, CA), using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). The primers had the following sequences: for TGFBIp, sense 5′-GCA GAC TCT GCG CTT GAG ATC-3′ and antisense 5′-GGG CTA GTC GCA CAG ACC TC-3′; and for αactin, sense 5′-TGA GAG GGA AAT CGT GCG TG-3′

Antiseptic Effects of Vicenin-2 and Scolymoside and antisense 5′-TTG CTG ATC CAC ATC TGC TGG-3′. The PCR settings were as follows: initial denaturation at 95 °C was followed by 35 cycles of amplification for 15 s at 95 °C and 20 s at 60 °C, with subsequent melting curve analysis, increasing the temperature from 72 to 98 °C. Quantification of gene expression was calculated relative to α-actin. Cell Viability Assay MTT was used as an indicator of cell viability. Cells were grown in 96-well plates at a density of 5×103 cells/ well. After 24 h, cells were washed with fresh medium, followed by treatment with each compound. After an incubation period of 48 h, cells were washed, and 100 μL of MTT (1 mg/mL) was added, followed by incubation for 4 h. Finally, DMSO (150 μL) was added in order to solubilize the formazan salt formed and the amount of formazan salt was determined by measuring the OD at 540 nm using a microplate reader (Tecan Austria GmbH, Austria). Expression of CAMs and Receptor Expression Expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin on HUVECs was determined by whole-cell ELISA, as described previously [7, 24]. Briefly, confluent monolayers of HUVECs were treated with TGFBIp (5 μg/ mL) for 6 h followed by each compound (20 μM) for another 6 h. The medium was removed, and cells were washed with PBS and fixed with 50 μL of 1 % paraformaldehyde for 15 min at room temperature. After washing, 100 μL of mouse anti-human monoclonal antibodies (VCAM-1, ICAM-1, and E-selectin, Temecula, CA, 1:50 each) were applied. After 1 h (37 °C, 5 % CO2), the cells were washed three times, followed by application of 100 μL of 1:2000 peroxidase-conjugated anti-mouse IgG antibodies (Sigma) for 1 h. The cells were washed again three times and developed using o-phenylenediamene substrate (Sigma). Colorimetric analysis was performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells. The same experimental procedures were used for monitoring the cell surface expression of αvβ3 and αvβ5 using specific antibodies obtained from EMD Millipore (MA). Immunofluorescence Staining HUVECs were grown to confluence on glass cover slips coated with 0.05 % poly-L-lysine in complete media

containing 10 % FBS and maintained for 48 h. Cells were then stimulated with HMGB1 (1 μg/mL) for 16 h with or without 6 h each compound (20 μM). For cytoskeletal staining, the cells were fixed in 4 % formaldehyde in PBS (v/v) for 15 min at room temperature, permeabilized in 0.05 % Triton X-100 in PBS for 15 min, and blocked in blocking buffer (5 % BSA in PBS) overnight at 4 °C. Then, the cells were incubated with F-actin labeled fluorescein phalloidin (F 432; Molecular Probes, Invitrogen) and visualized by confocal microscopy at a ×630 magnification (TCS-Sp5, Leica microsystem, Germany). Permeability Assay In Vitro Permeability was quantitated by spectrophotometric measurement of the flux of Evans blue-bound albumin across functional HUVEC monolayers using a modified two-compartment chamber model, as previously described [7]. Briefly, HUVECs were plated (5 × 10 4 /well) in Transwells with a pore size of 3 μm and a diameter of 12 mm for 3 days. The confluent monolayers were treated TGFBIp (5 μg/mL, for 6 h) followed by incubation with each compound for 6 h. Migration Assay In Vitro Migration assays were performed in Transwell plates with a diameter of 6.5 mm, with filters having a pore size of 8 μm. HUVECs (6×104) were cultured for 3 days in order to obtain confluent endothelial monolayers. Before addition of human neutrophils to the upper compartment, cell monolayers were treated with TGFBIp (5 μg/mL, for 6 h) followed by treatment with each compound for 6 h. Cells in the upper chamber of the filter were aspirated and non-migrating cells on top of the filter were removed using a cotton swab. Human neutrophils on the lower side of the filter were fixed with 8 % glutaraldehyde and stained with 0.25 % crystal violet in 20 % methanol (w/v). Each experiment was repeated in duplicate wells, and, within each well, nine randomly selected high-power microscopic fields (HPF, ×200) were counted and expressed as a migration index. In Vivo Permeability and Leukocyte Migration Assay For in vivo study, male mice were anesthetized with 2 % isoflurane (Forane, JW pharmaceutical, South Korea) in oxygen delivered via a small rodent gas anesthesia machine (RC2, Vetequip, Pleasanton, CA), first in a breathing chamber and then via a facemask. They were allowed to breathe spontaneously during the procedure. Mice were

Lee, Ku, and Bae treated with TGFBIp (0.1 mg/kg, i.v.) for 6 h followed by treatment with vicenin-2 or scolymoside (11.9. or 23.8 μg/ mouse) for 6 h. For in vivo permeability assay, 1 % Evans blue dye solution in normal saline was injected intravenously into each mouse. Thirty minutes later, mice were euthanized and peritoneal exudates were collected by washing cavities with 5 mL of normal saline and centrifuging at 200 g for 10 min. Absorbance of supernatants was read at 650 nm. Vascular permeabilities are expressed as micrograms of dye/mouse that leaked into the peritoneal cavity and were determined using a standard curve, as previously described [25, 26]. For assessment of leukocyte migration, mice were euthanized after 6 h and peritoneal cavities were washed with 5 mL of normal saline. Samples (20 μL) of peritoneal fluids obtained were mixed with 0.38 mL of Turk’s solution (0.01 % crystal violet in 3 % acetic acid), and numbers of leukocytes were counted under a light microscope.

Cell-Cell Adhesion Assay Adherence of human neutrophils to endothelial cells was evaluated by fluorescent labeling of human neutrophils as previously described [27–29]. Briefly, purified human neutrophils (1.5 × 106/mL, 200 μL/ well) were labeled with Vybrant DiD dye and then added to washed and stimulated HUVECs. HUVEC monolayers were treated with TGFBIp (5 μg/mL) for 6 h followed by treatment with each compound from 0 to 20 μM for 6 h.

Hematoxylin & Eosin Staining and Histopathological Examination Male C57BL/6 mice underwent CLP and were administered vicenin-2 or scolymoside (23.8 μg/mouse) intravenously at 12 and 50 h after CLP (n=5). Mice were euthanized 96 h after CLP. To analyze the phenotypic change of the lung in mouse, lung samples were removed from each mouse, washed three times in phosphate buffered saline (PBS, pH 7.4) to remove remaining blood, fixed in 4 % formaldehyde solution (Junsei, Tokyo, Japan) in PBS, pH 7.4 for 20 h at 4 °C. After fixation, the samples were dehydrated through ethanol series, embedded in paraffin, sectioned at 4 μm, and placed on a slide. The slides were deparaffinized in a 60 °C oven, rehydrated, stained with hematoxylin (Sigma). To remove overstaining, the slides were three quick dipped in 0.3 % acid alcohol and counterstained with eosin (Sigma). They were then removed, overstaining in ethanol series and xylene, then cover slipped. Light microscopic analysis of the lung specimens was done by blinded observation to evaluate pulmonary architecture, tissue edema, and infiltration of the inflammatory cells as previously defined [30]. The results were classified into four grades where grade 1 represented normal histopathology; grade 2 indicated minimal neutrophil leukocyte infiltration; grade 3 represented moderate neutrophil leukocyte infiltration, perivascular edema formation, and partial destruction of pulmonary architecture; and finally, grade 4 included dense neutrophil leukocyte infiltration, abscess formation, and complete destruction of pulmonary architecture. Measurements of Organ Injury

ELISA for Phosphorylated p38 Mitogen-Activated Protein Kinase, Nuclear Factor-κB, Tumor Necrosis-α, Extracellular Regulated Kinases 1/2, and Interleukin-6 Expression of phosphorylated p38 mitogen-activated protein kinase (MAPK) was quantified according to the manufacturer’s instructions using a commercially available ELISA kit (Cell Signaling Technology, Danvers, MA). Total and phosphorylated p65 nuclear factor (NF)-κB (#7174, #7173, Cell Signaling Technology) or total and phosphorylated extracellular regulated kinase (ERK) 1/2 (R&D Systems, Minneapolis, MN) activities in nuclear lysates were determined using ELISA kits. The concentrations of interleukin-6 (IL-6) and tumor necrosis (TNF)-α in cell culture supernatants were determined using ELISA kits (R&D Systems). Values were measured using an ELISA plate reader (Tecan, Austria GmbH, Austria).

Plasma levels of aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine were measured using commercial assay kits (Pointe Scientific, Lincoln Park, MI). Statistical Analysis All experiments were performed independently at least three times. Values are expressed as mean±standard error of the mean (SEM). The statistical significance of differences between test groups was evaluated by one-way analysis of variance (ANOVA) and Tukey’s post hoc test. Kaplan-Meier survival analysis was performed for evaluation of overall survival rates. SPSS for Windows, version 16.0 (SPSS, Chicago, IL) was used to perform statistical analysis, and statistical significance was accepted for p values 2 μM. However, in the absence of LPS pretreatment, VCN or SCL did not affect TGFBIp release (Fig. 1a). In order to confirm these effects in vivo, CLP-induced septic mice were used because this model more closely resembles human sepsis than LPS-induced endotoxemia [31]. As shown in Fig. 1b, treatment with VCN or SCL resulted in marked inhibition of CLP-induced release of TGFBIp. Assuming that the average weight of a mouse was 20 g, and the average blood volume was 2 mL, the amount of VCN or SCL injected (11.9 or 23.8 μg per mouse) was equivalent to 10 or 20 μM in peripheral blood. To determine the molecular mechanism by which VCN or SCL inhibited the release of LPS-mediated TGFBIp, we tested the effects of VCN or SCL on the transcriptional regulation of TGFBIp by LPS in HUVECs. Thus, we measured the effect of MTU on LPS-induced TGFBIp mRNA levels using real-time qRT-PCR. As shown in Fig. 1c, LPS induced an increase in the expression levels of TGFBIp mRNA and treatment with VCN or SCL that resulted in

decreased expression levels of LPS-induced TGFBIp mRNA. Next, we investigated the effects of VCN or SCL on expression of the TGFBIp receptors, integrin αvβ3 and αvβ5 in HUVECs [7]. As shown in Fig. 1c, treatment with LPS resulted in over fourfold increase in expression of αvβ5 in HUVECs, and treatment with VCN or SCL resulted in significantly inhibited expression of αvβ5. However, consistent to a previous report [7], the expression of integrin αvβ3 was not changed by LPS [7] nor each compound (Fig. 1d). Therefore, the inhibitory effects of VCN or SCL on release of TGFBIp were mediated by suppression of TGFBIp receptor (integrin αvβ5). To assess the cytotoxicity of VCN or SCL, cell viability assays were performed in HUVECs treated with VCN and SCL for 24 h. At concentrations up to 50 μM, VCN or SCL did not affect cell viability (Fig. 1e). High plasma concentrations of TGFBIp in patients with sepsis are known to be related to the severity of sepsis [7], and pharmacological inhibition of TGFBIp is known to improve survival in animal models of sepsis [6]. Therefore, prevention of CLP-induced release of TGFBIp by VCN

Fig. 2. Effects of VCN or SCL on TGFBIp-mediated permeability in vitro and in vivo. The effects of posttreatment with different concentrations of VCN (white bar) or SCL (black bar) for 6 h on the barrier disruptions caused by TGFBIp (a, 5 μg/mL, 6 h) were monitored by measuring the flux of Evans bluebound albumin across HUVECs. b The effects of VCN (white bar) or SCL (black bar) at 10 or 20 μM/mouse on TGFBIp-induced (0.1 mg/kg, i.v.) 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 cover slips were stimulated with HMGB1 for 1 h and then treated with each compound (20 μM) for 6 h and stained for F-actin. Arrows indicate intercellular gaps. d HUVECs were activated with TGFBIp (5 μg/mL, 6 h), followed by treated with different concentrations of VCN (white bar) or SCL (black bar) for 6 h. The effects of each compound on TGFBIp-mediated expression of phospho p38 were determined by ELISA. Results are expressed as the mean ± SEM of at least three independent experiments. D = 0.2 % DMSO is the vehicle control. *p < 0.05 versus TGFBIp alone.

Antiseptic Effects of Vicenin-2 and Scolymoside

Fig. 3. Effects of VCN or SCL on TGFBIp-mediated proinflammatory responses. a–c HUVECs were stimulated with TGFBIp (5 μg/mL) for 6 h, followed by treatment with each compound (a, 20 μM each) or (b, c) increasing concentrations of VCN (white bar) or SCL (black bar) for 6 h. 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 VCN (white bar) or SCL (black bar) at 10 or 20 μM/mouse on leukocyte migration into the peritoneal cavities of mice caused by TGFBIp (0.1 mg/kg, i.v.) were analyzed. All results indicate the mean ± SEM of three separate experiments (n = 5). D = 0.2 % DMSO is the vehicle control. *p < 0.05 vs. TGFBIp.

and SCL suggests the potential for use of VCN and SCL in treatment of vascular inflammatory diseases. Effect of VCN or SCL on TGFBIp-Mediated Vascular Barrier Disruption A permeability assay was performed to determine the effects of VCN or SCL on the barrier integrity of HUVECs. Treatment with 20 μM VCN or SCL alone did not alter barrier integrity (Fig. 2a). In contrast, TGFBIp is known to cause cleavage and disruption of endothelial barrier integrity [6, 7]. Thus, HUVECs were treated with various concentrations of each compound for 6 h after addition of TGFBIp (5 μg/mL). As shown in Fig. 2a, treatment with VCN or SCL resulted in a dose-dependent decrease in TGFBIp-mediated disruption of barrier integrity. TGFBIp-mediated vascular permeability in mice was assessed in order to confirm this vascular barrier protective effect in vivo. As shown in Fig. 2b, treatment with VCN or SCL resulted in markedly inhibited peritoneal leakage of dye induced by TGFBIp. Cytoskeletal proteins are important for the maintenance of cell integrity and shape [32]. In addition, redistribution of the actin cytoskeleton, detachment of cells, and loss of cell-cell contact due to cytokine stimulation are all

associated with increased endothelial monolayer permeability [33, 34]. Therefore, we next examined the effects of VCN or SCL on actin cytoskeletal arrangement in HUVECs by immunofluorescence staining of HUVEC monolayers with F-actin-labeled fluorescein phalloidin. Control HUVECs exhibited a random distribution of Factin throughout the cells, with some localization of actin filament bundles at the cell boundaries (Fig. 2c). Barrier disruption in HUVECs induced by HMGB1 treatment (1 μg/mL) was accompanied by the formation of paracellular gaps (shown by arrows). In addition, treatment with VCN or SCL (20 μM) inhibited the formation of HMGB1-induced paracellular gaps with the formation of dense F-actin rings (Fig. 2c). These results suggest that VCN or SCL treatment inhibited the HMGB1-mediated morphological changes and gap formation in endothelial cells, which are associated with F-actin redistribution, thereby increasing vascular barrier integrity. Sepsis induces, such as high mobility group box 1 (HMGB1) and LPS, are known to induce proinflammatory responses by promoting phosphorylation of p38 MAPK [35–38]. Therefore, we determined whether TGFBIp could also enhance the phosphorylation of p38 MAPK, if so, determined whether VCN or SCL inhibit TGFBIp-induced activation of p38 MAPK in HUVECs.

Lee, Ku, and Bae

Fig. 4. Effects of VCN or SCL on TGFBIp-stimulated production of TNF-α/IL-6 and activation of NF-κB/ERK 1/2. a, b HUVECs were stimulated with TGFBIp (5 μg/mL) for 6 h, followed by treatment with VCN or SCL for 6 h. TGFBIp-mediated production of TNF-α (a) or IL-6 (b) in HUVECs was analyzed after treatment of cells with VCN or SCL at 10 μM (white bar) or 20 μM (black bar) for 6 h. c–f Confluent HUVECs were activated with TGFBIp (5 μg/mL, 6 h), followed by incubation with VCN or SCL at 10 μM (c, e) or at 20 μM (d, f) for 6 h and TGFBIp-mediated activation of phospho-NF-κB p65 (white bar) or total NF-κB p65 (black bar) in HUVECs was analyzed (c, d) or phospho-ERK1/2 (white box) or total ERK1/2 (black box) in HUVECs was analyzed (e, f). D = 0.2 % DMSO is the vehicle control. *p < 0.05 vs. TGFBIp.

As shown in Fig. 2d, TGFBIp induced the activation of p38 MAPK, which was significantly inhibited by treatment with VCN or SCL. These findings demonstrate inhibition of TGFBIp-mediated endothelial disruption and maintenance of human endothelial cell barrier integrity by VCN and SCL in mice treated with TGFBIp. Effects of VCN or SCL on TGFBIp-Mediated CAMs Expression, Neutrophil Adhesion, and Migration Several studies have report that TGFBIp enhanced the expression of CAMs, such as ICAM-1, VCAM-1, and E-selectin, on the surfaces of human cells, thereby promoting adhesion and migration of leukocytes across the endothelium to sites of inflammation [4, 7, 39, 40]. According to our findings, TGFBIp induced upregulation of the surface expression of VCAM-1, ICAM-1, and E-Selectin (Fig. 3a) and VCN or SCL inhibited this effect, suggesting that the inhibitory effects of

VCN and SCL on expression of CAMs are mediated via attenuation of the TGFBIp signaling pathway by VCN or SCL. The optimized concentration of each compound for this experiment was 20 μM (data not shown). In addition, elevated expression of CAMs corresponded well with enhanced binding of human neutrophils to TGFBIp-activated endothelial cells, followed by their migration. In addition, treatment with VCN or SCL resulted in downregulation of human neutrophils adherence and their subsequent migration across activated endothelial cells in a concentrationdependent manner (Fig. 3b, c). These results suggest that VCN and SCL not only inhibit endotoxinmediated release of TGFBIp in endothelial cells but also downregulate the pro-inflammatory signaling effect caused by release of TGFBIp, thereby inhibiting amplification of inflammatory pathways by nuclear cytokines. To confirm this effect in vivo, we examined TGFBIp-induced migration of leukocytes in mice.

Antiseptic Effects of Vicenin-2 and Scolymoside

Fig. 5. Effects of VCN or/and SCL on lethality or organ injury after CLP. a Male C57BL/6 mice (n = 20) were administered VCN (23.8 μg/mouse, i.v., white square) or SCL (23.8 μg/mouse, i.v., black square) at 12 and 50 h after CLP. Animal survival was monitored every 6 h after CLP for 132 h. Control CLP mice (black circle) and sham-operated mice (white circle) were administered sterile saline (n = 20). Kaplan-Meier survival analysis was used for determination of overall survival rates versus CLP-treated mice. b Male C57BL/6 mice underwent CLP and were administered VCN (23.8 μg/mouse) or SCL (23.8 μg/ mouse) intravenously at 12 and 50 h after CLP (n = 5). Mice were euthanized 96 h after CLP. Histopathological scores of the lung tissue were recorded as described in methods. *p < 0.05 vs. CLP. c Photomicrographs of lung tissues (H&E staining, ×200). Sham group (grade 1); CLP group (grade 3); right, CLP + VCN and CLP + SCL group, (grade 2). Illustrations indicate representative images from three independent experiments. d–f The same as b, c except that mice were bled and euthanized. AST, ALT, BUN, and creatinine level in plasma was measured. All results indicate the mean ± SEM of three separate experiments. D = 0.2 % DMSO is the vehicle control. *p < 0.05 vs. CLP.

TGFBIp was found to stimulate migration of leukocytes into the peritoneal cavities of mice, and treatment with VCN or SCL resulted in a significant reduction of peritoneal leukocyte counts (Fig. 3d). Experiments on CAMs are widely performed in vitro for study of regulation of the interactions between leukocytes and endothelial cells [41, 42]. In the current study, treatment with VCN and SCL resulted in downregulation of TGFBIp-induced levels of VCAM-1, ICAM-1, and Eselectin, suggesting that VCN and SCL inhibit the adhesion and migration of leukocytes to inflamed endothelium. Effects of VCN or SCL on TGFBIp-Stimulated Production of TNF-α/IL-6 and Activation of NF-κB/ERK Pro-inflammatory cytokines (TNF-α, IL-1α and IL-1β, IL-12, and IL-6) are necessary for initiation of

an effective inflammatory process against infection, whereas their excess production has been associated with multiple organ-system dysfunction and mortality [43, 44]. Therefore, we determined the effects of VCN or SCL on TGFBIp-mediated production of proinflammatory cytokines. According to the results, levels of IL-1α, IL-1β (data not shown), TNF-α, and IL-6 increased in TGFBIp-stimulated endothelial cells; these increases were significantly reduced by VCN or SCL (Fig. 4a, b), indicating that VCN and SCL regulate the most important signals involved in induction of proinflammatory responses in human endothelial cells. In addition, activation of NF-κB and ERK1/2 is required for initiation of pro-inflammatory responses [45–47]. Therefore, we evaluated the effects of VCN or SCL on activation of NF-κB and ERK 1/2 by TGFBIp. As shown in Fig. 4c–f, treatment with TGFBIp resulted in increased activation of NF-κB and ERK 1/2 and

Lee, Ku, and Bae these increases were significantly reduced by treatment with VCN or SCL. The inflammatory response is an important component in the pathogenesis of vascular injury, and endothelial dysfunction is specifically related to leukocyte recruitment during formation of vascular inflammatory lesions [48, 49]. Tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) are two important mediators involved in vascular inflammatory processes. NF-κB is a well-known proinflammatory transcriptional factor [45, 46]. Activation of NF-κB occurs in response to pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) [45, 46]. There is considerable evidence suggesting that suppression of the NF-κB signaling pathway confers significant vascular protective effects [46, 50], which delay or prevent development of vascular diseases in animal models of disease [51, 52]. Therefore, preventing production of TNF-α and activation of NF-κB in vascular endothelial cells are considered promising therapeutic targets for treatment of vascular inflammatory diseases and VCN and SCL exert an anti-inflammatory effect via inhibition of production of inflammatory mediators and the NF-κB pathway. Protective Effect of VCN and/or SCL in CLP-Induced Septic Mortality Sepsis is a systemic response to serious infection and has a poor prognosis when it is associated with organ dysfunction, hypoperfusion, or hypotension [53, 54]. Based on the above-described findings, we hypothesized that treatment with VCN or SCL would result in reduced mortality in our CLP-induced sepsis mouse model. To investigate the question of whether VCN or SCL protects mice from CLP-induced sepsis lethality, VCN or SCL was administered to mice after CLP. Twenty-four hours after the operation, animals manifested signs of sepsis, such as shivering, bristled hair, and weakness. Administration of VCN or SCL (23.8 μg/mouse) 12 h after CLP did not prevent CLP-induced death (data not shown); therefore, they were administered two times (once 12 h after CLP and once 50 h after CLP), which resulted in an increase in the survival rate from 40 to 50 %, according to Kaplan-Meier survival analysis (p < 0.0001, Fig. 5a). This marked survival benefit achieved by administration of VCN or SCL suggests that suppression of TGFBIp release and of TGFBIp-mediated inflammatory responses provides a therapeutic strategy for management of sepsis and septic shock. We also tested the

addictive effects of VCN and SCL in CLP-induced death. To do this, VCN and SCL (23.8 μg/mouse each) were co-administrated two times (once 12 h after CLP and once 50 h) after CLP. Data showed that there were no additive effects by VCN and SCL (data not shown). Protective Effect of VCN or SCL in the CLP-Induced Organ Damage To confirm the protective effects of VCN or SCL on CLP-induced death, we determined the effects of each compound on CLP-induced pulmonary injury. There were no significant differences between the lungs of sham and sham + each compound in light microscopic observations (data not shown). In the CLP group, interstitial edema with massive infiltration of the inflammatory cells into the interstitium and alveolar spaces were observed and the pulmonary architecture was severely damaged (Fig. 5b, c). These morphological changes were less pronounced in the CLP + VCN and CLP + SCL group (Fig. 5b, c). Systemic inflammation during sepsis frequently causes multiple organ failure (MOF), in which the liver and kidney are major target organs [55]. CLP resulted in significant increases in the plasma level of ALT and AST (markers of hepatic injury, Fig. 5d) and BUN and creatinine (markers of renal injury, Fig. 5e. f), which are reduced by VCN and SCL. In summary, our results demonstrate that VCN and SCL inhibit both LPS- and CLP-mediated release of TGFBIp, expression of TGFBIp receptor (integrin αvβ5), and TGFBIp-mediated barrier disruption through increases in barrier integrity and inhibition of CAM expression. In addition, VCN and SCL reduce human neutrophil adhesion and migration toward HUVECs. These barrier protective effects of VCN and SCL were confirmed in a mouse model, in which treatment with VCN and SCL resulted in reduction of TGFBIp-induced mortality. Our findings indicate that VCN and SCL merit use as potential therapeutic agents for severe vascular inflammatory diseases, such as sepsis and septic shock.

ACKNOWLEDGMENTS This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government [MSIP] (Grant No. 2012R1A5A2A42671316).

Antiseptic Effects of Vicenin-2 and Scolymoside Conflict of interest. The authors declare that they have no competing interests.

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