Pharmacological Research Elsevier Editorial System(tm) for Manuscript Draft
Manuscript Number: YPHRS-D-15-00157R2
Title: beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation
Article Type: Regular Papers
Keywords: endothelial cells; beta-Naphthoflavone; inflammation; adhesion molecule
Corresponding Author: Dr. Shinn-Jong Jiang, Ph.D. Corresponding Author's Institution: Tzu Chi University First Author: Sheng-Yao Hsu
Order of Authors: Sheng-Yao Hsu; Je-Wen Liou; Tsung-Lin Cheng; Shih-Yi Peng; Chi-Chen Lin; Yuan-Yuan Chu; Wei-Cheng Luo; Zheng-Kai Huang; Shinn-Jong Jiang
Abstract: β-Naphthoflavone (β-NF), a ligand of the aryl hydrocarbon receptor, has been shown to possess anti-oxidative properties. We
investigated the anti-oxidative and anti-inflammatory potential of β-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (α). Pretreatment with β-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of
p67phox, and TNF-α-induced monocyte binding and transmigration. In addition, β-NF significantly inhibited TNF-α-induced ICAM-1and VCAM-1 expression. The mRNA expression levels of the inflammatory cytokines TNF-α and IL-6 were reduced by β-NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition of adhesion molecules was associated with suppressed nuclear translocation of NF-κB p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation of Egr-1, a downstream transcription factor involved in the MEK-ERK
signaling pathway, was suppressed by β-NF treatment. Our findings show that β-NF inhibits TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby suppressing the expression of adhesion molecules. This results in reduced adhesion and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes in a peritonitis model. Our findings also suggest that β-NF might prevent TNF-α-induced
Cover Letter
Sep 30, 2015
Professor Emilio Clementi Editor in Chief
Pharmacological Research
Ms. Ref. NO.: YPHRS-D-15-00157R1
Title: beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation
Authors: Hsu et al.
Dear Professor Emilio Clementi,
Thank you for your communication dated Sep 04, 2015 regarding our revised manuscript. We were pleased to know that our manuscript was rated as potentially acceptable for publication in Pharmacological Research, subject to adequate revision and response to the comments raised by the reviewer. The manuscript has been revised according to the reviewer’s suggestions. The rewritten text is highlight (in
broad letter) in the revised manuscript.
As you notice, we have revised the manuscript by modifying the Abstract, Introduction, Methods, Results, Discussion and Reference sections, based on the comments made by the reviewers.
Detailed responses to the reviewer are included in a separate document. As you notice, we agreed with all the comments raised by the reviewer. We would like to take this opportunity to express our sincere thanks to the reviewer who identified areas of our manuscript that needed corrections or modification. We would like also to thank you for allowing us to resubmit a revised copy of the manuscript.
I hope that the revised manuscript is accepted for publication in Pharmacological Research.
Sincerely Yours,
Assistant Professor
Department of Biochemistry
School of Medicine, Tzu Chi University Hualien 97004, Taiwan
Tel.: 886-3-8565301#2431 Fax: 886-3-8580641
*Response to Reviewers
Sep 30, 2015
Dear Professor Emilio Clementi:
Thank you for your communication dated Sep 04, 2015 regarding our revised manuscript. We are submitting our revised manuscript No. YPHRS-D-15-00157R1 entitled “beta-Naphthoflavone protects from peritonitis by reducing
TNF-alpha-induced endothelial cell activation” for reconsideration of publication in Pharmacological Research. The manuscript has been revised according to the
reviewer’s suggestions. The rewritten text is highlight (in broad letter) in the revised manuscript. All questions have been considered and answered as follows:
Reviewer#1:
1) Extra word “leukocytes” in Abstract is deleted in the new revised manuscript. 2) The paragraphs on Egr-1 and lack of mutagenesis in NF in Introduction section
are moved to Discussion section (page 23, paragraph 1, line 1-11 and page 18, line 9-21) according to reviewer’s suggestion.
3) According to reviewer’s suggestion, Methods section is shortened. The detail methods about Preparation of nuclear extracts, Western blotting, Immunofluorescent analysis, and RNA interference are described in supplementary Materials and Methods.
4) There is no Figure 1b in Figure legend. Actually, it is “Figure 1.” “-NF…” in the manuscript.
5) We are sure that Section 3.3 refers to figure 3 in the manuscript.
As reviewer argued, the values listed in the paragraph for fold changes are not very large. We repeated the western blotting about ICAM-1 and VCAM-1 as revised Figure 3 in 3 independent experiments and all original data are uploaded as supplementary data figure S8 and S9. The fold increase in TNF- then decreased by various concentrations of -NF was described in the section 3.3 (page 14, paragraph 2, line 5-11). The number of cell culture done for the westerns and the standard deviations on the graphs are also listed.
We thank the reviewer for helpful comments and hope the manuscript is now acceptable for publication in the Pharmacological Research. Thank you and best regards.
Shinn-Jong Jiang, Ph.D. Assistant Professor
Department of Biochemistry
School of Medicine, Tzu Chi University Hualien 97004, Taiwan
Tel.: 886-3-8565301#2431 Fax: 886-3-8580641
*Manuscript
Click here to view linked References
1 beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced
2 endothelial cell activation
3 Sheng-Yao Hsua, Je-Wen Lioub, Tsung-Lin Chengc,g, Shih-Yi Pengb, Chi-Chen Lind,
4 Yuan-Yuan Chuh, Wei-Cheng Luoe, Zheng-Kai Huangf, Shinn-Jong Jiangb,*
5
6 aDepartment of Ophthalmology, Tainan Municipal An-Nan Hospital-China Medical 7 University, Tainan, Taiwan
8 bDepartment of Biochemistry, School of Medicine, Tzu Chi University, Hualien,
9 Taiwan
10 cDepartment of Physiology, College of Medicine, Kaohsiung Medical University,
11 Kaohsiung, Taiwan
12 dInstitute of Biomedical Sciences, College of Life Sciences, National Chung Hsing
13 University, Taichung, Taiwan
14 eMaster program in Microbiology, Immunology and Biochemistry, School of
15 Medicine Master Thesis, Tzu Chi University, Hualien, Taiwan
16 fBachelor in Department of Molecular Biology and Human Genetics, College of Life
17 Sciences, Tzu Chi University, Hualien, Taiwan
18 gOrthopaedic Research Center, College of Medicine, Kaohsiung Medical University 19 Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
1 hPostgraduate program in Biochemistry, School of Medicine, Tzu Chi University, 2 Hualien, Taiwan 3 4 5 *Corresponding author: 6 Shinn-Jong Jiang, PhD 7 Assistant Professor
8 Department of Biochemistry, College of Medicine 9 Tzu Chi University
10 Hualien 97004, Taiwan 11 Tel.: +886-3-8565301-2431 12 Fax: +886-3-8580641
13 E-mail: sjjiang@mail.tcu.edu.tw
14
15 The authors have no conflicts of interest that they wish to declare.
16
17 Running title: -Naphthoflavone inhibits TNF--induced inflammation 18
1 Abstract
2 β-Naphthoflavone (-NF), a ligand of the aryl hydrocarbon receptor, has been shown 3 to possess anti-oxidative properties. We investigated the anti-oxidative and
4 anti-inflammatory potential of -NF in human microvascular endothelial cells treated 5 with tumor necrosis factor-alpha (TNF-). Pretreatment with -NF significantly 6 inhibited TNF--induced intracellular reactive oxygen species, translocation of 7 p67phox, and TNF--induced monocyte binding and transmigration. In addition, -NF 8 significantly inhibited TNF--induced ICAM-1and VCAM-1 expression. The mRNA 9 expression levels of the inflammatory cytokines TNF- and IL-6 were reduced by 10 -NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition 11 of adhesion molecules was associated with suppressed nuclear translocation of NF-κB
12 p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation
13 of Egr-1, a downstream transcription factor involved in the MEK-ERK signaling
14 pathway, was suppressed by -NF treatment. Our findings show that -NF inhibits 15 TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby
16 suppressing the expression of adhesion molecules. This results in reduced adhesion
17 and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes 18 in a peritonitis model. Our findings also suggest that -NF might prevent
1
2 Keywords
3 endothelial cells; -Naphthoflavone; inflammation; adhesion molecule 4
5 Abbreviations
6 -NF, -Naphthoflavone; Egr-1, early growth response gene-1; ERK, extracellular 7 signal-regulated kinase; HUVECs, human umbilical vein endothelial cells; ICAM-1,
8 intracellular adhesion molecule-1; LPS, lipopolysaccharide; MCP-1, monocyte
9 chemotactic protein-1; NF-B, nuclear factor-B; TNF-, tumor necrosis factor; 10 VCAM-1, vascular cell adhesion molecule-1.
1 1. Introduction
2 Vascular inflammatory responses are modulated by complex interactions between
3 circulating leukocytes, tissue-resident leukocytes and the vascular endothelium. 4 Recruitment of leukocytes to endothelium depends on the interactions of the 5 endothelial-cell surface proteins E- and P-selectins with their ligands presented on 6 leukocytes. Vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion 7 molecule-1 (ICAM-1) are most prominently involved in this process [1-3]. Enhanced 8 expression of endothelial cell adhesion molecules and have been proposed to be
9 related to a variety of diseases [4-9]. Exposure to pro-inflammatory molecules such
10 as lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-) and interleukin-1 11 (IL-1) also leads to a significant increase in the expression of adhesion molecules on 12 the surface of endothelial cells [10-12]. Nuclear factor-B (NF-B) may also have a 13 stimulatory effect on adhesion molecules [13,14], while oxidative stress is suggested 14 to be another factor that contributes to the regulation of VCAM-1 [15].
15 Reactive oxygen species (ROS) have a deleterious effect on vascular functions 16 [16,17]. Superoxide also provokes monocytes to secrete several pro-inflammatory 17 factors such as TNF-, interleukin-6 (IL-6), interleukin-10 (IL-10) and monocyte 18 chemoattractant protein-1 (MCP-1), and all these factors are mediators or modulators 19 of inflammatory reactions in the vascular wall [18]. Recent studies on atherosclerosis 20 suggested that the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 21 enzyme complex of vascular cells plays a critical role in the production of superoxide 22 in atherosclerotic lesions [19]. It is essential to decrease the production of ROS as 23 they are implicated in the pathogenesis of several diseases.
24 The compound β-naphthoflavone (-NF; 5,6-benzoflavone) is a synthetic derivative 25 of a naturally occurring flavonoid compound. It is known to strongly upregulate
1 cytochrome P-450 (CYP) 1A expression via activation of the aryl hydrocarbon
2 receptor (AhR) [20,21], best known for playing important role in regulating the
3 toxicity of xenobiotics such as halogenated aromatic hydrocarbons (HAHs) and
4 polycyclic aromatic hydrocarbons (PAHs). Interaction with the HAH
5 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), results in the AhR-dependent
6 upregulation of several species- and tissue-specific toxic and biological responses [22],
7 including the inhibition of T-cell dependent immune responses [23]. β-NF has also 8 been shown to mitigate dextran sulfate sodium -induced colitis [24].
9 On the other hand, -NF can increase the expression of a P450-derived
10 arachidonic acid metabolite, 5,6-epoxyeicosatrienoic acid (EET), which induces
11 hyperpolarization by activation of Ca2+-dependent K+ channels [25]. It is now known
12 that EETs are endothelium-derived hyperpolarizing factors (EDHFs). A recent report
13 by Node et al. outlined a new autocrine role for EETs in endothelial cells, suggesting
14 they act as anti-inflammatory mediators [26]. Node et al. reported that EETs are
15 predominant inhibitors of CAM expression promoted by TNF-, IL-1, and bacterial
16 LPS. Although EETs suppressed the expression of VCAM-1, E-selectin, and ICAM-1,
17 their effects upon VCAM-1 were the most pronounced. However, the effects of -NF
1 In this study, we demonstrate that -NF reduces the expression of adhesion 2 molecules in endothelial cells, and decreases mononuclear cell adhesion by
3 suppressing NF-B signaling. Our results suggest that -NF helps to reduce the risk 4 of vascular inflammation by decreasing plasma cytokine release and by directly acting
5 on the vascular endothelium.
6
7 2. Materials and methods 8 2.1. Cell culture
9 The human microvascular endothelial cells HMEC-1 (ATCC, No CRL-10636) 10 was obtained from American Type Culture Collection (Teddington, UK) and 11 cultured in MCDB131 medium containing endothelial cell growth supplement 12 (Millipore, Billerica, MA, USA) and 15% fetal bovine serum (FBS), as previously 13 described [27]. The human monocytic cell line THP-1 was maintained in
14 RPMI-1640 medium supplemented with 10% FBS.
15
16 2.2. Cell viability assays
17 HMEC-1 cells were grown to confluence in 96-well plates. Upon reaching 18 confluence, medium containing -NF at various concentrations was added. After a 19 48-h incubation, the viability of HMEC-1 cells was determined using a WST-1
1 assay (Roche, Indianapolis, IN, USA), according to the manufacturer’s 2 instructions.
3
4 2.3. Monocyte-HMEC-1 adhesion assays
5 HMEC-1 cells were grown to confluence in 24-well culture plates, treated with 6 -NF for 1 h, and then stimulated with 10 ng/ml TNF-α for 18 h. THP-1 monocytes 7 were suspended in RPMI 1640 containing 0.1% bovine serum albumin (BSA) and 8 labeled with 5 M Calcein-AM for 30 min at 37°C; they were then washed twice 9 with Hank’s buffered saline solution (HBSS). Fluorescently labeled monocytes 10 (2 × 105 cells/well) were then added and incubated with the -NF-treated HMEC-1 11 cells for 30 min at 37C. Non-adherent monocytes that had not adhered were
12 removed by gently washing three times with HBBS. Images of adherent THP-1 cells 13 (5 images/well) were obtained using fluorescence microscopy, with the number of 14 bound monocytes counted using AlphaImager 2200 software.
15
16 2.4. Transendothelial migration
17 Migration assays were performed in 24-well 6.5-mm diameter trans-well plates 18 containing polycarbonate membranes with filters (8-μm pore size) (BD Biosciences). 19 Briefly, HMEC-1 cells cultured on trans-well filters were pretreated with increasing
1 concentrations of -NF for 1 h and stimulated with 10 ng/mL TNF-α for 18 h. 2 THP-1 monocytes (2 × 105 cells/well) were added to the upper chambers of 3 trans-well inserts containing 50 μL of RPMI 1640. After a 4-h incubation at 4 37C/5% CO2, cells that had migrated to the lower chamber were harvested and 5 counted with a microscope. All experiments were conducted independently at least 6 three times, with the data representing the mean number of THP-1 cells that had 7 migrated.
8
9 2.5. Measurement of ROS production
10 Intracellular ROS production was assessed using 2′,7′-diclorofluorescein 11 diacetate (H2DCFDA; Invitrogen). HMEC-1 cells were treated with -NF for 1 h 12 and then 10 ng/mL TNF-α for 20 min. HMEC-1 cells were incubated with
13 H2DCFDA (10 M) for 5 min at 37°C. Images were obtained using a fluorescence 14 microscope (IX-71, Olympus). Fluorescence intensity was measured using Image J 15 software, averaged, and normalized to the control value. Three independent
16 experiments were performed.
17
18 2.6. Transient transfection and luciferase assays
1 transfected with plasmids using Lipofectamine (Invitrogen), according to the 2 manufacturer’s protocol. Briefly, transfection mixtures contained 0.5 μg of 3 pGL3-4κB-Luc or 0.1 μg of pCMV-β-gal and were mixed with the Lipofectamine 4 reagent before adding to cells for 6 h. Cells were washed and fresh medium was 5 added. After 18 h, cells were treated with -NF for 1 h and stimulated with TNF-α 6 for 6 h. After cells were lysed, luciferase and β-galactosidase activities were
7 determined using a luciferase assay kit (Promega, Madison, WI, USA) according to 8 the manufacturer’s instructions. Luciferase activity was normalized with respect to 9 β-galactosidase activity and expressed as a percentage of control activity.
10
11 2.7. RNA isolation and quantitative polymerase chain reaction (qPCR) assays 12 HMEC-1 cells were grown to confluence in 6-cm2 culture plates, treated with 13 -NF for 1 h, and stimulated with 10 ng/ml TNF-α for 8 h. Total RNA was isolated 14 using Trizol Reagent (Invitrogen), according to the manufacturer’s suggested 15 protocol. An aliquot (5 μg) of purified RNA was reverse transcribed into first-strand 16 complementary DNA (cDNA) with a 2720 Thermal Cycler (Applied Biosystems, 17 Grand Island, NY, USA), 200 U/μL M-MLV reverse-transcriptase (Invitrogen) and 18 0.5 mg/μL oligo(dT)-adapter primers (Invitrogen) in a 20-μL reaction mixture. The 19 qPCR assays for TNF-, IL-6, and glyceraldehyde 3-phosphate dehydrogenase
1 (GAPDH) were performed with a Roche LightCycler 480 System (Roche,
2 Indianapolis, IN, USA) and iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, 3 USA). The oligonucleotide primers used were specific for TNF- 5′-AGG GAC 4 CTC TCT CTA ATC AG-3′ and 5′-TGG GAG TAG ATG AGG TAC AG-3′), IL-6 5 (5′-GCC GCC CCA CAC AGA CA-3′ and 5′-CCG TCG AGG ATG TAC CGA 6 AT-3′), and GAPDH (5′-ACG GAT TTG GTC GTA TTG GG-3′ and 5′-TGA TTT 7 TGG AGG GAT CTC GC-3′). Thermal cycling conditions involved an initial
8 denaturation step at 95°C followed by 35 amplification cycles (15 s at 95°C and 20 s 9 at 60°C) and subsequent melt curve analysis (72°C–98°C). Quantitation of gene 10 expression was conducted relative to GAPDH expression levels.
11
12 2.8. Animals and Peritonitis model
13 The animal experiment was used 8- to 10-week-old male BABL/c mice to be 14 animal model. These mice were purchased from the National Laboratory Animal 15 Breeding and Research Center, Taipei. The mice were housed in a
16 temperature-controlled, light-cycled facility and this study was carried out in strict 17 accordance with the recommendations in the Guide for the Care and Use of 18 Laboratory Animals of the National Institutes of Health. This animal experiment 19 performed by Dr. Shih-Yi Peng was allowed by Tzu Chi University Institutional
1 Animal Care and Use Committee (Permit Number: 101070).
2 Peritonitis was induced by an intraperitoneal injection of 4% (w/v) thioglycollate 3 in 1 mL of sterile saline (Sigma Aldrich, USA). Treatment with DMSO or -NF 4 (20 mg/kg) was performed 1 h before the administration of thioglycollate by 5 intravenous administration. At 24 h after thioglycollate injection, mice were killed 6 by exposure to CO2, and 5 mL of HBSS was injected into the peritoneal cavity. Cells 7 were obtained by aspirating peritoneal lavage. Differential cell counts were
8 determined using a Hematology Analyzer (KX-21N; Sysmex, USA).
9
10 2.9. Statistical analysis
11 Results are expressed as the means ± S.D. from at least three independent 12 experiments. Differences between groups were assessed by one-way analysis of 13 variance (ANOVA). A P-value less than 0.05 was considered statistically 14 significant.
15
16 3. Results
17 3.1. Anti-inflammatory effects of -NF on a peritonitis model in vivo
18 We assessed the effects of -NF on inflammatory cell recruitment using an acute
1 elicited inflammatory cells were detected in the peritoneal cavity. Administration of
2 -NF 1 h before thioglycollate stimulation significantly reduced total white blood cell 3 infiltration in the peritoneal cavity (Fig. 1). Lymphoycte infiltration was also
4 somewhat reduced as a result of -NF treatment.
5 3.2. Monocyte adhesion and transmigration of endothelial cells
6 To test whether -NF inhibits cytokine-mediated vascular inflammation, the effects 7 of -NF on TNF-α-induced monocyte adhesion to HMEC-1 cells were assessed. 8 Normal, confluent HMEC-1 cells bound to THP-1 cells but to a minimal extent.
9 Treatment with TNF-α caused a marked increase in HMEC-1–THP-1 adhesion.
10 Treatment with -NF decreased the extent of monocyte adhesion to HMEC-1 cells 11 and was concentration dependent (Fig. 2A). -NF at 0.1 M significantly reduced the 12 extent of TNF-α-induced monocyte adhesion (0.7 ± 0.11-fold vs. TNF-α alone, n = 3,
13 P < 0.05). The effect of -NF on monocyte transendothelial migration was studied 14 using trans-well assays. HMEC-1 cells stimulated by TNF-α exhibited increased
15 levels of monocyte adhesion and migration across the endothelium as compared to
16 when TNF-α was absent. In contrast, the ability of THP-1 cells to migrate across
17 HMEC-1 cells was significantly decreased to 90% by the presence of -NF, in a 18 dose-dependent manner (Fig. 2B). These results show that -NF can inhibit 19 monocytic migration induced by inflammation. However, the inhibitory effects of
1 -NF on monocyte adhesion were not due to cytotoxicity, as -NF had no effect upon 2 cell viability up to 48 h (Fig. 2C).
3 3.3. Effects of β-NF on TNF-α-induced expression of cell adhesion molecules 4 We examined whether -NF could inhibit TNF-α-induced expression of adhesion
5 molecules that mediate leukocyte adhesion to HMEC-1 cells. -NF pretreatment
6 inhibited TNF-α-induced expressions of ICAM-1 (1.59 ± 0.18-fold relative to 7 control vs. 1.32 ± 0.04-fold relative to control for 1 M -NF and 1.17 ± 0.07-fold 8 relative to control for 10 M -NF, n = 3, P < 0.05; Fig. 3) and VCAM-1 expression 9 (1.53 ± 0.19-fold relative to control vs. 1.29 ± 0.09-fold relative to control for 1 M 10 -NF and 1.19 ± 0.06-fold relative to control for 10 M -NF, n = 3, P < 0.05; Fig.
11 3). 12
13 3.4. anti-oxidative effects of β-NF on ROS production and NADPH oxidase
14 activity
15 ROS have been shown to activate various transcription factors in cultured
16 endothelial cells and have been implicated as a common second messenger in various
17 pathways leading to NF-κB activation. To determine whether -NF could ameliorate
18 the oxidative stress induced by TNF-α, the level of intracellular ROS production was
1 ROS production (Fig. 4A). NADPH oxidase in cells comprises membrane-bound
2 (gp91phox and p22phox ) and cytosolic components (p47phox, p67phox, p40phox, and Rac
3 proteins). NADPH oxidase activation requires the membrane translocation of
4 cytosolic components to associate with membrane-bound components and assemble
5 into an active enzyme. Immunofluorescence staining demonstrated that -NF 6 pretreatment of HMEC-1 cells resulted in a significant reduction of membrane
7 expression of p67phox after TNF-α stimulation in a dose-dependent manner (Fig. 4B).
8
9 3.5. Anti-inflammatory effects of -NF by inhibiting NF-B activation
10 -NF exerted its anti-inflammatory effects by inhibiting TNF-α-induced oxidative 11 stress and the expression of cell adhesion molecules. The upregulation of cell
12 adhesion molecules and activation of NF-B can be induced by TNF-α; therefore, we 13 investigated the involvement of NF-B with respect to the anti-inflammatory effects 14 of -NF in endothelial cells. To determine whether NF-κB activation by TNF-α is 15 inhibited by treatment with -NF, western blotting, immunofluorescence staining, and 16 luciferase reporter assays were performed. From the total cell lysate, -NF was shown 17 to prevent TNF-α-induced phosphorylation of IκBα in a dose-dependent manner (Fig.
18 5A). In addition, immunofluorescence analysis revealed that the nuclear translocation
1 treatment. However, pretreatment with -NF prevented this activation in a
2 concentration-dependent manner (Fig. 5B). Transient transfections were performed
3 using an NF-κB-dependent luciferase reporter plasmid to further examine the effects
4 of -NF on NF-κB transcriptional activity. Treatment with TNF-α activated 5 NF-B-luc in endothelial cells, while -NF efficiently inhibited TNF-α-induced 6 NF-κB luciferase activity (Fig. 5C).
7 Activation of MAP kinases plays a crucial role during inflammatory responses. For
8 example, AP-1 is a redox-sensitive transcription factor, with the phosphorylation of
9 JNK and ERK occurring prior to AP-1 activation. We detected phosphorylation of
10 JNK, p38, and ERK, and found that phosphorylation levels of ERK and p38 were
11 increased by TNF-α. These increases could be abrogated by -NF to a certain degree 12 (Fig. 5A). However, -NF had no influence on phosphorylation of JNK (data not 13 shown). The anti-inflammatory effects of -NF occurred through the inhibition of 14 NF-B; however, the involvement of MAP kinases could not be excluded.
15 Egr-1, a downstream transcription factor involved in the MAPK/ERK signaling
16 pathway, was upregulated by TNF-α treatment (Fig. 5D). We found that pretreatment
17 with -NF reduced TNF-α-induced Egr-1 expression to 50% (Fig. 5D). To further 18 investigate the effects of -NF on Egr-1 signaling, we used immunofluorescence to 19 examine the nuclear translocation of Egr-1. Consistent with the expression results,
1 Egr-1 upregulation was inhibited when HMEC-1 cells were treated with -NF (Fig. 2 5E).
3
4 3.6. Inhibitory effects of β-NF on TNF-α-induced cytokine transcription
5 We used qPCR assays to quantify the effects of -NF at various concentrations 6 on the expression of TNF-α and IL6 mRNAs (Fig. 6). Compared with control cells,
7 mRNA levels were significantly increased when endothelial cells were cultured
8 with TNF-α. -NF significantly reduced TNF-α-mediated expression of TNF-α and 9 IL-6 mRNAs. In addition, there was no significant difference in the mRNA
10 expression levels between -NF-treated and control cells. 11
12 4. Discussion
13 To the best of our knowledge, this study is first to show that, in HMEC-1 cells, -NF,
14 an AhR activator, inhibits TNF-α-induced expression of cell adhesion molecules. Our
15 findings suggest that -NF possesses anti-inflammatory properties in endothelial cells.
16 Additionally, we have presented novel evidence to show that the inhibitory effect f
17 -NF is mediated by ROS, NF-κB and ERK1/2-Egr-1 pathways. AhR is a
18 ligand-dependent transcription factor that mediates environmental and immunotoxic
19 mechanisms [28]. The anti-inflammatory activity of AhR makes it a possible
20 therapeutic target for the treatment of autoimmune inflammation. For example,
21 activation of AhR by TCDD reduces inflammation in experimental autoimmune
1 suppress proinflammatory responses [24,30]. Selective AhR regulators can also show 2 anti-inflammatory activity, including alleviation of cytokine-mediated acute phase
3 genes, as seen in Huh 7 cells [31]. Recently, Singh et al. showed that AhR activation
4 facilitates epigenetic regulation, thus affecting reciprocal differentiation of Tregs and
5 Th17 cells and alleviating inflammation in murine (C57BL/6) colitis [32]. -NF has
6 also been known to alleviate DSS-induced colitis [24]. However, the activation of
7 AhR does not always correlate with anti-inflammatory effects. Several reports have
8 shown that -NF increases oxidative stress [33,34] and enhances
9 hepatocarcinogenesis [33,35,36]. The observation that -NF can upregulate the
10 cytochrome P-450-dependent monooxygenase system has led to extensive
11 investigation of -NF as a modifier of chemical carcinogenesis [37,38]. However, 12 Salmonella/microsome assays have shown that -NF is not mutagenic, with or 13 without metabolic activation induced by Aroclor-1254 [39,40]. Lee et al., also 14 reported that -NF has no cytotoxic effect on human colon cancer cells [41 ]. 15 Therefore, -NF can be considered a putative chemopreventive agent [42]. 16 Controversially, it has been reported that -NF may have hepatocellular
17 tumor-promoting activity as it increases the surface area and number of 18 preneoplastic foci, positive for placental glutathione S-transferase (GST-P), 19 following N-diethylnitrosamine treatment in rats [35,43]. This increase in
20 tumorigenicity is thought to arise due to stimulation of oxidative stress responses, 21 accompanied by lipid peroxidation and oxidative DNA damage [36,43]. Therefore,
22 the physiological functions of AhR appear to be complex, exhibiting
23 pro-inflammatory or anti-inflammatory characteristics depending on the affected
24 tissue. However, the hepatoma-promoting activity of -NF reported in previous
25 papers is observed in combination with the carcinogenic agent N-diethylnitrosamine,
1 we showed that -NF is beneficial in preventing inflammatory processes in
2 endothelial cells. The reports on the effects of -NF provide contradictory findings
3 and require further investigation.
4 Redox regulations in the cell conforms signal transductions that control
5 metabolism, energetics, survival, and cell death. Reactive species are known to act 6 as cell signaling molecules, supporting the double-edged sword of the oxidative 7 stress paradigm in cells [44,45]. Oxidative stress significantly contributes to the 8 pathogenesis of cardiovascular disease. The production of ROS (peroxides and free
9 radicals) is an especially harmful aspect of oxidative stress. Some of the potential
10 sources of ROS in the vasculature are xanthine oxidase, NADPH oxidase, and the
11 mitochondria. Several clinical studies have indicated that enhanced vascular oxidative
12 stress is strongly related to cardiovascular incidents in patients with coronary artery
13 disease [46]. Vascular NADPH oxidases seem to be the most significant source of
14 ROS in inflammatory responses in the vascular wall, causing the development of
15 vascular wall lesions [47,48]. Recent progress in our understanding of atherosclerosis
16 has provided supplementary proof that the NADPH oxidase enzyme complex plays a
17 critical role in the generation of superoxide in atherosclerotic lesions [19]. In an
18 earlier study, we have shown that reduction in the activating subunits of NADPH
19 oxidase, p47 phox and p67 phox, is related to decreased neo-intima formation after
20 carotid ligation in C57BL/6 mice [49]. In this study, we have seen an increase in ROS 21 and NADPH oxidase activity in endothelial cells treated with TNF-α. In cultured cells, 22 addition of NAC, a free radical scavenger, inhibited TNF-α-induced ROS levels. This 23 was an observation that was also made in controls (Fig. 4A). Similarly, treatment with 24 10 μM -NF again resulted in reduced ROS levels and NADPH oxidase activity in 25 HMEC-1 cells following TNF-α stimulation (Fig. 4A). Flavonoids are predominant
1 comprises a benzene ring with adjacent methoxy–hydroxyl groups [50]. The chemical
2 structure of β-NF is similar to that of flavonoids and is likely to have similar
3 inhibitory effects on NADPH oxidase activity. As shown in our results, -NF has
4 strong anti-oxidative activity and protects endothelial cells against activation and
5 injury by ROS.
6 Inflammation is one of the factors that increase the risk of developing
7 cardiovascular disease. The first step in the process of vascular inflammation is the
8 adhesion of monocytes to the endothelium. Subsequently, the monocytes infiltrate the
9 endothelial wall and differentiate into macrophages. This critical step is modulated by
10 interaction between monocytes and the surface molecules of endothelial cells [51,52].
11 The expression of cell adhesion molecules, such as VCAM-1 and ICAM-1, mediates
12 monocyte and macrophage adhesion as well as other processes that initiate
13 atherogenesis [53]. In our current study, we demonstrated that anti-inflammatory
14 effect of β-NF is related to fewer monocytes adhering to stimulated endothelial cells
15 and inhibition of monocyte transendothelial migration (Fig. 2). This occurs when the
16 expression of adhesion molecules such as ICAM-1 and VCAM-1 is inhibited. As
17 figure 3 shows, TNF- slightly increases ICAM-1 and VCAM-1 expression; 18 however, NF pretreatment decreases TNF- induced ICAM-1 and VCAM-1 19 expression. It was reported that NAC suppresses TNF-α-stimulated upregulation of 20 adhesion molecules by decreasing ROS and NF-κB activity [54,55]. n addition to this,
21 decline in ROS levels might further prevent cytokine generation in endothelial cells. 22 Treatment with -NF attenuated production of the pro-inflammatory cytokines TNF-α
23 and IL-6 following TNF-α stimulation (Fig. 6). In our animal model of peritonitis, we
24 observed the inhibition of infiltration of white blood cells and lymphocytes, which
25 confirmed the anti-inflammatory properties of -NF (Fig. 1). Our results are
26 supported by numerous previous studies that have shown that decreasing intracellular
1 ROS production inhibits the adhesion of monocytes to endothelial cells [56,57].
2 The redox-sensitive transcription factor NF-κB can be activated by oxidative stress
3 and participates in the production of pro-inflammatory cytokines [58]. The
4 ROS-mediated NF-κB pathway is needed for the transcriptional activation of
5 endothelial ICAM-1and VCAM-1 [59]. NF-κB activation contributes to the
6 development of many chronic diseases, especially atherosclerosis [60].
7 Phosphorylated IκB-α and subsequently nuclear translocation of NF-κB p65 lead to
8 the activation of specific target genes, including ICAM-1 and VCAM-1. Therefore,
9 we propose that the anti-inflammatory effects of -NF are due to the inhibition of
10 NF-κB activation. Western blotting analysis and immunofluorescence assays showed
11 that pretreatment with -NF inhibited TNF-α-stimulated nuclear translocation of
12 NF-κB p65 (Fig. 5). Consistent with this finding, the luciferase reporter assay showed
13 that pretreatment with -NF attenuated TNF-α-induced NF-κB promoter activity.
14 Oxidative stress enhances NF-κB translocation and the decrease that we observed in
15 NF-κB could be a secondary effect of ROS production suppressed by -NF [61]. It
16 was discovered that -NF suppresses the production of TNF-α-induced ROS,
17 indicating its anti-inflammatory function in the ROS-NF-κB pathway in vascular
18 endothelial cells. The effective concentration of -NF (10 M) required to suppress
19 the ROS-NF-κB pathway and the effective concentration for reducing the expression
20 of cell adhesion molecules are the same. This finding is supported by the results from
21 a previous in vitro study that demonstrated free radical scavenging activity for
22 flavonoids from the fruits of T. orientalis [62]. Perhaps, the protective role of -NF as
23 a free radical scavenger contributes to its overall anti-inflammatory effects by
24 inhibiting NF-κB activation in vascular endothelial cells. To further confirm whether
25 the observed effects are due to AhR-activation rather than direct interaction with 26 and inhibition of NOX, we performed these experiments using selective AhR
1 antagonist -Naphthoflavone (-NF) and AhR siRNA [63]. Effect of AhR siRNA on
2 inhibition of AhR expression was examined and the result is shown in
3 supplementary data figure S 3. According to the result, AhR siRNA partially
4 inhibited AhR expression. Effect of -NF on TNF--induced cytokine responses,
5 ROS and NF-kB translocation and effect of -NF on TNF--induced cytokine
6 responses, ROS and NF-kB translocation in AhR silicing were estimated as
7 supplementary data figure S 4-7. The effect of AhR silencing alone on
8 TNF--induced various responses was also examined. In these experiments, -NF
9 had no effect on TNF--induced cytokine responses, ROS, and NF-kB
10 translocation. Meanwhile, AhR silencing indeed increased TNF--induced cytokine
11 responses, ROS, and NF-kB translocation in comparison with negative control
12 siRNA. -NF also showed inhibitory ability on TNF--induced various responses in
13 both AhR and negative control silencing since the AhR silencing is partially.
14 According to these results, we speculate the observed effects are due to
15 AhR-activation rather than -NF direct interaction with and inhibition of NADPH
16 oxidase in our system.
17 Other than the NF-κB/IκB pathway, Akt associated with ERK and p38 MAPK 18 plays a significant role in the signal transduction pathways that modulate the 19 expression of cell adhesion molecules in response to external stimuli such as TNF- 20 [64,65]. Moreover, PI3K-Akt and p38 MAPK signaling also contribute to NF-κB 21 activation and increased expression of adhesion molecules in response to TNF- 22 [66,67]. Our results indicate that -NF greatly suppresses the phosphorylation of Akt, 23 ERK, and p38 MAPK in TNF--stimulated endothelial cells (Fig. 5A). These results 24 demonstrate that the anti-inflammatory effects of -NF are partially due to the 25 inhibition of adhesion molecules through suppression of ERK, p38, MAPK, and 26 PI3K-Akt activation.
1 An early zinc-finger transcription factor, early growth response gene-1 (Egr-1), 2 contributes to the formation of atherosclerotic lesions [68,69]. Its expression is 3 induced in response to various extracellular stimuli such as growth factors,
4 cytokines, hypoxia, and other harmful stimuli [70, 71]. Egr-1 transcription depends 5 on the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated 6 kinase (ERK) signaling (MAPK/ERK) pathway and serum-response elements of the 7 Egr-1 promoter [72]. Cytokines such as the pro-inflammatory cytokine TNF- 73 , 8 anti-inflammatory cytokine transforming growth factor- (TGF-) [74], and basic 9 fibroblast growth factor (bFGF) [75] also have the Egr-1 sequence in their
10 promoter sites [76]. Additionally, Egr-1 transcriptionally modulates ICAM-1,
11 VCAM-1, and coagulation elements such as tissue factors [77]. Several factors
12 rapidly increase the expression of the transcription factor Egr-1 during the progression 13 of atherosclerosis. Once activated, Egr-1 modulates a variety of pro-inflammatory and 14 pro-atherogenic genes in both mice and humans [78]. ERK activation upregulates 15 phosphorylation, which in turn leads to the activation of the transcription factors c-jun, 16 c-fos, and Egr-1 [79-81]. In our study, the level of stimulus-induced Egr-1 was
17 reduced in endothelial cells treated with -NF (Fig.5D and 5E). To our knowledge, 18 this study is the first to show that -NF inhibits TNF--induced expression of 19 VCAM-1 and ICAM-1 through the suppression of the ERK and Egr-1 signaling 20 pathways. However, our results do not exclude other transcription factors and 21 signaling pathways, such as AP-1, SP-1, GATA-2, and IRF-1, that are also involved 22 in the induction of adhesion molecules [82-85]. These findings suggest that the 23 suppressive effects of -NF on stimulus-induced expression of adhesion molecules
24 and on activation of NF-B and ERK-Egr-1 and Akt signaling might be due to AhR 25 activation.
26
1 5. Conclusion
2 In summary, our findings indicate that -NF blocks TNF-stimulated NADPH
3 oxidase activity and ROS production, alleviates TNF-induced adhesion
4 molecules expressions and monocyte binding, and prevents the infiltration of white
5 blood cells in a peritonitis model. To the best of our knowledge, we have provided
6 the first evidence of the potential benefits of -NF as a protective agent against
7 vascular inflammation in HMEC-1 cells. Our findings could therefore provide new
8 insights into the pathophysiological mechanisms with regard to the 9 anti-inflammatory properties of -NF in TNF- induced endothelial stimulation.
10
11 Acknowledgments
12 We appreciate the technical assistance provided by Mr. Hong-Kai Su and Mr.
13 Hao-Jhih Yang. This work was supported by Tzu Chi University ( TCMRC-P-102001
14 for SJJ) and Tainan Municipal An-Nan Hospital-China Medical University
15 (ANHRF103-13 for SYH)
16
17 Conflicts of interest
18 The authors have declared that there are no conflicts of interest.
19
20
21
22
1
2
3
4
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14 15 16 17 18 19 20 21 22
1 Figure legends
2 Figure 1. -NF alleviates peritonitis inflammation. Cell counts from peritoneal fluid 3 24 h after intraperitoneal injection of thioglycollate in mice are shown. Values are the
4 mean ± S.E.M. (n = 10 for control vs. n = 14 for the -NF treatment). *
P < 0.05 vs.
5 DMSO control.
6
7 Figure 2. -NF inhibits TNF--induced adhesion of monocytes to HMEC-1 cells and 8 the transmigration of monocytes. (A) Adhesion of fluorescent THP-1 monocytes was
9 visualized by microscopy and quantified using AlphaImager. Values are the
10 mean ± S.D. *P < 0.05 for three independent experiments. (B) THP-1 cells were
11 allowed to transmigrate through the HMEC-1 monolayer towards a TNF- gradient. 12 Values are the mean ± S.D. of transmigrated monocytes compared with that of
13 controls from three independent experiments. *P < 0.05. (C) HMEC-1 cells in 96-well
14 microplate were treated with various concentrations of -NF. After a 48-h incubation, 15 cell proliferation was evaluated using the colorimetric WST-1 assay. Values are the
16 mean ± S.D. from three independent experiments.
17
18 Figure 3. -NF inhibits the expression of TNF--induced adhesion molecules. 19 Protein expression levels of ICAM-1 and VCAM-1 were measured by western
1 blotting with GAPDH used as a loading control as described in supplementary
2 Materials and Methods. Values are the mean ± S.D. of the protein normalized to 3 GAPDH from three independent experiments. **p < 0.01 vs. control and #p < 0.05 vs. 4 cells stimulated with TNF- in the absence of -NF.
5
6 Figure 4. -NF inhibits production of TNF--induced ROS and p67phox
membrane
7 translocation in endothelial cells. (A) ROS production. (B) Immunofluorescence of
8 p67phox membrane translocation as described in supplementary Materials and
9 Methods. Fluorescent intensity was quantified using Image J. Values are the
10 mean ± S.D. of fluorescent intensities vs. control from three independent experiments.
11 *p < 0.05 vs. control and ##p < 0.01 vs. cells stimulated with TNF- in the absence of
12 -NF. 13
14 Figure 5. The anti-inflammatory effects of -NF are mediated through inhibition of
15 the NF-κB and ERK-Egr-1 pathways. (A) Expression levels of pIκB, pERK, ERK,
16 pAKT, AKT, pp38, and GAPDH were analyzed by western blotting as described in
17 supplementary Materials and Methods. Results are representative of three
18 independent experiments. (B) HMEC-1 cells were treated with various concentrations
