Protective effect of sinomenine on cartilage degradation and chondrocytes apoptosis.

Abstract

Sinomenine (SIN), an alkaloid extracted from the stem of the Chinese medicinal plant sinomenium acutum, has been used for treating rheumatoid arthritis. But little is known whether SIN has a protective effect on osteoarthritis (OA). In this study, we investigated the protective effect of SIN on IL-1beta-induced proteoglycan degradation and apoptosis in rabbit articular cartilage and chondrocytes. Treatment with 10 ng/ml IL-1beta increased the level of glycosaminoglycan (GAG) released into the culture media, and up-regulated the activity and mRNA expression of matrix metalloproteinase 13 (MMP-13) and down-regulated the activity and mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in cartilage explants, as confirmed by the methods of GAG quantitation, MMP-13/TIMP-1 enzyme-linked immunosorbent assay (ELISA) and real-time quantitative RT-PCR. Treatment with 10 ng/ml IL-1beta resulted in marked apoptosis in chondrocytes, as demonstrated by decreased cell viability, occurrence of DNA laddering and increased caspase-3 activity and annexin V binding of phosphatidylserine. However, simultaneous treatment with SIN (10, 50 or 250 microM) inhibited the GAG release and the activity and mRNA expression of MMP-13, and enhanced the activity and mRNA expression of TIMP-1 in a dose-dependent manner in cartilage explants. Furthermore, DNA fragment, caspase-3 activity and apoptosis rate were down-regulated, and cell viability was up-regulated dose-dependently in chondrocytes. Thus, SIN has the protective capacity to antagonize cartilage degradation and chondrocyte apoptosis, which suggest that SIN may act as an agent for pharmacological intervention in the progress of OA.

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10531053
 e-mail: dengmin1706＠yahoo.com.cn
YAKUGAKU ZASSHI 130(8) 1053―1060 (2010)  2010 The Pharmaceutical Society of Japan
―Regular Article―
Protective EŠect of Sinomenine on Cartilage Degradation and Chondrocytes Apoptosis
Xiao-dong JU,aMin DENG,
Jian-quan WANG,aJia-kuo YU,aGuo-qing CUI,aand Yue-lin HUa
,bYing-fang AO,aChang-long YU,a
aInstitute of Sports Medicine, andbDepartment of Neurology, Peking University Third Hospital,
49 North Garden Road, Beijing 100191, China
(Received March 25, 2010; Accepted May 6, 2010)
Sinomenine (SIN), an alkaloid extracted from the stem of the Chinese medicinal plant sinomenium acutum, has
been used for treating rheumatoid arthritis. But little is known whether SIN has a protective eŠect on osteoarthritis
(OA). In this study, we investigated the protective eŠect of SIN on IL-1b-induced proteoglycan degradation and apop-
tosis in rabbit articular cartilage and chondrocytes. Treatment with 10 ng/ml IL-1b increased the level of glycosamino-
glycan (GAG) released into the culture media, and up-regulated the activity and mRNA expression of matrix metallo-
proteinase 13 (MMP-13) and down-regulated the activity and mRNA expression of tissue inhibitor of metalloproteinase
1 (TIMP-1) in cartilage explants, as conˆrmed by the methods of GAG quantitation, MMP-13/TIMP-1 enzyme-linked
immunosorbent assay (ELISA) and real-time quantitative RT-PCR. Treatment with 10 ng/ml IL-1b resulted in marked
apoptosis in chondrocytes, as demonstrated by decreased cell viability, occurrence of DNA laddering and increased
caspase-3 activity and annexin V binding of phosphatidylserine. However, simultaneous treatment with SIN (10, 50 or
250 mM) inhibited the GAG release and the activity and mRNA expression of MMP-13, and enhanced the activity and
mRNA expression of TIMP-1 in a dose-dependent manner in cartilage explants. Furthermore, DNA fragment, caspase-
3 activity and apoptosis rate were down-regulated, and cell viability was up-regulated dose-dependently in chondrocytes.
Thus, SIN has the protective capacity to antagonize cartilage degradation and chondrocyte apoptosis, which suggest that
SIN may act as an agent for pharmacological intervention in the progress of OA.
Key words―sinomenine; cartilage; chondrocyte; apoptosis; matrix metalloproteinase-13; tissue inhibitor of metal-
loproteinase-1
INTRODUCTION
Osteoarthritis (OA) is one of the most common
chronic diseases and a major source of functional dis-
ability in elderly persons. With worldwide population
ageing, the incidence of OA is rapidly increasing, and
it is anticipated that OA will become the fourth lead-
ing cause of disability in the coming decades.1)OA is
characterized by progressive degradation of articular
cartilage and chondrocytes apoptosis and death.2)
Chondrocytes apoptosis has been identiˆed as a vital
reason for cell loss and is now considered as an im-
portant factor contributing to degradation of ex-
tracellular matrix (ECM) in OA cartilage.3)Many
researches have also demonstrated that enzymatic
cleavage by matrix metalloproteinases (MMPs)
together with cytokines plays critical roles in the initi-
ation and progression of cartilage destruction.4,5)An
imbalance between MMPs and tissue inhibitors of
metalloproteinases (TIMPs) is an determinant of the
extent of ECM turnover.6)Hence, a therapeutic ap-
proach regulating the MMPs/TIMPs balance and
apoptosis may be eŠective in the treatment of OA.
Currently, most treatments for OA are directly to
targeted symptoms of disease rather than the under-
lying causes. As therapeutic agents, analgesics and
non-steroidal anti-in‰ammatory drugs (NSAIDs)
represent the mainstay.7)Limitations of these drugs
due to great side eŠects, and lack of speciˆcity and
beneˆt to the underlying cartilage changes suggest a
need to develop therapeutic strategies focusing on
natural agents or supplements that could modify or
reverse the progression of cartilage degradation in the
aŠected joints. Sinomenine (7,8-didehydro-4-hydrox-
y-3,7-dimethoxy-17-methylmorphinan-6-one) (SIN)
is an alkaloid extracted from the stem of the Chinese
medicinal plant sinomenium acutum.8)Previous stu-
dies have demonstrated that sinomenine has sig-
niˆcant anti-in‰ammatory, analgesic and immuno-
suppressive properties.9)However, all these resear-
ches mainly focused on the eŠect of SIN on rheumatic
or rheumatoid arthritis, little is known about its eŠect

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10541054 Vol. 130 (2010)
on cartilage and chondrocytes in OA.
The proin‰ammatory cytokine IL-1b, which is one
of the most potent catabolic factors and plays an vital
role in the pathogenesis of OA,10)can induce the en-
hanced production of MMPs and inhibit the level of
TIMPs11,12)and also suppress synthesis of ECM and
increase the apoptosis of chondrocytes thus inhibiting
the repair process in cartilage.5)It is always used on
cartilage and chondrocyte to establish an OA
model.13,14)Thus, in the present study, we investigat-
ed the potential of SIN to protect rabbit cartilage and
chondrocytes against imbalance of MMP-13/TIMP-1
and apoptosis in an IL-1b-mediated OA model.
MATERIALS AND METHODS
Meterials
nan Zhengqing Pharmaceutical Group. 1,9-dimethyl-
methylene blue (DMMB), collagenase were obtained
from Sigma (St. Louis, USA). Dulbecco's Modiˆed
Eagle Medium (DMEM) and Fetal Bovine Serum
(FBS)werepurchasedfromGIBCO(Carlsbad,USA).
Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2F
(Z-DEVD-FMK) was purchased from Calbiochem
(La Jolla, USA). MMP-13 and TIMP-1 enzyme-
linked immunosorbent assay (ELISA) kits were pur-
chased from R&D (Minneapolis, USA). Reverse
transcription-polymerase chain reaction (RT-PCR)
kit was purchased from Invitrogen (Paisley, UK).
The annexin V ‰uorescein isothcocyanate (FITC)
apoptosis detection kit was purchased from Boehrin-
ger Mannheim (Mannheim, Germany). DNA extrac-
tion kit and caspase-3 assay kit was obtained from
Promega (Madison, USA). All other reagents or
drugs were of analytical grade.
Animals New Zealand rabbits (ˆve-week-old,
1200-1400 g) were provided by Peking University Ex-
perimental Animal Center. All rabbit experiments
and care were performed according to the Guide for
the Care and Use of Laboratory Animals (National
Academy Press, Washington DC, 1996).
Cartilage Explants Culture
were obtained from the knee joints of rabbits. Brie‰y,
after the articular surfaces were exposed surgically
under sterile conditions, articular cartilage was re-
moved and steeped in complete medium (DMEM,
supplemented with heat-inactivated 10％ FBS; peni-
cillin 100 U/ml; streptomycin 100 mg/ml). The sam-
ples were then rinsed several times with complete
medium and incubated for 2 days at 37° C in a humidi-
Sinomenine was provided from Hu-
Articular cartilages
ˆed 5％ CO2/95％ air incubator for stabilization. The
complete medium was replaced with a basal medium
(DMEM, supplemented with heat-inactivated 1％
FBS, 10 mM HEPES, 100 U/mlpenicillin and 100 mg/
ml streptomycin). The cartilage explants were treated
with 10 ng/ml IL-1b alone or with 10 ng/ml IL-1b＋
various concentrations of SIN (10, 50 or 250 mM) or
SIN for 72 h. Explants cultured in the absence of IL-
1b and SIN were used as controls.
GAG Quantitation in Cartilage Explants Cultures
　Approximately 50 mg cartilage pieces were placed
in 24-well plates and incubated in 10 ng/ml IL-1b
with or without various concentrations of SIN (10, 50
or 250 mM). The culture media were harvested 72 h
later and stored at －80° C for the followed assays.
The amount of GAG in the media at the end of reac-
tion re‰ect the amount of proteoglycan degradation.
GAG levels in the culture media were determined by
the amount of polyanionic material reacting with
DMMB, using shark chondroitin sulfate as the
standard.15)Samples were examined spectrophoto-
metrically at 540 nm (Spectramax, Molecular De-
vices, Sunnyvale, CA, USA). Results were expressed
as mg GAG released into the medium per mg wet
weight of the cartilage
Activity of MMP-13 and TIMP-1 in Cartilage Ex-
plants CulturesThe levels of MMP-13 and
TIMP-1 activity in the harvested media were evaluat-
ed using ELISA kits according to the manufacturer's
instructions. Plates were read at 450 nm with
microplate reader. Results are expressed as -fold
changes, considering the value of control as 1.
Expression of MMP-13 and TIMP-1 mRNA in
Cartilage Explants To investigate the mRNA ex-
pression of MMP-13 and TIMP-1 in cartilage ex-
plants, real-time quantitative RT-PCR was per-
formed. Cartilage explants were lysed with Trizol and
total RNA was extracted in accordance with the
manufacturer's instructions. First, 2 mg RNA was
subjected to RT reaction for 60 min at 37° C and was
stopped by incubation at 95° C for 5 min. Then 1 ml
cDNA was subjected to real-time quantitative PCR to
assess MMP-13 and TIMP-1 mRNA expression levels
with an ABI 7700 Sequence Detection System, in ac-
cordance with the manufacturer's instructions (Ap-
plied Biosystems, Warrington,
threshold cycle (CT) values of each sample were nor-
malized by that of glyceraldehyde-3-phosphate de-
hydrogenase (GAPDH) gene, and relative expression
UK). Obtained

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10551055 No. 8
level was expressed mean value of control group as 1.
Isolation and Culture of Chondrocytes
drocytes were obtained from articular of rabbit tibial
plateaus and femoral condyle. Cartilage slices were
digested in serum-free medium with sequential treat-
ments of 0.2％ pronase for 1 h, and then overnight
with 0.025％ collagenase type II in medium sup-
plemented with 5％ FBS in a humidiˆed atmosphere
of 5％ CO2at 37° C with continuous agitation. After
removing undigested cartilage using a 70 mm nylon
sieve, the chondrocytes were collected by centrifuga-
tion, and then cultured at 105cells/ml in DMEM with
10％ Fetal Bovine Serum, 100 IU/ml penicillin and
100 mg/ml streptomycin. Chondrocytes were treated
with 10 ng/ml IL-1b alone or with 10 ng/ml IL-1b＋
various concentrations of SIN (10, 50 or 250 mM) or
SIN for 24 h. Chondrocytes cultured in the absence of
IL-1b and SIN were used as controls.
Cell Viability Assay
mined by use of an MTT assay.16)Chondrocytes were
seeded in 96-well plates at 1×104cells per well and
grown to 70％ con‰uence in culture medium. The
medium was replaced by medium containing various
concentrations of SIN and/or 10 ng/ml IL-1b. A
total of 5 mg/ml MTT was added to each well after 24
h, and the culture continued to incubate for another 4
h at 37° C. After the medium had been removed, cells
and dyecrystalswere
dimethylsulfoxide (DMSO), and optical density was
measured at 570 nm by use of a model ELX-800
microplate assay reader (One Lambda Inc.).
DNA Fragmentation in Chondrocytes
was extracted by use of a DNA extraction kit accord-
ing to manufacturer's instructions after treated with
various concentrations of SIN and/or 10 ng/ml IL-1b
for 24 h. Brie‰y, a 10 ml DNA sample was loaded
onto 1.5％ horizontal agarose gels containing ethidi-
um bromide. Gels were run at 60 V for 1 h submerged
in Tris acetic acid-EDTA buŠer and DNA fragments
were visualized using ultraviolet(UV) illumination.17)
Chondrocytes Apoptosis
to various concentrations of SIN and/or 10 ng/ml IL-
1b for 24 h, We used annexinV-FITC apoptosis kit to
detect chondrocytes apoptosis.18)In addition, to exa-
mine the apoptotic pathways involved in IL-1b-in-
duced apoptosis and the anti-apoptosis mechanism of
SIN, chondrocytes also were treated with 100 mM Z-
DEVD-FMK (caspase-3-speciˆc inhibitor) alone or
Z-DEVD-FMK＋IL-1b for 24 h. Chondrocytes were
Chon-
Cell viability was deter-
solubilizedwith200 ml
DNA
After being exposed
harvested and suspended in binding buŠer at a ˆnal
cell concentration of 106cells/ml. Approximately 105
cells were incubated in the dark with annexin V and
propidium iodide for 15 min. Then the suspension
was analyzed with FACS scan ‰ow cytometer (Becton
Dickinson, Heidelberg, Germany). Annexin V FITC
and propidum iodide-related ‰uorescence was record-
ed on FL1-H (525 nm) and FL2-H (575 nm) ˆlters,
respectively.
Caspase-3 Activity in Chondrocytes
activity was detected with Apo-ONETM Homogene-
ous Caspase-3 Assay kit, according to the manufac-
turer's instructions. Brie‰y, chondrocytes were seed-
ed into 96-well plates at 104cells/well. After being ex-
posed to 10 ng/ml IL-1b and/or various concentra-
tions of SIN, 100 mM Z-DEVD-FMK for 24 h, cells
were washed with ice-cold PBS. Then, 1 ml Z-DEVD-
R110 and 99 ml caspase buŠer were mixed to make the
homogeneous caspase-3 reagent. A total of 100 ml
homogeneous caspase-3 reagent was added to each
well. The contents were gently mixed at 300 500 rpm
for at least 30 s and incubated for 4 h at room temper-
ature in the dark. The intensity of ‰uorescence was
measured at an excitation wavelength of 498 nm and
an emission wavelength of 521 nm with microplate
spectro‰uorometer (Wallac Victor2TM 1420 Multi-
label Counter, USA).
Statistical AnalysisResults are expressed as the
mean±S.E.M of triplicate values for each experi-
ment. The signiˆcance of statistical diŠerences among
groups was determined using ANOVA and post hoc
test. Values at p＜0.05 were considered to be statisti-
cally signiˆcant.
Caspase-3
RESULTS
EŠect of SIN on Proteoglycan Degradation in Car-
tilage Explants Cultures
aŠects proteoglycan degradation, cartilage explants
were cultured with 10 ng/ml IL-1b and/or various
concentrations of SIN for 72 h. The results showed
that untreated cartilage explants also had a basal level
of GAG release, however it was also partly blocked
by 50 or 250 mM SIN. This indicated that SIN on its
own could not induce the enhanced GAG release and
has no toxic eŠect on cartilage in the scope of 0 250
mM (Fig. 1). After incubation with IL-1b, the
amount of GAG released into the culture media in-
creased signiˆcantly compare to the control group
(6.79±0.88 mg/mg vs. 1.26±0.21 mg/mg). How-
To study whether SIN

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1056 1056
Fig. 1.
tilage Explants Cultures
Cartilage explants were treated with 10 ng/ml IL-1b or/and various con-
centrations of SIN (10 250 mM) for 72 h. Then GAG concentrations in cul-
ture media were determined by the DMMB assay. Results were expressed as
mg GAG released into the medium per mg wet weight of the cartilage. n＝8.
Data are mean±S.E.M.  p＜0.05,   p＜0.01 as compared to control;#p＜
0.05,##p＜0.01 as compared to IL-1b.
EŠect of SIN on Proteoglycan Degradation in Car-
Fig. 2.
TIMP-1(B) in IL-1b-induced Cartilage Explants Cultures
Cartilage explants were treated with 10 ng/ml IL-1b or/and various con-
centrations of SIN (10 250 mM) for 72 h, followed by ELISA. Relative lev-
els of activities were expressed mean value of control group as 1. n＝8. Data
are mean±S.E.M.  p＜0.01 as compared to control;#p＜0.01 as compared
to IL-1b.
EŠect of SIN on the Activity of MMP-13 (A) and
Vol. 130 (2010)
ever, SIN treatment caused a signiˆcant and dose-
dependent reduction in IL-1b-induced release of
GAG (Fig. 1). Thus, the critical observation of the
present studies is that SIN appeared to be an eŠective
agent for blocking the IL-1b-induced release of GAG
from cartilage explants.
EŠect of SIN on the Activity of MMP-13 and
TIMP-1 in Cartilage Explants Cultures
clearly showed an increase of MMP-13 activity and a
decline of TIMP-1 activity in IL-1b-induced cartilage
culture media compared to control. SIN at three con-
centrations markedly suppressed MMP-13 activity in
a dose-dependent manner. Furthermore, TIMP-1 ac-
tivities were signiˆcantly increased dose-dependently
in three SIN groups. 250 mM SIN alone had no obvi-
ous eŠect on the MMP-13 and TIMP-1 activity (Fig.
2).
EŠect of SIN on the mRNA Expression of MMP-
13 and TIMP-1 in Cartilage Explants
the eŠect of SIN on IL-1b-induced mRNA expression
of MMP-13 and TIMP-1, we treated cartilage ex-
plants with IL-1b alone or with IL-1b plus various
concentrations of SIN for 24 h. As shown in Fig. 3,
analysis of real-time quantitative RT-PCR revealed
that the levels of mRNA in cartilages treated with IL-
1b alone were about 3.9-fold for MMP-13 and 0.6-
fold for TIMP-1 when compared with the level of
control. Importantly, IL-1b-mediated increase of
MMP-13 mRNA was suppressed by SIN in a dose-
dependent manner, and SIN enhanced the production
of TIMP-1 mRNA in IL-1b-treated cartilages in a
ELISA
To evaluate
dose-dependent manner. In addition, 250 mM SIN a-
lone had no obvious eŠect on the mRNA expression
of MMP-13 and TIMP-1 (Fig. 3).
EŠect of SIN on Cell Viability in Chondrocytes
　After incubation with IL-1b, approximately 49.3％
of chondrocytes underwent death. Treatment with
SIN (10, 50 or 250 mM) decreased the cell death rate
in a dose-dependant manner; but three concentration
of SIN alone did not cause any apparent cytotoxicity
(Fig. 4). Thus, SIN showed a good chondroprotec-
tive eŠect on IL-1b-induced cell death.
EŠect of SIN on DNA Fragmentation in Chondro-
cytesExposed to IL-1b for 24 h, typically pro-
nounced DNA laddering was observed. However,
treatment with SIN inhibited IL-1b-mediated DNA
laddering, especially at the concentration of 250 mM,
which almost completely inhibit DNA fragmentation.
In addition, 250 mM SIN alone had no obvious eŠect
on the DNA fragmentation (Fig. 5).
EŠect of SIN on Apoptosis in Chondrocytes
anti-apoptotic eŠect of SIN was also conˆrmed using
annexin V staining to detect the presence of phos-
The

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1057 1057
Fig. 3.
(A) and TIMP-1(B) in IL-1b-induced Cartilage Explants
Cartilage explants were treated with 10 ng/ml IL-1b or/and various con-
centrations of SIN (10 250 mM) for 72 h. Then mRNA expression of
MMP-13 and TIMP-1 were detected with real-time quantitative RT-PCR.
Relative expression levels of mRNA were expressed mean value of control
group as 1. n＝8. Data are mean±S.E.M.  p＜0.05,   p＜0.01 as compared
to control;#p＜0.05,##p＜0.01 as compared to IL-1b.
EŠect of SIN on the Gene Expressions of MMP-13
Fig. 4.
cytes Viability
Chondrocytes were treated with 10 ng/ml IL-1b or/and various concen-
trations of SIN (10 250 mM) for 24 h, followed by MTT method. n＝8 Data
are mean±S.E.M.  p＜0.01 as compared to control;#p＜0.05,##p＜0.01 as
compared to IL-1b.
EŠect of SIN on IL-1b-induced Decrease in Chondro-
Fig. 5.
in Chondrocytes
Chondrocytes were treated with 10 ng/ml IL-1b or/and various concen-
trations of SIN for 24 h. Then 10 ml DNA sample of each group was loaded
onto agarose gels and run at 60 V for 1 h. DNA fragments were visualized us-
ing ultraviolet (UV) illumination. (A) control; (B) treatment with 250 mM
SIN; (C) treatment with 10 ng/ml IL-1b; (D) treatment with 10 ng/ml IL-
1b and 10 mM SIN; (E) treatment with 10 ng/ml IL-1b and 50 mM SIN; (F)
treatment with 10 ng/ml IL-1b and 250 mM SIN.
EŠect of SIN on IL-1b-induced DNA Fragmentation
No. 8
phatidylserine on the cell membrane. Treatment with
IL-1b signiˆcantly increased the percentage of apop-
totic chondrocytes to 46.7％, but SIN markedly
decreased the percentage of cell apoptosis to 25.1％,
14.8％ and 5.2％ at 10, 50 or 250 mM respectively
(Fig. 6). To further support these results and also to
understand the apoptotic pathways, caspase-3 inhibi-
tion experiments was performed. Z-DEVD-FMK a-
lone had no toxic eŠect on chondrocytes apoptosis
(data not shown), however this caspase-3-special in-
hibitor greatly blocked IL-1b-induced apoptosis,
showing that it was a caspase-3 dependent apoptosis.
EŠect of SIN on Caspase-3 Activity in Chondro-
cytes Figure 7 showed that caspase-3 activity was
increased by 7.4-fold compared with control after IL-
1b incubation. In contrast, chondrocytes treated with
10, 50 or 250 mM SIN showed a signiˆcant decrease in
caspase-3 activity compared with IL-1b-treated cells
at the same time point. And Z-DEVD-FMK also com-
pletely inhibited the caspase-3 activity induced by IL-
1b. Combined with the results of chondrocytes apop-
tosis, these data exhibited that SIN did block chon-
drocytes apoptosis by inhibiting IL-1b-induced acti-
vation of caspase-3.
DISCUSSION
Sinomenium acutum has been used for the treat-
ment of joint pain and arthritis by Chinese medical
doctors for over 2000 years.19)SIN is the most phar-
macologically active compound in sinomenium acu-
tum in terms of anti-in‰ammatory, analgesic, anti-ar-
thritic, and immunosuppressive properties, and is a
promising drug that has been used for treating rheu-