Effect of a novel synthesized sulfonamido-based gallate-SZNTC on chondrocytes metabolism in vitro

Article abstract of DOI:10.1016/j.cbi.2014.08.002

The ideal therapeutic agent for treatment of osteoarthritis (OA) should have not only potent anti-inflammatory effect but also favorable biological properties to restore cartilage function. Gallic acid (GA) and its derivatives are anti-inflammatory agents

Full text of DOI:10.1016/j.cbi.2014.08.002

Chemico-Biological Interactions 221 (2014) 127–138
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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Effect of a novel synthesized sulfonamido-based gallate-SZNTC on
chondrocytes metabolism in vitro
Qin Liu a,b,1, Mu-Yan Li a,1, Xiao Lin c,d, Cui-Wu Lin , Bu-Ming Liu c,d, Li Zheng a,b, , Jin-Min Zhao b
c
⇑
a
The Medical and Scientific Research Center, Guangxi Medical University, Nanning 530021, China
Research Center for Regenerative Medicine, Guangxi Medical University, Nanning 530021, China
School of Chemistry and Chemical Engineering, Guangxi University, Nanning, Guangxi 530004, China
Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Traditional Medical and Pharmaceutical Sciences, Nanning 530022, China
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 May 2014
Received in revised form 30 July 2014
Accepted 7 August 2014
Available online 15 August 2014
The ideal therapeutic agent for treatment of osteoarthritis (OA) should have not only potent anti-inflam-
matory effect but also favorable biological properties to restore cartilage function. Gallic acid (GA) and its
derivatives are anti-inflammatory agents reported to have an effect on OA (Singh et al., 2003) [1]. How-
ever, GA has much weaker antioxidant effects and inferior bioactivity compared with its derivatives. We
modified GA with the introduction of sulfonamide to synthesize a novel sulfonamido-based gallate
named sodium salt of 3,4,5-trihydroxy-N-[4-(thiazol-2-ylsulfamoyl)-phenyl]-benzamide (SZNTC) and
analyzed its chondro-protective and pharmacological effects. Comparison of SZNTC with GA and sulfat-
hiazole sodium (ST-Na) was also performed. Results showed that SZNTC could effectively inhibit the
Interleukin-1 (IL-1)-mediated induction of metalloproteinase-1 (MMP-1) and MMP-3 and could induce
the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), which demonstrated ability to reduce
the progression of OA. SZNTC can also exert chondro-protective effects by promoting cell proliferation
and maintaining the phenotype of articular chondrocytes, as evidenced by improved cell growth,
enhanced synthesis of cartilage specific markers such as aggrecan, collagen II and Sox9. Expression of
the collagen I gene was effectively down-regulated, revealing the inhibition of chondrocytes dedifferen-
tiation by SZNTC. Hypertrophy that may lead to chondrocyte ossification was also undetectable in SZNTC
Keywords:
Osteoarthritis
Gallate acid
Sulfathiazole sodium
Modification
Chondro-protective effect
Pharmacological effect
groups. The recommended dose of SZNTC ranges from 3.91
lg/ml to 15.64 lg/ml, among which the most
profound response was observed with 7.82 g/ml. In contrast, its source products of GA and ST-Na have a
l
weak effect in the bioactivity of chondrocytes, which indicated the significance of this modification. This
study revealed SZNTC as a promising novel agent in the treatment of chondral and osteochondral lesions.
Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1
. Introduction
Cartilage defects are usually characterized by a structural
(MMPs) and down-regulation of metalloproteinase-1 (TIMP-1) [5],
which combined may simulate cartilage senescence and destruc-
tion in OA patients. The ideal therapeutic agent for OA would not
only reduce joint inflammation but would also maintain normal
cartilage function [6]. Gallic acid (GA) and its derivatives are a
group of polyphenol compounds that have been known to have
strong anti-oxidant [7] and anti-inflammatory [1,8,9] properties
through the modulation of several important pharmacological
and biochemical pathways. Gallic acid has been reported to induce
apoptosis of rheumatoid arthritis (RA) fibroblast-like synoviocytes
(FLS) by regulating the expression of apoptosis-related proteins
and reducing the expression of pro-inflammatory mediators, such
as pro-inflammatory cytokines, chemokines, COX-2 and MMP-9
breakdown due to trauma or disease, which may lead to chronic
disabilities. After injury, catabolic factors are activated, such as
pro-inflammatory cytokines that can inhibit chondrogenic differ-
entiation and induce a gradual self-destruction of cartilage finally
resulting in secondary osteoarthritis (OA) [2]. Interleukin-1 (IL-1),
a well-known monocyte/macrophage product, inhibits the synthe-
sis of proteoglycans and collagen and enhances their degradation
[
3,4]. IL-1 is known to mediate up-regulation of metalloproteinases
⇑
Corresponding author. Address: The Medical and Scientific Research Center,
Research Center for Regenerative Medicine, Guangxi Medical University, Nanning
[
10]. Another investigation revealed that gallic acid attenuates pro-
inflammatory and pro-oxidant effects [11], In addition, the bioac-
tivity of GA is compromised because it is much more hydrophilic
than its esters, resulting in much weaker anti-oxidant effects than
5
30021, China. Tel.: +86 07715358132; fax: +86 07715350975.
E-mail address: zhengli224@163.com (L. Zheng).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cbi.2014.08.002
009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
0

1
28
Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
its esters in cell systems [7]. GA has also been reported to suppress
cell proliferation [12]. Therefore, the introduction of certain lipo-
philic compounds onto GA may improve its bioactivity and phar-
macological effects.
6.94(s, 2H, Ar–H), 6.73 (s, 1H, Py–H), and 2.23(s, 6H, 2 ꢁ –CH
3
).
1
3
C-NMR (125 MHz, DMSO) d 165.99, 156.30, 145.57, 143.39,
137.29, 129.05, 124.42, 118.99, 107.84 and 22.95.
SZNTC was dissolved in dimethylsulfoxide (DMSO, Sigma, USA).
ST-Na and GA were dissolved in double distilled water respec-
tively. The stock solutions were stored at ꢀ4 °C.
The sulfonamide family, possessing a broad spectrum of syn-
thetic bacteriostatic antibiotics, has been commonly used in the
last century in human and veterinary medicine for therapeutic
and prophylactic purposes for their ability to easily penetrate
through membranes and into body fluids and tissues [13].
Recently, a new series of arylsulfonamido-based hydroxamates
were synthesized and evaluated and were reported to be effective
in blocking ex vivo cartilage degradation without side effects on
cytotoxicity [14]. These compounds contained several phenyl
groups and a sulfonamide group, which may illustrate the need
to synthesize GA derivatives with the addition of sulfonamide
groups. The work of Lin et al. showed favorable bioactivities of
GA derivatives [15]. Thus, GA modified with sulfonamide may
exhibit easy absorption properties that may promote its pharma-
cological activity.
In this study, we synthesized a sulfonamido-based gallate,
sodium salt of 3,4,5-trihydroxy-N-[4-(thiazol-2-ylsulfamoyl)-phe-
nyl]-benzamide (SZNTC) and tested its effect on IL-1-stimulated
chondrocytes and the restoration of chondrocytes. Comparison of
SZNTC with its substrates, GA and sulfathiazole sodium (ST-Na),
was also performed. We hypothesized that GA-sulfonamide
composites may enhance the bioactivity of GA. This study may
be helpful in developing a new agent for the treatment of OA.
2.2. Articular chondrocytes culture
Articular chondrocytes were harvested from knee joint cartilage
slices of 1-week-old New Zealand rabbits by enzymatic digestion.
In brief, cartilage slices from two rabbits were dissociated enzy-
matically with 0.25% trypsin (Solarbio, China) for 30 min and then
with 2 mg/ml collagenase type II (Gibco, USA) in alpha-modified
Eagle’s medium (
the chondrocytes were resuspended. Cells were cultured with
alpha-modified Eagle’s medium ( -MEM, Gibco, USA) containing
a-MEM, Gibco, USA) for 3 h. After centrifugation,
a
20% (v/v) fetal bovine serum (FBS) (Gibco, USA) and 1% (v/v)
penicillin/streptomycin (Solarbio, China) in a 5% CO2 humidified
incubator at 37 °C with the culture medium replaced every other
day after plating. Articular chondrocytes at passage 2 were used
for further studies.
2.3. Cytotoxicity assay
Cytotoxicity in chondrocytes was assessed by the 3-(4,5)-dim-
ethylthiahiazo(-z-y1)-3,5-di-phenytetrazolium-romide (MTT, Gibco,
USA) method. Articular chondrocytes were cultured with 200 l of
l
2
. Materials and methods
culture medium in 96-well microplates with preliminary treat-
ment of various concentrations of SZNTC, ST-Na and GA for 3 d.
Twenty microliter of 5 mg/ml MTT was added, and the plates were
incubated in the dark at 37 °C for 4 h. After removal of the MTT,
2.1. The synthesis of SZNTC
Electrospray ionization mass spectrometry (ESI-MS) was
recorded on a Shimadzu LC-MS 2010A. H and C NMR spectra
were obtained from a Bruker Advance III 300 at 400 and
cells were treated with 150 ll dimethyl sulfoxide (DMSO, Gibco,
1
13
USA) to dissolve the formazan product. The absorbance was deter-
mined at 570 nm using an enzyme-labeled instrument (Thermo
Fisher Scientific, UK).
1
25 MHz, respectively.
The compound sodium salt of 3,4,5-trihydroxy-N-[4-(thiazol-2-
As determined by MTT analysis, the concentrations of SZNTC at
ylsulfamoyl)-phenyl]-benzamide (SZNTC) was prepared from gallic
acid (GA) and sulfathiazole sodium (ST-Na). The synthetic strategy
is presented in Fig. 1 in detail. After reactions, distilled water was
added to the mixture, and then the raw product precipitated and
was separated by vacuum filtration. The raw product was recrys-
tallized in a THF-methanol solvent system.
3.91, 7.82 and 15.64
and of GA at 1.95, 3.9 and 7.8
presented, were chosen for further investigation.
l
g/ml; of ST-Na at 1.95, 3.9 and 7.8
lg/ml;
lg/ml, among which a peak was
2.4. IL-1 induced chondrocytes
SZNTC has the following properties: pale yellow powder,
yield 59%, m.p. > 255 °C, MS-ESI: m/z: 529.2[M-H] , H–NMR
400 MHz, DMSO) d 10.22(s, 1H, –CO–NH), 7.91 (m, 4H, 2 ꢁ Ar–H),
To investigate the effects of SZNTC, ST-Na and GA on Interleu-
kin-1 (IL-1) on the induction of chondrocytes, five groups were
divided: (1) control group, chondrocytes without any treatment;
ꢀ
1
(
Fig. 1. Schematic description of R synthesis route and synthetic procedures of SZNTC. Reagents and conditions: (a) acetyl oxide, oil bath, 120 °C (b) SOCl
sulfathiazole sodium, THF, pyridine, ice bath (d) HCl, THF, 60 °C.
2
, oil bath, 80 °C (c)

Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
129
(
(
2) OA model group, chondrocytes treated with 10 ng/ml IL-1b
10 ng/ml, Gibco, USA); (3) SZNTC treatment groups, chondrocytes
fixed by 95% alcohol for 30 min were successively incubated with
0.1% Safranin O (Sigma, USA) for 10 min. Subsequently, the cells
were rinsed with tap water and then dried naturally. Eventually,
the cells were sealed with neutral gum, observed and
photographed by an inverted phase contrast microscope (Zeiss
Corporation, Germany).
pre-incubated with various concentrations of SZNTC (3.91, 7.82
and 15.64 g/ml) for 1 h followed by stimulation with IL-1b for
4 h; (4) ST-Na treatment groups, chondrocytes pre-incubated
with various concentrations of ST-Na (1.95, 3.9 and 7.80 g/ml)
l
2
l
for 1 h followed by stimulation with IL-1b for 24 h; (5) GA
treatment groups, chondrocytes pre-incubated with various
concentrations of GA (1.95, 3.9 and 7.80 lg/ml) for 1 h followed
2.8. Cell viability assay
by stimulation with IL-1b for 24 h. The concentrations of SZNTC,
ST-Na and GA were derived from the cytotoxicity assay.
Cell viability was determined with a live-dead viability assay kit
(
Invitrogen, USA). In brief, cells were quickly rinsed with PBS, and
then, 1 M calcein-AM and 1 M PI were added to the cell cultures
and incubated in the dark for 5 min at 37 °C. After rinsing with PBS,
l
l
2.5. Cell proliferation analysis and biochemical assay
the images were captured using
microscope (Nikon A1, Japan).
a laser scanning confocal
Chondrocytes were then continuously treated with SZNTC, ST-
Na and GA for 2, 4 and 6 d with the culture medium changed every
d. Cells treated for 2, 4 and 6 d were digested with proteinase K
Sigma, USA) for the following biochemical assay. Intracellular gly-
2
(
2.9. Immunohistochemical staining
cosaminoglycan (GAG) secretion was assayed with 1,9-dimethylm-
ethylene blue (DMMB; Sigma, USA) dye, and the DNA content was
quantified by Hoechst 33258 dye (Sigma, USA) assessment. The
absorbance value of total intracellular DNA content in each sample
was measured with a spectrofluorometer using Hoechst 33258 dye
at 460 nm with calf thymus DNA as a standard. The total intracel-
lular glycosaminoglycan (GAG) secretion was quantified spectro-
photometrically at 525 nm with chondroitin sulfate (Sigma, USA)
as a standard. Finally, the GAG content was normalized to the total
DNA content of the chondrocytes.
The secretion of collagen types I and II, MMP-1 and TIMP-1
were performed immunohistochemically with an immunohisto-
chemical staining kit (Bioss, China). To visualize protein, cells were
fixed in 4% (w/v) paraformaldehyde and treated with Triton X-100.
To exclude endogenous peroxidase activity, cells were incubated
2 2
with 3% H O for 10 min at room temperature. Cells were blocked
with normal goat serum for 10 min at room temperature. After a
1:200 dilution of rat anti-rabbit antibody (collagen type I and II)
was added, cells were then incubated with the second antibody
and biotin labeled horseradish peroxidase. Subsequently, the anti-
0
body binding was visualized with a 3,3 -diaminobenzidine tetrahy-
2.6. Morphological examination
drochloride (DAB) kit (Boster, China) before brief counterstaining
with hematoxylin. Eventually, cells were gradually dehydrated,
sealed with neutral gum, observed and photographed with an
inverted phase contrast microscope (Zeiss Corporation, Germany).
Cells of the control, SZNTC, ST-Na and GA groups were removed
from the incubator at 6 d respectively, and were then fixed in 95%
alcohol for subsequent Hematoxylin-eosin (HE, JianCheng Biotech,
China) staining. Cells were incubated with a nuclear dye for 3 min
and then with a cytoplasmic dye for 5 s. Subsequently, the cells
were rinsed by PBS, naturally dried and sealed with neutral gum.
Cells were then examined and photographed utilizing an inverted
phase contrast microscope (Zeiss Corporation, Germany).
2.10. Real-time quantitative PCR (qRT-PCR) analysis
The genetic information was detected by qRT-PCR for type I, II
and X collagen, aggrecan, Sox9, MMP-1, MMP-3 and TIMP-1. Total
intracellular RNA was extracted with an RNA isolation kit (Tiangen
Biotechnology; Beijing, China) according to the manufacturer’s
instructions. Approximately 300 ng of total RNA was used as a
template and reverse transcribed into cDNA using a reverse tran-
scription kit (Fermentas Company, USA). The qRT-PCR reactions
were performed using a Quantitative PCR Detection System (Real-
plex 4, Eppendorf Corporation, USA) with a FastStart Universal
SYBR Green Master (Mix, Roche company, Germany) under the
condition of 10 min at 95 °C, 15 s at 95°Cand 1 min at 60 °C. The
primers used for PCR were designed as follows (Table 1). The melt-
ing curve data were collected to verify PCR specificity. Each gene
was analyzed in triplicate to diminish operation errors. The relative
Anotherportion ofthe chondrocyteswas used for the detectionof
actin filaments. In brief, cells were fixed with 4% paraformaldehyde
(
PFA, Sigma, USA) for 10 min at room temperature. After a rinse with
PBS, cells were treated with 0.5% Triton X-100 (Sigma Aldrich, USA)
for 5 min. Cells were treated with rhodamine phalloidin (Invitrogen,
USA) for 30 min at room temperature in the dark to label the cellular
matrix. After double-staining with Hoechst 33258 (Beyotime, USA)
away from light for 5 min, fluorescence was detected with a laser
scanning confocal microscope (Nikon A1, Japan).
2.7. Safranin O staining
ꢀDDCt
Histology was performed to assess the synthesis of glycosami-
gene expression levels were calculated using the 2
method
noglycans (GAGs) using Safranin O staining. The cells after being
using glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Table 1
Primer sequences used in qRT-PCR experiments.
mRNA
Forward primer
Reverse primer
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
GAPDH
5 -GTCATCATCTCAGCCCCCTC-3
5 -GGATGCGTTGCTGACAATCT-3
0
0
Aggrecan
Type I collagen
Type II collagen
Type X collagen
Sox9
MMP-1
MMP-3
TIMP-1
5 -TTGCCTTTGTGGACACCAGT-3
5 -GAGCCAAGGACGTAAACCCA-3
0
0
0
0
5 -CCCAGCCACCTCAAGAGAAG-3
5 -CGGGGCTCTTGATGTTCTCA-3
0
5 -TCCGGAAACCAGGACCAAAG-3
5 -CTTTGTCACCACGGTCACCT-3
0
5 -CTACGCTGAGCGGTACCAAA-3
5 -GGCTTCCCAGTGGCTGATAG-3
0
0
5 -GACGCACATCTCGCCCAAC-3
5 -TCTCGCTTCAGGTCAGCCTT-3
0
0
5 -GGCATTGGAGGGGATGTTCA-3
5 -GGCTGACTGGGATTTTGGGA-3
0
0
5 -TTCCAACCCTGCTACTGCTG-3
5 -TCACCTCCAAGCCAAGGAACA-3
0
0
0
0
5 -CTGCGGGTACTCCCACAAAT-3
5 -CCTAGGAGGAGCTGGTCTGT-3

1
30
Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
2.11. Statistical analysis
Results were presented as the means ± SD. Significant
differences were determined using one way analysis of variance
ANOVA) followed by Dunnett’s post hoc test. The level of
(
significance was set to P < 0.05.
Fig. 3. Quantitative comparison of ECM-related gene expression of MMP-1 (A),
MMP-3 (B) and TIMP-1 (C) by qRT-PCR. The chondrocytes were cultured with
different concentrations of SZNTC, GA and ST-Na on the induction by IL-1b for 24 h
(
1
Control: 0
5.64 g/ml; G-1: 1.95
ST-2: 3.9 g/ml; ST-3: 7.8
levels in SZNTC, GA and ST-Na media relative to the control group were analysed by
l
g/ml; IL-1b: 10 ng/mL; TC-1: 3.91
g/ml; G-2: 3.9 g/ml; G-3: 7.8
g/ml) (n = 3 for each experiment). The gene expression
l
g/ml; TC-2: 7.82
l
g/ml; TC-3:
l
l
l
lg/ml; ST-1: 1.95
l
g/ml;
l
l
ꢀ
DDCT
the 2
method using GAPDH as the internal control. The data represent the
Fig. 2. Cytotoxicity of SZNTC, GA and ST-Na on chondrocytes after 3 d (mean ± SD,
n = 4). p < 0.05, p < 0.01, p < 0.001.
mean ± SD of three independent culture experiments. Bars with different letters are
significantly different from each other at P < 0.05.
⁄
⁄⁄
⁄⁄⁄

Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
131
3
. Results
3.2. Effects of SZNTC, ST-Na and GA on IL-1b-induced chondrocytes
3
.1. Cytotoxicity assay
To investigate the effects of SZNTC, ST-Na and GA on arthritis,
chondrocytes were pre-incubated with SZNTC, ST-Na and GA for
1 h prior to stimulation with IL-1b for 24 h. Chondrocytes stimu-
lated by IL-1b exhibited upregulation of MMP-1 and MMP-3 gene
expression, and downregulation of TIMP-1 expression. In contrast,
SZNTC inhibited the IL-1b-mediated induction of MMP-1 and
MMP-3 gene expression and induced the expression of TIMP-1.
However, ST-Na and GA could not effectively prevent the induction
of MMP-1 and MMP-3 gene expression or upregulate TIMP-1
expression (Fig. 3). We next examined the effects of IL-1b, SZNTC,
ST-Na and GA on protein secretion of MMP-1 and TIMP-1 in
chondrocytes. Treatment with IL-1b resulted in the upregulation
of MMP-1and the downregulation of TIMP-1 at the protein level.
In agreement with the results of RT-PCR, SZNTC (but not ST-Na
and GA) could downregulate MMP-1 expression and upregulate
TIMP-1 expression, as demonstrated with higher levels of staining
This study examined the cytotoxicity of different drugs on
articular chondrocytes by MTT assay. Articular chondrocytes were
treated with SZNTC, ST-Na or GA in increasing concentrations
(
1.95–37.5
l
g/ml). As shown in Fig. 2, SZNTC concentrations rang-
g/ml were comparable to the control and
ing from 1.95 to 18.75
therefore nontoxic to cells. However, SZNTC exhibited an inhibitive
effect on chondrocytes at the concentrations ranging from 25 to
3
or inhibitive effects on chondrocyte growth at different levels
ranging from 1.95 to 37.5
over the range 3.90–15.62
the cell proliferation were used in subsequent assays, whereas
concentrations of ST-Na and GA ranging from 1.95 to 7.80 g/ml
and 1.95 to 7.80 g/ml, respectively, were utilized.
l
7.5
lg/ml. Comparatively, ST-Na and GA exhibited insignificant
l
l
g/ml. Hence, concentrations of SZNTC
g/ml which can significantly improve
l
l
Fig. 4. Immunohistochemical staining images revealed the presence of MMP-1 (a) and TIMP-1(b). Chondrocytes cultured in vitro with different concentrations of SZNTC, GA
and ST-Na on the induction by IL-1b for 24 h: (A) 3.91 g/ml; (B) 7.82 g/ml; (C) 15.64 g/ml; (D) control (0 g/ml); (E) 1.95 g/ml; (F) 3.9 g/ml; (G) 7.8 g/ml; (H) IL-1b
10 ng/ml); (I) 1.95 g/ml; (J) 3.9 g/ml; (K) 7.8 m.
g/ml; cell seeding density: 2 ꢁ 10 /ml (original magnification ꢁ 100). Scale bar = 200
l
l
l
l
l
l
l
4
(
l
l
l
l

1
32
Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
for MMP-1 and lower levels of staining for TIMP-1. These results
suggested that the IL-1b-mediated induction of MMPs and
downregulation of TIMP-1 were effectively blocked by SZNTC
whereas these effects could not be prevented by ST-Na or GA
showed that GAGs gradually accumulated in all groups. Compara-
tively, GAG production in culture media treated with SZNTC was
significantly improved over that in the control at the same time
point, whereas reduced tendency was obviously observed in ST-
Na and GA groups compared with the control. Particularly, SZNTC
(Fig. 4, Table 2).
at a concentration of 7.82 lg/ml exhibited the strongest promotion
3.3. Cell proliferation
GAG synthesis among the three concentrations.
The Safranin O-positive stain (Fig. 6b) in the SZNTC group indi-
cated that GAGs were abundant and homogeneously distributed
around the chondrocytes. Comparison of ST-Na and GA groups
with the control revealed indigent GAGs. The result of Safranin O
staining conformed to GAG production by biochemical analysis
(Fig. 5B).
In this study, the cell proliferation in experimental groups and
control groups was analyzed by measurements of DNA content.
Comparatively, cells cultured with SZNTC grew faster than those
in the control group (P < 0.05), as shown by DNA values obviously
higher than the control in the same culture period. Oppositely, cells
treated with ST-Na and GA grew slower than those in the control
group (P < 0.05), which agreed with DNA values results. Further-
more, among all SZNTC groups, the highest cell proliferation was
3.5. Cell morphology
achieved at a concentration of 7.82
that SZNTC exhibited the strongest promoting effect on chondro-
cyte growth, especially at the concentration of 7.82 g/ml (Fig. 5A).
l
g/ml. These data indicated
We assessed the morphology of articular chondrocytes with an
inverted microscope after treatment with SZNTC (3.91, 7.82 and
l
15.64
l
g/ml), ST-Na (1.95, 3.9 and 7.8
lg/ml) and GA (1.95, 3.9
and 7.8
l
g/ml) (Fig. 6a). There was no significant difference in car-
3
.4. Secretion of GAGs
tilaginous morphology in all any group after 6 d of culture. Com-
pared with the control, the chondrocytes in the presence of
SZNTC grew better and had a distinctive proliferation tendency
that gradually increased with time. In addition, at the concentra-
To determine if extracellular GAG production was affected by
SZNTC, ST-Na and GA, biochemical assays were performed after
, 4 and 6 d of culture. Results of intracellular GAG production trea-
2
tion of 7.82 lg/ml, SZNTC could better enhance the proliferation
ted by different concentrations of SZNTC, ST-Na and GA (Fig. 5B)
of chondrocytes over that of the other two concentrations.
Nevertheless, these effects were not exhibited by the other two
experimental groups treated with ST-Na and GA. Fig. 7b shows
the actin filaments of chondrocytes by staining with rhodamine
phalloidin/Hoechst 33258, which was in agreement with the HE
analysis. The cells in the SZNTC treated groups grew in clumps
with a densely distributed ECM. In the ST-Na and GA groups, fewer
cells and less ECM were present compared with the control.
Table 2
MMP-1 and TIMP-1 expression.
MMP-1 expression
Positive area
TIMP-1 expression
Positive area
A (3.91
B (7.82
C (15.64
l
l
l
g/ml)
g/ml)
g/ml)
g/ml)
E (1.95 g/ml)
g/ml)
g/ml)
3.5%
2.1%
2.5%
67.1%
83.5%
79.2%
84.7%
29.3%
33.6%
28.3%
40.1%
33.7%
38.4%
35.6%
3.6. Cell viability assay
D (0
l
1.6%
l
41.4%
30.6%
34.9%
23.2%
30.4%
27.5%
32.3%
F (3.9
G (7.8
l
l
Viable cells and dead cells were determined using calcein-AM/
PI staining (Fig. 7a). The results demonstrated that SZNTC exerted
potent effects, whereas ST-Na and GA demonstrated an inhibitory
effect on chondrocytes survival under identical culture conditions.
Calcein-AM/PI staining images displayed that survival in SZNTC
groups was higher than observed in the control; however, viable
H (10 ng/ml)
I (1.95 g/ml)
g/ml)
g/ml)
l
J (3.9
l
K (7.8
l
Fig. 5. Quantification of cell proliferation (DNA) and matrix production (glycosaminoglycan (GAG)) of cells by biochemical assays: (A) the proliferation of chondrocytes
cultured with different concentrations of SZNTC, GA and ST-Na in vitro for 2, 4 and 6 d (control: 0 g/ml; TC-1: 3.91 g/ml; TC-2: 7.82 g/ml; TC-3: 15.64 g/ml; G-1:
.95 g/ml; G-2: 3.9 g/ml; G-3: 7.8 g/ml; ST-1: 1.95 g/ml; ST-2: 3.9 g/ml; ST-3: 7.8 g/ml); (B) GAG (mg) normalized to DNA (mg). Data from four independent
experiments were evaluated, and the mean ± SD is shown. Bars with different letters are significantly different from each other at P < 0.05.
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Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
133
chondrocytes in the ST-Na and GA groups was significantly less
than that in the control. Consistent with the result of cell prolifer-
ation (Fig. 5A), more viable cells were found than dead cells in the
SZNTC groups, implying that SZNTC could better support cell
growth compared with ST-Na and GA. Among the SZNTC groups,
groups, not only was collagen II staining weaker than the control
but collagen I staining was also stronger than the control. These
results indicated that SZNTC may more effectively inhibit de-dif-
ferentiation of chondrocytes cultured in vitro than ST-Na and GA.
SZNTC at a concentration of 7.82
evidenced by more viable cells.
lg/ml was superior to others, as
3.8. Gene expression
The effects of SZNTC, ST-Na and GA on chondrocyte ECM syn-
3
.7. Secretion of type I and type II collagen
thesis were further inspected through examination of gene expres-
sion of collagen I, collagen II, collagen X, Sox9, aggrecan (a
proteoglycan composed of GAGs) after 2, 4 and 6 d of culture. As
shown in Fig. 9, cartilage specific gene expressions, such as aggre-
can, collagen II and Sox9, were significantly boosted by SZNTC at
concentrations ranging from 3.91 to 15.64 lg/ml but were mark-
edly reduced by ST-Na and GA. Moreover, the highest collagen II,
aggrecan and Sox9 expressions in the SZNTC group were accompa-
Expression of type I and type II collagen in the cytoplasm at dif-
ferent levels with and without drugs-treated culture media was
shown in Fig. 8 and Table 3. Strongly positive staining with large
areas was evident for cartilage-specific type II collagen and only
very sparse and light staining was seen for type I collagen in the
SZNTC groups compared with control groups after incubation for
6
d, confirming the maintenance of the chondrocytic phenotype
nied with the 7.82
lg/ml concentration. The presence of SZNTC up-
after treatment with SZNTC. In addition, in the ST-Na and GA
regulated collagen II, aggrecan and Sox9 expressions, suggesting
Fig. 6. Hematoxylin-eosin staining and Safranin O/fast green staining images respectively showing the morphology (a) and GAG production (b) of chondrocytes cultured
in vitro with different concentrations of SZNTC, GA and ST-Na for 6 d: (A) 3.91 g/ml; (B) 7.82 g/ml; (C) 15.64 g/ml; (D) control (0 g/ml); (E) 1.95 g/ml; (F) 3.9 g/ml; (G)
.8 g/ml; (H) 1.95 g/ml; (I) 3.9 g/ml; (J) 7.8 m.
g/ml; cell seeding density: 2 ꢁ 10 /ml (original magnification ꢁ 100). Scale bar = 200
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4
7
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34
Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
that SZNTC either delayed or prevented the chondrocytes from
dedifferentiating into a hypertrophic phenotype; correspondingly,
ST-Na and GA may insignificantly influence the differentiation of
chondrocytes. At the same time, collagen X expression was scar-
cely detectable in all groups, suggesting that cell hypertrophy
was not prominent.
control group after being cultured for 2, 4 and 6 d. Moreover, the
levels of collagen I at a concentration of 7.82 g/ml were lower
than that of the other two concentrations in the SZNTC group.
These results further hinted that SZNTC could inhibit the dediffer-
entiation of chondrocytes but ST-Na and GA could not do so.
l
Therefore, SZNTC ranging from 3.91 to 15.64 lg/ml could
SZNTC at different concentrations induced lower collagen I
expression, whereas higher or similar collagen I expressions were
surveyed in the ST-Na and GA group when compared with the
upregulate the synthesis of cartilage specific markers while down-
regulating dedifferentiation related genes. The effects of ST-Na and
GA are the opposite of SZNTC. Among all of the groups, SZNTC at
Fig. 7. Confocal laser scanning microscopy images showing the viability (a) and actin filaments (b) of chondrocytes cultured in vitro with different concentrations of SZNTC,
GA and ST-Na for 6 d: (A) 3.91 g/ml; (B) 7.82 g/ml; (C) 15.64 g/ml; (D) control (0 g/ml); (E) 1.95 g/ml; (F) 3.9 g/ml; (G) 7.8 g/ml; (H) 1.95 g/ml; (I) 3.9 g/ml; (J)
.8 m.
g/ml; cell seeding density: 2 ꢁ 10 /mL (original magnification ꢁ 100). Scale bar = 200
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Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
135
Fig. 8. Immunohistochemical staining images revealed the presence of type I (a) and type II (b) collagen. Chondrocytes cultured in vitro with different concentrations of
SZNTC, GA and ST-Na for 6 d: (A) 3.91 g/ml; (B) 7.82 g/ml; (C) 15.64 g/ml; (D) Control (0 g/ml); (E) 1.95 g/ml; (F) 3.9 g/ml; (G) 7.8 g/ml; (H) 1.95 g/ml; (I) 3.9 g/
g/ml; cell seeding density: 2 ꢁ 10 /ml (original magnification ꢁ 100). Scale bar = 200 m.
l
l
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l
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l
4
ml; (J) 7.8
l
l
4
. Discussion
GA was reported to have an effect on OA. However, GA has
Table 3
Type I collagen and type II collagen expression.
Type I collagen expression
Positive area
Type II collagen expression
Positive area
much weaker antioxidant effects than its esters and inferior
bioactivity, which may be due to its hydrophilicity. Based on the
hypothesis that synthetic compounds of gallates and sulfonamides
may enhance its chondro-protective and pharmacological effects,
we synthesized SZNTC and examined its effects.
Many studies have demonstrated that IL-1 inhibits chondrocyte
compensatory biosynthesis pathways, which can further compro-
mise cartilage repair [16]. Chondrocytes stimulated with IL-1b
in vitro have been exploited to imitate the microenvironment that
occurs in osteoarthritis (OA) [17]. IL-1b is known to exert a key role
in cartilage degradation, through the induction of MMPs secreted
by chondrocytes. Targeting MMPs is a promising approach to the
treatment of OA because the MMPs, particularly MMP-1 and
MMP-13, are interstitial collagenases that degrade type II collagen
in the cartilage, which is a critical step in the progression of OA. In
A (3.91
l
l
l
g/ml)
g/ml)
g/ml)
g/ml)
E (1.95 g/ml)
g/ml)
g/ml)
18.9%
10.2%
5.1%
90.9%
94.5%
93.5%
79.4%
36.8%
59.5%
48.7%
31.2%
67.3%
37.7%
B (7.82
C (15.64
D (0
l
7.7%
l
23.9%
20.1%
22.1%
25.8%
21.0%
24.1%
F (3.9
l
G (7.8
l
l
g/ml)
g/ml)
H (1.95
I (3.9
J (7.8
g/ml)
l
l
the concentration of 7.82
aggrecan and collagen II expression, which was in agreement with
the results of GAG production (Fig. 5B, Fig. 6b).
lg/ml demonstrated the strongest

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36
Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
Fig. 9. Quantitative comparison of ECM-related gene expression of aggrecan (A, B, C), collagen II (D, E, F), Sox9 (G, H, I) and collagen I (J, K, L) by qRT-PCR. The chondrocytes
were cultured with different concentrations of SZNTC, GA and ST-Na for 2, 4 and 6 d (Control: 0 g/ml; TC-1: 3.91 g/ml; TC-2: 7.82 g/ml; TC-3: 15.64 g/ml; G-1: 1.95 g/
ml; G-2: 3.9 g/ml; G-3: 7.8 g/ml; ST-1: 1.95 g/ml; ST-2: 3.9 g/ml; ST-3: 7.8 g/ml) (n = 3 for each experiment). The gene expression levels in SZNTC, GA and ST-Na media
relative to the control group were analyzed by the 2 method using GAPDH as the internal control. The data represent the means ± SD of three independent culture
experiments. Bars with different letters are significantly different from each other at P < 0.05.
l
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l
ꢀ
DDCT
the present study, we utilized IL-1b to induce MMP gene expres-
sion, and then assessed the effects of SZNTC, ST-Na and GA on
MMP induction in rabbit articular chondrocytes. We observed that
SZNTC inhibited the IL-1b-mediated induction of MMP-1 and
MMP-3 and induced the expression of TIMP-1, a metalloproteinase
inhibitor, in rabbit articular chondrocytes (Fig. 3). However, its
substrates, including GA and ST-Na, exhibit weaker effects. These
results demonstrated that SZNTC has the potential to be developed
as potent anti-inflammatory agent in the treatment of OA.
The results indicated that SZNTC could observably promote
chondrocytes proliferation (Fig. 5A). SZNTC also markedly pro-
moted GAGs deposition in cultured chondrocytes, which was
shown with a biochemical assay (Fig. 5B). Proteoglycans (PGs)
are important components of extracellular matrices [18]. For all
PGs, glycosaminoglycans (GAGs) constitute a major component
of their molecular mass; moreover, GAG and a large number of
water molecules generate the expansion pressure and make the
cartilage flexible, which plays an important role in maintaining
cartilage load-bearing capacity [19]. Consistent with the increase
in GAG production, SZNTC could upregulate the gene expression
of cartilage-specific aggrecan, collagen II and Sox9 (Fig. 9). Chon-
drogenic transcription factor Sox9 played a major role in an
increased level of chondrogenesis [20,21], in particular activating
co-expression with collagen type II [22–24]. In addition, extensive
gene therapy approaches using viral methods to over-express Sox9
resulted in marked improvements in the secretion of cartilaginous
matrix by articular chondrocytes, bone marrow–derived stem cells
and nucleus pulposus cells [25–27]. These data hinted that SZNTC
could facilitate chondrocytes proliferation and stimulate exuberant
cartilage matrix secretion.
SZNTC. Dedifferentiation happens when the differentiated pheno-
type of chondrocytes, primarily composed of type II collagen and
cartilage-specific proteoglycans, is bereaved and replaced by a
complex collagen phenotype consisting of the vast majority of type
I collagen and a low level of proteoglycan synthesis [28–30]. Fur-
thermore, collagen type X, which is specifically associated with
hypertrophic chondrocytes and precedes the onset of endochon-
dral ossification [31], was nearly undetectable in SZNTC groups,
implying that the hypertrophy of chondrocytes would not be
induced by SZNTC. As a consequence, the decreasing collagen I
expressions and the inconspicuous expressions of collagen X might
suggest that SZNTC may be preventing the dedifferentiation and
hypertrophy of chondrocytes.
Because GA possesses inferior pharmacological effects and bio-
logical properties, modification of GA may be meaningful. Epigallo-
catechin-3-gallate (EGCG), the ester of epigallocatechin and gallic
acid, was found to repress the degradation of human cartilage pro-
teoglycan and type II collagen and selectively inhibit ADAMTS-1,
ADAMTS-4 and ADAMTS-5 [32,33]. Another study revealed that
EGCG ameliorates IL-1b-mediated suppression of TGF-b synthesis,
and enhances type II collagen and aggrecan core protein synthesis
in human articular chondrocytes [34]. A study reported that
sulfonamides could also inhibit cell wall synthesis [35]. At the
same time, another study showed that sulfonamides were slightly
cytotoxic in human keratinocytes and rat hepatocytes [36]. In
agreement with this work, Lin et al. showed the enhanced bioactiv-
ity of GA derivatives, but comparison with substrates and effect on
arthritis have not been performed [15]. In this study, SZNTC, as a
novel derivative of GA, can also support the chondrocyte growth
and maintain their phenotype. This implied that suitable modifica-
tion of GA may lead to the improvement of its pharmacological
effects.
In addition, the expression of collagen type I, which marks
dedifferentiation of chondrocytes, was effectively inhibited by

Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
137
Our results demonstrated that the concentration of SZNTC with
respect to enhancing chondrocytes proliferation ranged from 1.95
to 18.75 lg/ml (Fig. 2). DNA production of rabbit articular chon-
[6] M.M. Hardy, K. Seibert, P.T. Manning, M.G. Currie, B.M. Woerner, D. Edwards, A.
Koki, C.S. Tripp, Cyclooxygenase 2-dependent prostaglandin E2 modulates
cartilage proteoglycan degradation in human osteoarthritis explants, Arthritis
Rheum. 46 (2002) 1789–1803.
drocytes was enhanced in a dose-dependent manner when chon-
drocytes were cultured in the medium containing SZNTC at
concentration of 3.91–15.64 lg/ml, and the 7.82 lg/ml group sup-
ported the strongest cell proliferation and stimulated the greatest
[7] Z. Lu, G. Nie, P.S. Belton, H. Tang, B. Zhao, Structure–activity relationship
analysis of antioxidant ability and neuroprotective effect of gallic acid
derivatives, Neurochem. Int. 48 (2006) 263–274.
[
8] B.H. Kroes, A.J. van den Berg, H.C. Quarles van Ufford, H. van Dijk, R.P. Labadie,
Anti-inflammatory activity of gallic acid, Planta Med. 58 (1992) 499–504.
9] C.Y. Hsiang, Y.C. Hseu, Y.C. Chang, K.J. Kumar, T.Y. Ho, H.L. Yang, Toona sinensis
and its major bioactive compound gallic acid inhibit LPS-induced
[
matrix secretion.
This study is a preliminary exploration of the effect of deriva-
tives of GA on chondrocytes metabolism. Further studies are
needed to elucidate the underlying mechanism of chondrogenesis
induced by SZNTC. Besides, the application of SZNTC on arthritis
should also been carried out.
inflammation in nuclear factor-
jB transgenic mice as evaluated by in vivo
bioluminescence imaging, Food Chem. 136 (2013) 426–434.
[
10] C.H. Yoon, S.J. Chung, S.W. Lee, Y.B. Park, S.K. Lee, M.C. Park, Gallic acid, a
natural polyphenolic acid, induces apoptosis and inhibits proinflammatory
gene expressions in rheumatoid arthritis fibroblast-like synoviocytes, Joint
Bone Spine 80 (2013) 274–279.
[
[
[
[
11] G. Kuppan, J. Balasubramanyam, F. Monickaraj, G. Srinivasan, V. Mohan, M.
Balasubramanyam, Transcriptional regulation of cytokines and oxidative
stress by gallic acid in human THP-1 monocytes, Cytokine 49 (2010) 229–234.
12] T.T. Ou, M.C. Lin, C.H. Wu, W.L. Lin, C.J. Wang, Gallic acid attenuates oleic acid-
induced proliferation of vascular smooth muscle cell through regulation of
AMPK-eNOS-FAS signaling, Curr. Med. Chem. 20 (2013) 3944–3953.
5
. Conclusion
In conclusion, sulfonamido-based gallate SZNTC may relieve IL-
destruction as a means to reduce the progression of OA. This
1
´
13] W. Baran, E. Adamek, J. Ziemianska, A. Sobczak, Effects of the presence of
sulfonamides in the environment and their influence on human health, J.
Hazard. Mater. 196 (2011) 1–15.
novel compound also exhibits a chondro-protective effect by pro-
moting cell proliferation and maintaining the phenotype of articu-
lar chondrocytes. In contrast, its source products of GA and ST-Na
have weak effects, not only in the inhibition of OA but also in the
bioactivity of chondrocytes, which indicated the significance of this
modification. SZNTC as a novel agent is promising in the treatment
of chondral and osteochondral lesions.
14] E. Nuti, S. Santamaria, F. Casalini, K. Yamamoto, L. Marinelli, V. La Pietra, E.
Novellino, E. Orlandini, S. Nencetti, A.M. Marini, S. Salerno, S. Taliani, F. Da
Settimo, H. Nagase, A. Rossello, A. Rossello, Arylsulfonamide inhibitors of
aggrecanases as potential therapeutic agents for osteoarthritis Synthesis and
biological evaluation, Eur. J. Med. Chem. 62 (2013) 379–394.
15] X. Lin, L. Zheng, Q. Liu, B. Liu, B. Jiang, X. Peng, C. Lin, In vitro effect of a
synthesized sulfonamido-based gallate on articular chondrocytemetabolism,
Bioorg. Med. Chem. Lett. 24 (2014) 2497–2503.
[
[
[
16] M. Lotz, Cytokines in cartilage injury and repair, Clin. Orthop. Relat. Res. (2001)
S108–S115.
17] F. Moldovan, J.P. Pelletier, F.C. Jolicoeur, J.M. Cloutier, J. Martel-Pelletier,
Diacerhein and rhein reduce the ICE-induced IL-1b and IL-18 activation in
human osteoarthritic cartilage, Osteoarthritis Cartilage 8 (2000) 186–196.
Conflict of Interest
The authors confirm that this article content has no conflicts of
interest.
[18] M.D. Buschmann, A.J. Grodzinsky,
associated electrostatic forces in cartilage mechanics, J. Biomech. Eng. 117
1995) 179–192.
[19] H. Ash, Z. Nevo, D. Aviezer, Characteristics of cartilage biopsies used for
autologous chondrocytes transplantation, Cell Transplant. 10 (2001) 203–208.
A molecular model of proteoglycan-
(
Transparency Document
[
[
[
20] H. Akiyama, Transcriptional regulation in chondrogenesis by Sox9, Clin.
Calcium 21 (2011) 845–851.
The Transparency document associated with this article can be
found in the online version.
21] S.R. Tew, P.D. Clegg, Analysis of post transcriptional regulation of SOX9 mRNA
during in vitro chondrogenesis, Tissue Eng. Part A 17 (2011) 1801–1807.
22] L.J. Ng, S. Wheatley, G.E. Muscat, J. Conway-Campbell, J. Bowles, E. Wright,
D.M. Bell, P.P. Tam, K.S. Cheah, P. Koopman, SOX9 binds DNA, activates
transcription, and coexpresses with type II collagen during chondrogenesis in
the mouse, Dev. Biol. 183 (1997) 108–121.
Acknowledgments
[23] O.J. Marshall, V.R. Harley, Molecular mechanisms of SOX9 action, Mol. Genet.
Metab. 71 (2000) 455–462.
[
24] S.R. Davies, L.W. Chang, D. Patra, X. Xing, K. Posey, J. Hecht, G.D. Stormo, L.J.
Sandell, Computational identification and functional validation of regulatory
motifs in cartilage-expressed genes, Genome Res. 17 (2007) 1438–1447.
25] S.R. Tew, Y. Li, P. Pothacharoen, L.M. Tweats, R.E. Hawkins, T.E. Hardingham,
Retroviral transduction with SOX9 enhances re-expression of the chondrocyte
phenotype in passaged osteoarthritic human articular chondrocytes,
Osteoarthritis Cartilage 13 (2005) 80–89.
This work has been financially supported by National Science &
Technology Pillar Program of China (Grant No. 2012BAI42G00),
Guangxi Scientific Research and Technological Development
Foundation (Grant No. Guikehe 14125008-2-14), Guangxi
Science Fund for Distinguished Young Scholars (Grant No.
[
2
014GXNSFGA118006).
[26] R. Paul, R.C. Haydon, H. Cheng, A. Ishikawa, N. Nenadovich, W. Jiang, L. Zhou, B.
Breyer, T. Feng, P. Gupta, T.C. He, F.M. Phillips, Potential use of Sox9 gene
therapy for intervertebral degenerative disc disease, Spine 28 (2003) 755–763.
[27] H. Tsuchiya, H. Kitoh, F. Sugiura, N. Ishiguro, Chondrogenesis enhanced by
overexpression of Sox9 gene in mouse bone marrow-derived mesenchymal
stem cells, Biochem. Biophys. Res. Commun. 301 (2003) 338–343.
[28] P.D. Benya, J.D. Shaffer, Dedifferentiated chondrocytes reexpress the
differentiated collagen phenotype when cultured in agarose gels, Cell 30
(1982) 215–224.
[29] M. Schnabel, S. Marlovits, G. Eckhoff, I. Fichtel, L. Gotzen, V. Vécsei, J. Schlegel,
Dedifferentiation-associated changes in morphology and gene expression in
primary human articular chondrocytes in cell culture, Osteoarthritis Cartilage
10 (2002) 62–70.
[30] T.A. Karlsen, A. Shahdadfar, J.E. Brinchmann, Human primary articular
chondrocytes, chondroblasts-like cells, and dedifferentiated chondrocytes:
differences in gene, microRNA, and protein expression and phenotype, Tissue
Eng. Part C Methods 17 (2010) 219–227.
[31] K.M. Kwan, M.K. Pang, S. Zhou, S.K. Cowan, R.Y. Kong, T. Pfordte, B.R. Olsen,
D.O. Sillence, P.P. Tam, K.S. Cheah, Abnormal compartmentalization of
cartilage matrix components in mice lacking collagen X: implications for
function, J. Cell Biol. 136 (1997) 459–471.
References
[
1] R. Singh, S. Ahmed, Epigallocatechin-3-gallate selectively inhibits interleukin-
beta-induced activation of mitogen activated protein kinase subgroup c-Jun
N-terminal kinase in human osteoarthritis chondrocytes, J. Orthop. Res. 21
2003) 102–109.
2] M.B. Goldring, Anticytokine therapy for osteoarthritis, Exp. Opin. Biol. Ther. 1
2001) 817–829.
1
(
[
[
(
3] J. Qin, Y.S. Liu, J. Liu, J. Li, Y. Tan, X.J. Li, J. Magdalou, Q.B. Mei, H. Wang, L.B.
Chen, Effect of Angelica sinensis polysaccharides on osteoarthritis in vivo and
in vitro: a possible mechanism to promote proteoglycans synthesis, Evid.
Based Complement Altern. Med. 2013 (2013). 794-761.
4] N. Ishiguro, T. Ito, H. Ito, H. Iwata, H. Jugessur, M. Ionescu, A.R. Poole,
Relationship of matrix metalloproteinases and their inhibitors to cartilage
proteoglycan and collagen turnover: analyses of synovial fluid from patients
with osteoarthritis, Arthritis Rheum. 42 (1999) 129–136.
5] J.A. Mengshol, M.P. Vincenti, C.I. Coon, A. Barchowsky, C.E. Brinckerhoff,
Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene
expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear
factor kappaB: differential regulation of collagenase 1 and collagenase 3,
Arthritis Rheum. 43 (2000) 801–811.
[
[
[32] C. Adcocks, P. Collin, D.J. Buttle, Catechins from green tea (Camellia sinensis)
inhibit bovine and human cartilage proteoglycan and type II collagen
degradation in vitro, J. Nutr. 132 (2002) 341–346.

1
38
Q. Liu et al. / Chemico-Biological Interactions 221 (2014) 127–138
[
[
33] M.N. Vankemmelbeke, G.C. Jones, C. Fowles, M.Z. Ilic, C.J. Handley, A.J. Day, C.G.
Knight, J.S. Mort, D.J. Buttle, Selective inhibition of ADAMTS-1, -4 and -5 by
catechin gallate esters, Eur. J. Biochem. 270 (2003) 2394–2403.
34] R. Andriamanalijaona, M. Kypriotou, C. Baugé, E. Renard, F. Legendre, M.
Raoudi, K. Boumediene, H. Gatto, P. Monginoux, J.P. Pujol, Comparative effects
of 2 antioxidants, selenomethionine and epigallocatechin-gallate, on catabolic
and anabolic gene expression of articular chondrocytes, J. Rheumatol. 32
[35] F. Daschner, Inhibition of cell wall synthesis by sulfonamides and
trimethoprim, Chemotherapy 22 (1976) 12–18.
[36] M. Patoux-Pibouin, B. Hirel, C. Chesne, E. Watier, J. Chevrant-Breton, A.
Guillouzo, Cytotoxicity testing of beta-lactam antibiotics, non-steroidal anti-
inflammatory drugs and sulfonamides in primary human keratinocyte
cultures, Toxicol. In Vitro 9 (1995) 493–497.
(
2005) 1958–1967.