A Soft Coral-Derived Compound, 11-epi-Sinulariolide

Acetate Suppresses Inflammatory Response and Bone

Destruction in Adjuvant-Induced Arthritis
Yen-You Lin1., Yen-Hsuan Jean2., Hsin-Pai Lee1,2, Wu-Fu Chen3, Yu-Min Sun4, Jui-Hsin Su5,6, Yi Lu1,
Shi-Ying Huang1, Han-Chun Hung4, Ping-Jyun Sung5,6, Jyh-Horng Sheu1*, Zhi-Hong Wen1*
1 Department of Marine Biotechnology and Resources, Asia-Pacific Ocean Research Center, National Sun Yat-Sen University, Kaohsiung, Taiwan, Republic of China,
2 Section of Orthopedic Surgery, Pingtung Christian Hospital, Pingtung, Taiwan, Republic of China, 3 Department of Neurosurgery, Chang Gung Memorial Hospital-
Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan, Republic of China, 4 Doctoral Degree Program in Marine Biotechnology,
National Sun Yat-Sen University, Kaohsiung, Taiwan, Republic of China, 5 Graduate Institute of Marine Biotechnology, National Dong Hwa University, Pingtung, Taiwan,
Republic of China, 6 Taiwan Coral Research Center, National Museum of Marine Biology and Aquarium, Pingtung, Taiwan, Republic of China

Abstract
In recent years, a significant number of metabolites with potent anti-inflammatory properties have been discovered from
marine organisms, and several of these compounds are now under clinical trials. In the present study, we isolated 11-epi-
sinulariolide acetate (Ya-s11), a cembrane-type compound with anti-inflammatory effects, from the Formosa soft coral
Sinularia querciformis. Preliminary screening revealed that Ya-s11 significantly inhibited the expression of the
proinflammatory proteins induced nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated murine
macrophages. We also examined the therapeutic effects of Ya-s11 on adjuvant-induced arthritis (AIA) in female Lewis rats,
which demonstrate features similar to human rheumatoid arthritis (RA). Animal experiments revealed that Ya-s11
(subcutaneously 9 mg/kg once every 2 days from day 7 to day 28 postimmunization) significantly inhibited AIA
characteristics. Moreover, Ya-s11 also attenuated protein expression of cathepsin K, matrix metalloproteinases-9 (MMP-9),
tartrate-resistant acid phosphatase (TRAP), and tumor necrosis factor-a (TNF-a) in ankle tissues of AIA-rats. Based on its
attenuation of the expression of proinflammatory proteins and disease progression in AIA rats, the marine-derived
compound Ya-s11 may serve as a useful therapeutic agent for the treatment of RA.

Citation: Lin Y-Y, Jean Y-H, Lee H-P, Chen W-F, Sun Y-M, et al. (2013) A Soft Coral-Derived Compound, 11-epi-Sinulariolide Acetate Suppresses Inflammatory
Response and Bone Destruction in Adjuvant-Induced Arthritis. PLoS ONE 8(5): e62926. doi:10.1371/journal.pone.0062926
Editor: Yong Jiang, Southern Medical University, China
Received November 30, 2012; Accepted March 27, 2013; Published May 13, 2013
Copyright: ß 2013 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported, in part, by grants from the National Science Council, Taiwan (NSC100-2325-B-110-001 and NSC99-2313-B110-003-MY03), and
Pingtung Christian Hospital, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] (Z-HW); [email protected] (J-H Sheu)
. These authors contributed equally to this work.

Introduction causes hyperplasia of the synovial tissue, with clusters of large

numbers of infiltrating cells, and a tumor-like structure called
Rheumatoid arthritis (RA) is an autoimmune and chronic pannus invades the joint lining and destroys local articular
erosive inflammatory disease characterized by chronic edema and structure [9,10]. Although the precise etiology of RA remains
inflammation of the synovial tissue that lines joints. RA affects unclear, macrophages, neutrophils, lymphocytes, and synovial
approximately 1% of the adult population worldwide, and RA fibroblasts in hyperplastic synovial tissue have been identified as
patients present an average of 43% of maximum possible joint major participants in the initiation and development of RA
destruction after 20 years of suffering from the disease [1–3]. As [11,12]. These infiltrating cells can release proinflammatory
RA progresses, the lining of the joints degenerates, leading to cytokines such as tumor necrosis factor alpha (TNF-a) that
articular destruction and decreased joint mobility with radiological mediate synovial inflammation and joint destruction [8,13]. In
evidence of erosive damage and significantly impacting quality of addition, proinflammatory cytokines activate synovial fibroblasts
life within 2 years of disease onset [4,5]. Over the past 2 decades, and chondrocytes and lead to upregulation of osteoclast-related
more effective therapeutic strategies for RA including synthetic proteins including cathepsin K, matrix metalloproteinase-9
modifying antirheumatic drugs and/or biologic agents have been (MMP-9), and tartrate-resistant acid phosphatase (TRAP), which
developed, but they carry potential risks. Additionally, nonsteroi- also participate in inflammatory arthritis resulting in joint
dal medications and corticosteroids are required as adjunctive destruction [14].
therapy [6]. Therefore, new drug development for RA remains Lipopolysaccharide (LPS)-challenged murine macrophages are
essential. widely used for in vitro anti-inflammatory screening of terrestrial-
Prior studies have indicated that inflammatory processes are and marine-derived natural compounds [15–17]. LPS activates
critical to the development of RA [2,7,8]. Synovial inflammation the nuclear factor-kB pathway to massively upregulate the

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 Effect of Ya-s11 in Adjuvant-Induced Arthritis

proinflammatory proteins inducible nitric oxide synthase (iNOS) cells for 18 h in DMEM containing only LPS (0.01 mg/ml)
and cyclooxygenase-2 (COX-2) in murine macrophages [18]. without the other added compounds. For the anti-inflammatory
Several studies have indicated that macrophages play a critical role activity assay, Ya-s11 (1, 10, 25, or 50 mM) was added to the
in RA by expressing pro-inflammatory proteins in inflamed medium 10 min before LPS treatment of RAW264.7 murine
synovial tissue and at the cartilage-pannus junction [11,19]. They macrophages. Cell pellets were collected by washing with ice-cold
also release cytokines and chemokines that mediate inflammation phosphate-buffered saline (PBS) and then lysed in 4 uC lysis buffer
and form a complex cytokine network with neutrophils, lympho- (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 100 mg/
cytes, and synovial fibroblasts in RA [13]. Macrophages are also ml phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin). After
important for their capacity to differentiate into osteoclasts, centrifuging at 14,000 rpm for 30 min at 4uC, the supernatant
multinucleated giant cells, or mononuclear precursor cells. A was obtained from the pellet and prepared for western blot
number of previous studies have employed macrophage-related analysis of pro-inflammatory proteins, iNOS and COX-2. Protein
cell lines to investigate RA-related mechanisms of action and concentrations were quantified using the DC protein assay kit
identify the anti-arthritic activity of compounds [8,20–22]. (Bio-Rad, Hercules, CA, USA) modified from the assay of Lowry
Adjuvant-induced arthritis (AIA) is also widely used as an animal et al. (1951) [30]. Western blotting was performed as described in
model for the study of clinical RA. Several potential anti-RA our previous study [15]. An equal volume of sample buffer (2%
agents have been discovered from this model [1,8,23]. sodium dodecyl sulfate (SDS), 10% glycerol, 0.1% bromophenol
In recent years, a significant number of metabolites with potent blue, 2% 2-mercaptoethanol, and 50 mM Tris–HCl, pH 7.2) was
bioactive properties have been discovered in marine organisms, added to the supernatant and then loaded into a tricine SDS-
and several of these compounds are now under clinical trials polyacrylamide (7% or 10%) gel for electrophoresis at 120 V for
[24,25]. In this study, we aimed to examine an anti-inflammatory 120 min. The proteins were transferred to a polyvinylidene
cembrane-type compound for its ability to suppress RA progres- difluoride membrane (PVDF; 0.45- mM pore size, Immobilon-P,
sion. We isolated 11-epi-sinulariolide acetate (Ya-s11), a known Millipore, Bedford, MA, USA) at 135 mA overnight at 4 uC in
cembrane-type compound, from the soft coral Sinularia querciformis. transfer buffer (50 mM Tris–HCl, 380 mM glycine, 1% SDS, and
We found that Ya-s11 inhibited expression of the pro-inflamma- 20% methanol). The membrane was blocked for 40 min at room
tory proteins iNOS and COX-2 in LPS-induced murine temperature with 5% non-fat dry milk in Tris-buffered saline with
RAW264.7 macrophages. We also evaluated the anti-inflamma- Tween-20 (TTBS; 0.1% Tween 20, 20 mM Tris–HCl, 137 mM
tory and anti-rheumatic effects of Ya-s11 in AIA rats. NaCl, pH 7.4) and then incubated overnight at 4 uC with primary
antibodies directed against iNOS (1:2,000 dilution; BD Pharmin-
Materials and Methods gen, San Diego, CA, USA; catalog no. 6103322; polyclonal
antibody), COX-2 (1:2,000, Cayman Chemical, Ann Arbor, MA,
Preparation of 11-epi-sinulariolide acetate USA; catalog no. 160106; polyclonal antibody), and b-actin
In the present study, 11-epi-sinulariolide acetate (Ya-s11, (1:2,000, Sigma, St. Louis, MO, USA; catalog no. A5316-2ML;
structure shown in Fig. 1) was isolated and purified from non- monoclonal antibody). The iNOS, COX-2, and b-actin antibodies
protected soft coral, S. querciformis, collected from the Dongsha recognized bands at ,135, ,72, and 45 kDa, respectively. The
Islands, Taiwan in 2008. We state that no specific permissions immunoreactive bands were visualized by enhanced chemilumi-
were required for sample collection in Dongsha Islands. Moreover, nescence (Millipore, Billerica, MA, USA) and the Biochemi
we also state that the area was not privately owned or protected. Imaging System, and relative densitometric quantification was
The method of 11-epi-sinulariolide acetate extraction was modified performed using LabWorks 6.2 software (UVP, Upland, CA,
from that of Lu et al. [26,27]. The soft coral specimens were USA).
minced and exhaustively extracted with 95% ethanol (8 liters). The
crude extract was concentrated to an aqueous suspension and In vivo study
further partitioned between n-hexanes, ethyl acetate, and H2O. Animals. Female Lewis rats (180–220 g) were used for the
The n-hexaneethyl acetate layer was separated over normal phase experiments and obtained from National Laboratory Animal
silica gel by column chromatography and eluted with n-hexane, Center, Taiwan. The rats were maintained in plexiglass cages in a
ethyl acetate, acetone, and methanol to yield 29 fractions. Fraction temperature-controlled (2461 uC) room on a 12-h light/dark
11 was further eluted with n-hexanes and acetone (1:1) over cycle and given free access to food and water. Each rat was used
normal phase silica gel to generate 5 subfractions. Subfraction 4 only once during the experiment. All drug injections were
was further separated by reverse-phase RP18 gel elution with H2O performed under isoflurane anesthesia. The use of animals
and acetonitrile (2:1) by column chromatography to yield Ya-s11. accorded to the Guiding Principles in the Care and Use of
The structure of Ya-s11 was confirmed by nuclear magnetic Animals of the American Physiology Society and was approved by
resonance spectroscopy (NMR) [28]. The purity (.98%) of Ya-s11 the institutional animal care and use committee of National Sun
was determined and verified by 1H-NMR and 13C-NMR spectra Yat-sen university. Every effort was made to minimize the number
(Varian Mercury Plus 400 FT-NMR at 400 MHz, Varian, CA, U of animals used and their suffering.
SA).
Adjuvant induce arthritis (AIA) and compound treatment
In vitro, anti-inflammatory activity assay The method of generating AIA rats was modified from that of
The anti-inflammatory activity assay was modified from Jean et Sano et al. (1992) and Turull and Queralt (2000) [31,32]. Heat-
al. (2008) and Chen et al. (2011) [15,29]. RAW 264.7 murine killed and lyophilized Mycobacterium butyricum was suspended in
macrophages were supplied by the American Type Culture incomplete Freund’s adjuvant 10 mg/ml (Sigma, St. Louis, MO,
Collection (ATCC, No. TIB-71) and grown in DMEM (including USA) on ice. Rats were immunized with an adjuvant injection of
10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM 10 mg/ml M. butyricum in incomplete Freund’s adjuvant. On day
pyruvate, 4.5 g/l glucose, 50 mg/ml streptomycin, and 100 U/ml 0, rats were injected intradermally at the base of the tail with
penicillin G) at 37 uC in a humidified 5% CO2: 95% air incubator. 0.1 ml of adjuvant, and the development of arthritis was
Inflammatory response was induced by incubating RAW264.7 monitored from day 0 to day 28. Lewis rats were randomly

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 Effect of Ya-s11 in Adjuvant-Induced Arthritis

Figure 1. Chemical structure and source of 11-epi-sinulariolide acetate (Ya-s11). (A) The chemical structure of 11-epi-sinulariolide acetate.
Molecular formula, C22H32O5, Molecular weight, 376. (B) The soft coral sample, Sinularia querciformis, was collected from Dongsha Island.
doi:10.1371/journal.pone.0062926.g001

divided into 5 groups: AIA (n = 7), AIA plus Ya-s11 (3 mg/kg) nized with xylene, and dehydrated in a graded series of alcohol,
(n = 6), AIA plus Ya-s11 (9 mg/kg) (n = 6), naı̈ve (n = 6), and Ya- after which endogenous peroxidase activity was quenched by 30-
s11 (9 mg/kg) treatment alone (n = 6). In the AIA plus Ya-s11 min incubation in 0.3% hydrogen peroxide. The antigen was
groups, rats received 10 times of Ya-s11 at 2-day intervals between retrieved by enzymatic digestion with proteinase K (20 mM;
days 7 and 28. All rats underwent measurement of hindpaw Sigma) in PBS for 20 min. After washing in ice-cold PBS, slides
edema and clinical evaluation at day 0 and before every Ya-s11 were incubated at 4 uC for 48 h with anti-cathepsin K (1:100,
injection between days 7 and 28, and then rats were sacrificed on Abcam, Cambridge, MA, USA; catalog no. ab19027; polyclonal
day 28 for histopathological analysis and immunochemical antibody), anti-MMP9 (1:100, Abcam; catalog no. ab76003;
staining. The extent of edema in the foot and hindpaw was monoclonal antibody.), anti-TNF-a (1:100, AnaSpec, Fremont,
measured from day 0 (baseline) to day 28 after AIA using a CA, USA; catalog no. 55383; polyclonal antibody), or anti-TRAP
plethysmometer (Paw Volume Meter, Singa, Taiwan). Rats were (1:100, Santa Cruz, Delaware Avnue Santa Cruz, CA, USA,
evaluated for arthritic processes every 2 days using a macroscopic catalog no sc-28204; polyclonal antibody) in a humidified
scoring system, with score 0 = no signs of arthritis, 1 = swelling chamber. The sections were incubated for 90 min with biotiny-
and/or redness of the paw or 1 digit, 2 = two tow joints involved, lated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA)
3 = more than two joints involved, 4 = severe arthritis of the entire diluted 1:200 in 1% bovine serum albumin (BSA) in PBS. Sections
paw and all digits [9,33,34]. The macroscopic score for each rat were then immunohistochemically labeled with the avidin-biotin
was calculated by adding the scores of each individual paw [9,34]. complex technique using an ABC kit (Vectastain ABC kit; Vector
Laboratories). Finally, the sections were treated with 3,39-
Histopathological examination and diaminobenzidine tetrahydrochloride (DAB; Peroxidase substrate
immunohistochemical staining kit, Vector Laboratories) for 1–5 min. All slides for immunohis-
Rats were sacrificed by perfusion with ice-cold PBS and 4% tochemistry were analyzed under a light microscope (DM 600B,
paraformaldehyde on day 28 after immunization, and ankle joints Leica Inc. Wetzlar, Germany) with microscope digital image
were removed and fixed in 10% neutral formalin for 4 days. The output system.
ankle joints were decalcified with 12.5% ethylenediaminetetraace-
tic acid (EDTA) in 10% neutral formalin for 2 weeks and then Statistical analysis
sectioned on the sagittal plane through the center of samples. The All data are presented as mean 6 SEM. For the immunore-
specimens were dehydrate in a graded series of alcohol (Tissue- activity data, each test band is shown as the integrated optical
Tek, Sakura Finetek Japan Co., Ltd, Japan), embedded with density (IOD) computed with respect to the average optical density
paraffin, and cut into 2- mm sections (Microm HM550, Microm, of the corresponding control (LPS-only treatment) band. The data
Waldorf, Germany for hematoxylin and eosin (H&E) and were analyzed using 1-way analysis of variance (ANOVA) followed
immunohistochemical staining. General and pathological changes by Student-Newman-Keuls post hoc test (SigmaStat 3.5 for
in morphology were assessed by microscopic examination using an Windows). Differences resulting in P values less than 0.05 were
upright microscope for higher magnification (DM 6000B, Leica considered significant.
Inc., Germany) and a stereomicroscope for lower magnification
(APO Z16, Leica Inc., Singapore) with a microscope digital image Results
output system (SPOT idea 5.0 Mp Color Digital Camera,
Diagnostic Instruments Inc., Sterling Height, MI., USA). To The inhibitory effects of Ya-s11 on iNOS and COX-2
quantitatively evaluate joint destruction in the ankle, the degree of protein expression
morphologic changes in each group was scored on photomicro- The dose inhibition of 11-epi-sinulariolide acetate (Ya-s11) on
graphs of tissue sections, with score 0 = no damage, 1 = edema, LPS-induced pro-inflammatory 130-kDa iNOS and 71-kDa
2 = presence of inflammatory cells, and 3 = cartilage and bone COX-2 protein expression is shown in Figure 2. At 1, 10, 25,
damage (Cuzzocrea et al., 2005). Infiltrating cells were further and 50 mM doses of Ya-s11, iNOS levels were significantly
quantified according to the histopathological features of neutro- reduced to 84.8968.23%, 39.8965.64%, 11.861.03%, and
phils, lymphocytes, macrophages, and synovial fibroblasts [35–37]. 1.461.74% relative protein expression, respectively, compared
Ankle joint specimens were processed for immunohistochemical to the cells treated only with LPS (Fig. 2B). At 10, 25, and 50 mM
analysis as described in previous studies [38–40]. Paraffin- concentrations, Ya-s11 significantly reduced COX-2 levels to
embedded ankle joint sections were placed on slides, deparaffi- 82.8961.63%, 65.9364.22%, 52.6364.76%, and 42.1363.25%

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 Effect of Ya-s11 in Adjuvant-Induced Arthritis

relative protein expression compared to the cells treated only with or arthritis score was observed in the group treated with Ya-s11
LPS (Fig. 2C). Levels of b-actin protein (internal control) (9 mg/kg) (Fig. 3E-F).
demonstrated no significant difference between concentrations of
1, 10, 25, and 50 mM Ya-s11 or compared with LPS only. Thus, The effect of Ya-s11 on histological features of AIA in the
Ya-s11 demonstrated concentration-dependent inhibition of LPS- ankle joint
induced iNOS and COX-2 protein expression in RAW 264.7 Rats were sacrificed on day 28 after immunization, and paraffin
murine macrophages. Additionally, at the experimental concen- sections of ankle joints were subjected to H&E staining for
trations, Ya-s11 did not induce cytotoxicity as determined through histopathological analysis. Representative photographs of ankle
trypan blue staining and Annexin-V/PI double-staining assay joint sections are shown from control (Fig. 4A, E, I), AIA (Fig. 4B,
(Figure S1). F, J), AIA plus Ya-s11 (3 mg/kg) (Fig. 4C, G, K) and AIA Plus Ya-
s11 (9 mg/kg) rats (Fig. 4D, H, L), respectively. Similar to previous
Effect of Ya-s11 on the clinical features of AIA studies [4,41], in the AIA group, synovial tissue demonstrated
AIA developed rapidly in rats immunized with heat-killed M. synovial hyperplasia, cartilage and bone erosion (Fig. 4I), and
butyricum, and Figure 3 show the square of typical representative moderate to severe infiltration of immune cells into subchondral
macroscopic photographs. Both AIA and AIA plus Ya-s11 (3 mg/ bone marrow (Fig. 4J). The AIA plus Ya-s11 (3 mg/kg) group did
kg) groups demonstrated edema and erythema on the ankle joints not show attenuation of the synovial hyperplasia or cartilage and
and hindpaws (Fig. 3B and C). Ya-s11 (9 mg/kg) significantly bone erosion, but moderated bone resorption in bone marrow
attenuated the AIA-induced edema and erythema of the hindpaw (Fig. 4G, K). By contrast, the AIA plus Ya-s11 (9 mg/kg) group
(Fig. 3D). Figure 3E illustrates the time-dependent increase of paw demonstrated significantly inhibited AIA-induced joint destruction
edema in immunized rats. Paw edema significantly increased to and synovial hyperplasia, cartilage and bone erosion, pannus
approximately 121.4–127.4% of baseline values from day 19 to formation, and bone resorption (Fig. 4H, J). Histological damage
day 23 in the AIA group, and the AIA plus Ya-s11 (9 mg/kg) scores were significantly higher in the AIA group compared to the
group demonstrated a dose-dependent inhibition of paw edema naı̈ve group. Those of the AIA plus Ya-s11 (9 mg/kg) group were
compared with the AIA-only group. Quantitative analysis using a significantly lower than in the AIA and AIA plus Ya-s11 (3 mg/kg)
macroscopic scoring system (Fig. 3F) revealed a significant groups. No significant differences were observed between the AIA
reduction in the AIA-induced upregulation of arthritis score in and AIA plus Ya-s11 (3 mg/kg) groups (Fig. 4M).
the AIA plus Ya-s11 (9 mg/kg) group. The AIA plus Ya-s11
(3 mg/kg) group demonstrated a slight but not significant
attenuation in arthritis score (Fig. 3F). No change in paw edema

Figure 2. Effect of Ya-s11 on pro-inflammatory iNOS and COX-2 protein expression in LPS-stimulated macrophage cells. (A)
immunoreactive bands corresponding to iNOS, COX-2, and b-actin protein from RAW 264.7 cells; (B) relative density of iNOS immunoreactive bands;
(C) relative density of COX-2 immunoreactive bands. The relative intensity of the LPS group was set to 100%. Band intensities were quantified by
densitometry and are indicated as the percent change relative to that of the LPS group. Western blotting with b-actin was performed to verify that
equivalent amounts of protein were loaded in each lane. Ya-s11 significantly inhibited LPS-induced iNOS and COX-2 protein expression in murine
Raw 264.7 macrophage cells. The experiment was repeated 4 times. *P,0.05, significantly different from the LPS-induced group.
doi:10.1371/journal.pone.0062926.g002

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 Effect of Ya-s11 in Adjuvant-Induced Arthritis

Figure 3. Effect of Ya-s11 on AIA rats. Typical representative macroscopic photographs of ankle and paw from the (A) naı̈ve, (B) AIA, (C)
AIA+Ya2s11 (3 mg/kg), and (D) AIA+Ya2s11 (9 mg/kg) groups. The AIA and AIA+Ya2s11 (3 mg/kg) groups displayed significant the edema on ankle
joints and erythema on hindpaws (red square) compared to the naı̈ve group (C). The AIA+Ya2s11 (9 mg/kg) group demonstrated apparent reduction
of AIA-induced edema and erythema (D). Quantitative analysis of the effect of Ya-s11 at doses of 3 or 9 mg/kg on AIA-induced paw edema (E).
Baseline values for the paw volume of each rat were set to 100%, and changes in edema level were calculated as a percentage increase from the
control (pre-drug) volume. Ya-s11 demonstrated dose-dependent inhibition of AIA-induced paw edema in rats. Clinical evaluation of the effect of Ya-
s11 on AIA-induced clinical signs in rats (F). Ya-s11 demonstrated a dose-dependent effect on AIA-induced clinical signs. Ya-s11 (3 or 9 mg/kg) was
subcutaneously injected every 2 days between day 7 and day 28. Values reflect the mean 6 SEM for each group. *P,0.05 compared with the naı̈ve
group. #P,0.05 compared with the AIA group.
doi:10.1371/journal.pone.0062926.g003

Effect of Ya-s11 on infiltrating cells in synovial tissue Effect of Ya-s11 on cathepsin K, MMP-9, TRAP, and TNF-a
Pannus formation is accompanied by the infiltration of in AIA
inflammatory cells, including lymphocytes, monocytes/macro- Bone erosion in RA is primarily the result of activated
phages, neutrophils, and synovial fibroblasts, into synovial tissue osteoclasts that express enzymes related to bone resorption, such
[2]. Representative photographs show synovial tissue stained with as MMP9, cathepsin K, and TRAP [11,42]. Figure 6 shows the
H&E from the naı̈ve group (Fig. 5A), AIA group (Fig. 5B), AIA distribution of cathepsin K immunoreactivity in the ankle joint in
plus Ya-s11 (3 mg/kg) group (Fig. 5C), and AIA plus Ya-s11 the naı̈ve (Fig. 6A), AIA (Fig. 6B), AIA plus Ya-s11 (3 mg/kg)
(9 mg/kg) group (Fig. 5D). The synovial tissue of the naı̈ve group (Fig. 6C), and AIA plus Ya-s11 (9 mg/kg) groups (Fig. 6D).
demonstrated synovial fibroblasts with few immune cells (Fig. 5A), Cathepsin K immunoreactivity appeared to increase in the ankle
and marked upregulation of infiltrating cells was apparent in joint in the AIA group (Fig. 6B). In the AIA plus Ya-s11 (9 mg/kg)
synovial tissue from the AIA group (Fig. 5B). Ya-s11 (9 mg/kg) group, inhibited AIA-induced upregulation of cathepsin K was
significantly inhibited AIA-induced upregulation of infiltrating observed (Fig. 6D). Higher-magnification images of cathepsin K
cells in synovial tissue (Fig. 5D). Neutrophils, lymphocytes, immunoreactivity in the synovial tissue and cartilage with
macrophages, and synovial fibroblasts all significantly increased subchondral bone in the naı̈ve (Fig. 6F, K), AIA (Fig. 6G, L),
between the naı̈ve and AIA groups (Fig. 5E–R), and the AIA plus AIA plus Ya-s11 (3 mg/kg) (Fig. 6H, M), and AIA plus Ya-s11
Ya-s11 (9 mg/kg) group demonstrated a significant decrease in the (9 mg/kg) groups (Fig. 6I, N) are taken from Figures 6A, 6B, 6C,
number of neutrophils, lymphocytes, and macrophages as well as and 6D, respectively. In the AIA group, cathepsin K immunore-
inhibition of synovial fibroblast proliferation. No significant activity was observed in synovial tissue-infiltrating cells (Fig. 6G),
change was found in the AIA plus Ya-s11 (3 mg/kg) group cartilage chondrocytes, and subchondral bone marrow (Fig. 6I) of
compared with the AIA group. the ankle joint. In the AIA plus Ya-s11 (9 mg/kg) group, cathepsin
K immunoreactivity was inhibited in synovial tissue and

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 Effect of Ya-s11 in Adjuvant-Induced Arthritis

Figure 4. Histopathological assessments of the effect of Ya-s11 on the AIA rat ankle joint. Representative sections of ankle joint from the
(A, E, I) naı̈ve, (B, F, J) AIA, (C, G, K) AIA+Ya-s11 (3 mg/kg), and (D, H, L) AIA+Ya-s11 (9 mg/kg) groups stained with H&E. Normal joint structure showing
calcaneus-talus articulation with the distal tibia and normal synovial tissue was observed in the naı̈ve group (A). Marked joint destruction with bone
damage, synovial tissue hyperplasia, and increased relative size of the marrow cavity of the subchondral bone marrow was observed in the AIA group
(B). A higher-magnification view of AIA group shows pannus formation (arrow) and bone erosion over the rim of articular bone (F), and synovial tissue
cells infiltration into subchondral bone marrow through the erosive orifice (arrowhead) into the calcaneus (J). The AIA+Ya-s11 (3 mg/kg) group
demonstrated marked joint destruction with bone damage and synovial tissue hyperplasia (C). A higher-magnification view of AIA+Ya-s11 (3 mg/kg)
group shows less severe pannus formation (arrow) and cartilage and bone destruction and resorption on the calcaneus compared with the AIA group
(G). In the AIA+Ya-s11 (9 mg/kg) group, no morphological change in the ankle joint was apparent (H, L). The representative histopathological scores
(M) of each group were analyzed to assess the degree of morphological changes, and the AIA+Ya-s11 (9 mg/kg) group demonstrated a significant
decrease in the degree of arthritis. SB, subchondral bone marrow; Ca, calcaneus; Ta, talus; Ti, tibia; CD, cartilage destruction; BD, bone destruction; E–
L, scale bar = 500 mm. *P,0.05 compared with the naı̈ve group. #P,0.05 compared with the AIA group.
doi:10.1371/journal.pone.0062926.g004

subchondral bone marrow (Fig. 6I) but not cartilage (Fig. 6N). The inhibition of cathepsin K immunoreactivity in synovial tissue or
AIA plus Ya-s11 (3 mg/kg) group demonstrated reduced cathep- cartilage (Fig. 6H, M).
sin K immunoreactivity in subchondral bone marrow without Figure 7 shows the distribution of MMP-9 immunoreactivity in
the ankle joint in the naı̈ve (Fig. 7A), AIA (Fig. 7B), AIA plus

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 Effect of Ya-s11 in Adjuvant-Induced Arthritis

Figure 5. Effect of Ya-s11 on cell infiltration to the synovial tissue in AIA rats. Representative photographs of H&E-stained synovial tissue
from the (A) naı̈ve, (B) AIA, (C) AIA+Ya-s11 (3 mg/kg), and (D) AIA+Ya-s11 (9 mg/kg) groups. The synovial tissue of the naı̈ve group demonstrated
synovial fibroblasts with few immune cells (arrow) (A). Upregulation of neutrophils (red arrow), lymphocytes (red head arrow), macrophages (black
head arrow), and synovial fibroblasts (black arrow) was observed in synovial tissue from the AIA group (B) and AIA+Ya-s11 (3 mg/kg) group (C). Ya-
s11 (9 mg/kg) appeared to attenuate AIA-induced upregulation of immune cells. The numbers of neutrophils (E), lymphocytes (F), macrophages (G),
and synovial fibroblasts (H) were analyzed in synovial tissue from each group. Higher-magnification views of synovial tissue from the AIA group
shows histopathological features of infiltrating cells (E-H). Cell numbers significantly increased between the naı̈ve group and AIA group and were
significantly decreased in the AIA+Ya-s11 (9 mg/kg) group compared with the AIA group. *P,0.05 compared with the naı̈ve group. #P,0.05
compared with the AIA group. A–D, scale bar = 100 mm; E–H, scale bar = 5 mm.
doi:10.1371/journal.pone.0062926.g005

Figure 6. Effect of Ya-s11 on cathepsin K protein expression in the ankle of AIA rats. Cathepsin K protein immunoreactivity is shown in
red-brown (arrow) in ankle joint sections from the (A, F, K) naı̈ve, (B, G, L) AIA, (C, H, M) AIA+Ya-s11 (3 mg/kg), and (D, I, N) AIA+Ya-s11 (9 mg/kg)
groups. (F)–(J) show cathepsin K immunoreactivity in the synovial tissue of ankle joints outlined in boxes in (A)–(E), respectively. (K)–(O) show
cathepsin K immunoreactivity in the articular cartilage outlines in boxes in (A)–(E), respectively. The immunostaining results indicate the upregulation
of cathepsin K protein expression in the ankle joint (synovial tissue and cartilage) in AIA rats (B, G, L) and the inhibition of cathepsin K protein
expression in synovial tissue and cartilage by treatment with 9 mg/kg but not 3 mg/kg Ya-s11. (E, J, O) A sample from the AIA group incubated
without primary antibody for cathepsin K showed no specific staining. Scale bar = 100 mm.
doi:10.1371/journal.pone.0062926.g006

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 Effect of Ya-s11 in Adjuvant-Induced Arthritis

Ya-s11 (3 mg/kg) (Fig. 7C), and AIA plus Ya-s11 (9 mg/kg) Discussion
groups (Fig. 7D). In the AIA group, MMP-9 immunoreactivity
appeared to increase in the ankle joint (Fig. 7B). Administration of This study employed LPS-induced RAW264.7 murine macro-
9 mg/kg but not 3 mg/kg Ya-s11 inhibited AIA-induced MMP-9 phage cells and AIA as in vitro and in vivo models, respectively, to
expression in the ankle joint (Fig. 7C, D). Higher-magnification assess the anti-inflammatory and anti-arthritic effects of Ya-s11.
images of MMP-9 immunoreactivity in the synovial tissue and Marine-derived Ya-s11 significantly down-regulated expression of
cartilage with subchondral bone in the naı̈ve (Fig. 7F, K), AIA the proinflammatory proteins iNOS and COX-2 in LPS-induced
(Fig. 7G, L), AIA plus Ya-s11 (3 mg/kg) (Fig. 7H, M), and AIA RAW 264.7 murine macrophage cells. Administration of Ya-s11
plus Ya-s11 (9 mg/kg) groups (Fig. 7I, N) are taken from also significantly inhibited AIA-induced paw edema and the
Figures 7A, 7B, 7C, and 7D, respectively. No obvious increase upregulation of arthritis score in a dose-dependent manner.
in MMP-9 immunoreactivity in synovial tissue (Fig. 7G), cartilage, Histopathological and immunohistochemical examination further
or subchondral bone marrow (Fig. 7I) was observed in the AIA demonstrated that AIA-induced histological features in the ankle
group. Compare with the AIA group, the AIA plus Ya-s11 (9 mg/ joint and the osteoclast-related proteins, cathepsin K, MMP-9,
kg) group demonstrated a noticeable decrease in MMP-9 TRAP, and TNF-awere upregulated in ankle joint tissue in the
immunoreactivity in synovial tissue, cartilage, and subchondral AIA group. Systemic injection of Ya-s11 (9 mg/kg) not only
attenuated AIA-induced pathological changes in the ankle joint,
bone marrow (Fig. 7G, I, L, N). The AIA plus Ya-s11 (3 mg/kg)
but also significantly reduced the osteoclast-related protein
group demonstrated reduced MMP-9 immunoreactivity in sub-
expression.
chondral bone marrow (Fig. 7M) without any apparent inhibition
in synovial tissue or cartilage (Fig. 7H, M).
Figure 8 shows the distribution of TRAP immunoreactivity in Effect of Ya-s11 anti-inflammatory activity in vitro and in
the subchondral bone marrow of the ankle joint in the naı̈ve vivo
(Fig. 8A), AIA (Fig. 8B), AIA plus Ya-s11 (3 mg/kg) (Fig. 8C), and RA is a synovial inflammatory disease characterized by
AIA plus Ya-s11 (9 mg/kg) groups (Fig. 8D). In the AIA group, proliferative synovial fibroblasts and infiltrating cells [2]. Previous
TRAP immunoreactivity appeared to increase in bone marrow. studies have highlighted the important role played by macro-
Administration of 9 mg/kg but not 3 mg/kg Ya-s11 inhibited phages in the process of RA [11,19]. Macrophages mediate
AIA-induced TRAP expression in ankle joint bone marrow synovial inflammation in RA by forming complex cytokine
(Fig. 8C, D). Figure 9 illustrates TNF-a immunoreactivity in networks with neutrophils, lymphocytes, and synovial fibroblasts
synovial tissue from the naı̈ve (Fig. 9A), AIA (Fig. 9B), AIA plus and are also critical to osteoclast differentiation [1,2,13,23,41]. Ya-
Ya-s11 (3 mg/kg) (Fig. 9C), and AIA plus Ya-s11 (9 mg/kg) s11 was able to downregulate iNOS and COX-2 protein
groups (Fig. 9D). TRAP immunoreactivity appeared to increase in expression in LPS-stimulated macrophage RAW 264.7 cells
synovial tissue in the AIA group. (Fig. 2), a well-established in vitro model for assessing the anti-
inflammatory activity of compounds. In the in vivo study, AIA rats
demonstrated a significantly increased number of macrophages in

Figure 7. Effect of Ya-s11 on MMP-9 protein expression in the ankle joint of AIA rats. MMP-9 protein immunoreactivity is shown in red-
brown (arrow) in ankle joint sections from the (A, F, K) naı̈ve, (B, G, L) AIA, (C, H, M) AIA+Ya2s11 (3 mg/kg), and (D, I, N) AIA+Ya2s11 (9 mg/kg)
groups. (F)–(J) show MMP-9 immunoreactivity in the synovial tissue of ankle joints outlined with boxes in (A)–(E), respectively. (K)–(O) show MMP-9
immunoreactivity in the articular cartilage outlined with boxes in (A)–(E), respectively. The immunostaining results indicate the upregulation of MMP-
9 protein expression in the ankle joint (synovial tissue and cartilage) in AIA rat (B, G, L) and inhibition of the AIA-induced upregulation of MMP-9
protein expression in synovial tissue and cartilage by treatment with 9 mg/kg but not 3 mg/kg Ya-s11. (E, J, O) A sample from the AIA group
incubated without primary antibody for MMP-9 showed no specific staining. Scale bar = 100 mm.
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 Effect of Ya-s11 in Adjuvant-Induced Arthritis

Figure 8. Effect of Ya-s11 on TRAP protein expression in subchondral bone marrow of AIA rats. TRAP protein immunoreactivity is shown
in red-brown (arrow) in the bone marrow of ankle joint sections from the (A) naı̈ve, (B) AIA, (C) AIA+Ya2s11 (3 mg/kg), and (D) AIA+Ya2s11 (9 mg/
kg) groups. The immunohistochemical results indicate upregulation of TRAP protein expression in the bone marrow of AIA rats (B). Treatment with
9 mg/kg but not 3 mg/kg Ya-s11 appeared to inhibit the AIA-induced upregulation of TRAP protein expression in bone marrow of the ankle joint. (E)
A sample from the AIA group reacted without the primary antibody for TRAP showed no specific staining. Scale bar = 100 mm.
doi:10.1371/journal.pone.0062926.g008

ankle joint synovial tissue as well as AIA-induced increased of AIA-induced joint destruction with osteoclast-related
neutrophils and lymphocytes with fibroblast proliferation (Fig. 5). protein expression
Although subcutaneous injection of 3 mg/ml Ya-s11 did not Many studies have clearly illustrated the mediation of joint
significantly decrease the number of infiltrating cells in synovial inflammation, pannus formation, and invasion of infiltrating cells
tissue, synovial hyperplasia was reduced in this treatment group into cartilage and bone by continuous release of osteoclast-related
(Fig. 4). Treatment with 9 mg/ml Ya-s11 inhibited synovial proteins [43–45]. TNF-a is a cytokine produced by macrophages
inflammation with reduced cell infiltration in AIA-rats. that also plays an important role in RA joint destruction and may
mediate MMP-9 and cathepsin K expression in RA by upregulating

Figure 9. Effect of Ya-s11 on TNF-a protein expression in synovial tissue of AIA rats. TNF-aÆprotein immunoreactivity is shown in red-
brown (arrow) in the synovial tissue of ankle joint sections from the (A) naı̈ve, (B) AIA, (C) AIA+Ya2s11 (3 mg/kg), and (D) AIA+Ya2s11 (9 mg/kg)
groups. The immunohistochemical results indicated upregulation of TNF-a protein expression in synovial tissue in AIA rats (B).Treatment with 9 mg/
kg but not 3 mg/kg Ya-s11 appeared to inhibit the AIA-induced upregulation of TNF-a protein expression. (E) A sample from the AIA group reacted
without the primary antibody for TRAP showed no specific staining. Scale bar = 100 mm.
doi:10.1371/journal.pone.0062926.g009

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 Effect of Ya-s11 in Adjuvant-Induced Arthritis

the transcription factor c-Fos/AP-1 [10,11]. MMP-9 and cathepsin RA activity in AIA model rats, in which Ya-s11 inhibited TNF-a
K in turn play important roles in osteoclastogenesis and osteoclastic cathepsin K, TRAP, and MMP-9 expression and decreased the
activity [38,40,44] and are expressed by leukocytes, synovial major features of RA pathogenesis. Ya-s11 is a cembrane-type
fibroblasts, chondrocytes, and osteoclasts [39,40,42,43,46,47]. natural compound of marine origin originally isolated from the
Our immunohistochemical analyses revealed AIA-induced MMP- Red Sea [28,49], which we isolated from the soft coral Sinularia
9 and cathepsin K expression in synovial tissue, cartilage, and bone querciformis. A cembrane-type compound was first isolated from
marrow (Fig. 6–7). TRAP, which is considered a marker of Sinularia querciformis in 1985 [50], and since then 8 types of
osteoclasts and plays important roles in osteoclast activity [4], was cembrane-type compound with anti-inflammatory activity have
also observed in bone marrow (Fig. 7). Previous studies have been isolated from this species by Lu et al. [26,27]. However, Ya-
implicated MMP-9, cathepsin K, and TRAP in the bone resorption s11 can also be isolated and purified from the same genus of soft
process in bone marrow [42,48], and our histopathological coral, Sinularia flexibilis, which can be cultured in a culture tank
assessment of AIA rats also indicated significant bone resorption [29], and an increasing number of anti-inflammatory compounds
in the bone marrow (Fig. 4). Hence, the continuous formation of have also been isolated from Sinularia sp. [29,51–54]. The main
these destructive enzymes in joints affected by RA, with the increase structure of Ya-s11 is a cembranolide analogue, the chemical
of infiltrating cells, pannus formation, and cartilage and bone skeleton of which differs from that of steroids, which may further
erosion and resorption, leads to the development of severe arthritis highlight the potential of Ya-s11 as a useful therapeutic agent for
with joint edema [40,44]. Accordingly, in the present study, the AIA rheumatic diseases and other inflammatory disease.
group demonstrated significant differences in foot and paw edema
and clinical evaluation between day 11 and day 28, with edema and
Conclusions
erythema of the ankle joint apparent on typical representative
macroscopic photographs (Fig. 3). In this study, we isolated and purified Ya-s11 from the soft coral
Sinularia querciformis. In vitro study revealed that Ya-s11 significantly
Effect of Ya-s11 on AIA-induced joint destruction inhibited the expression of the proinflammatory proteins iNOS
We demonstrated that Ya-s11 exerts a therapeutic effect on and COX-2 in LPS-challenged murine macrophages cell model.
joint destruction in a rat model of AIA. The therapeutic efficacy of In vivo study revealed that Ya-s11 significantly reduced AIA
Ya-s11 was not limited to general anti-inflammatory effects, but characteristics. Moreover, using histological analysis, we had
included substantial inhibition of cartilage and bone destruction found that Ya-s11 also improved the histopathologic features of
compared to AIA rats, as well as inhibition of osteoclast-related RA. Immunohistochemical analysis showed that Ya-s11 also
protein expression. The AIA plus Ya-s11 (9 mg/kg) group attenuated protein expression of cathepsin K, MMP-9, TRAP,
demonstrated inhibition of MMP-9 and cathepsin K expression and TNF-a in ankle tissues of AIA-rats. We concluded that Ya-s11
in the synovial tissue and bone marrow (Fig. 6) as well as inhibited ameliorated the infiltration of inflammatory cells and bone
MMP-9 protein expression in articular cartilage. Although destruction and downregulated the expression of osteoclast-related
cathepsin K expression was not inhibited in chondrocytes, the proteins in the ankle tissue of AIA rats. Hence, the soft coral-
histological features of the joint display did not display significant derived compound Ya-s11 may serve as a useful therapeutic agent
morphologic changes in the AIA+Ya2s11 (9 mg/kg) group. for the treatment of RA.
TRAP expression in the bone marrow was also inhibited by
treatment with Ya-s11 (9 mg/kg). Immunohistochemical analysis Supporting Information
further demonstrated an increase in TNF-a in the synovial tissue
of the AIA group and its reduction by treatment with Ya-s11 Figure S1 Annexin-V/PI double-staining assay. After
(9 mg/kg). Thus, osteoclast-related protein expression and syno- treating with different does of Ya-s11, RAW264.7 cells induced
vial inflammation were inhibited by Ya-s11, which also demon- by LPS (0.01 mg/ml) were analyzed by fluorescence microscopy by
strated a dose-dependent effect on foot and paw edema and the staining with annexin V-FITC and propidium iodide. (A, G)
clinical evaluation of arthritis and delayed the onset of arthritis. Control group; (B, H) LPS alone group; (C, I) LPS+ Ya-s11
Rats in the Ya-s11 (9 mg/kg) group demonstrated only approx- (1 mM); (D, J) LPS+ Ya-s11 (10 mM); (E, K) LPS+ Ya-s11 (25 mM);
imately 10% increase of foot and paw edema compared to (F, L) LPS+ Ya-s11 (50 mM). The above observations suggest that
baseline, with typical representative macroscopic photographs of different does of Ya-s11 did not induce RAW264.7 cells stimulated
the paw implying that edema was not apparent in the with LPS apoptosis or necrosis significantly (red: stained with
photographs, only erythema. In summary, Ya-s11 demonstrates Annexin V-FITC, green: stained with PI, scale bar = 100 mm).
anti-RA activity, with reduced expression of osteoclast activity- (TIF)
related proteins TNF-a, MMP-9, cathepsin K, and TRAP and
effective reduction of the clinical features of AIA. Author Contributions
Conceived and designed the experiments: Y-YL Y-HJ Z-HW. Performed
Ya-s11 as a potential anti-rheumatoid arthritis agent for
the experiments: Y-YL Y-MS YL S-YH H-CH. Analyzed the data: Y-YL
drug development H-PL W-FC. Contributed reagents/materials/analysis tools: P-JS J-H
The present study demonstrated the efficacy of Ya-s11 as a Sheu J-H Su. Wrote the paper: Y-YL Y-HJ Z-HW.
potential anti-inflammatory compound. We also illustrated its anti-

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