Lan Gấm 1 - Nghiên cứu khoa học

Journal of Ethnopharmacology 135 (2011) 440–449
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Kinsenoside, a high yielding constituent from Anoectochilus formosanus, inhibits
carbon tetrachloride induced Kupffer cells mediated liver damage
Wen-Tsong Hsieha
, Chia-Tzu Tsaia
, Jin-Bin Wub
, Hung-Bo Hsiaoa
, Li-Chan Yanga
, Wen-Chuan Lina,∗
a
School of Medicine and Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC
b
Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan, ROC
a r t i c l e i n f o
Article history:
Received 5 November 2010
Received in revised form 11 March 2011
Accepted 18 March 2011
Available online 4 April 2011
Keywords:
Kinsenoside
Kupffer cell
Liver damage
NF-␬B
a b s t r a c t
Aim: In the present study, we have evaluated the hepatoprotective ability of kinsenoside, a major com-
ponent of Anoectochilus formosanus, in vitro and in vivo.
Materials and methods: The inhibitory action of kinsenoside on lipopolysaccharide (LPS)-stimulated RAW
264.7 macrophage cells and Kupffer cells were investigated. Mice hepatic injury was produced by CCl4
twice a week for 3 weeks. Mice in the three CCl4 group were treated daily with water and kinsenoside
throughout the experimental period.
Results: In LPS-stimulated macrophage RAW 264.7 cells and Kupffer cells, kinsenoside inhibited nitric
oxide (NO) production and also blocked LPS-induced inducible NO synthase expression. Furthermore,
kinsenoside inhibited the NF-␬B activation induced by LPS, and this is associated with the abrogation
of I␬B␣ degradation, with subsequent decreases in nuclear p65 and p50 protein levels. Moreover, the
phosphorylations of p38, ERK and JNK in LPS-stimulated RAW 264.7 cells were suppressed by kinsenoside.
In the in vivo study, kinsenoside significantly protected the liver from injury, by reducing the activities
of plasma aminotransferase, and by improving the histological architecture of the liver. kinsenoside
inhibited Kupffer cell activation by reducing the CD 14 mRNA and protein expressions.
Conclusion: These results indicate that kinsenoside alleviates CCl4-induced liver injury, and this protection
is probably due to the suppression of Kupffer cell activation.
© 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Kupffer cells are the resident macrophages of the liver, involved
in several types of chemical-induced liver damage, including dam-
age related to carbon tetrachloride (CCl4) (Qiu et al., 2005). Kupffer
cells are activated in response to the administration of CCl4 leading
to tissue damage through the release of biologically active medi-
ators, such as, reactive oxygen species and cytokines (Muriel et
al., 2001; Luckey and Petersen, 2001). Rivera et al. (2001) demon-
strated that the destruction of Kupffer cells using gadolinium
chloride attenuated CCl4-induced hepatic fibrosis. Kupffer cells are
considered essential to CCl4-induced liver damage.
Anoectochilus formosanus Hyata (Orchidaceae) is one of the orig-
inal plants of the precious crude drugs, used as a folk medicine in
Taiwan. This herb is also referred to as “the Medicine of Kings”
due to its diverse pharmacological effects, such as antihyper-
glycemia (Shih et al., 2002), antiosteoporosis (Shih et al., 2001),
anti-adiposity (Du et al., 2008), antifatigue (Ikeuchi et al., 2005)
and hepatoprotection (Du et al., 2008). The vegetative propagation
∗ Corresponding author. Tel.: +886 4 22053366x2229; fax: +886 4 22053764.
E-mail address: wclin@mail.cmu.edu.tw (W.-C. Lin).
of A. formosanus by tissue culture has been achieved in Taiwan, and
this technology has been used in commercial agricultural applica-
tions. A. formosanus extract is used as a health supplement for liver
disease in Taiwan.
Previously, Du et al. (2000) investigated the glycosidic con-
stituents of the whole plants of A. formosanus, as propagated by tis-
sue culture. A new compound, 2-(␤-d-glucopyranosyloxymethyl)-
5-hydroxymethylfuran, in conjunction with known compounds,
such as [3-(R)-3-␤-d-glucopyranosyloxybutanolide; Kinsenoside
Fig. 1] and 3-(R)-3-␤-d-glucopyranosyloxy-4-hydroxybutanoic
acid, have been isolated from the plant (Du et al., 2000). Among
these compounds, the biologically active compound kinsenoside is
yielded in higher quantities (Du et al., 2001). Zhang et al. (2007)
showed that kinsenoside provides protection against damage to
␤ cells in streptozotocin diabetic rats, and contributes to antihy-
perglycemic function. In an anti-adiposity assay using rats on a
high-fat diet and aurothioglucose-induced obese mice, kinsenoside
suppressed an increase in the body and liver weights, significantly
ameliorating the level of triglycerides on the liver (Du et al., 2008).
Currently, we have shown that kinsenoside inhibits the
production of inflammatory mediators and enhances the gen-
eration of anti-inflammatory cytokines in lipopolysaccharide
(LPS)-stimulated mouse peritoneal lavage macrophages (Hsiao
0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.03.040

W.-T. Hsieh et al. / Journal of Ethnopharmacology 135 (2011) 440–449 441
Fig. 1. Structure of kinsenoside.
et al., 2010). Furthermore, kinsenoside inhibits the secretion of
tumor necrosis factor ␣ from LPS-stimulated Kupffer cells (Wu
et al., 2010). This anti-inflammatory mechanism might explain
the ameliorative effect of crude extracts of A. formosanus on liver
fibrosis induced by CCl4 and thioacetamide (Shih et al., 2005; Wu
et al., 2007, 2010). However, the hepatoprotective mechanisms of
kinsenoside are still unclear. We hypothesized that kinsenoside
may protect the liver from CCl4-induced injury by inhibiting the
activation of Kupffer cells, which encouraged us to evaluate the
anti-hepatitis effect of kinsenoside. Furthermore, we investigated
the inhibition of LPS-induced inducible NO synthase (iNOS) expres-
sion in RAW 264.7 cells by kinsenoside.
2. Materials and methods
2.1. Preparation of kinsenoside
Fresh A. formosanus Hayata (Orchidaceae) was purchased from
Yu-Jung Farm (Pu-Li, Taiwan) where it was cultivated. The plants
were identified by the Institute of Chinese Pharmaceutical Sciences,
China Medical University, where vocher specimens (CMPC 1253)
have been deposited.
Fresh whole plants of A. formosanus (10 kg) were extracted using
water, and the filtrate was successively partitioned using ethyl
acetate. Water-soluble portions (AFEW) were evaporated under
reduced pressure, yielding 218.4 g of red residue. AFEW (210 g)
was applied to a DIAION HP-20 column (Nippon Ressui Co., Japan)
and eluted with H2O, 10%, 20%, and 50% methanol in water, and
100% methanol to provide five fractions (AFEW-1–AFEW-5). The
dry weight of fraction AFEW-2 was 22.1 g.
Fraction AFEW-2 (10 g) was further purified using silica gel
(Si 60 F245; Merck, Germany) with chloroform/ethanol (15:8)
as the mobile phase to provide four fractions. Fraction 4 (4.5 g)
was applied to preparative high-performance liquid chromatog-
raphy (HPLC) to yield a pure compound (4.1 g). Conditions used for
the preparation of HPLC were as follows: pump, Shimadzu LC-8A
(Kyoto, Japan); mobile phase, water; column, Mightysil ODS RP-18
Aqua column (i.d. 20 mm, 250 mm long; 5 ␮m particle size; Kanto
Chemical Co., Tokyo, Japan). The pure compound was identified
by mass spectroscopy (Jeol GCmate, Tokyo, Japan). Extensive NMR
analysis (1H, 13C, DEPT, COSY, HMQC, HMBC; Jeol 400 MHz, Tokyo,
Japan) identified the compound as kinsenoside, previously isolated
by Ito et al. (1993).
The content of kinsenoside was measured using HPLC. The con-
ditions of HPLC were the same as those described in a previous
study (Wu et al., 2007). The purity of the kinsenoside was approx-
imately 85%.
2.2. Animals
Male Wistar male rats and ICR male mice were purchased
from BioLASCO Co. Ltd. (Taipei, Taiwan). The experimental ani-
mals received humane care, and the study protocols complied well
with the institutional guidelines of the China Medical University
for the use of laboratory animals. The animals were housed in an
air-conditioned room (21–24 ◦C) under 12 h of light (7:00–19:00),
and were allowed free access to food pellets and water throughout
the study.
2.3. Primary cell culture
Rat kupffer cells were isolated according to the method of Froh
et al. (2002). The freshly isolated cells were suspended in RPMI-
1640 medium (Hyclone, Logan, UT, USA) supplemented with 10%
heat-inactivated fetal bovine serum, 2 mM glutamine, penicillin
(100 U/ml), and streptomycin (100 ␮g/ml). The cells were plated
onto 96-well (5 × 104 cells) or 6-well (1 × 106) culture dishes for NO
detection or RNA extract, respectively. They were maintained in an
incubator at 37 ◦C in a humidified atmosphere of 90% air – 100 ml/l
CO2. The non-adherent cells were removed after a 15 min culture.
The adherent cells were used for the experiments. Purity of Kupffer
cell fraction was consistently >80% as determined by CD68 staining
(flow cytometry). All adherent cells were analyzed for their abil-
ity to phagocytosis, which indicated that they were viable Kupffer
cells.
For the experiments, cells cultured for 24 h were washed and
cultured in fresh medium with various concentrations of kinseno-
side (10, 25, and 50 ␮M) 2 h before LPS (0.1 ␮g/ml) treatment. For
NO detection, the supernatants were collected 24 h after LPS treat-
ment.
For RNA extract, the cells were collected 4 h after LPS treat-
ment. RT-PCR analyses for iNOS and CD14 were performed as
described a little later in the text (section of RT-PCR analysis). The
PCR primers for rats iNOS, CD14, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were 5 -GAATTATACACGGAAGGGCCAA-
3 and 5 -AAATGAACCACCCGACTGAAG-3 (product
size, 161 bp), 5 -GTTCACAGAGGAAGGGACAG-3 and
5 -TGAGAAGTTGCAGTAGCAGC-3 (product size,
300 bp), and 5 -TGTGTCCGTCGTGGATCTGA-3 and 5 -
CCTGCTTCACCACCTTCTTGA-3 (product size, 76 bp), respectively.
2.4. Culture of RAW 264.7 cells
RAW 264.7 cells, derived from murine macrophages and
obtained from the Food Industry Research and Development
Institute (Hsinchu, Taiwan), were cultured in DMEM (Hyclone),
supplemented with 10% endotoxin free, heated-inactivated fetal
bovine serum, 100 U/ml of penicillin, and 100 ␮g/ml of strepto-
mycin.
2.5. Measurement of nitrite and cytotoxicity assay
For the NO assay, the cells (3 × 104 cells/well) were preincu-
bated for 2 h with various concentrations of kinsenoside and further
cultured for 24 h with 0.1 ␮g/ml of LPS in 96-well plates. The super-
natants were removed at the allotted time and NO production
was quantified by Griess reagent (Sigma–Aldrich, St. Louis, MO)
(Minghetti et al., 1997).
The viability of the Kupffer cells or RAW 264.7 cells was also
detected by a MTS assay (CellTiter 96 Aqueous One Solution Cell
Proliferation assay, Promega Corporation, Madison, WI, USA). The
result was expressed as an optical density.
2.6. Western blot analysis
The cytoplasmic and nuclear protein extracts were described
previously (Chen et al., 1998). Harvested proteins were separated
by SDS-polyacylamide gels electrophoresis, and transferred to the
nitrocellulose membrane (Amersham Biosciences, Inc., Piscataway,

442 W.-T. Hsieh et al. / Journal of Ethnopharmacology 135 (2011) 440–449
Fig. 2. Effects of kinsenoside on CD14 and iNOS mRNA expression in LPS-stimulated Kupffer cells. Kupffer cells were pre-incubated for 2 h with indicated concentrations
of kinsenoside, and then activated for 24 h with 0.1 ␮g/ml LPS. The expression levels of CD14 and iNOS mRNA were measured and quantified densitometrically. Values
were normalized to GAPDH mRNA expression. Values were means ± SD (n = 3). ###
P < 0.001 as compared with the control group. *P < 0.05, **P < 0.01as compared with the
LPS + vehicle group. Con: control; Veh: vehicle.
NJ, USA). After blocking, the membrane was incubated with pri-
mary antibodies overnight at 4 ◦C. The primary antibodies were
obtained from the following sources: p65, I␬B␣, phosphorylated
I␬B␣ (P-I␬B␣), p38, phosphorylated p38 (P-p38), extracellular sig-
nal regulated kinase (ERK), phosphorylated ERK (P-ETK), c-Jun
N-terminal kinase (JNK), phosphorylated JNK (P-JNK) from Cell
signaling (Danvers, USA); and proliferating cell nuclear antigen
(PCNA), ␣-tubulin, p50 from Santa Cruz (CA, USA). Thereafter, the
blot was washed, exposed to horseradish peroxidase-conjugated
secondary antibodies for 1 h, and then developed by enhanced
chemiluminescence (Thermo, Rockford, USA). PCNA and ␣-tubulin
were used as an internal control in nuclear and cyoplasmic exper-
iments, respectively.
2.7. Electrophoretic mobility shift assay (EMSA) for nuclear
factor-ÄB (NF-ÄB)
To determine NF-␬B activation, the sequence of the NF-
␬B–binding oligonucleotide used as a fluorescence DNA probe was
cy5.5-5 -TCGACCAACTGGGGACTCTCCCTTTGGGAACA-3 , cy5.5-5 -
5 -TCGATGTTCCCAAAGGGAGAGTCCCCAGTTGG-3 (Protech Tech-
nology Enterprise, Taipei, Taiwan). The DNA binding reaction was
performed at room temperature in a volume of 20 ␮l, which con-
tained the binding buffer (10 mM Tris–HCl pH 7.5, 50 mM NaCl,
1 mM DTT), 1 ␮g of poly (dI-dC), 50 nM cy5.5 labeled probe, 0.5%
Tween-20, and 15 ␮g of nuclear extracts. After incubation for
30 min, the protein–DNA complexes were separated from the
unbound DNA by electrophoresis through a 5% nondenaturing poly-
acrylamide gel at 100 V for 1 h in a 0.5 X TBE buffer (Amresco, Solon,
Ohio). Subsequently the gel was transferred to and imaged on a
LI-COR Odyssey infrared imaging system at 700 and 800 nm chan-
nels and 169 ␮m resolution. The density of fluorescence in each
band was measured in triplicate with the use of the LI-COR imaging
software.
2.8. CCl4-induced liver injury
Mice weighing 24–27 g, were randomly allocated to four groups
(a control group and three CCl4-treated groups) 1 day prior to
administration of the test substance. Liver damage was induced
in three groups of 8 mice each by oral administration of CCl4
(0.1 ml/10 g in body weight). CCl4 dissolved in olive oil (diluted
1:9) was administered 2 times per week, for 3 weeks. The animals
received CCl4 with distilled water (0.1 ml/10 g in body weigh) or
kinsenoside (50 and 150 mg/kg, p.o., daily). The control group was
orally administered olive oil (0.1 ml/10 g body weight) 2 times per
week, and distilled water (0.1 ml/10 g body weight, p.o., daily) for
3 weeks. During CCl4 administeration, the time-interval between
the administration of CCl4 and kinsenoside was at least 5 h to
avoid disturbance of the absorption of each substance. The drug
was administered when the injury model was induced. The drug
treatment duration was 3 weeks in total. At the end of the experi-
mental period, the mice were sacrificed under CO2 anesthesia and
blood was withdrawn from the abdominal vein. The livers was
quickly removed, washed with cold normal saline, blotted dry, and
weighed. The largest lobe of the liver was divided into two parts:
one part was submerged in 10% neutral formalin, for the prepa-
ration of pathological sections, and the second part was stored at
−80 ◦C for RT-PCR analyses.
2.9. Assessment of liver functions
Blood samples were centrifuged at 4700 rpm at 4 ◦C for 15 min,
to separate the plasma. The plasma alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) were assayed using
clinical test kits (Roche Diagnostics, Mannheim, Germany) for spec-
trophotometric determination (Cobas Mira Plus, Roche, Rotkreuz,
Switzerland).

Fig. 3. Effect of kinsenoside on LPS-induced iNOS protein expression in RAW
264.7 cells. RAW 264.7 cells were pre-incubated for 2 h with indicated concentra-
tions of kinsenoside, and then activated for 24 h with 0.1 ␮g/ml LPS. The ratio of
immunointensity between the iNOS and the loading control ␣-tublin was calcu-
lated. Values were means ± SD (n = 3). ###
P < 0.001 as compared with the control
group. ***P < 0.001 as compared with the LPS + vehicle group. Con: control; Veh:
vehicle.
2.10. RT-PCR analysis
Total RNA was isolated from mice liver and from Kupffer
cells, cultured using the acid guanidinium thiocyanate-phenol-
chloroform extraction method, as described by Chomczynski
and Sacchi (1987). Total RNA (5 ␮g) from each liver sample
was subjected to reverse transcription using moloney murine
leukemia virus reverse transcriptase (RT) in a 50-␮l reaction
volume. Aliquots of the reverse transcription mix were used
for amplification of fragments specific to CD14 and iNOs by
the polymerase chain reaction (PCR). The levels of expres-
sion of all the transcripts were normalized to that of GAPDH
mRNA in the same tissue samples. The PCR primers for mouse
iNOS, CD14 and GAPDH were 5 -TGGGAATGGAGACTGTCCCAG-
3 and 5 -GGGATCTGAATGTGATGTTTG-3’ (product size,
306 bp), 5 -CCTAGTCGGATTCTATTCGGAGCC-3 and 5 -
AACTTGGAGGGTCGGGAACTTG-3 (product size, 375 bp), and 5 -
TGT GTCCGTCGTGGATCTGA-3 and 5 -CCTGCTTCACCACCTTCTTGA-
3 (product size, 76 bp), respectively. The identities of the resulting
PCR products were confirmed by sequence analysis. The PCR
products were run on 2% agarose gel, recorded on Polaroid film,
and the bands quantitated by densitometry. The mean ratio of
each group was calculated as the average from eight animals.
Fragments shown in Fig. 8 reflect the pooled data of eight samples.
2.11. Light microscopy and immunohistochemistry
After formalin fixation, the tissue samples were sliced, embed-
ded using a standard protocol, and stained with hematoxylin/eosin
(HE). The expression and localizations of CD14 in the liver were
detected by immunohistochemical staining as previously described
elsewhere (Anan et al., 2006). For the single staining of CD14,
deparaffinized tissue sections were incubated with a monoclonal
anti-CD14 (Santa Cruz, Santa Cruz, CA) antibody and a secondary
biotinylated mouse antimouse IgG (Santa Cruz) fragment. The
specific staining was visualized using an immunodetection kit
(SuperSensitive link-label IHC detection system, BioGenex, San
Ramon, USA) and 3,3 -diaminobenzidine. With an automated
Fig. 4. Kinsenoside inhibited LPS-induced NF-␬B activation by EMSA. RAW 264.7
cells were either incubated alone or in the presence of kinsenoside for 2 h, treated
with 0.1 ␮g/ml LPS for 1 h, and then tested for nuclear NF-␬B by EMSA as described.
Data show phosphorimaging analysis of EMSA experiments and were expressed
as percentage of values for LPS treatment only. Vlaues were means ± SD (n = 3).
##
P < 0.01 as compared with the control group. **P < 0.01 as compared with the
LPS + vehicle group. Con: control; Veh: vehicle
software analysis program (Image-Pro Plus version 5.1; Media
Cybernetics, MD, USA), the percent immunostained/field area of
digital photomicrographs were quantified.
2.12. Statistical analysis
Results are expressed as the mean ± SD. All experimental data
were analyzed by one-way analysis of variance following the
Dunnet test. A P value <0.05 was considered statistically signifi-
cant.
3. Results
3.1. Kinsenoside inhibited NO production and mRNA expression
of CD14 and iNOS in LPS-stimulated Kupffer cell
As shown in Table 1, the culture treatment of Kupffer cells with
0.1 ␮g/ml LPS for 24 h caused a dramatic increase in NO production.

Fig. 5. Effects of kinsenoside on LPS-induced NF-␬B nuclear translocation in RAW 264.7 cells. RAW 264.7 cells were pre-incubated for 2 h with indicated concentrations of
kinsenoside, and then activated for 1 h with 0.1 ␮g/ml LPS. The ratios of immunointensity between the p65, p50 and the loading control PCNA were calculated. Values were
means ± SD (n = 3). ##
P < 0.001 as compared with the control group. *P < 0.05, **P < 0.01 as compared with the LPS + vehicle group. Con: control; Veh: vehicle
Table 1
Effect of kinsenoside on viability and production of NO in Kupffer cells after LPS
stimulation.
Group Concentration
(␮M)
Cell viability
(optical density)
NO (␮M)
Control 1.08 ± 0.05 4.9 ± 0.4
LPS + vehicle +
kinsenoside
– 1.18 ± 0.07 23.7 ± 2.1###
10 1.15 ± 0.02 20.0 ± 4.1
50 1.28 ± 0.04 10.0 ± 0.8***
100 1.29 ± 0.12 7.0 ± 0.7***
Values were means ± SD (n = 3).
###
P < 0.001 as compared with the control group.
***
P < 0.001 compared with LPS + vehicle group.
Table 2
Effect of kinsenoside on viability and production of NO in Raw 264.7 cells after LPS
stimulation.
Group Concentration
(␮M)
Cell viability
(optical density)
NO (␮M)
Control 1.31 ± 0.15 1.1 ± 1.1
LPS + vehicle +
kinsenoside
– 1.21 ± 0.16 22.3 ± 1.8###
10 1.22 ± 0.27 14.5 ± 1.4***
50 1.34 ± 0.17 11.3 ± 1.6***
100 1.36 ± 0.37 8.2 ± 1.8***
Values are means ± SD (n = 3).
###
***
P < 0.001 compared with LPS + vehicle group.
Kinsenoside inhibited NO generation in a concentration-dependent
manner. Kupffer cells did not undergo any change in viability after
exposure to LPS + kinsenoside (Table 1).
The fragments speciﬁc to CD14 and iNOS were ampliﬁed by
RT-PCR (Fig. 2). The values from densitometric analysis were nor-
malized to the corresponding GAPDH transcript and were expressed
as CD14/GAPDH, iNOS/GAPDH ratios. As shown in Fig. 2, the Kupffer
cells expressed high levels of CD14 and iNOS mRNA when stimu-
lated with LPS for 4 h, while the expression of CD14 and iNOS mRNA
were barely detectible in unstimulated cells. LPS-activated Kupf-
fer cells treated with kinsenoside showed a suppression of CD14
and iNOS mRNA expression in a concentration-dependent manner
(Fig. 2).
Fig. 6. Effects of kinsenoside on the LPS-induced protein expression of I␬B␣ and P-
I␬B␣ in RAW 264.7 cells. RAW 264.7 cells were pre-incubated for 2 h with indicated
concentrations of kinsenoside, and then activated for 15 min with 0.1 ␮g/ml LPS.
The ratio of immunointensity between the P-I␬B␣ and the loading control ␣-tublin
was calculated. Vlaues were means ± SD (n = 3). ###
P < 0.001 as compared with the
control group. ***P < 0.001 as compared with the LPS + vehicle group. Con: control;
Veh: vehicle

Fig. 7. Effects of kinsenoside on the LPS-induced protein expressions of P-JNK, P-ERK and P-p38 in RAW 264.7 cells. RAW 264.7 cells were pre-incubated for 2 h with indicated
concentrations of kinsenoside, and then activated for 15 min with 0.1 ␮g/ml LPS. The ratios of immunointensity between the P-JNK, P-ERK, P-38 and the corresponding loading
controls were calculated. Vlaues were means ± SD (n = 3). ###
P < 0.001 as compared with the control group. *P < 0.05 compared with the LPS + vehicle group. Con: control;
Veh: vehicle
3.2. Kinsenoside inhibited NO production and iNOS protein
expression in LPS-stimulated RAW 264.7 macrophages
As shown in Table 2, the culture treatment of Raw 264.7
cells with 0.1 ␮g/ml LPS, for 24 h, caused a dramatic increase
in NO production. Kinsenoside inhibited NO generation in a
concentration-dependent manner. Raw 264.7 cells did not undergo
any change in viability after exposure to LPS + kinsenoside
(Table 2).
As shown in Fig. 3, Raw 264.7 cells expressed high level of iNOS
when stimulated with LPS (0.1 ␮g/ml) for 24 h. Western blot analy-
sis of LPS-activated Kupffer cells treated with kinsenoside showed
a suppression of iNOS expression in a concentration-dependent
manner (Fig. 3).
3.3. Kinsenoside inhibited NF-ÄB activation induced by LPS
Raw 264.7 cells were pretreated with kinsenoside for 2 h, and
then treated with LPS (0.1 ␮g/ml) for 1 h. prepared nuclear extracts,
and assayed NF-␬B activation by EMSA. Kinsenoside signiﬁcantly
attenuated the LPS-induced DNA binding activity of NF-␬B (Fig. 4).
Raw 264.7 cells were incubated with LPS in the presence or
absence of kinsenoside for 2 h. Western blot analysis showed that
negligible levels of p50 and p65 protein were detected in control
cell nuclei, but treatment with LPS for 1 h caused their nuclear
translocations. It was found that pretreatment with kinseno-
side concentration-dependently attenuated p50 and p65 levels in
nuclear fractions (Fig. 5). PCNA was used as an internal control in
these experiments.

Fig. 8. Effect of kinsenoside on the hepatic mRNA expression of iNOS and CD14 in CCl4-treated mice. The expression levels of iNOS and CD14 mRNA were measured and
quantified densitometrically. Values were normalized to GAPDH mRNA expression. Values were means ± SD (n = 8). #
P < 0.05, ###
*P < 00.5, **P < 0.01 as compared with the CCl4 + H2O group. Con: control.
3.4. Kinsenoside inhibited IÄB˛ and mitogen-activated protein
kinase (MAPKs) phosphorylation induced by LPS in RAW 264.7
cells
Western blot analysis of cytoplasmic extracts with antibodies
specific to I␬B␣ showed that kinsenoside inhibited LPS-mediated
P-I␬B␣ in a concentration-dependent manner (Fig. 6), while LPS
alone caused a remarkable increase in the level of P-I␬B␣. However,
non-phosphorylated I␬B␣ expression was unaffected by LPS or LPS
plus kinsenoside.
Kinsenoside suppressed the LPS-stimulated P-ERK, P-JNK, and
P-p38 MAPKs (Fig. 7). However, non-phosphorylated ERK, JNK, and
p38 kinase expression were unaffected by LPS or LPS plus kinseno-
side.
3.5. Effects of kinsenoside on biochemical parameters
As shown in Table 3, CCl4 treatment resulted in a significant
increase in the plasma AST and ALT activities, compared to the
control group. Oral administration of kinsenoside (50, 150 mg/kg)
significantly reduced the CCl4-induced increase in AST and ALT
activities.
3.6. RT-PCR analysis of liver tissue
As shown in Fig. 8, fragments of iNOS and CD14 were amplified by
RT-PCR. The iNOS/GAPDH and CD14/GAPDH ratios in the CCl4 group
were 170% and 220%, respectively, greater than those in the control
group. Treatment with kinsenoside (50, 150 mg/kg) reduced the
ratio of iNOs/GAPDH and CD14/GAPDH (Fig. 8).
3.7. Pathological changes
CCl4 administration caused liver morphological changes, evi-
denced by marked necrosis (Fig. 9B). Kinsenoside significantly
reduced CCl4-induced necrosis (Fig. 9C and D).
In the control livers, when stained with CD14 antibodies. CD14
immunoreactivity was increased 37-fold in CCl4-treated mice
when compared with control mice (Fig. 10B and E). In kinsenoside-
treated mice liver, CD14 immunoreactivity was reduced (Fig. 10C
and E).
4. Discussion
In the present study we found that kinsenoside reduced NO
accumulation in cultivated RAW 264.7 macrophages and Kupffer
cells, which were stimulated by LPS. The inhibitory action of kin-
senoside on the NO production seemed to be mediated via NF␬B
and MAPKs pathways. In addition, CCl4 intoxication induced liver
damage, Kupffer cells were activated, with inflammatory cytokines
production. Kinsenoside treatment prevented the alterations pro-
duced by CCl4.
Most liver diseases are accompanied by inflammatory processes.
Therefore pharmacological strategies focus on attenuating this
inflammatory response exerted by immune cells, such as Kupffer
cells (Kolios et al., 2006). Upon inflammatory stimuli, such as LPS,
the Kupffer cells trigger signals for the production of diverse inflam-
matory mediators, such as NO (Valatas et al., 2004). NO exerts its
role in host defense. However, if NO production gets out of con-
trol, damage of host cells occurs due to the cytotoxic potential of
NO (Kolios et al., 2006). Therefore, NO is discussed as an impor-
tant regulator in states of inflammatory disease, including hepatic
inflammatory conditions (Sass et al., 2001).
It is well known that LPS stimulates Kupffer cells to secrete NO
by triggering the CD14 receptor (Saito et al., 2000). In the present
study, kinsenoside has decreased LPS-induced NO production in
isolated rat Kupffer cells. In addition, LPS-stimulated mRNA expres-
sion of CD14 and iNOS in Kupffer cells and kinsenoside pretreatment
efficiently decrease the expression of CD14 and iNOS. These results
indicate that the inhibitory effect of kinsenoside on NO secre-
tion from Kupffer cells to LPS stimulation may be derived at least
partly from the downregulation of the CD14 signaling. Kinsenoside

Table 3
Effect of kinsenoside on the plasma ALT and AST activities in CCl4-treated mice.
Drugs Doses (mg/kg) AST (U/L) ALT (U/L)
Control – 59.4 ± 13.3 31.5 ± 4.1
CCl4 + H2O – 1783.3 ± 344.4###
1992.0 ± 468.1###
CCl4 + kinsenoside 100 1363.7 ± 389.8**
1182.4 ± 686.3*
300 1032.9 ± 359.4***
1035.9 ± 422.7**
All values are means ± SD (n = 8).
###
*
P < 0.05 compared with CCl4 + H2O group.
**
***
reduces NO production and iNOS mRNA expression in the Kupffer
cells, indicating that this observation may be of special impotance
in order to elucidate the mechanisms of the action of kinsenoside
as a hepatoprotective compound.
Kupffer cells as part of the mononuclear phagocyte system
share many functions with macrophages. The actions of kinseo-
side inhibiting NO production and iNOS protein expression were
also observed in RAW 264.7 macrophage cells, therefore, we have
used RAW 264.7 cells to examine the mechanisms of kinsenoside.
Activation of the NF-␬B protein plays a central role in inflam-
mation through the regulation of genes encoding proinflammation
molecules such as inducible enzymes iNOS (Surh et al., 2001). NF-
␬B (a heterodimer of p65 and p50) is located in the cytoplasm as
an inactive complex, bound to I␬B␣, which is phosphorylated and
subsequently degraded, and then dissociates to produce activated
NF-␬B (Ghosh and Hayden, 2008). In the present study, it has been
found that the translocation of NF-␬B was inhibited by kinseno-
side in a concentration-dependent manner, and phosphorylation
and degradation of I␬B-␣, which are required for NF-␬B activa-
tion, were abolished in cells treated with kinsenoside. It provided
evidence that kinsenoside inhibits the activation of NF-␬B.
MAPKs are a highly conserved family of protein serine/threonine
kinases and include the p38, ERK, and JNK subgroups (Jeffrey
et al., 2007). MAPKs are involved in the signaling pathway for LPS-
induced iNOS expression (Hambleton et al., 1996; Bhat et al., 1998;
Alizian et al., 1999). Moreover, there is evidence that MAPKs are
involved in the activation of NF-␬B (Guha and Mackman, 2001).
Thus, the activation of p38, JNK, and ERK is used as a hallmark
of LPS-induced signal transduction in Raw 264.7 cells. Therefore,
to further confirm the inhibitory mechanism of NF-␬B activation
by kinsenoside, we have investigated the effects of kinsenoside on
p38, JNK, and ERK phosphorylation in Raw 264.7 cells stimulated
with LPS, and it has been found that these phosphorylations were
suppressed by kinsenoside. Although we have demonstrated that
kinsenoside impaired phosphorylation of I␬B␣ and MAPKs, I␬B␣
phosphorylation was more severely affected by kinsenoside than
the MAPKs in LPS-stimulated RAW 264.7 cells.
Kupffer cells are the resident macrophages of the liver, which
upon activation, release toxic cytokines and reactive oxygen species
that participate in CCl4-induced liver injury (Luckey and Petersen,
2001; Muriel and Escobar, 2003). Thus, agents that selectively block
Kupffer-cell activation or depletion of Kupffer cells may provide
effective prevention against the progression of liver injury (Muriel
and Escobar, 2003; Xu et al., 2008).
CD14, one of the most important LPS receptors, plays an impor-
tant role in the activation of Kupffer cells (Su, 2002). In this study,
immunohistochemistry and RT-PCR methods have been employed
to determine the changes in CD14 expression. The expression of
Fig. 9. Treatment with kinsenoside improved the histology of CCl4-treated mice liver. HE staining of liver sections from: (A) control group; (B) CCl4 + H2O group, showing
gross necrosis around the central vein. (C) CCl4 + kinsenoside (50 mg/kg) group, (D) CCl4 + kinsenoside (150 mg/kg) group, showing a reduction in gross necrosis around the
central vein.

Fig. 10. Immunostaining of CD14 in the liver section. (A) Control group, (B) CCl4 + H2O group, CD14-positive cells expressing. (C) CCl4 + kinsenoside (50 mg/kg) group, (D)
Cl4 + kinsenoside (150 mg/kg) group, showing a marked reduction in CD14-positive cells. (E) Histogram representing image-quantitation of the mean percentage CD14
area/total area of the liver (n = 8). ###
P < 0.001 as compared with the control group. *P < 0.05, **P < 0.01 as compared with the CCl4 + H2O group.
the CD14 mRNA and CD14 protein in the liver tissue increased
markedly when chronically stimulated by CCl4. This is in agreement
with the previous study that chronic CCl4 administration increases
CD14 expression in the liver (Qiu et al., 2005). Kinsenoside treat-
ment suppressed mRNA and protein expression of CD14 induced
by CCl4 in mice. In addition, kinsenoside also reduced the hepatic
iNOS mRNA expression induced by CCl4 in mice. The results of the
in vivo study also demonstrated that kinsenoside inhibited iNOS
mRNA expression via the inactivation of Kupffer cells.
Plasma ALT and AST activities are the most commonly used bio-
chemical markers of hepatitis (Sturgill and Lambert, 1997). In the
present study, plasma ALT and AST activities were noted to increase
in cases of CCl4-induced liver injury. Kinsenoside reduced plasma
ALT and AST activities, which had been increased with CCl4 admin-
istration. These results confirmed that kinsenoside could protect
against CCl4-induced liver damage. Histological examination using
the HE stain also showed that kinsenoside reduced CCl4-induced
liver injury.
Taken together, these results indicate that kinsenoside pro-
tected the mouse liver from CCl4-causing injury by suppressing
hepatic inflammation. These results confirm and extend our in vitro
observation.
Acknowledgement
This study was supported by grants from the National Sciences
Council of the Republic of China (NSC 98-2320-B-039-028-MY3)
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