Botanical Studies (2008) 49: 301-309.

*

Corresponding author: Present address: Department of

Microbiology and Immunology, Taipei Medical University,

# 250 Wu-Hsing Street, Taipei 110, Taiwan. E-mail:

cmbsycl@tmu.edu.tw; Tel: +886-2-27361661 ext. 3414;

FAX: +886-2-23778620.

INTRODUCTION

Herbal remedies used in traditional folk medicines

provide an interesting and yet largely unexplored source

for the discovery and development of potential new drugs.

Moreover, traditional medicinal methods still play vital

roles in serving basic health needs in developing countries.

Therefore, it is of great interest to screen these plants in

order to validate their use in folk medicine while at the

same time seeking to reveal their active principles vis-a-

vis the isolation and characterization of their constituents.

Chinese herbal medicines (CHMs) are considered useful

for the treatment of a variety of human deficiencies. CHMs

are classified into many categories, and these include heat-

clearing, blood-regulating, Qi-regulating, drain-damping,

wind-damp-dispelling, and exterior releasing (Chongyun

et al., 2005).

The matrix metalloproteinases (MMPs) are

a family of zinc-dependent endopeptidases that play a

key role in the turnover of the extracellular matrix in skin

(Fisher et al., 1996). Aging and exposure to environmental

insults, such as ultra-violet (UV) irradiation, increase the

expression of MMPs (Fisher et al., 1998; Brenneisen et al.,

2002). Excessive MMP activity, which causes the collapse

of the meshwork in the extracellular matrix, produces UV

irradiation-like skin damage, such as wrinkling, a loss

of elasticity, and the dilation of surface micro-capillary

vessels (Bolognia, 1993). MMP-1, which belongs to

the subfamily of collagenases, is the key enzyme in the

The effect of Chinese herbal medicines on TNF-£\

induced matrix metalloproteinase-1, -9 activities and

interleukin-8 secretion

Mei-Hsien LEE

1

, Yi-Yuan YANG

2,3

, Yu-Hui TSAI

3,4

, Yueh-Lun LEE

5

, Po-Yuan

HUANG

4

,

I-Jen HUANG

6

, Kur-Ta CHENG

7

, and Sy-Jye LEU

3,4,5,

*

1

Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan

2

School of Medical Laboratory Science and Biotechnology, Taipei Medical University, Taipei 110, Taiwan

3

Center for Reproductive Medicine and Sciences, Taipei Medical University Hospital, Taipei 110, Taiwan

4

Graduate Institute of Cell and Molecular Biology, Taipei Medical University, Taipei 110, Taiwan

5

Department of Microbiology and Immunology, Taipei Medical University, Taipei 110, Taiwan

6

Applied Bioscience Division, Taiwan Sugar Research Institute, Tainan, Taiwan

7

Department of Biochemistry, Taipei Medical University, Taipei 110, Taiwan

(Received August 31, 2007; Accepted May 9, 2008)

ABSTRACT.

Matrix metalloproteinases (MMPs) play an important role in normal physiological functions

and pathological processes. They are involved in normal skin functions as well as in the aging, healing, em-

bryonic development, reproduction, and inflammatory responses of skin. Previous studies report that both

high MMP-1 and MMP-2 activities were found in the skin of patients with dermatitis, and large amounts of

MMP-9 have been reported to be accumulated in unhealed wounds. Interleukin-8 (IL-8), a C-X-C chemokine,

may mediate neutrophil recruitment and activation and is involved in various inflammatory skin diseases. In

this study, eleven Chinese herbal medicines were analyzed for their potential as anti-inflammatory agents us-

ing human fibroblast WS-1 cell lines. The results indicate MMP-1 and -9, but not MMP-2, were induced by

TNF-

£\

treatment in WS-1 cells. However, when WS-1 cells were pre-treated with eleven Chinese herbal

medicines before TNF-

£\

stimulation, all these herbal medicines suppressed TNF-

£\

-stimulated MMP-1 activity

in WS-1 cells as analyzed by casein zymography. In addition,

the suppression of MMP-9 activity

was also ob-

served when WS-1 cells were treated with either Paeonia suffruticosa, Scutellaria baicalensis, Saposhnikovia

divaricata, Dioscorea opposita, Rubus chingii, or Salvia miltiorrhiza. Of which, R. chingii significantly inhib-

ited IL-8 secretion induced by TNF-

£\

treatment as well. These results revealed that some novel components

present in these Chinese herbal medicines may be used for the treatment of inflammatory responses in skin

cells.

Keywords: Chinese herbal medicines; MMP-1,-2,-9; IL-8.

BIOChemISTRy

302

Botanical Studies, Vol. 49, 2008

collagen turnover that is required for remodeling of the

dermal matrix. MMP-2, and -9, also known as gelatinases

A and B, are important in clearing collagen fragments

generated by collagenases. MMP proteolysis is regulated

by the actions of endogenous inhibitors such as the tissue

inhibitor of metalloproteinase (TIMP), which is involved

in the binding of the active sites of both active and latent

MMPs, forming stable, but inactivated, enzyme-inhibitor

complexes (Kahari and Saarialho-Kere, 1997).

Interleukin-8 (IL-8), which belongs to the C-X-C sub-

family of chemokines, is a chemotactic attractant for both

neutrophiles and T cells. IL-8 and other chemokines can

activate or regulate the inflammatory activity of skin dis-

eases like atopic dermatitis (Kimata and Lindley, 1994;

Hatano et al., 1999; Nomura et al., 2003; Park et al., 2006),

psoriasis (Fukuoka et al., 1998b), and bullous pemphigoid

(Inaoki and Takehara, 1998). IL-8 is produced not only by

monocytes and lymphocytes, which are the major targets

of the chemotactic activity of this chemokine, but also by

epithelial cells, fibroblasts, keratinocytes, and endothelial

cells (Schroder, 1995; Noso et al., 1996; Fukuoka et al.,

1998a). IL-8 directly enhances MMP-2, -9 production on

the endothelial cells which regulate angiogenesis (Li et al.,

2003) and on the endometrial stromal cells which promote

tumor invasiveness (Mulayim et al., 2004).

To date, the relationship between these Chinese herbal

medicines and the MMP and IL-8 activities in human fi-

broblasts has been little investigated. In the present study,

we chose the CHMs usually used to dispel pathogenic

factors from the exterior of the body by diaphoresis, as

well as those used in febrile or inflammatory conditions

to invigorate blood circulation and for their tonic actions.

Therefore, the aim of this study was to investigate the ef-

fects of CHMs on the regulation of MMP-1, -2, -9 as well

as TIMP-1 activities and IL-8 secretion in TNF-

£\

treated

human fibroblast WS-1 cells.

mATeRIALS AND meThODS

materials

The tested Chinese herbal medicines (Table 1) were

purchased from local medicinal markets in Taipei.

Their identities were authenticated by Professor Chang,

H.C. at the Bureau of Food and Drug Analysis, of the

Department of Health in Taiwan. After authentication, the

specimens were then deposited in the Graduate Institute

of Pharmacognosy, Taipei Medical University, Taipei,

Taiwan.

Preparation of plant extracts

The dried leaves of the plants selected for this study

were pulverized and extracted using 95% ethanol twice.

After filtering, the combined filtrates were concentrated

under reduced pressure. The final residues were then

freeze-dried and stored in a closed container until use. We

calculated the yields of plant extracts using the following

formula:

Yield (%) = (mass of the extract/mass of the dried raw

plant material) ¡Ñ 100%.

Cell Culture

Human skin fibroblast WS-1 cells were obtained from

CCRC 6003, Hsinchu, Taiwan. The cells were maintained

in a 37¢XC, 5% CO

2

humidified incubator as monolayers in

75 cm

3

culture flasks. They were grown in Minimal Essen-

tial Medium (MEM, Eagle) with 2 mM L-glutamine, 0.1

mM non-essential amino acids, 10% inactivated fetal calf

serum, 50 U/mL penicillin and 50 £gg/mL streptomycin.

We cultured the cells until confluence and harvested them

with a trypsin-EDTA solution. Subsequently, the WS-1

cells (1 ¡Ñ 10

5

cells/well in 0.5 mL medium) were distrib-

uted and cultured in 24-well plates. Samples of CHM (100

£gg/mL) were added to the cultures and incubated at 37¢XC

for 48 h. After a 48-h incubation period, the medium was

removed, and a fresh serum-free medium with 10 ng/ml of

TNF-

£\

in combination with CHMs (100 £gg/mL) was add-

ed for an additional 24-h period. Ultimately, the culture

supernatants were collected and frozen at -70¢XC

for further

analysis while the cells were harvested for MTT assay.

mTT assay for cell viability

Mitochondrial dehydrogenase activity, which reduces

3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl tetrazolium

bromide (MTT, Sigma, St. Louis, USA) in active mito-

chondria to purple formazan, was used to determine cell

survival in a colorimetric assay. Cell viability was calcu-

lated accordingly:

Cell viability = Absorbance (sample tested) / Absor-

bance (medium only) ¡Ñ 100ƒs

mmP activity by Zymography

Analyses of MMP-1 activity by casein zymography,

MMP-2 and -9 activities by gelatin zymography were as

described (Windsor, 2002) with minor modification. WS-1

cells were cultured and treated with eleven CHM (100 £gg/

ml) separately for 48 h. After incubation, the culture me-

dia were replaced with a serum-free MEM containing 10

ng/ml of TNF-

£\

and each of the CHMs for an additional

24 h. Five £gg of total proteins in the cultured serum-free

media were subjected to SDS-PAGE analyses on 10%

gels containing casein (for MMP-1 activity) or gelatin

(for MMP-2 and -9 activity) substrate (1 mg/mL) under

non-reducing conditions. Recombinant MMP-9 (0.5 ng)

(Chemicon, Temecula, USA) were used as the standard

control. After electrophoresis, the gels were washed with

distilled water containing 2.5% Triton X-100 with gentle

shaking for 1 h. The gels were then incubated at 37¢XC for

18 h in a buffer (50 mM Tris-HCl [pH 7.5], 0.2 M NaCl,

5 mM CaCl

2

) and subsequently stained with Coomassie

Blue. Clear bands of protein degradation were visualized

by destaining in 30% methanol containing 10% glacial

acetic acid. We then scanned photographs of the gel with

an imaging densitometer system (Kodak 1D Image Analy-

sis System 3.5). The MMP activity was calculated from

LEE et al. ¡X CHMs modulating TNF-

£\

induced activities

303

the intensity value (area x intensity) for each band by Ko-

dak 1D Image Analysis Software and normalized with the

intensity value with the medium alone and multiplied by

100%.

For an inhibition percentage calculation, the band inten-

sity for the CHM treated group was normalized with the

TNF-

£\

treated group and shown as relative to the control

(TNF-

£\)

activity.

IL-8 and TImP-1 analysis by enzyme-linked

immunosorbent assay (eLISA)

The WS-1 cells were treated as described above.

Concentrations of IL-8 and TIMP-1 in the supernatants

were quantified using a commercially available duoset

ELISA development system (R&D Systems; Minneapolis,

MN). In principle, ELISA plates were coated with specific

mouse anti-human IL-8 antibody (4 £gg/ml) or mouse

anti-human TIMP-1 antibody (2 £gg/ml). Properly diluted

cell-free supernatants were then added to the wells in

duplicate and incubated for 2 h, after which time, the

secondary biotinylated goat anti-human IL-8 antibody (20

ng/ml) or goat anti-human TIMP-1 antibody (50 ng/ml)

were added. After washing to remove unbound reagent,

streptavidin-conjugated horseradish-peroxidase was added

and incubated for 20 min. After washing, a 1:1 solution of

H

2

O

2

and tetramethylbenzidine was added and developed

until the desired color was reached. The intensity of the

color was measured at 450 nm in an ELISA plate reader

(Emax, Molecular Device). The data was then calculated

according to a standard curve using a Softmax computer

program. The detecting sensitivity for IL-8 and TIMP-1

ranged from 31.25 to 2000 pg/ml.

Table 1. Ethnobatanical data of the selected Chinese herbal medicines.

No.

Species

Family

Traditional uses (Xie and Huang, 1994)

Classification

1 Paeonia suffruticosa Andr. Ranunculaceae It is us ed to elim inat e hea t from the blood for

treatment of bleeding in high febrile conditions

Heat-clearing and

blood-cooling agent

2 Scutellaria baicalensis Gergi Labiatae It is used to eliminate heat in the lung for cough

with yellow thick phlegm, used for the treatment

of pyogenic infections of skin, hypertension, and

threatened abortion.

Heat-clearing and

damp-drying agent

3 Saposhinkovia divaricata

(Turcz.) Schischk

Umbelliferae It is used as diaphoretic for affection due to wind

and cold and rheumatic pain.

Wind-cold-effusing

agent

4 Dioscorea opposita Thunb Dioscoreaceae It is used to invigorate the functions of spleen and

stom ach for the treatment of poor appetite and

chronic diarrhea.

Qi-supplementing

agent

5 Rubus chingii Hu

Rosaceae It is us ed a s a n as tringent for the t re atm ent of

frequent micturition and seminal emission.

Astringent agent

6 Angelica sinensis Diels Umbelliferae It is used to nourish the blood and to invigorate the

blood circulation for the treatment of menstrual

disorders.

Blood-nourishing agent

7 Angelica dahurica Benth Umbelliferae It is used in the treatment of affection due to

wind, cold, or dampness, as anodyne for frontal

headache, etc.

Wind-cold-effusing

agent

8 Glycyrrhiza uralensis Fisch. Leguminosae It is used to invigorate the functions of the heart

and spleen for the treatme nt of s ymptom s due

to deficiency of vital energy of these viscera; as

antitoxicant for drug poisoning.

Qi-supplementing

agent

9 Ligusticum chuanxiong Hort. Umbelliferae It is used to invigorate blood circulation and

promote the flow of vital energy for the treatment

of abnormal menstruation, dysmenorrheal,

amenorrhea, and coronary heart diseases.

Blood-quickening and

stasis-dispelling agent

10 Astragalus membranaceus

(Fisch.)

Leguminosae It is used to replenish the vital energy and to stop

perspiration for the treatm ent of s ponta neous

perspiration, night sweat, prolapse of uterus and

anus.

Qi-supplementing

agent

11 Salvia miltiorrhiza Bge Labiatae It is used to promote blood circulation and

to remove blood stasis for the treatment of

dysmenorrheal, amenorrhea, abdominal mass es

due to stagnation of blood, carbuncles, and ulcers.

Blood-quickening and

stasis-dispelling agent

304

Botanical Studies, Vol. 49, 2008

Data Analysis

The data of each triplet test was presented as the mean

¡Ó standard error bar (SEM). The student¡¦s t-test was used

to analyze the significance of differences between the non-

TNF-

£\

treated group control (medium alone) and the TNF-

£\

-treated group and between the CHM-treated groups and

the TNF-

£\

-treated group (relative to control). A P-value

of < 0.05 was considered statistically significant.

ReSULTS

Chinese herbal medicines do not affect WS-1

cell viability

The MTT assay was performed to investigate whether

Chinese herbal medicines affect the growth of WS-1 cells.

The viabilities of human WS-1 cells at 100 £gg/ml of these

CHM extracts were assayed by MTT. As shown in Figure

1, the cell viabilities after being treated with each of the

eleven CHM were above 80% at the examined concentra-

tion, indicating no significant toxic effects as compared to

the control (100%).

Chinese herbal medicines inhibit mmP-1 activity

To further investigate the potential anti-aging effects

of Chinese herbal medicines on WS-1 cells, the MMP-1

activity was analyzed by casein zymography. As shown

in Figure 2A, TNF-

£\

significantly up-regulated MMP-1

activity (p < 0.001) as compared to the medium alone. All

the tested CHMs at 100 £gg/ml concentration suppressed

the TNF-

£\

- induced MMP-1 activity of WS1 cells to

a varying degree. The inhibitory effect on the MMP-1

activity was semi-quantified and expressed as shown in

Figure 2B. Among which, R. chingii exhibited the most

significant inhibitory activity (94.46 ¡Ó 4.73%, p < 0.001),

followed by S. miltiorrhiza (77.50 ¡Ó 4.28%, p < 0.001), A.

sinensis (57.58 ¡Ó 9.05%, p < 0.05), S. baicalensis (52.97 ¡Ó

5.61%, p < 0.001), S. divaricata (48.26 ¡Ó 7.31%, p < 0.05),

A. membranaceus (44.92 ¡Ó 10.57%, p < 0.05), A. dahurica

(42.76 ¡Ó 5.92%, p < 0.05), D. opposita (40.62 ¡Ó 8.78%,

p < 0.05), L. chuanxiong (32.37 ¡Ó 10.62%, p < 0.05) and

P. suffruticosa (30.39 ¡Ó 7.14%, p < 0.05). In contrast, G.

uralensis had a minimal effect (29.22 ¡Ó 10.23%, p < 0.05).

Figure 1. Effect of Chinese herbal medicines on WS-1 cell vi-

ability. WS-1 cells were cultured and treated with eleven CHMs

(100 £gg/ml) separately for 48 h. After incubation, the culture

media were replaced with a serum-free MEM containing 10

ng/ml of TNF-

£\

and each of the CHMs for an additional 24 h.

Cell viability was determined according to the MTT assay as de-

scribed in Materials and Methods. Results are expressed as mean

¡Ó standard error of three independent experiments.

Figure 2. Effect of Chinese herbal medicines on MMP-1 activ-

ity by casein zymography gel analysis. WS-1 cells were cultured

and treated with eleven CHMs (100 £gg/ml) separately for 48 h.

After incubation, the culture media were replaced with a serum-

free MEM containing 10 ng/ml of TNF-

£\

and each of the CHMs

for an additional 24 h. The supernatants were used for MMP-1

activity assay by casein zymography gel as described in Materi-

als and Methods. (A) Lanes 1-14 represent the results of mo-

lecular weight marker, medium alone without treatment, TNF-

£\

treatment, and eleven CHM treatments, respectively; (B) The

MMP-1 activity was semi-quantified by densitometer and repre-

sented as its value of treated groups over that of TNF-

£\

treated

cells (relative to control). ++ represents p < 0.001 as compared

to medium alone (non-TNF-

£\

treated cells); * and ** represent p

< 0.05 and < 0.001, respectively, as compared to TNF-

£\

-treated

cells.

Values are expressed as mean ¡Ó standard error of six inde-

pendent experiments.

LEE et al. ¡X CHMs modulating TNF-

£\

induced activities

305

effect of Chinese herbal medicines

on mmP-2

and 9 activities

The MMP-9 and -2 activities were measured by gela-

tin zymography. As shown in lane three of Figure 3A,

the TNF-

£\

treatment significantly increased MMP-9 (p <

0.05), but not MMP-2, activities as compared to the me-

dium alone.

Among the eleven Chinese herbal medicines

tested, six extracts showed different degrees of an inhibi-

tory effect on the MMP-9 activity of TNF-

£\

-stimulated

cells, including R. chingii (59.23 ¡Ó 10.94%, p < 0.05), S.

baicalensis (56.83 ¡Ó 3.71%, p < 0.05), S. divaricata (51.96

¡Ó 10.67%, p < 0.05), S. miltiorrhiza (42.33 ¡Ó 10.35%, p

< 0.05), P. suffruticosa (28.93 ¡Ó 10.06%, p < 0.05), and

D. opposita (24.23 ¡Ó 7.69%, p < 0.05). These results are

shown in Figure 3B. However, only S. divaricata (52.11 ¡Ó

12.98%, p < 0.05) and R. chingii (50.30 ¡Ó 7.16%, p < 0.05)

showed a significant inhibitory effect on the MMP-2 activ -

ity of cells treated with TNF-

£\

alone (Figure 3).

Effect of Chinese herbal medicines

on TNF-

£\

-

induced TIMP-1 secretion

TNF-

£\

did not have a significant effect on the TIMP-1

production of WS-1 cells as shown in Figure 4. Among

the eleven samples tested, both A. dahurica and L. chuanx-

iong significantly increased TIMP-1 secretion by 1.29 fold

and 1.78 fold, respectively. While R. chingii showed sig-

nificant inhibition, P. suffruticosa-, S. baicalensis-, and S.

divaricata-treated cells showed an inhibitory trend in the

TIMP secretion.

Effect of Chinese herbal medicines

on TNF-

£\

-

induced IL-8 secretion

IL-8 secretion was significantly enhanced in the TNF-

£\

-stimulated WS-1 cells as compared to the control cells.

When treated with the eleven Chinese herbal medicines,

the TNF-

£\

-induced IL-8 secretion was downregulated dra-

matically by 64.30 ¡Ó 7.25% (p < 0.05) in R. chingii-treated

cells. In contrast, a synergistic/additive stimulated effect

was detected in the groups treated with P. suffruticosa and

S. divaricata.

DISCUSSION

Recent evidence has indicated that chronological aging

and UV irradiation alter the signal transduction pathways

of human skin, promote MMPs expression, decrease pro-

collagen synthesis, and cause connective tissue damage

(Herrmann et al., 1993; Brenneisen et al., 1996; Chung et

al., 2001; Fisher et al., 2001; Kang et al., 2001; Brenneisen

et al., 2002; Chung et al., 2002; Rittie and Fisher, 2002;

Choi et al., 2007). These results indicate that the downreg-

ulation of MMPs activities could be beneficial to human

skin and have great commercial potential. MMP-1 is the

most abundant MMP produced in fibroblast cells. Studies

conducted by many laboratories indicated that collagen

synthesis becomes lower with increased age, but MMP-1

levels become higher in sun-protected human skin in vivo

(Varani et al., 2000; Moon et al., 2006). In this study, we

examined eleven CHMs and found

that all the tested sam-

ples have inhibitory effects on TNF-

£\

-induced MMP-1 ac-

tivity of WS-1 cells. Notably, R. chingii exhibited a potent

inhibition of TNF-

£\

-induced MMP-1, -2, and -9 activities.

In addition, the secretion of TIMP-1 and IL-8 induced by

TNF-

£\

treatment was also inhibited by R. chingii extract

using ELISA analysis. The inhibitory effect on TNF-

£\

in-

duced MMP-9 activity was also observed in the treatment

of R. chingii, S. baicalensis, S. divaricata, S. miltiorrhiza,

P. suffruticosa, and D. opposite as measured by gelatin

zymography. However, we were unable to detect any dif-

ference among the total MMP-9 protein expression in cells

treated with various tested Chinese herbal medicines by

ELISA analysis as observed by gelatin zymography meth-

od (data not shown). Nonetheless, our results indicate that

the inhibition of MMP activities by these tested CHMs

might be a potential strategy for the prevention and/or the

treatment of UV-induced skin damage.

Figure 3. Effect of Chinese herbal medicines on MMP-2 and -9

activities analyzed by gelatin zymography gel. WS-1 cells were

cultured and treated as described in Figure 2. The supernatants

were used to determine the MMP-2 and -9 activities by gelatin

zymography gel analysis. (A) Lanes 1-14 represent recombinant

MMP-9 (0.5 ng) treatment, medium alone without treatment,

TNF-

£\

treatment, and c ells treated with each of the eleven

CHMs, res pectively; (B) The MMP -2 and -9 activities were

semi-quantified by densitometer and represented as the values of

treated groups over that of TNF-

£\

treated cells (relative to con-

trol). + represents p < 0.05 as compared to medium alone (non-

TNF-

£\

treated cells); * represents p < 0.05

as compared with the

levels of TNF-

£\

-treated cells.

Values are expressed as mean ¡Ó

standard error of six independent experiments.

306

Botanical Studies, Vol. 49, 2008

Several studies have indicated that UV-irradiation in-

creases MMPs activities while the TIMP-1 synthesis is

not altered in cultured human fibroblast (Herrmann et al.,

1993; Brenneisen et al., 2000; Naru et al., 2005). Hence,

unbalanced synthesis of MMP and TIMPs may promote

proteolysis and lead to cutaneous photoaging. Our study

shows that TNF-

£\

does not regulate TIMP-1 secretion of

WS-1 cells. A. dahurica and L. chuanxiong significantly

enhanced TIMP-1 expression which may contribute to

further inhibition of MMP-1 activities. This stimulatory

effect was not seen in WS-1 cells treated with the other

herbal medicines. Rubus chingii even showed a significant

inhibitory effect on TIMP-1 expression. Thus, A. dahurica

and L. chuanxiong might contribute to anti-aging effects

on the human skin fibroblast, and they might have poten-

tial as new ingredients in natural cosmetics.

We also evaluated the effects of CHMs on IL-8 produc-

tion by TNF-

£\

-stimulated WS-1 cells. The IL-8 level was

significantly increased in the psoriatic patients compared

with the control (Okubo and Koga, 1998). The IL-8 was

expressed in the neutrophils of psoriasis (Duan et al.,

2001). TNF-

£\

is well known to stimulate IL-8 produc-

tion by human dermal fibroblasts (NHDF) as reported by

Fukuoka et al. (Fukuoka et al., 1998a). Studies have also

demonstrated that IL-8 expression enhances angiogenic

activity through the induction of MMP-9 and subsequently

regulates the tumorigenesis and production of spontane-

ous metastases of human transitional cell carcinoma and

human prostate cancer (Inoue et al., 2000a; Inoue et al.,

2000b). Moreover, an anti-IL-8 antibody inhibits the

growth of bladder cancer xenografts via the downregula-

tion of MMP-2 and MMP-9 expression (Mian et al., 2003).

Among the Chinese herbal medicines tested in this study,

R. chingii significantly downregulated IL-8 secretion (Fig-

ure 4B). It is possible that R. chingii also inhibits MMP-2

and MMP-9 expression in WS-1 cells via the similar

mechanisms triggered by the anti-IL-8 antibody. Taken

together, these data suggest that R. chingii may inhibit the

migration of neutrophiles into skin lesions by reducing the

IL-8 autocrine and/or paracrine functions which are secret-

ed by dermal fibroblasts and keratinocytes. It is noted that

P. suffruticosa and S. divaricata promoted IL-8 secretion

significantly, but inhibited MMP-1 and 9 activities. These

results suggest that the IL-8 may modulate the MMPs¡¦ ex-

pression indirectly. Furthermore, IL-8 secretion and MMP

activities may not be positively co-related due to the major

compounds interacting synergistically or antagonistically.

Rubus chingii has traditionally been used for the treat-

ment of frequent micturition, urorrhea, and seminal emis-

sion (Xie and Huang, 1994). In a recent literature, R.

chingii was reported to protect against tert-Bu hydroper-

oxide-induced oxidative damage in primary rat hepato-

cytes (Yau et al., 2002). It is also reported to decrease the

contents of luteinizing hormone (LH), follicle stimulating

hormone (FSH) and prostaglandin E

2

as well as increase

the amount of LHRH secreted by the thymus gland and the

level of testosterone in blood (Chen et al., 1996). Rubus

chingii extract also showed an inhibitory effect on tyrosi-

nase activity in rats (Hwang and Lee, 2007). Traditionally,

S. divaricata has been used as a diaphoretic for effects

related to the wind and cold and for rheumatic pain as well

as a spasmolytic for tetanus (Xie and Huang, 1994). The

literature shows that S. divaricata exhibits anti-prolifera-

tive properties against several human tumor cell lines as

well as potent antioxidant, anti-inflammatory, and protec-

tive properties on LPS-activated RAW 264.7 cells (Chang

et al., 2007; Tai and Cheung, 2007; Chang et al., 2007).

It also exhibited the effects of anti-fever and analgesia,

sedation, antibacterial effects, regulation of immunologi-

cal function, anticoagulation, and others (Gao, 2004). A.

Figure 4. Effect of Chinese herbal medicines on TIMP-1 and IL-8 secretion by ELISA. WS-1 cells were cultured and treated as de-

scribed above. The supernatants were used to assay the TIMP-1 (A) and IL-8 (B) secretion by ELISA as described in Materials and

Methods. The results are expressed as mean ¡Ó standard error of 3-5 independent experiments. ++ represents p < 0.001 as compared to

medium alone (non-TNF-

£\

treated cells). * and ** represent p < 0.05 and < 0.001, respectively, as compared to TNF-

£\

-treated cells.

LEE et al. ¡X CHMs modulating TNF-

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induced activities

307

dahurica inhibited cyclooxygenase-2 (COX-2) expression

(Ban and Ohuchi, 2003) and had the potential to modulate

cytochrome P450 activity (Ioannides, 2002; Saita, 2006).

Ligusticum chuanxiong was thought to activate the blood,

promote qi, expel wind and relieve pains (Zhang, 2005).

It was also used to treat cardiovascular and cerebrovascu-

lar diseases (Fang, 2006; Lim and Yong, 2004; Wang and

Ou-Yang, 2005; Wang et al., 2006). However, despite the

many medical applications that have been described, noth-

ing in the literature has previously investigated the effects

of R. chingii, S. divaricata, A. dahurica, and L. chuanxiong

on skin fibroblast cells. To the best of our knowledge, the

present study is the first to show the differential regulatory

effects of the extracts from R. chingii, S. divaricata, A. da-

hurica and L. chuanxiong on human fibroblast WS-1 cells.

These results may justify the search for active components

from these specimens, which may be used for treating the

inflammatory responses of skin cells.

Acknowledgments. This study was supported in part

by the National Science Council of Taiwan, Grant NSC

93-2314-B-038-049 (Dr. Leu SJ).

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