lsevier.com/locate/ytaap

Toxicology and Applied Pharmac

Inhibition of tumor necrosis factor-a-induced expression of adhesion

molecules in human endothelial cells by the saponins derived from roots

of Platycodon grandiflorum

Ji Young Kim a, Dong Hee Kim b, Hyung Gyun Kim a, Gyu-Yong Song c, Young Chul Chung d,

Seong Hwan Roh e, Hye Gwang Jeong a,*

a Department of Pharmacy, College of Pharmacy, Research Center for Proteineous Materials, Chosun University, Kwangju, South Koreab Department of Pathology, College of Oriental Medicine, Daejeon University, Daejeon, South Korea

c Department of Pharmacy, College of Pharmacy, Chungnam National University, Taejon, South Koread Division of Food Science, Chinju International University, Chinju, South Korea

e Jangsaeng Doraji Research Institute of Biotechnology, Jangsaeng Doraji Co., Ltd., Chinju, South Korea

Received 6 September 2005; revised 30 September 2005; accepted 30 September 2005

Available online 4 November 2005

Abstract

Adhesion molecules play an important role in the development of atherogenesis and are produced by endothelial cells after being

stimulated with various inflammatory cytokines. This study examined the effect of saponins that were isolated from the roots of

Platycodon grandiflorum A. DC (Campanulaceae), Changkil saponins (CKS), on the cytokine-induced monocyte/human endothelial cell

interaction, which is a crucial early event in atherogenesis. CKS significantly inhibited the TNFa-induced increase in monocyte adhesion

to endothelial cells as well as decreased the protein and mRNA expression levels of vascular adhesion molecule-1 and intercellular cell

adhesion molecule-1 on endothelial cells. Furthermore, CKS significantly inhibited the TNFa-induced production of intracellular reactive

oxygen species (ROS) and activation of NF-nB by preventing InB degradation and inhibiting InB kinase activity. Overall, CKS has

anti-atherosclerotic and anti-inflammatory activity, which is least in part the result of it reducing the cytokine-induced endothelial

adhesion to monocytes by inhibiting intracellular ROS production, NF-nB activation, and cell adhesion molecule expression in

endothelial cells.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Saponins; Platycodon grandiflorum; Adhesion molecules; Endothelial cells; NF-nB

Introduction

Atherosclerosis is a chronic inflammatory process as a

result of increased oxidative stress. The activation of the

vascular endothelium, the increased adhesion of circulating

monocytes to the injured endothelial layer, followed by their

infiltration into the vessel wall and differentiation into

macrophages are key stages in the development of athero-

sclerosis (Springer, 1994; Price and Loscalzo, 1999; Ross,

1999; Iiyama et al., 1999; Glass and Witztum, 2001).

Endothelial cells recruit monocytes by selectively expressing

0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.taap.2005.09.015

* Corresponding author. Fax: +82 62 230 6639.

E-mail address: hgjeong@chosun.ac.kr (H.G. Jeong).

cell surface adhesion molecules such as vascular cell adhesion

molecule-1 (VCAM-1), intercellular cell adhesion molecule-1

(ICAM-1), and endothelial cell selectin (E-selectin) (Price and

Loscalzo, 1999). Proinflammatory cytokines such as tumor

necrosis factor-alpha (TNFa), which is commonly found in

atherosclerotic lesions, can induce chemotactic factors, other

cytokines, and cell adhesion molecules, all of which can

contribute to the inflammatory process (DiDonato et al., 1997;

Price and Loscalzo, 1999; Ross, 1999; Glass and Witztum,

2001). TNFa can activate the redox-sensitive eukaryotic

transcription factors such as nuclear factor-nB (NF-nB) (Senand Paker, 1996; DiDonato et al., 1997; Tak and Firestein,

2001). NF-nB activation is induced by a cascade of events

leading to inhibitor nB (InB) kinase (IKK) activation. IKK

phosphorylates InB leading to its degradation and finally to

ology 210 (2006) 150 – 156

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J.Y. Kim et al. / Toxicology and Applied Pharmacology 210 (2006) 150–156 151

the translocation of NF-nB to the nucleus (Tak and Firestein,

2001; Chen et al., 2002). Therefore, the pathways leading to

NF-nB activation are frequent targets for many anti-inflam-

matory drugs (Tak and Firestein, 2001). Reactive oxygen

species (ROS) may be a common intracellular messenger for a

variety of redox-sensitive transcription pathways that lead to

the expression of cell adhesion molecules in vascular

endothelial cells (Ross, 1999; Griendling et al., 2000; Harrison

et al., 2003). Accumulating in vitro and in vivo evidence

suggests that substances with antioxidant activity can scav-

enge intracellular ROS and inhibit the endothelial adhesive-

ness to monocytes by reducing the expression of various cell

adhesion molecules. Moreover, the actions of antioxidants on

the cell adhesion molecules expression may play an important

role in preventing atherogenesis (Martin et al., 1997; Wu et al.,

1999; Noguchi et al., 2003; Chen et al., 2003; Choi et al.,

2004).

Some plants contain non-nutritional constituents that may

have beneficial biological activities (Bellisle et al., 1998).

Platycodi Radix is the root of Platycodon grandiflorum A. DC

(Campanulaceae) (4 years old) and has been used as a food

and in traditional oriental medicine to treat adult diseases such

as bronchitis, asthma and pulmonary tuberculosis, and

inflammatory diseases, as well as being taken as a sedative.

In addition, its biological significance has previously been

reviewed (Lee, 1973). Previous studies found that Changkil

(CK), which is the aqueous extract from the root from 20-

year-old P. grandiflorum plants (Lee, 1991, Patent on the

method of cultivating the perennial balloon flower, Patent No.

045971, Korea), prevented hypercholesterolemia and hyper-

lipidemia (Kim et al., 1995). Recently, it was shown that CK

had protective effects against acetaminophen- and carbon

tetrachloride-induced hepatotoxicity and inhibits the progress

of hepatic fibrosis in rats (Lee and Jeong, 2002; Lee et al.,

2001, 2004a). In addition, it was also reported that CK and the

saponin fraction (CKS) derived from CK have potent

antioxidant effects, such as superoxide radical scavenging

activity via the xanthine and xanthine oxidase system and the

inhibition of ROS production by tert-butyl hydroperoxide in

hepatocytes and the liver (Lee and Jeong, 2002; Lee et al.,

2004b).

Although it has been reported that CK prevents hypercho-

lesterolemia and hyperlipidemia (Kim et al., 1995) and CKS

derived from CK has antioxidant effects both in vivo and in

vitro (Lee and Jeong, 2002; Lee et al., 2004b), there are no

reports on the influence of CKS on the expression of the cell

adhesion molecules and the adhesion of monocytes to

endothelial cells. Because the transcriptional activation of cell

adhesion molecules is sensitive to the intracellular redox status

and the actions of antioxidants on cell adhesion molecules

expression might play an important role in preventing

atherogenesis (Martin et al., 1997; Wu et al., 1999; Noguchi

et al., 2003; Chen et al., 2003; Choi et al., 2004), this study

investigated the effect of CKS on the adhesion of monocytes to

endothelial cells and the expression levels of the cell adhesion

molecules. In addition, this study examined the mechanism(s)

involved in the anti-atherosclerotic effect of CKS.

Materials and methods

Chemicals and materials. The chemicals and cell culture materials were

obtained from the following sources: 2V,7V-bis-(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) and 2V,7V-dichlorofluorescin diacetate (DCFDA) from

Molecule Probe; MTT-based colorimetric assay kit from Roche Co.;

LipofectAMINE Plus, Dulbecco’s modified Eagle’s medium (DMEM), fetal

bovine serum (FBS), and penicillin – streptomycin solution from Life

Technologies, Inc.; pGL3-4nB-Luc and the luciferase assay system from

Promega; pCMV-h-gal from Clonetech; recombinant human TNFa, mouse

anti-humanVCAM-1, ICAM-1, and E-selectin antibodies fromBD Pharmingen;

GST-InBa and the antibodies to h-actin, IKKh, InBa, and the phosphory-

lated form of InBa (Ser 32) from Santa Cruz Biotechnology, Inc.; Western

blotting detection reagents (ECL) from Amersham Pharmacia Biotech. All

other chemicals were of the highest commercial grade available.

Preparation of CKS. CK refers to the aqueous extract obtained from the 22-

year-old roots of P. grandiflorum, which was supplied by Jang Saeng Doraji

Co., Jinju, South Korea. The composition of the P. grandiflorum root is

reported elsewhere (Kim et al., 1995). The CK and CKS were prepared using

the method described elsewhere (Lee and Jeong, 2002; Lee et al., 2001, 2004a).

CK was subjected to column chromatography over amberlite XAD-2, Diaion

MCl Gel HP20, or Kogel BG4600. After removing the saccharides and amino

acids with water, the column was eluted with methanol to obtain the saponin

fraction of CK, CKS, as described previously (Tada et al., 1975).

Cell cultures. ECV304 cells, which are a spontaneously transformed

immortal endothelial cell line established from the vein of apparently normal

human umbilical cord, as well as U937 cells, a human monocytic leukemia cell,

were obtained from the American Type Culture Collection (Bethesda, MD).

The ECV304 and U937 cells were grown in DMEM supplemented with 10%

FCS, 100 U/ml penicillin, and 100 Ag/ml streptomycin at 37 -C in a 5% CO2

humidified incubator. The CKS was dissolved in the culture media. The cell

viability was determined using a MTT assay. The MTT assay was performed

according to the manufacturer’s instructions.

Monocyte–endothelial cell adhesion assay. The endothelial cellswere grown

to confluence in 48-well plates, pretreated with CKS for 1 h, and stimulated with

TNFa for 6 h. The cells were then washed twice with PBS. The U937 cells were

labeled with 10 AM BCECF for 1 h at 37 -C and washed twice with the growth

medium. This was followed by adding 2.5 � 105 of the labeled cells to the

endothelial cells and incubated them in a CO2 incubator for 1 h. The non-adherent

cellswere removed from the plate bywashingwith PBS, and theU937 cells bound

to the endothelial cells were lysed with 50 mM Tris–HCI, pH 8.0, containing

0.1% SDS. The fluorescent intensity was measured using a spectrofluorometer

(Varioskan, Thermo Electron Co., Vantaa, Finland) at an excitation and emission

wavelength of 485 nm and 535 nm, respectively. The adhesion data are

represented in terms of the fold change compared with the control values.

Cell ELISA. ELISA was used to determine the level of ICAM-1, VCAM-1,

and E-selectin expression on the cell surface, as previously described with

minor modifications (Noguchi et al., 2003). Briefly, the endothelial cells on the

96-well plates were pretreated with or without CKS for 1 h, which was

followed by a treatment with TNFa for 6 h at 37 -C. After the treatments, the

cells were fixed by 1% glutaraldehyde and exposed to the mouse anti-human

VCAM-1, ICAM-1, or E-selectin antibody at 1:1000 dilution in the PBS

containing 1% skim milk for 2 h at room temperature. The cells were washed

and incubated with a horseradish-peroxidase-conjugated secondary antibody.

The expression of VCAM-1, ICAM-1, or E-selectin was quantified by adding a

peroxidase substrate solution and measuring the absorbance of each well at 490

nm using a microplate reader.

RNA isolation and reverse transcriptase polymerase chain reaction

(RT-PCR). The endothelial cells were pretreated with CKS for 1 h and

stimulated with TNFa for 3 h, which was followed by washing twice with PBS.

The total RNAwas isolated from the endothelial cells using the Trizol reagent.

The procedure for cDNA synthesis, semiquantitative RT-PCR for VCAM-1,

ICAM-1, E-selectin, and GAPDH mRNA, and the analysis of the results were

Fig. 1. Effects of CKS on the adhesion of monocytes to endothelial cells. The

endothelial cells were pretreated with CKS for 1 h and then stimulated with

TNFa (10 ng/ml) for 6 h. Fluorescence-labeled monocytic U937 cells were

added to the monolayer endothelial cells and allowed to adhere for 1 h. The

adherent cells were measured, as described in Materials and methods. The

values are expressed as a mean T SD of three individual experiments

performed in triplicate. *P < 0.01 compared with TNFa alone.

J.Y. Kim et al. / Toxicology and Applied Pharmacology 210 (2006) 150–156152

all performed as described elsewhere (Scholzen et al., 2003). The cycle number

was determined to be within the linear amplification range from a linear

amplification curve. The PCR reactions were electrophoresed through a 1.5%

agarose gel and visualized by ethidium bromide staining and UV irradiation.

Immunoblot analysis. The endothelial cells were pretreated with CKS for 1

h and stimulated with TNFa for 6 h followed by washing twice with PBS. The

whole-cell lysates were prepared and resolved by 10% SDS-polyacrylamide gel

electrophoresis (SDS-PAGE) followed by electroblotting onto a polyvinylidene

diflouride membrane. The membranes were probed with antibodies to ICAM-1,

VCAM-1, E-selectin, or h-actin and incubated with the horseradish-peroxidase-conjugated secondary antibody. The blots were probed with the ECL Western

blot detection system according to the manufacturer’s instructions.

Intracellular ROS production assay. The fluorescent probe, DCFDA, was

used to determine the intracellular generation of ROS by TNFa, as described

elsewhere (Wang and Joseph, 1999). Briefly, the confluent endothelial cells in

the 48-well plates were pretreated with CKS for 1 h. After removing the CKS

from the wells, the cells were incubated with 20 AM DCFDA for 30 min. The

cells were then stimulated with 10 ng/ml TNFa, and the fluorescence intensity

(relative fluorescence units) was measured at an excitation and emission

wavelength of 485 nm and 530 nm, respectively, using a fluorescence

spectrophotometer for 1 h.

Preparation of nuclear extracts and the electrophoretic mobility shift assay

(EMSA). Endothelial cells were pretreated with CKS for 1 h and stimulated

with TNFa for 30 min then washed twice with PBS. The cells were harvested,

and the nuclear extracts were prepared, as described previously (Kim et al.,

2004). The double-stranded deoxyoligonucleotide containing the NF-nB (5V-AGT TGA GGG GAC TTT CCC AGG C-3V) was end-labeled with

[g-32P]dATP. The nuclear extracts (15 Ag) were incubated with 2 Ag of poly

(dI–dC) and the 32P-labeled DNA probe in a binding buffer (100 mM NaCl, 30

mM HEPES, 1.5 mM MgCl2, 0.3 mM EDTA, 10% glycerol, 1 mM

dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 Ag/ml concentration

each of aprotinin and leupeptin) for 20 min on ice. The specificity of binding

was examined by competition with the unlabeled oligonucleotide. The DNA

protein complex was separated using 6% non-denaturing acrylamide gels.

Following electrophoresis, the gel was dried and autoradiographed.

Transient transfection and luciferase and b-galactosidase assays. The

endothelial cells were grown to 60–80% confluence, and the cells were

transiently co-transfected with the plasmids using LipofectAMINE Plus

according to the manufacturer’s protocol. Briefly, the transfection mixture

containing 0.5 Ag of either the pGL3-4nB-Luc and 0.2 Ag of pCMV-h-gal wasmixed with the LipofectAMINE Plus reagent and added to the cells. After 18 h,

the cells were treated with CKS for 1 h and stimulated with TNFa for 12 h and

then lysed. The luciferase and h-galactosidase activities were determined as

described elsewhere (Kim et al., 2004). The luciferase activity was normalized

with respect to the h-galactosidase activity and is expressed as a percentage of

the activity of the TNFa group.

IjBa degradation and IKK assay. The cytoplasmic extracts were prepared

from the endothelial cells treated with CKS for 1 h and stimulated with TNFa

for 30 min. The extracts were resolved in 10% SDS-PAGE and analyzed by

immunoblotting using an antibody against InBa, as described above. For the

IKK assay, equal amounts of the total cellular protein (800 Ag) were

immunoprecipitated with the IKKh antibody and protein A/G-PLUS agarose

for 12 h at 4 -C. The kinase assay was carried out in a kinase buffer containing

5 AM cold ATP, 10 ACi [g-32P]ATP (5000 Ci/mmol), and 1 Ag of the GST-InBafusion protein as a substrate and incubated for 20 min at 25 -C. The reaction

was quenched by adding the Laemmli buffer followed by boiling for 5 min. The

samples were subjected to 10% SDS-PAGE, transferred to polyvinylidene

difluoride membranes, immunoblotted, and analyzed by autoradiography.

Statistical analyses. All the experiments were repeated at least three times.

The results are expressed as a mean T SD, and the data were analyzed using

one-way ANOVA followed by a Dunnett’s test or Student’s t test to determine

any significant differences. A P value < 0.05 was considered significant.

Results

CKS inhibits monocyte adhesion to TNFa-activated endothelialcells

This study examined the effect of CKS on the adhesion of

monocytes to endothelial cells. The endothelial cells were

pretreated with CKS for 1 h before treating them with TNFa for

6 h, which was followed by an adhesion process and the

quantification of adhered fluorescent monocytes. As shown in

Fig. 1, TNFa significantly increased the adhesion of monocytes

to the endothelial cells compared with the untreated (control)

cells. However, CKS significantly inhibited the adhesion of

monocytes to the endothelial cells in a dose-dependent manner.

The cell viability was assessed using a MTT assay. An

examination of the cytotoxicity of CKS in the endothelial cells

indicated that these compounds did not adversely affect the cell

viability (>90% cell viability, Fig. 1). Therefore, inhibition of the

adhesion of monocytes on the endothelial cells by CKS was not

the result of its cytotoxicity against the cells.

CKS inhibits cell surface expression of cell adhesion molecules

The effects of CKS on the TNFa-induced VCAM-1, ICAM-

1, and E-selectin expression in endothelial cells were deter-

mined by cell ELISA (Fig. 2). Exposure of the cells to TNFa

for 6 h induced the strong up-regulation of the cell surface

expression of VCAM-1, ICAM-1, and E-selectin. The TNFa-

induced cell surface expression of VCAM-1 and ICAM-1 was

significantly inhibited by pretreating the cells with CKS,

whereas there was no change in E-selectin expression.

Effects of CKS on ICAM-1, VCAM-1, and E-selectin protein

and mRNA expression

The effects of CKS on the expression of cell adhesion

molecules in endothelial cells were confirmed by immunoblot-

,

Fig. 2. Effects of CKS on the endothelial cell surface expression of VCAM-1,

ICAM-1, and E-selectin. The endothelial cells were pretreated with CKS for 1

h and then stimulated with TNFa (10 ng/ml) for 6 h. The cells were fixed with

glutaraldehyde, and the cell surface expressions of VCAM-1, ICAM-1, and E-

selectin were analyzed by cell ELISA, as described in Materials and methods.

The values are expressed as a mean percentage of TNFa-induced adhesion

molecule expression T SD of three individual experiments, performed in

triplicate. *P < 0.01 compared with the TNFa alone.

Fig. 3. Effects of CKS on TNFa-induced expression of VCAM-1, ICAM-1,

and E-selectin protein and mRNA. The endothelial cells were pretreated with

CKS for 1 h and stimulated with TNFa (10 ng/ml). (A) Immunoblot analysis.

After 6 h of incubation, the cell lysates (30 Ag protein) were separated by SDS-

PAGE, transferred to a nitrocellulose membrane, and blotted with an anti-

ICAM-1, VCAM-1, E-selectin, or h-actin antibody. (B) RT-PCR analysis. After

3 h of incubation, the total RNAwas prepared, and RT-PCR was performed, as

described in the Materials and methods. The PCR products were separated on a

1.5% agarose gel and stained with ethidium bromide. These blots (A, B) are a

representative of three independent experiments. One of the three representative

experiments is shown.

J.Y. Kim et al. / Toxicology and Applied Pharmacology 210 (2006) 150–156 153

ting of the cell lysate protein. Pretreating the cells with CKS

significantly inhibited the TNFa-induced VCAM-1 and

ICAM-1 expression but had little effect on E-selectin expres-

sion (Fig. 3A). The observed changes in the level of the cell

adhesion molecules might reflect the change in protein

synthesis or degradation. The mRNA levels of the cell

adhesion molecules were measured by RT-PCR analysis in

order to further examine the mechanism responsible for the

changes in the amount of the cell adhesion molecules. The

VCAM-1 and ICAM-1 mRNA levels were markedly decreased

by pretreating the cells with CKS but had little effect on the E-

selectin induced by TNFa (Fig. 3B). This suggests that CKS

suppresses VCAM-1 and ICAM-1 expression at the transcrip-

tional level and contributes to decreasing the production of

their protein and monocytes adhesion.

Effect of CKS on TNFa-induced NF-jB activation

NF-nB and activation are essential for inducing ICAM-1

and VCAM-1 by TNFa or other inflammatory cytokines

(Marui et al., 1993; Collins et al., 1995; Ledebur and Parks,

1995; Neish et al., 1995; Price and Loscalzo, 1999; Ross, 1999;

Glass and Witztum, 2001), and CKS is effective at inhibiting

the expression of these cell adhesion molecules induced by

TNFa in endothelial cells. Therefore, this study examined

whether or not CKS could suppress NF-nB activation in the

TNFa-activated in endothelial cells using EMSA and transient

transfections. As shown in Fig. 4A, the TNFa-stimulated

endothelial cells had a higher NF-nB binding activity.

However, pretreating the cells with CKS for 1 h markedly

reduced the TNFa-stimulated NF-nB activation in a dose-

dependent manner. The addition of an excess of unlabeled NF-

nB oligonucleotide completely prevented the NF-nB binding,

demonstrating the binding specificity of the NF-nB complexes.

Therefore, transient transfections were performed using the

NF-nB-dependent luciferase reporter plasmid in order to

further examine the effects of CKS on the NF-nB transcrip-

tional activity in the TNFa-activated endothelial cells. CKS

inhibited the TNFa-activated NF-nB transcriptional activities

(Fig. 4B). Overall, these results suggest that the suppression of

ICAM-1 and VCAM-1 expression by CKS occurs via the

inhibition of NF-nB activation.

The prevention of InB degradation, which indicates that

CKS inhibits NF-nB activation, was also examined because it

is well documented that NF-nB activation correlates with the

rapid proteolytic degradation of InB. TNFa induced the

transient degradation of InBa in these cells, whereas CKS

prevented the degradation of InBa (Fig. 4C). The effect of

CKS on cellular IKK activation was determined because InBais phosphorylated by IKK (Fig. 4D). The cells were pretreated

with CKS for 30 min and then activated by TNFa. The IKK

complex was immunoprecipitated from the cell lysates and

analyzed for its IKK activity using GST-InBa as a substrate.

The blot used for the autoradiogram was then probed for IKKhby immunoblot analysis. CKS significantly inhibited the IKK

activity induced by TNFa (Fig. 4D). However, CKS had little

or no effect on the IKK protein level (Fig. 4D, upper panel).

This suggests that the inhibition of TNFa-induced IKK activity

by CKS was not due to the decreased IKK expression level.

J.Y. Kim et al. / Toxicology and Applied Pharmacology 210 (2006) 150–156154

Additional experiments were performed to more stringently

examine the effect of CKS on the IKK activity. IKKh was

immunoprecipitated from the cell lysates of the TNFa-

activated cells, and CKS was added at the beginning of the

assay for IKK activity. CKS had no effect on the activity of

IKK (Fig. 4D, lower panel). This suggests that the inhibition of

ICAM-1 and VCAM-1 expression by CKS occurs via the

suppression of IKK activity, which results in the prevention of

NF-nB activation.

Fig. 5. Effects of CKS on TNFa-induced ROS production. The endothelial

cells were pretreated with CKS for 1 h and incubated with 20 AM DCFDA for

30 min. This was followed by stimulation with TNFa (10 ng/ml). The

fluorescence intensity of cells was measured using a fluorescence microplate.

The values are expressed as a mean percentage of the fluorescence intensity TSD of three individual experiments performed in triplicate. *P < 0.01 compared

with the TNFa alone.

CKS inhibits TNFa-induced intracellular ROS production

ROS has been shown to activate various transcription

factors in cultured endothelial cells and has been implicated

as a common second messenger in various pathways leading to

NF-nB activation (Martin et al., 1997; Muller et al., 1997;

Ross, 1999; Kunsch and Medford, 1999). Therefore, the level

of intracellular ROS production was assessed by monitoring

the fluorescence in order to determine if CKS can reduce the

level of TNFa-induced oxidative stress in endothelial cells.

Fig. 5 shows the effects of CKS on the production of

intracellular ROS in endothelial cells induced by TNFa.

Pretreatment with CKS significantly inhibited the TNFa-

Fig. 4. Effects of CKS on (A) TNFa-induced NF-nB activity determined by

EMSA, (B) pNF-nB-Luc reporter plasmid, (C) InB degradation and (D) TNFa

induced InB kinase activity. (A) The endothelial cells were pretreated with CKS

for 1 h and then stimulated with TNFa (10 ng/ml) for 30 min. The nuclea

extracts were prepared, and EMSAwas performed. The arrow indicates the NF

nB binding complex. Excess NF-nB; 200-fold molar excess of the non-labeled

NF-nB probe. (B) The endothelial cells were transiently transfected with pGL3

4nB-Luc and pCMV-h-gal. After 18 h, the cells were pretreated with CKS for 1

h and then stimulated with TNFa (10 ng/ml) for 12 h. This was followed by

harvesting and determining their luciferase, and h-galactosidase activities weredetermined. The luciferase activities are expressed relative to the TNFa. The

values are expressed a mean T SD of three individual experiments, performed in

triplicate. *P < 0.01 compared with the TNFa alone. (C) The endothelial cells

were pretreated with CKS for 1 h and then stimulated with TNFa (10 ng/ml) fo

30 min. The total cellular protein (50 Ag) was separated on 10% SDS

polyacrylamide gels and blotted with the antibody specific for InB. (D) Theendothelial cells were pretreated with CKS for 1 h and then stimulated with

TNFa (10 ng/ml) for 30 min. The IKK was immunoprecipitated from the cel

lysates using IKKh Ab. The activity of the immunoprecipitated IKK was

measured using GST-InBa as substrate, and the GST-p-InBa was visualized by

autoradiography. The relative amount of IKKh in the precipitated complex was

determined by immunoblotting (upper panel). The IKK was immunoprecipi

tated from the TNFa-activated cells, and the IKK activity was measured in the

presence or absence of CKS added to the assay mixture. The IKK activity was

assessed using GST-InBa as described for the upper panel (lower panel). These

blots (A, C, D) are a representative of each of three independent experiments

-

r

-

-

r

-

l

-

.

J.Y. Kim et al. / Toxicology and Applied Pharmacology 210 (2006) 150–156 155

induced ROS production in endothelial cells. This demon-

strates that CKS has significantly antioxidant activity.

Discussion

P. grandiflorum is commonly used in traditional oriental

herbal medicine and has beneficial effects on inflammatory

diseases (Lee, 1973). Previous studies have demonstrated that

CKS isolated from P. grandiflorum has antioxidant and anti-

inflammatory effects (Kim et al., 2001; Lee and Jeong, 2002;

Lee et al., 2004b). However, it is unclear if CKS inhibits

cytokine-induced cell adhesion molecules expression and

reduces the adhesion of monocytes to endothelial cells. This

study investigated the effects of CKS on the expression of

endothelial cell adhesion molecules and the adhesion of

monocyte to human endothelial cells. The results suggest that

a pretreatment with CKS significantly suppressed the follow-

ing in cultured endothelial cells: the adhesion of TNFa-

induced intracellular ROS formation; the activation of the

adhesion of redox-sensitive transcription factor NF-nB, via thesuppression of IKK activity; the expression of VCAM-1 and

ICAM-1; and the adhesion to monocytes. These results suggest

that CKS can inhibit vascular inflammation and prevent in

vitro atherogenesis.

One of the earliest events in atherogenesis is the adhesion of

monocytes to the endothelium, which is followed by their

infiltration and differentiation into macrophages. This key step

is mediated by an interaction between monocytes and the

molecules expressed on the endothelial cell surface (Springer,

1994; Price and Loscalzo, 1999; Ross, 1999; Iiyama et al.,

1999; Glass and Witztum, 2001). These cell adhesion

molecules primarily mediate the adhesion of monocytes

specifically found in atherosclerosis lesions to the vascular

endothelium (Price and Loscalzo, 1999; Ross, 1999). It was

found that TNFa-induced VCAM-1, and ICAM-1 expression

at both the protein and mRNA level was blocked by pretreating

the cells with CKS in a dose-dependent manner, but there was

no effect on the secretion of E-selectin. Moreover, the adhesion

of monocytes to the endothelial cells was markedly inhibited. It

has been demonstrated that atherosclerosis is a chronic

inflammatory disease associated with increased oxidative stress

in the vascular endothelium (Diaz et al., 1997; Ross, 1999).

Oxidative stress up-regulates the expression of the cell

adhesion molecules via the activation of redox-sensitive

transcriptional factors such as NF-nB (Marui et al., 1993;

Ledebur and Parks, 1995; Sen and Paker, 1996; Price and

Loscalzo, 1999; Ross, 1999; Tak and Firestein, 2001; Glass

and Witztum, 2001). NF-nB is involved in the development of

atherosclerosis (Price and Loscalzo, 1999; Ross, 1999; Glass

and Witztum, 2001) as well as in the signal transduction

pathways for the TNFa-induced cell adhesion molecules such

as ICAM-1 and VCAM-1 (Marui et al., 1993; Ledebur and

Parks, 1995; Collins et al., 1995; Neish et al., 1995). The

results showed that the activation of TNFa-stimulated NF-nBwas inhibited by CKS, indicating that CKS has some inhibitory

effect on the NF-nB pathways specific to the cytokine-

stimulated induction of VCAM-1 and ICAM-1 expression.

This study demonstrated that CKS suppresses the TNFa-

activated NF-nB binding and transcription activity of endothe-

lial cells. Furthermore, it was found that CKS inhibited the

TNFa-induced activation of IKK, InBa phosphorylation, and

degradation. The phosphorylation of InBa is regulated by IKK,

which in turn is regulated by many upstream kinases, including

NIK, Akt, and mitogen-activated protein kinase kinase kinase 1

(Tak and Firestein, 2001; Chen et al., 2002). CKS did not

directly affect the activity of IKK (Fig. 4D), which suggests

that CKS inhibits the TNFa-induced IKK activity through an

indirect mechanism. Therefore, CKS might inhibit IKK

activation by inhibiting one or more of the upstream kinases

responsible for IKK activation. Although the down-regulatory

ability of CKS on ICAM-1 and VCAM-1 expression was

demonstrated by the inhibition of NF-nB activation in the

TNFa-stimulated endothelial cells, the precise mechanism(s) is

unclear. The activation of the MAPK members such as the

ERK1/2, JNK1/2, and p38 MAP kinases is involved in the

stimulation of NF-nB activity and the subsequent expression of

cell adhesion molecules in the TNFa-activated endothelial cells

(Sethi et al., 2002). CKS might inhibit the activity of these

kinases, leading to NF-nB activation before or during the InBphosphorylation step. The suppression of NF-nB activation by

CKS might account for this. Therefore, further studies on the

effects of CKS will be needed to understand the regulation of

the expression of the cell adhesion molecules by CKS and to

clarify the mechanisms involved.

It was shown that cytokine-activated endothelial expression

of the cell adhesion molecules and monocytes adhesion to

endothelial cells are inhibited by natural antioxidants via the

suppression of NF-nB activation (Marui et al., 1993; Martin et

al., 1997; Wu et al., 1999; Noguchi et al., 2003; Choi et al.,

2004). Therefore, the antiatherogenic effects of antioxidants

might be due to their antioxidant properties. Previous studies

have shown that CKS is effective in protecting hepatocytes

against tert-butyl hydroperoxide-induced oxidative damage

both in vivo and in vitro (Lee et al., 2004b). CKS scavenged

the superoxide anion, hydroxyl radicals, and reduced the level

of cellular ROS production and lipid peroxidation in tert-butyl

hydroperoxide-treated hepatocytes (Lee et al., 2004b). There-

fore, the inhibitory effect of CKS on the TNFa-induced

activation of NF-nB might be due to its antioxidant properties.

This finding, in conjunction with a report showing that CK

prevents hypercholesterolemia and hyperlipidemia (Kim et al.,

1995), suggests that CKS can play an important role in

preventing antiartherosclerosis. During early atherogenesis,

cytokines such as TNFa may activate the membrane-bound

NADPH oxidase and increase the level of intracellular ROS

production, which can activate redox-sensitive transcriptional

pathway such as NF-nB and induce the expression of the cell

adhesion molecules in vascular endothelial cells (Martin et al.,

1997; Muller et al., 1997; Ross, 1999; Kunsch and Medford,

1999). In this study, the ability of CKS to inhibit the

production of intracellular ROS might be partially related to

the inhibition of NF-nB activation and its downstream

adhesion molecule expression in TNFa-stimulated endothelial

cells.

J.Y. Kim et al. / Toxicology and Applied Pharmacology 210 (2006) 150–156156

In conclusion, CKS inhibits the TNFa-induced expression

of the inflammatory cell adhesion molecules and the adhesion

of monocytes to endothelial cells by inhibiting the redox-

sensitive NF-nB activation through the suppression of IKK

activity through its antioxidant properties. The findings suggest

a new insight into the mechanism(s) responsible for the anti-

inflammatory and anti-atherosclerotic properties of CKS.

Acknowledgments

This work was supported by grants from the Plant Diversity

Research Center of 21st Century Frontier Research Program

(PF0320505-00), Korea Health 21 R&D Project of Ministry of

Health & Welfare (02-PJ9-PG3-21600-0005), and RIC(R)

grants from Traditional and Bio-Medical Research Center,

Daejeon University (RRC04730, RRC04710, RRC04722,

2005) by ITEP.

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