A novel synthesized sulfonamido-based gallic acid - LDQN-C: Effects on chondrocytes growth and phenotype maintenance

Article abstract of DOI:10.1016/j.bioorg.2014.09.005

Chondrocyte based therapy is promising to treat symptomatic chondral and osteochondral lesions. Growth factors to accelerate the proliferation and retain the phenotype of chondrocytes in vitro are imperative. However, the high cost and rapid degradation o

Full text of DOI:10.1016/j.bioorg.2014.09.005

Bioorganic Chemistry 57 (2014) 99–107
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
A novel synthesized sulfonamido-based gallic acid – LDQN-C: Effects
on chondrocytes growth and phenotype maintenance
Zhenhui Lu a,e, Shixiu Wei a,1, Huayu Wu b,1, Xiao Lin c,d, Cuiwu Lin d, Buming Liu c, Li Zheng a,e,
,
⇑
Jinmin Zhao e
a The Medical and Scientific Research Center, Guangxi Medical University, Nanning 530021, China
b Department of Cell Biology & Genetics, School of Premedical Sciences, Guangxi Medical University, Nanning, Guangxi 530021, China
c Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Traditional Medical and Pharmaceutical Sciences, Nanning 530022, China
d School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China
e Research Center for Regenerative Medicine, Guangxi Medical University, Nanning 530021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2014
Available online 30 September 2014
Chondrocyte based therapy is promising to treat symptomatic chondral and osteochondral lesions.
Growth factors to accelerate the proliferation and retain the phenotype of chondrocytes in vitro are
imperative. However, the high cost and rapid degradation of growth factors limited their further applica-
tion. Therefore, it is significant to find substitutes that can preserve chondrocytes phenotype and ensure
sufficient cells for cytotherapy. Antioxidant and anti-inflammatory agents or their derivatives that have
effect on arthritis may be an alternative. In this study, we synthesized sulfonamido-based gallate – LDQN-
C and investigated its effect on rat articular chondrocytes through examination of the cell proliferation,
morphology, viability, glycosaminoglycans (GAGs) synthesis and cartilage specific gene expression.
Results showed that LDQN-C could enhance secretion and synthesis of cartilage extracellular matrix
(ECM) by up-regulating expression levels of aggrecan, collagen II and Sox9 genes compared to the GA
treated group and control group. Expression of collagen type II was effectively up-regulated while colla-
gen I was down-regulated, which demonstrated that the inhibition of chondrocytes dedifferentiation by
LDQN-C. Range of 1.36 ꢀ 10ꢁ9 M to 1.36 ꢀ 10ꢁ7 M is recommended dose of LDQN-C, among which the
Keywords:
Chondrocyte based therapy
Gallic acid
Sulfachloropyrazine sodium
Pro-chondrogenesis
Phenotype maintenance
most profound response was observed with 1.36 ꢀ 10ꢁ8 M. GA at concentration of 0.125
lg/mL was com-
pared. This study might provide a basis for the development of a novel agent for the treatment of articular
cartilage defect.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
chondrocytes during expansion in vitro [4,5]. To retain the pheno-
type and accelerate the proliferation of chondrocytes culture
Articular cartilage has regenerative capacity after injury, which
may lead to morbidity and functional impairment [1,2]. Traditional
cartilage repair including subchondral drilling, microfracture,
osteotomy and etc. were not satisfactory. Chondrocyte based
approaches like autologous chondrocyte implantation (ACI), allog-
enous chondrocyte implantation are useful to treat symptomatic
chondral and osteochondral lesions [3]. But several issues are
involved: (1) the number of chondrocytes is limited; (2) chondro-
cytes are subject to age-related changes; (3) loss of phenotype
which is called dedifferentiation, is the main problem confronted
in vitro, lots of growth factors like transforming growth factor
(TGF-b1) and insulin-like growth factor (IGF) are employed [6].
However, the high cost, lose of activity and rapid degradation of
growth factors limited their application in clinic. Therefore, to
exploit low-cost and stable agents that will substitute for growth
factors in the maintenance of phenotype of chondrocytes is of
significance.
Gallic acid (GA) and its derivatives are a group of polyphenol
compounds which have strong anti-oxidant effect, and have been
known to impact several pharmacological and biochemical path-
ways [7]. It was reported that a lyophilized extract of wine which
contains large amounts of phenolic components have the protec-
tive effects in cartilage alteration [8]. However, bioactivity of GA
was compromised because it is much more hydrophilic than its
esters, resulting in much weaker antioxidant effects than its esters
⇑
Corresponding author. Address: The Medical and Scientific Research Center,
Research Center for Regenerative Medicine, Guangxi Medical University, Nanning
530021, China. Fax: +86 07715350975.
E-mail address: zhengli224@163.com (L. Zheng).
Shixiu Wei and Huayu Wu contributed equally to this work.
1
http://dx.doi.org/10.1016/j.bioorg.2014.09.005
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

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Z. Lu et al. / Bioorganic Chemistry 57 (2014) 99–107
in cell systems [7]. It was reported that GA suppressed cell prolif-
eration [9]. Therefore, to introduce some lipophilic compounds
may improve the bioactivity of GA and broaden its application.
Several derivatives of GA showed excellent biological proper-
ties. A sulfonamide-based GA derivatives can promote articular
chondrocytes metabolism [10]. Epigallocatechin-3-gallate (EGCG),
a derivative of GA, enhances aggrecan and type II collagen synthe-
sis and ameliorates IL-1b-mediated suppression of TGF-b synthesis
in human chondrocytes [11]. Recently, a series of new N-isopro-
poxy-arylsulfonamide hydroxamate inhibitors which contain sev-
eral phenyl group and sulfonamide group were proved to be
effective in inhibiting ex vivo cartilage degradation [12]. This
implied that incorporating sulfonamides to GA may enhance the
bioactivity and therefore support the growth of chondrocytes.
In this study, we synthesized sulfonamido-based gallate – N-[4-
(6-Chloro-pyridazin-3-ylsulfamoyl)-phenyl]-3,4,5-trihydroxy-benz-
mide (LDQN-C) and investigated its chondroprotective effect
through examination of the cell proliferation and expression of
cartilage specific genes and proteins of chondrocytes compared
2.3. LDQN-C treatment
LDQN-C was dissolved in sodium hydroxide solution (NaOH,
Sigma, USA) of 0.1 mg/mL as stock solution and stored at 4 °C.
The stock solution of LDQN-C was then added to the cell cultures
to provide various concentrations. Culture medium containing
various concentrations of LDQN-C was replaced every 3 days. As
control, stock solution of GA with the concentration of 0.125
mL was prepared.
lg/
2.4. Cytotoxicity test
The cytotoxicity of LDQN-C on chondrocytes was performed by
the 3-(4, 5)-dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazolium-
romide (MTT, Gibco, USA) method. Chondrocytes were seeded on
96-plates. The final cell number in each well was 5 ꢀ 103. Concen-
trations of LDQN-C ranged from 1.36 ꢀ 10ꢁ10 M to 54.5 ꢀ 10ꢁ3
M
and GA ranged from 6.25 ꢀ 10ꢁ8 ꢂ25
lg/mL were added to cell
cultures.
with GA at concentration of 0.125 lg/mL.
After 3 days of culture, cytotoxicity test was carried out by MTT
analysis. Briefly, a solution of MTT in PBS was added into each well
with the final concentration of 5 mg/mL and incubated at 37 °C for
2. Materials and methods
4 h. After the media was removed, 200 lL dimethylsulfoxide
(DMSO; Sigma) was added, which was used to dissolve MTT forma-
zan formed by metabolically viable cells. The absorbance was
detected by an enzyme-labeled meter (Thermo Fisher Scientific,
UK) at 570 nm. All experiments were performed in triplicate.
As determined by MTT analysis, the concentrations of LDQN-C
at 1.36 ꢀ 10ꢁ9, 1.36 ꢀ 10ꢁ8 and 1.36 ꢀ 10ꢁ7 M and of GA at
2.1. Synthesis of LDQN-C
LDQN-C was prepared from GA and Sulfachloropyrazine
sodium. The synthetic procedure is presented in Fig. 1.
Electrospray ionization mass spectrum (ESI-MS) was recorded
on a Shimadzu LC-MS 2010A. 1H and 13C NMR spectra were
obtained from a Bruker Advance III 300 at 400 and 125 MHz,
respectively.
0.125 lg/mL, among which a peak was presented, were chosen
for further investigation. Three groups were divided: (1) control
group: chondrocytes without any treatment; (2) LDQN-C treat-
ment groups: chondrocytes treated with various concentrations
of LDQN-C (1.36 ꢀ 10ꢁ9, 1.36 ꢀ 10ꢁ8 and 1.36 ꢀ 10ꢁ7 M); (3) GA
treatment groups: chondrocytes treated with GA at concentration
2.2. Articular cartilage cells culturec
Joint cartilage were dissected from new-born SD rats followed
by enzymatic digestion with 0.25% trypsin (Solarbio, China) for
30 min and then with 2 mg/mL collagenase type II (Gibco, USA)
of 0.125 lg/mL.
in Minimum Essential Medium Alpha Medium (
USA) for 3 h. After centrifuge (1000r, 5 min), the cells were resus-
pended in a basal culture medium of -MEM containing 20% Fetal
Bovine Serum (FBS, Gibco, USA) and 1% antibiotics (penicillin
100U/mL, streptomycin 100 U/mL). Culture conditions in a incuba-
tor (Thermo Fisher Scientific, UK) at 5% CO2 37 °C humidified
atmosphere.
a
-MEM, Gibco,
2.5. Cell viability assay
a
The cell viability of chondrocytes was determined by
Fluorescein diacetate (FDA, Genway Biotech, Inc, USA)/propidium
iodide (PI, Sigma, USA) staining at day 2, 4 and 6 respectively.
Briefly, FDA and PI stock solutions were added to the cells at a final
concentration of 2 lmol/L and 2 lg/L respectively and incubated in
Fig. 1. Synthesis route of LDQN-C. Reagents and conditions: (a) acetyl oxide, oil bath, 120 °C (b) SOCl2, oil bath, 80 °C (c) Sulfachloropyrazine sodium, THF, Pyridine, ice bath.

Z. Lu et al. / Bioorganic Chemistry 57 (2014) 99–107
101
the dark for 5 min at 37 °C. Images were captured via a laser
2.9. Safranin O staining
scanning confocal microscope (Nikon, Japan).
Safranin O-stained cells were scored for glycosaminoglycans
(GAGs) and Rhodamine phalloidin (Cytoskeleton, USA)/Hoechst
33258. The cells were fixed with 95% alcohol for 30 min and then
stained with 0.1% Safranin O (Sigma, USA) for 10 min. Subse-
quently, cells were rinsed with water and scaled with neutral
gum. Finally, the cells were observed and photographed under an
inverted phase contrast microscope equipped with a computer
(Zeiss Corporation, German).
2.6. Cell morphological analysis
After being culture with different concentrations of LDQN-C for
2, 4 and 6 days, the cells were washed by PBS for three times and
fixed with 95% alcohol for 30 min. Then the cells were washed by
PBS again and stained with hematoxylin and eosin kit (HE,
JianCheng Biotech, China) to observe the cell morphology. Images
were photographed by an inverted phase contrast microscope
(Zeiss Corporation, German).
2.10. Immunohistochemical examination
Cells were stained for 30 min at room temperature with rhoda-
mine phalloidin (Invitrogen, USA), followed by Hoechst 33258
(Beyotime, China) for 5 min to visualize nuclei. Imaging was
performed using scanning confocal microscope (Nikon, Japan).
For immunohistochemical examination, monoclonal antibody
to type II collagen (Boster, China) and type I collagen (Boster,
China) were used according to the instructions. Cells were incu-
bated with a primary antibody at a dilution of 1:200 for 2 h.
Whereafter, second antibody and biotin labeled horseradish perox-
idase were added. Subsequently, 30-diaminobenzidine tetrahydro-
chloride (DAB) kit (Boster, China) was used according to the
instructions with cells counterstained with haematoxylin. Finally,
cells were gradually dehydrated and sealed with neutral gum. An
inverted phase contrast microscope (Zeiss Corporation, German)
was used to evaluate and photograph the cells.
2.7. RNA extraction and qRT-PCR analysis
The real-time quantitative polymerase chain reaction (qRT-PCR)
was to analysis the gene expression of aggrecan, collagen II, Sox9,
collagen I and collagenX. The primer sequences and genbank acces-
sion numbers used for qRT-PCR are summarized in Table 1. Chon-
drocytes were seeded in 6-well plates and cultured alone or with
LDQN-C (concentrations of 1.36 ꢀ 10ꢁ9
,
1.36 ꢀ 10ꢁ8 and
2.11. Statistical Analysis
1.36 ꢀ 10ꢁ7 M) and GA at concentration of 0.125
lg/mL. After 2,
4 and 6 days, total RNA was successively extracted with a Total iso-
lation RNA kit (Invitrogen, USA) according to the instructions of
manufactures. Reverse transcription of RNA was carried out at
25 °C for 5 min, 42 °C 60 min and then 72 °C for 5 min using a
reverse transcription kit (Fermentas Company, USA). The qRT-
PCR reactions were performed using a Quantitative PCR Detection
System (realplex 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 °C and 1 min at
60 °C. The dissociation curve of each primer pair was analyzed to
Results were demonstrated as means SD. Statistical signifi-
cance was 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.
3. Results
3.1. Preparation of LDQN-C
The procedure of synthesis of GA and Sulfachloropyrazine
sodium was shown in Fig. 1 LDQN-C has the following properties:
light yellow powder, yield 55%, MS-ESI: m/z: 457.1[M-H]ꢁ, 1H NMR
confirm the primer specificity. Marker gene expression of
DDCT
chondrocytes was analyzed by the 2ꢁ
method using b-actin.
Each sample was repeated three times for each gene.
(400 MHz, DMSO)
d 10.27 (s, 1H, –CO–NH), 7.94–7.85 (dd,
J = 8.9 Hz, 4H, Ar–H), 7.77 (d, J = 9.5 Hz, 1H, Py–H), 7.55 (d,
J = 9.5 Hz, 1H, Py–H), 6.94 (s, 2H, Ar-H).13C NMR (125 MHz, DMSO)
d 166.06, 154.33, 145.58, 143.83, 137.34, 127.96, 124.35, 119.66,
107.45.
2.8. Cell proliferation analysis and biochemical assay
After cultured for 2, 4 and 6 days respectively, chondrocytes
were washed by PBS, and then digested with 0.25% trypsin/ETDA.
Cells were centrifuged and then responded in PBS containing
3.2. Cell cytotoxicity
60 lg/mL proteinase K (Sigma, USA) for 16 h at 60 °C. The DNA
content was determined by spectrofluorometer using Hoechst
33258 dye at 460 nm with calf thymus DNA as standard and the
absorption of Hoechst 33258 dye as the baseline (13). The total
secretion of glycosaminoglycan (GAG) was measured using 1,
9-Dimethylmethylene Blue (DMMB, SIGMA-ALORICH, USA) with
Chondroitin Sulfate as standard. The GAG content was qualified
on standard curve and accordingly normalized to the total DNA
content. All the experiments were carried out in triplicate.
The cytotoxicity of different concentrations of LDQN-C on chon-
drocytes was detected by MTT assay. Articular cartilage cells of SD
rats were treated with LDQN-C of various concentrations
(1.36 ꢀ 10ꢁ10–54.5 ꢀ 10ꢁ3 M) and GA (6.25 ꢀ 10ꢁ8 ꢂ25
lg/mL).
As shown in Fig. 2A, LDQN-C which ranged from 1.36 ꢀ 10ꢁ10 to
1.36 ꢀ 10ꢁ5 M exhibited nearly no cytotoxicity to chondrocytes.
Especially, LDQN-C from 1.36 ꢀ 10ꢁ9 to 1.36 ꢀ 10ꢁ7 M promoted
cell growth evidently (p < 0.05). In contrast, LDQN-C at the
Table 1
Primers for RT-PCR performance.
Gene name
Forward primer
Reverse primer
B-actin
50-CCCATCTATGAGGGTTACGC-30
50- CCGCTGGTCTGATGGACACT-30
50- CTGGTCCTTCCGGCCCTAGA-30
50-TCCAGCAAGAACAAGCCACA-30
50- CATGAGCCGAAGCTAACCC-30
50- TCTGCTGCTAGTGTCCTTGACG-30
50-TTTAATGTCACGCACGATTTC-30
50- AGGTGTTGGGGTCTGTGCAA-30
50- GGATCGGGGCCCTTCTCTCT-30
50- CGAAGGGTCTCTTCTCGCTC-30
50- CTCCTATGACTTCTGCGTCTGG-30
50- GGAATGCCTTGTTCTCCTCTTACT-30
Aggrecan
Collagen II
Sox9
Collagen I
Collagen X

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Z. Lu et al. / Bioorganic Chemistry 57 (2014) 99–107
Fig. 2. Cytotoxicity analysis of chondrocytes treated by different concentrations of LDQN-C (A) and GA (B) after 3 days (mean SD, n = 20).
concentrations of 1.36 ꢀ 10ꢁ4–54.5 ꢀ 10ꢁ3 M showed an inhibitive
effect on chondrocytes. Therefore, the concentrations of LDQN-C
ranged from 1.36 ꢀ 10ꢁ9 M to 1.36 ꢀ 10ꢁ7 M were chosen for fur-
ther investigation. The cytotoxicity of GA was showed in Fig. 2B.
Among the chosen concentrations, 0.125 lg/mL of GA promoted
chondrocytes growth the most significantly.
3.4. Cell morphology
Fig. 4 showed the morphology of articular chondrocytes cul-
tured for 2, 4 and 6 days. The chondrocytes treated by LDQN-C
grew better in the same period compared with the control and
GA groups. More round cells that represent typical morphology
of chondrocytes were found in LDQN-C groups. Especially at the
concentration of 1.36 ꢀ 10ꢁ8 M, LDQN-C facilitated cell prolifera-
tion more than others. Fig. 5 showed the actin filaments of chon-
drocytes by the rhodamine phalloidin/Hoechst 33258 staining,
which was in agreement with the HE analysis. It was shown that
cells in LDQN-C treated groups grew in clumps with densely dis-
tributed ECM. While in control and GA groups, less cells and ECM
were present.
3.3. Cell viability assay
Cell viability was determined by FDA/PI staining (Fig. 3). The
results demonstrated that LDQN-C exerted potent effect on chon-
drocytes survival compared to control and GA groups. The FDA/PI
staining images indicated that live cells in LDQN-C groups were
more than the ones in the control and GA groups, which was con-
sistent with the result of cell proliferation. The results implied that
LDQN-C has a positive effect on cell growth. Among the experi-
mental groups, concentration of 1.36 ꢀ 10ꢁ8 M was superior to
others.
3.5. Cartilage specific gene expression
The effect of LDQN-C on chondrocytes relative gene expression
of collagen I, collagen II, collagen X, Sox9, aggrecan (a proteoglycan
Fig. 3. Confocal laser scanning microscopy images showing the viability of chondrocytes cultured in vitro alone (control) or with GA (GA = 0.125
l
g/mL) and LDQN-C
(C1 = 1.36 ꢀ 10ꢁ9 M, C2 = 1.36 ꢀ 10ꢁ8 M, C3 = 1.36 ꢀ 10ꢁ7 M) for 2, 4 and 6 days. Cell seeding density: 2 ꢀ 104/mL (original magnification 100ꢀ, scale bar is 100
lm).

Z. Lu et al. / Bioorganic Chemistry 57 (2014) 99–107
103
Fig. 4. Hematoxylin-eosin staining images showing the morphology of chondrocytes cultured in vitro alone (control) or with GA (GA = 0.125
l
g/mL) and LDQN-C
(C1 = 1.36 ꢀ 10ꢁ9 M, C2 = 1.36 ꢀ 10ꢁ8 M, C3 = 1.36 ꢀ 10ꢁ7 M) for 2, 4 and 6 days. Cell seeding density: 2 ꢀ 104/mL (original magnification 100ꢀ, scale bar is 100
lm).
composed of GAGs) after 2, 4 and 6 days culture was detected by
Real time-PCR. As shown in Fig. 6, aggrecan, collagen II and
Sox9 were notably promoted by LDQN-C compared to the control
and GA groups. Particularly, LDQN-C at concentration of
1.36 ꢀ 10ꢁ8 M showed the highest collagen II, aggrecan and Sox9
expression. The results indicated that LDQN-C could upregulate
collagen II, aggrecan and Sox9 expressions relative to control and
GA groups. In contrast, collagen type I was down-regulated by
LDQN-C but not in GA and control groups. The results suggested
that LDQN-C may either delay or prevent the chondrocytes from
dedifferentiation. In addition, collagen X expression was scarcely
detectable in all groups, which suggested that cell hypertrophy
could not be detected. Among all the groups, concentration of
1.36 ꢀ 10ꢁ8 M showed the best performance, as demonstrated by
the highest expression of aggrecan and collagen II expression.
3.6. Cell proliferation
In this study, the cell proliferation in LDQN-C- treaded groups,
GA group and control group were analyzed by DNA content mea-
surements in 2, 4 and 6 days respectively. As shown in Fig. 7A,
the chondrocytes cultured with LDQN-C grew faster than control
Fig. 5. Phalloidin/Hoechst 33258 staining images showing the morphology of chondrocytes and actin filaments after treated alone (control) or with GA (GA = 0.125 lg/mL)
and LDQN-C (C1 = 1.36 ꢀ 10ꢁ9 M, C2 = 1.36 ꢀ 10ꢁ8 M, C3 = 1.36 ꢀ 10ꢁ7 M) for 2, 4 and 6 days. Cell seeding density: 2 ꢀ 104/mL (original magnification 100ꢀ, scale bar is
100 lm).

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Z. Lu et al. / Bioorganic Chemistry 57 (2014) 99–107
3.7. GAGs synthesis
Biochemical assays were used for the quantitative investigation
of the production of GAG after 2, 4 and 6 days of culture. The his-
togram showed the GAG production given as a ratio of GAG to DNA
in Fig. 7B. Quantitatively, GAG production in LDQN-C-treated
groups was significantly enhanced than that in control and GA
groups at the same time point. The effect of LDQN-C is dose-
dependent.
3.8. ECM secession
Qualitative assessment by using Safranin-O staining also
showed a deeper staining in LDQN-C groups compared with con-
trol and GA groups (Fig. 8). At concentration of 1.36 ꢀ 10ꢁ8 M,
LDQN-C exhibited the most GAG synthesis among the three
concentrations.
3.9. The production of collagen type I and type II
Expression of type I and type II collagen by immunohistochem-
ical staining with untreated, GA and LDQN-C-treated culture media
was shown in Fig. 9. Large areas of positive staining for cartilage-
specific type II collagen was shown in Fig. 9B, while only very sparse
and light staining for type I collagen was observed in Fig. 9A in
LDQN-C-treated groups. It is converse in control and GA groups,
especially in GA group. The results confirmed the maintenance of
chondrocytic phenotype after treated by LDQN-C (especially at
1.36 ꢀ 10ꢁ8 M), which indicated that LDQN-C may effectively inhi-
bit de-differentiation of chondrocytes cultured in vitro.
4. Discussion
It has been reported that GA derivatives appeared to play an
important role in protection of chondrocytes. In this study,
LDQN-C was synthesized by coupling sulfonamide groups with
GA and its effects on the chondrocytes growth and phenotype
maintenance were investigated, Comparison with GA was also con-
ducted. The results indicated that LDQN-C could enhance chondro-
cytes growth, as evidenced by more rapid cell proliferation than
control and GA groups (Fig. 7A). LDQN-C could obviously promote
GAGs deposition in cultured chondrocytes which was shown by
biochemical assay, but not in control and GA groups (Fig. 7B). As
the main component of cartilage matrix, GAG is crucial in main-
taining cartilage load-bearing capacity [13]. Consistent with the
increase of GAG production, LDQN-C could upregulate the expres-
sion of aggrecan, collagen II and Sox9 (Fig. 6). Sox9 was considered
as the primary chondrogenic marker that enhanced the production
of collagen and aggrecan [14] and acted a key role in chondrogen-
esis [15]. The results suggested that LDQN-C could facilitate chon-
drocytes growth and stimulate exuberant cartilage matrix
secretion through regulating the key activator of the chondro-
cyte-specific enhancer.
As is known to all that dedifferentiation occurred while the dif-
ferentiated phenotype of chondrocytes consists primarily of type II
collagen and cartilage-specific proteoglycan is lost and replaced by
a complex collagen phenotype consisting predominately of type I
collagen and a low level of proteoglycan synthesis [16,17]. Results
of PCR, biochemical and immunohistochemical assay, collagen
type I expression all showed downregulation of collagen type I,
which suggested that LDQN-C could effectively delay or prevent
dedifferentiation of chondrocytes. On the other hand, collagen type
X that is specifically associated with hypertrophic chondrocytes
and precedes the onset of endochondral ossification could not be
detected in all the LDQN-C-treated groups. This implied that
Fig. 6. Quantitative compare of ECM-related gene expression by qRT-PCR. The
chondrocytes were cultured alone (control) or with GA (GA = 0.125 lg/mL) and
LDQN-C (C1 = 1.36 ꢀ 10ꢁ9 M, C2 = 1.36 ꢀ 10ꢁ8 M, C3 = 1.36 ꢀ 10ꢁ7 M) for 2 days
(A), 4 days (B) and 6 days (C) (n = 3 for each experiment). The gene expression levels
in LDQN-C media relative to the control group were analyzed by the 2ꢁ CT method
DD
using GAPDH as the internal control. The data represent the mean SD of three
independent culture experiments.
and GA groups, which was evidenced by the significantly higher
DNA contents (P < 0.05) in the same culture period. The results also
indicated that LDQN-C at the concentration of 1.36 ꢀ 10ꢁ8 M pro-
moted cell growth the most in all the LDQN-C groups.

Z. Lu et al. / Bioorganic Chemistry 57 (2014) 99–107
105
Fig. 7. Quantification of cell proliferation by detected of DNA content (A) and matrix production by glycosaminoglycan (GAG) analysis (B). A. the proliferation of chondrocytes
cultured in vitro alone (control) or with GA (GA = 0.125
(mg). Data from four independent experiments were evaluated, and the mean SD is shown.
l
g/mL) and LDQN-C (C1 = 1.36 ꢀ 10ꢁ9 M, C2 = 1.36 ꢀ 10ꢁ8 M, C3 = 1.36 ꢀ 10ꢁ7 M) B. GAG (mg) normalized to DNA
Fig. 8. Safranin O staining showing the ECM synthesis of chondrocytes culture in vitro alone (control) or with GA (GA = 0.125
l
g/mL) and LDQN-C (C1 = 1.36 ꢀ 10ꢁ9 M,
C2 = 1.36 ꢀ 10ꢁ8 M, C3 = 1.36 ꢀ 10ꢁ7 M) for 2, 4 and 6 days. Cell seeding density: 2 ꢀ 104/mL (original magnification 100ꢀ, scale bar is 100
lm).
hypertrophy of chondrocytes would not be induced by LDQN-C.
Therefore, LDQN-C has the ability to maintain the phenotype of
chondrocytes, as proved by the reduced collagen I messages and
the barely evident messages of collagen X indicating the inhibition
of the dedifferentiation and hypertrophy.
derivatives of GA, Epigallocatechin-3-gallate (EGCG) was found to
inhibit the degradation of human cartilage proteoglycan and type
II collagen, and selectively inhibit ADAMTS-1, ADAMTS-4, and
ADAMTS-5 [20,21]. It was also found 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 [11]. Furthermore, recent studies indicated that sul-
fonamide-based gallates effectively inhibited IL-1b induced osteo-
arthritis [22] and exerted effects on cartilage growth [23–25]. In
agreement with these studies, LDQN-C which is a novel derivative
of GA, can also support the chondrocytes growth and maintain the
phenotype. As sulfonamide-based gallate, LDQN-C may be poten-
tial candidate for treatment of cartilage degenerative diseases. This
implied that suitable modification of GA may lead to the improve-
ment of the pharmacological effects in cartilage defect and may be
osteoarthritis.
As for the recommended dose of LDQN-C, our results showed that
the concentration of LDQN-C concerning enhancing chondrocytes
proliferation ranged from 1.36 ꢀ 10ꢁ10
M
to 1.36 ꢀ 10ꢁ5
M
(Fig. 2A). DNA synthesis of rat chondrocytes was increased in a
dose-dependent manner when chondrocytes were cultured in the
medium containing LDQN-C at the concentration of
1.36 ꢀ 10ꢁ9 M, 1.36 ꢀ 10ꢁ8 M and 1.36 ꢀ 10ꢁ7 M groups could sup-
port the greatest cell proliferation and stimulate the most matrix
secretion.
Due to inferior pharmacological effects and biological property
of GA [18,19], the modification is of significance. As one of the

106
Z. Lu et al. / Bioorganic Chemistry 57 (2014) 99–107
Fig. 9. Immunohistochemical staining images revealed the presence of type I (A) and type II (B) collagen. Chondrocytes cultured in vitro alone (control) or with GA
(GA = 0.125
100ꢀ, scale bar is 100
l
g/mL) and LDQN-C (C1 = 1.36 ꢀ 10ꢁ9 M, C2 = 1.36 ꢀ 10ꢁ8 M, C3 = 1.36 ꢀ 10ꢁ7 M) for 2, 4 and 6 days. Cell seeding density: 2 ꢀ 104/mL (original magnification
lm).
[7] Z. Lu, G. Nie, P.S. Belton, H. Tang, B. Zhao, Neurochem. Int. 48 (4) (2006) 263–
274.
Acknowledgments
[8] A.M. Panico, V. Cardile, S. Avondo, F. Garufi, B. Gentile, C. Puglia, F. Bonina, N.A.
Santagati, G. Ronsisvalle, Phytomedicine 13 (7) (2006) 522–526.
[9] T.T. Ou, M.C. Lin, C.H. Wu, W.L. Lin, C.J. Wang, Curr. Med. Chem. 20 (31) (2013)
3944–3953.
[10] X. Lin, L. Zheng, Q. Liu, B. Liu, B. Jiang, X. Peng, C. Lin, Bioorg. Med. Chem. Lett.
24 (11) (2014) 2497–2503.
[11] Rina Andriamanalijaona, Madeleine Kypriotou, Catherine Baugé, Emmanuelle
Renard, Florence Legendre, Maha Raoudi, Karim Boumediene, Hugues Gatto,
Patricia Monginoux, Jean-Pierre Pujol, J. Rheumatol. 32 (10) (2005) 1958–
1967.
This work has been financially supported by National Science &
Technology Pillar Program of China (Grant No. 2012BAI42G00),
National Natural Science Foundation of China (Grant No.
81260277), Guangxi Scientific Research and Technological Devel-
opment Foundation (Grant No. Guikehe 14125008-2-14), Guangxi
Science Fund for Distinguished Young Scholars (Grant No.
2014GXNSFGA118006).
[12] 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, Eur. J. Med. Chem. 62 (2013) 379–394.
[13] D. Robinson, H. Ash, A. Yayon, Z. Nevo, D. Aviezer, Cell Transplant. 10 (2)
(2001) 203–208.
[14] S.R. Tew, Y. Li, P. Pothacharoen, L.M. Tweats, R.E. Hawkins, T.E. Hardingham,
Osteoarthr. Cartilage 13 (1) (2005) 80–89.
[15] Shyni Varghese, Nathaniel S. Hwang, Adam C. Canver, Parnduangji
Theprungsirikul, Debora W. Lin, Jennifer Elisseeff, Matrix Biol. 27 (1) (2008)
12–21.
References
[1] S.W. O’Driscoll, F.W. Keeley, R.B. Salter, J. Bone Joint. Surg. Am. 70 (4) (1988)
595–606.
[2] J.A. Buckwalter, H.J. Mankin, Instr. Course Lect. 47 (1997) 477–486.
[3] C. Chung, J.A. Burdick, Adv. Drug Delivery Rev. 60 (2) (2008) 243–262.
[4] A.L. Aulthouse, M. Beck, E. Griffey, J. Sanford, K. Arden, M.A. Machado, W.A.
Horton, In vitro Cell. Dev. Biol. 25 (7) (1989) 659–668.
[5] A. Määttä, V. Glumoff, P. Paakkonen, D. Liska, R.P. Penttinen, K. Elima, Biochem.
J. 294 (1993) 365–371.
[6] L. Uebersax, H.P. Merkle, L. Meinel, J. Controlled Release 127 (1) (2008) 12–21.
[16] P.D. Benya, J.D. Shaffer, Cell 30 (1) (1982) 215–224.
[17] T.A. Karlsen, A. Shahdadfar, J.E. Brinchmann, Tissue Eng. Part C: Methods 17 (2)
(2010) 219–227.

Z. Lu et al. / Bioorganic Chemistry 57 (2014) 99–107
107
[18] M.S. Kang, H.S. Jang, J.S. Oh, K.H. Yang, N.K. Choi, H.S. Lim, S.M. Kim, J.
Microbiol. 47 (6) (2009) 760–767.
[22] Qin Liu, Mu-Yan Li, Xiao Lin, Cui-Wu Lin, Bu-Ming Liu, Li Zheng, Jin-Min Zhao,
Chem. Biol. Interact. (2014). 08.002.
[19] G. Kuppan, J. Balasubramanyam, F. Monickaraj, G. Srinivasan, V. Mohan, M.
Balasubramanyam, Cytokine 49 (2) (2010) 229–234.
[20] Clair Adcocks, Peter Collin, David J. Buttle, J. Nutr. 132 (3) (2002) 341–346.
[21] 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, Eur. J. Biochem. 270 (11) (2003) 2394–2403.
[23] L. Liu, Q. Liu, X. Lin, Q.J. Wei, L. Zheng, In Vitro Cell Dev. Biol. Anim. (2014). Agu 15.
[24] G.J. Xu, Zh. Lu, X. Lin, C.W. Lin, L. Zheng, J.M. Zhao, Braz. J. Med. Biol. Res. 47 (8)
(2014) 637–645.
[25] X. Lin, L. Zheng, Q. Liu, B. Liu, X. Peng, C. Lin, Bioorg. Med. Chem. Lett. 24 (11)
(2014) 2497–2503.