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The FASEB Journal article fj.201700265R. Published online October 25, 2017.
THE
JOURNAL
• RESEARCH •
www.fasebj.org
Ghrelin protects against osteoarthritis through
interplay with Akt and NF-kB signaling pathways
Ruize Qu,*,†,1 Xiaomin Chen,*,†,1 Wenhan Wang,†,‡,1 Cheng Qiu,†,1 Miaomiao Ban,† Linlin Guo,†
Krasimir Vasilev,§ Jianying Chen,{ Weiwei Li,*,2 and Yunpeng Zhao‡,3
*Department of Pathology and ‡Department of Orthopedics, Qilu Hospital, and †Medical School of Shandong University, Shandong
University, Jinan, China; §School of Engineering, University of South Australia, Mawson Lakes, South Australia, Australia; and {Institute of
Biopharmaceuticals of Shandong Province, Jinan, China
ABSTRACT: Osteoarthritis (OA) is a common chronic degenerative disease characterized by degeneration in the joints
and subsequent destruction of cartilage and bone, yet much remains to be elucidated regarding its molecular
mechanism. Ghrelin is a recently discovered neuropeptide with anti-inflammatory actions, but it is unknown
whether ghrelin is involved in OA. Human primary chondrocyte and cartilage samples were collected from patients
with OA, and the expression pattern of ghrelin was assessed. Human chondrocyte and cartilage samples were
stimulated with IL-1b and TNF-a, and exogenous ghrelin-alleviated disorganization of catabolism and anabolism
were mediated by IL-1b and TNF-a. Destabilization of the medial meniscus and anterior cruciate ligament transection models were established in wild-type mice that were administered ghrelin or PBS. Severity of inflammation and degeneration in the joints were determined by measuring the levels of various inflammatory cytokines
and degeneration-associated molecules. Ghrelin down-regulated the production of various inflammatory
cytokines, inhibited apoptosis of chondrocytes, decreased the levels of metalloproteinases (including matrix
metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin motif-5), and maintained
the expression of critical matrix components, such as aggrecan and collagen 2. Moreover, suppression of the Akt
signaling pathway and activation of NF-kB signaling in chondrocytes during OA development was antagonized
by ghrelin administration. This supports the assessment of ghrelin as a potential therapeutic approach to treat
degenerative cartilage diseases, including OA.—Qu, R., Chen, X., Wang, W., Qiu, C., Ban, M., Guo, L., Vasilev, K.,
Chen, J., Li, W., Zhao, Y. Ghrelin protects against osteoarthritis through interplay with Akt and NF-kB signaling
pathways. FASEB J. 32, 000–000 (2018). www.fasebj.org
KEY WORD:
inflammatory cytokines
•
IL-1b
•
TNF-a
•
Osteoarthritis (OA), characterized by destruction of cartilage, an inflammatory reaction of synovium, and abnormal turnover of subchondral bone, is a prevalent type of
arthritis and has become a main cause of disability in aging
people. OA affects millions of patients all over the world
ABBREVIATIONS: ACLT, anterior cruciate ligament transection; ADAMTS,
ADAM metallopeptidase with thrombospondin type 1 motif; Col, collagen; DMM, destabilization of medial meniscus; GAG, glycosaminoglycan;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase; micro-CT, microcomputed tomography; MMP, matrix metallopeptidase; OA, osteoarthritis; OARSI, Osteoarthritis Research Society International; WT, wild type
1
These authors contributed equally to this work.
Correspondence: Department of Pathology, Qilu Hospital, Shandong University, 107 Wenhuaxi Rd., Jinan 250012, China. E-mail: [email protected]
163.com
3
Correspondence: Department of Orthopaedic Surgery, Qilu Hospital,
Shandong University, 107 Wenhuaxi Rd., Jinan 250012, China. E-mail:
[email protected]
2
doi: 10.1096/fj.201700265R
This article includes supplemental data. Please visit http://www.fasebj.org to
obtain this information.
0892-6638/18/0032-0001 © FASEB
cartilage
•
chondrocyte
(1–3). However, much is left to be elucidated regarding the
inductive factors and underlying mechanisms, and there is
no efficient cure for OA in clinic (4, 5). The drugs used in
clinic mainly attenuate symptoms, and arthroplasty surgery is almost inevitable for many patients (3).
Extensive studies have reported that maintenance of
homeostasis in chondrocytes might provide a potential
target for treatment of OA (6, 7). Recent data have reported that various degenerative and inflammatory cytokines are involved in OA (8–10). Among them, IL-1b
and TNF-a are critical proinflammatory cytokines that
can induce other inflammation-associated molecules
(11–13). IL-1b and TNF-a are known to induce critical
catabolic biomarkers in cartilage, such as matrix metallopeptidase (MMP)-13 and ADAM metallopeptidase
with thrombospondin type 1 motif (ADAMTS)-5 (8, 14).
Moreover, these cytokines mediate activation of the
NF-kB signaling pathway, which has been widely reported
to disturb chondrocyte homeostasis (15, 16). Furthermore,
IL-1b and TNF-a impair chondrocyte anabolism, which
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1
leads to diminished production of extracellular matrix in
cartilage (17, 18). Impaired chondrocyte anabolism in OA
has been reported, which is achieved partially through
antagonization of the Akt signaling pathway (19). IL-1b
and TNF-a have become key molecular targets for OA
investigation (20, 21).
Ghrelin is a recently discovered neuropeptide that
plays a potential role in various conditions, including
neuronal activity and cell proliferation (22, 23). Ghrelin
is expressed widely in humans, and previous data have
implied that ghrelin might be a regulatory factor in
several systems (24). Ghrelin is expressed in chondrocytes and plays an important role in chondrocyte
differentiation (25). Previous studies have indicated that
ghrelin is closely associated with various rheumatic
diseases (26, 27). Furthermore, ghrelin is reported to
antagonize the expression and function of IL-1b and
TNF-a in several diseases (28–30). Additionally, ghrelin
is reported to suppress activation of the NF-kB signaling
pathway mediated by IL-1b and TNF-a (30, 31). However, whether ghrelin is involved in OA is unknown.
The objectives of this research are to demonstrate the
expression pattern and potential therapeutic effect of
ghrelin in OA development. In our study, we found that
treatment with ghrelin suppresses OA development.
The therapeutic effect of ghrelin was exerted at multiple
levels and was associated with the alleviation of inflammatory and degenerative response in OA.
MATERIALS AND METHODS
ligament and the anterior cruciate ligament of the right knee joint
were transected. PBS or ghrelin (1 mg/g body weight, i.p.) was
administered every day as previously reported with minor
modification (33, 34). Sham treatment was performed in the left
knee without transection of any ligament. Seven mice were used
at each time point in every group. The mice were euthanized 4 or
8 wk later. Knee joint tissues were isolated and processed for
further evaluation.
Histology and immunohistochemistry
Human cartilage samples and knee joints of mice in each experimental group were collected and fixed with 4% paraformaldehyde for 72 h, decalcified in 10% w/v EDTA for 14 d, dehydrated,
and embedded with paraffin. Sections (5 mm thick) were cut, and
serial sections of each sample were stained with Safranin-O/fast
green/iron hematoxylin. For immunohistochemistry of the indicated biomarkers, sections were pretreated with 0.1% trypsin
for 30 min at 37°C. The other matrix proteins in the cartilage
sections were pretreated with chondroitinase ABC (0.25 U/ml)
(Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 37°C and with
hyaluronidase (1 U/ml) (Sigma-Aldrich) for 60 min at 37°C. To
reduce nonspecific staining, 10% normal goat serum was used for
protein blocking at room temperature for 30 min. Thereafter,
slices were incubated with MMP-13 antibody (1:200 dilution)
(ab3208; Abcam, Cambridge, United Kingdom), ADAMTS-5
pAb (1:100 dilution) (ab41037; Abcam), anti-ghrelin antibody
(1:200 dilution) (ab129383; Abcam), collagen (Col) 10 pAb
(diluted 1:100) (Santa Cruz Biotechnology), and anti–p-IkBa
antibody (diluted 1:100) (Santa Cruz Biotechnology) overnight
at 4°C. Detection was performed using the Vectastain Elite
ABC kit (Vector, Burlingame, CA, USA), and 0.5 mg/ml
3,3-diaminobenzidine in 50 mM Tris-Cl substrate (SigmaAldrich) was used for visualization. Sections were counterstained with 1% hematoxylin.
Mice
All of the animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee of
Shandong University. C57BL/6 background age-matched male
wild-type (WT) mice were purchased from the Laboratory Animal Centre of Shandong University for in vivo experiments of the
current study.
Destabilization of medial meniscus–induced
OA model
In the surgically induced destabilization of medial meniscus (DMM) model, surgery was performed in 10-wk-old WT
mice. Animals were anesthetized with isoflurane. The right knee
joint was destabilized by transection of the medial meniscotibial
ligament to generate destabilization of the medial meniscus, as
previously reported (32). Thereafter, intra-articular injection of
PBS or 2 mg ghrelin (sc-364689; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) was performed every 3 d as previously reported
with minor modification (8). The left knee joint was sham treated,
in which the joint was prepared using the same approach as that
for the right knee joint but without ligament transection. Nine
mice were used per time point in each group. Mice were killed at
4 or 8 wk after surgery. Knee joint tissues were processed for
histologic evaluation or other indicated experiments.
Histopathological and quantificational evaluation
of OA
To grade the proteoglycan content of the articular cartilage on
Safranin-O–stained sections, the Osteoarthritis Research Society
International (OARSI) histology scoring system was performed
for medial tibia plateau as previously reported (8, 35, 36). To
further determine whether the OA changes in mice of each group
were associated with a loss of chondrocytes in cartilage, articular
chondrocytes were counted per unit area, and the articular cartilage thickness was analyzed through Adobe Photoshop 7.0
(Adobe Systems, San Jose, CA, USA). Proteoglycan loss of articular cartilage in surgically induced OA models was scored using
a scoring system as previously reported (37). Briefly, proteoglycan loss was graded as follows: 0, normal; 1, minimal loss; 2,
moderate loss; 3, marked loss; 4, severe, diffuse loss. Five random regions of interest were chosen from each sample, and the
thickness of cartilage within each region of interest was determined. All the mentioned parameters were determined and
averaged in all sections from each mouse, and 4 mice were analyzed from each group. For each mentioned parameter of
the present study, at least 5 sections of each sample underwent
double-blind examinations by 2 authors independently (R.Q.
and X.C.).
ELISA assays for circulating TNF-a and IL-6
Anterior cruciate ligament transection OA model
To demonstrate the protective effect of ghrelin in OA, 10-wk-old
WT mice were generally anesthetized, and the medial collateral
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Vol. 32
February 2018
In the current study, serum was collected from each group of
mice in the OA models, and circulating levels of TNF-a and IL-6
were measured through ELISA as previously reported (38).
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QU ET AL.
Briefly, TNF-a and IL-6 were assessed using a commercial kit
(eBioscience, San Diego CA, USA) according to the manufacturer’s instructions. All the samples were assayed in triplicate and
repeated at least 3 times.
Primary cultures of human chondrocytes
Human cartilage samples were harvested from patients undergoing total knee joint replacement surgery for OA at Qilu
Hospital, Shandong University. Informed consent was collected
from the patients in this study before surgery. The process was
approved by the Institutional Review Board of Shandong University, and all experiments were performed in accordance with
the relevant guidelines and regulations. Primary human articular
chondrocytes were isolated from the articular cartilage by enzymatic digestion as previously reported (39). Briefly, cartilage
slices were minced finely and washed several times with 1% PBS.
They were then digested in a mixture of 0.25% collagenase II in
DMEM without FBS and Penn Strep for 3–5 h in a spinner flask in
an incubator at 37°C and in an atmosphere of 5% CO2. The cell
suspension was used to establish cultures in the T175 dishes.
Within 2–3 d of harvesting, primary chondrocytes were replated
at 80% confluence in 6-well plates before being used in the
experiments.
and 59-TTGTCTCCAAGGGACCAGG-39 (iNOS); 59-CAGTGAGAAGGGCCGAAAGAC-39 and 59-CAGGGGCAGGGAGAAGGAG-39 for NF-kB2; 59-AGAAGGCTGGGGCTCATTTG-39 and
59-AGGGGCCATCCACAGTCTTC-39 (GAPDH). For mice: 59TGAGCCCTGAACACCAGAGAG-39 and 59-AAAGCCAGATGAGCGCTTCTA-39 (ghrelin); 5-AATGCTGGTACTCCAAACCC-3 and 5-CTGGATCGTTATCCAGCAAACAGC-3 (aggrecan);
59-ACTAGTCATCCAGCAAACAGCCAGG-39 and 59-TTGGCTTTGGGAAGAGAC-39 (Col II); 59-AATCTCACAGCAGCACATCA-39 and 59-AAGGTGCTCATGTCCTCATC-39 (IL-1b); 59CCTTCCTACCCCAATTTCCAAT-39 and 59-GCCACTCCTTCTGTGACTCCAG-39 for IL-6; 59-ACAGGAGGGGTTAAAGCTGC-39 and 59-TTGTCTCCAAGGGACCAGG-39 (iNOS); 59GCATTGACGCATCCAAACCC-39 and 59-CGTGGTAGGTCCAGCAAACAGTTAC-39 (ADAMTS-5); 59-ACTTTGTTGCCAATTCCAGG-39 and 59-TTTGAGAACACGGGGAAGAC-39
(MMP-13); 59-AATGCTGACTATGGCTACAAAA-39 and 59AAAACTGATGCGTGAAGTGCTG-39 (COX-2); 59-CAGTGAGAAGGGCCGAAAGAC-39 and 59-CAGGGGCAGGGAGAAGGAG-39 (NF-kB2); 59-AGAACATCATCCCTGCATCC-39 and 59-AGTTGCTGTTGAAGTCGC-39 (GAPDH).
The production of a single specific PCR product was measured through melting curve analysis. The experiments were
repeated 3 times for each indicated molecule.
Western blot
Cartilage explant cultures
Cartilage explants from humans were isolated and cultured as
previously reported (40). Briefly, cartilage samples were isolated
from human tibia plateau cartilage during total knee arthroplasty, which would otherwise be discarded. The collected
samples were then dissected into tiny pieces (diameter, 1 mm;
thickness, 1–2 mm). Thereafter, the cartilage pieces were cultured
in tissue-culture flasks with serum-free DMEM (containing
25 mM HEPES, 2 mM glutamine, 100 mg/ml streptomycin,
100 IU/ml penicillin, and 2.5 mg/ml gentamicin) supplemented with or without IL-1b (10 ng/ml) and ghrelin
(50 ng/ml). After the indicated incubating time, the conditioned
medium was collected for glycosaminoglycan (GAG) synthesis
analysis and other indicated experiments.
All protein extracts were collected from human articular cartilage
or cultured primary chondrocytes of each treatment group, resolved on a 10% SDS polyacrylamide gel, and electroblotted onto
a nitrocellulose membrane. After blocking in 5% nonfat dry milk
in Tris-buffered saline–Tween 20 (10 mM Tris-HCl, pH 8.0;
150 mM NaCl; and 0.5% Tween 20), blots were incubated with
polyclonal ghrelin [diluted 1:1000 (1:200 dilution, ab129383;
Abcam), iNOS (diluted 1:1000, ab3523; Abcam), p-Akt (diluted
1:1000, ab38449; Abcam), total Akt (diluted 1:1000, sc-8312;
Santa Cruz Biotechnology), COX-2 (diluted 1:1000, Santa
Cruz Biotechnology), p-IkBa (diluted 1:1000, Cell Signaling
Technology), or Col 2 (diluted 1:1000, Santa Cruz Biotechnology)] antibody for 1 h. After washing, the horseradish
peroxidase–conjugated secondary antibody (1:2000 dilution) was
added, and bound antibody was detected through an enhanced
chemilumescent system.
Real-time PCR
Total RNA was collected from the articular cartilage or cultured primary chondrocytes of each experimental group
with the RNeasy kit (Qiagen, Germantown, MD, USA) as
previously reported, and first-strand cDNA was established
through the ImProm-II reverse transcription system (Promega, Madison, WI, USA). For mice experiments, several
pieces of cartilage tissue from 3 mice of the same group were
pooled to perform real-time PCR successfully. Real-time
PCR was performed with SYBR Green I dye to monitor DNA
synthesis. Data from each sample were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primers
used for real-time RT-PCR were designed to generate
products between 100 and 200 bp in length. Oligonucleotides used as the specific primers to amplify genes are as follows. For humans: 59-TGAGCCCTGAACACCAGAGAG-39
and 59-AAAGCCAGATGAGCGCTTCTA-39 (ghrelin); 59-AATGCTGGTACTCCAAACCC-39 and 59-CTGGATCGTTATCCAGCAAACAGC-39 (aggrecan); 59-ACTAGTCATCCAGCAAACAGCCAGG-39 and 59-TTGGCTTTGGGAAGAGAC-39 (collagen 2; Col 2); 59-GCAGTATGACAAGTGCGGAGT-39 and
59-CAGGGCTAAATAGGCAGTGAA-39 (ADAMTS-5); 5-ACTTTGTTGCCAATTCCAGG-3 and 5-TTTGAGAACACGGGGAAGAC-3 (MMP-13); 59-ACAGGAGGGGTTAAAGCTGC-39
ROLE OF GHRELIN IN OSTEOARTHRITIS
Immunofluorescence staining
Primary human chondrocytes were cultured on coverslips, and
immunofluorescence staining of ghrelin was then performed on
these cells. Chondrocyte was stimulated with 10 ng/ml IL-1b in
the presence or absence of ghrelin for 1 h. Immunofluorescence
staining of NF-kB p65 was then performed on these cells, as
previously described, and cells were examined using a confocal
fluorescence microscope system (38).
TUNEL staining
TUNEL staining of knee joint articular cartilage from each
treatment group of the DMM model was performed according to
previous reports (8) by using the Promega DeadEnd Colorimetric
TUNEL System (Promega) following the manufacturer’s protocol.
GAG assay for cartilage explants
Cultured human cartilage explants were cultured as indicated. Conditioned media were collected from the human
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3
cartilage explant culture of each group, and the level of GAG
released from explants was assessed using dye DMMB (Polysciences, Warrington, PA, USA). Culture medium was pretreated with 0.5 U/ml of hyaluronidase (Seikagaku, Tokyo,
Japan) at 37°C for 3 h to remove exogenous hyaluronic acid,
which interferes with the DMMB assay. Digests (in duplicate)
were mixed with DMMB in 96-well plates and read at 520 nm
using SpectraMax 384 Microplate Reader (Molecular Devices,
Sunnyvale, CA, USA). The amount of GAG in the conditioned
medium was extrapolated using chondroitin-6-sulfate sodium
salt from shark cartilage (Sigma-Aldrich) as a standard. The
average values of the duplicates were normalized to the wet
weights of cartilage explants. The data were shown as the mean
of GAG released into the condition media from three explants
(treated in separate wells).
Statistical analysis
For comparison of various treatment groups, unpaired MannWhitney t tests, paired Student t tests, and 1-way or 2-way
ANOVA (where appropriate) were performed. For ANOVA,
Bonferroni post hoc analysis was used to compare treatment
groups. Each statistical analysis was performed using GraphPad
Prism, v.4.01 (GraphPad Software, La Jolla, CA, USA). Statistical
significance was achieved at P , 0.05.
RESULTS
Ghrelin is detected in cartilage and is downregulated in IL-1b–stimulated chondrocytes
To date, it is unknown whether the ghrelin expression level is associated with the development of OA. To
investigate the expression pattern of ghrelin, human
cartilage tissue was collected from patients with OA
undergoing arthroplasty. Articular cartilage from patients
with trauma undergoing amputation was used as a control.
Immunohistochemistry for ghrelin was performed in cartilage samples, and ghrelin was detected in cartilage tissue.
Ghrelin was largely expressed within the cytoplasm of
chondrocytes (Fig. 1A, insert). Cartilage tissue was collected
from patients with OA and from the control group, and the
ghrelin expression level was determined using Western
blot. The expression level of ghrelin was diminished in OA
cartilage (Fig. 1B, C). Moreover, real-time PCR performed in
human cartilage showed that the mRNA level of ghrelin
was remarkably decreased in degenerative cartilage when
compared with the control group (Fig. 1D). To further study
the expression pattern of ghrelin in chondrocytes, immunostaining was performed for ghrelin in human chondrocytes. Ghrelin was expressed mainly in the cytoplasm
of chondrocyte (Fig. 1E). To determine whether inflammatory cytokine IL-1b is associated with the expression
level of ghrelin, primary human chondrocytes were
stimulated with 10 ng/ml IL-1b. Thereafter, mRNA and
protein were collected from chondrocytes to test ghrelin
levels. As a result, IL-1b significantly reduced protein
(Fig. 1F, G) and mRNA levels (Fig. 1H) of ghrelin in
chondrocytes. Moreover, an anterior cruciate ligament
transection (ACLT) mouse model was established, and
mRNA as well as protein levels of ghrelin were detected
by real-time PCR (Fig. 1I), Western blot (Fig. 1J, K), and
4
Vol. 32
February 2018
immunohistochemistry (Fig. 1L). The expression level of
ghrelin was decreased in cartilage of ACLT mouse model.
Exogenous ghrelin protects against OA
development in vivo
To investigate whether ghrelin has a protective effect
against OA, an ACLT posttraumatic OA model was
established. Intraperitoneal injection of PBS or ghrelin
(1 mg/g body weight) was performed once every day for 4
or 8 wk. At the 4-wk time point, the knee joint of each
group was collected from the PBS and ghrelin groups, and
immunohistochemistry and Western blot were performed
to detect ghrelin expression. The ghrelin level was elevated
in the ghrelin treatment group compared with the PBS
group, which implied that systemic administration of
ghrelin might reach the articular cartilage of the knee joint
(Supplemental Fig. S1). To determine the protective function
of ghrelin in OA, micro-CT was performed in each group at
the 8-wk time point. Microcomputed-tomography (microCT) analysis showed disorganization of bone structure
and formation of osteophytes after ACLT surgery, which
was attenuated by additional administration of ghrelin
(Fig. 2A). The ACLT model displayed destruction of
articular cartilage in the PBS treatment group, and
ghrelin administration protected the structure of articular cartilage, which was detected by Safranin-O staining (Fig. 2B). Moreover, ghrelin significantly improved
the OARSI score, attenuated proteoglycan loss, and
protected chondrocyte number and cartilage thickness
in cartilage, which were assessed by histologic grading
analysis (Fig. 2C–F). Furthermore, mRNA was isolated and
collected from articular cartilage of the PBS and ghrelin
treatment groups at the 8-wk time point, and levels of
MMP-13, ADAMTS-5, TNF-a, IL-6, iNOS, and COX-2
were tested through real-time PCR. All the mentioned
degeneration-associated molecules induced in the ACLT
OA model were abolished by ghrelin (Fig. 3A–F).
Cartilage samples were collected, and immunohistochemistry was performed for critical metalloproteinases,
including MMP-13 and ADAMTS-5. Expression levels of
both molecules were enhanced in cartilage of the ACLT
model, whereas ghrelin diminished their expression levels
(Fig. 3G). Serum was collected from each group, and circulating levels of IL-6 and TNF-a were assayed with
ELISA. Treatment of ghrelin markedly decreased the
levels of both molecules in the ACLT model (Fig. 3H).
Additionally, total protein was isolated from articular cartilage of the ACLT model, and iNOS and COX-2
expression levels were detected through Western blot.
Ghrelin greatly attenuated induction of iNOS and COX-2
in cartilage tissue of the ACLT model (Fig. 3I–K).
To further investigate the role of exogenous ghrelin in
OA, the DMM model was established in WT mice, and
local delivery of 2 mg ghrelin or PBS was performed
through intra-articular injection every 3 d. Knee joint
samples were collected from the PBS and ghrelin treatment groups, and Safranin-O staining implied that ghrelin
protected the cartilage structure in the DMM model (Fig.
4A). OARSI score, proteoglycan loss, and chondrocyte
number in cartilage as well as cartilage thickness were
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QU ET AL.
Figure 1. Detection and expression pattern of ghrelin chondrocyte and articular cartilage. A) Detection of ghrelin in cartilage
tissue as assayed by immunohistochemistry (n = 4). Scale bar, 200 mm. B, C ) Detection of ghrelin in primary human cartilage as
assayed by Western blot. Significant differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). **P , 0.01 vs.
control group. D) Dampened expression of ghrelin in primary human cartilage measured by real-time PCR. Significant
differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). **P , 0.01 vs. control group. E ) Diminished level
of ghrelin in chondrocyte after treatment of IL-1b as detected by immunostaining (n = 4/group). Scale bars, 50 mm. F, G)
Detection of ghrelin in primary human chondrocyte stimulated by IL-1b as assayed by Western blot. Significant differences are
indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). *P , 0.05 vs. control group. H ) Reduced level of ghrelin in
primary human chondrocytes stimulated by IL-1b as compared with the control group as measured by real-time PCR. Significant
differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). *P , 0.05 vs. control group. I ) Dampened level of
ghrelin in articular cartilage of the ACLT model as compared with the sham control group measured by real-time PCR.
Significant differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). **P , 0.01 vs. control group. J, K )
Decreased expression of ghrelin in cartilage after ACLT operation as detected through Western blot. Significant differences are
indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). **P , 0.01 vs. control group. L) Reduced level of ghrelin in
articular cartilage of the ACLT model as detected by immunohistochemistry (n = 4/group). Scale bars, 100 mm.
assessed by histologic grading analysis (Fig. 4B–E), and
ghrelin treatment improved all the mentioned parameters
in the DMM mouse model. Moreover, cartilage samples
were collected from the sham control group and from
the DMM groups, and immunohistochemistry was performed for ADAMTS-5 and MMP-13. Both molecules
were elevated in the DMM model, and ghrelin suppressed
this alteration in cartilage tissue (Supplemental Fig. S2A).
Furthermore, protein was collected from each group, and
Western blot was performed for iNOS. An enhanced iNOS
level in the DMM model was inhibited by local delivery of
ghrelin (Supplemental Fig. S2B, C).
It has been reported that ghrelin suppresses cell apoptosis (41). In this study, cell death of chondrocytes in the
DMM model was detected through TUNEL staining,
which showed that ghrelin antagonized apoptosis of
ROLE OF GHRELIN IN OSTEOARTHRITIS
chondrocytes in the DMM model (Supplemental Fig. S3A,
upper panels). Hypertrophy of chondrocytes is a feature of
cartilage degeneration in OA development. In this study,
chondrocyte hypertrophy in the DMM model was assayed
through immunohistochemistry for Col 10 and Western
blot for Col 2. As a result, ghrelin treatment inhibited expression of Col 10 (Supplemental Fig. S3A, lower panels)
and maintained the expression level of Col 2 (Supplemental Fig. S3B, C).
Ghrelin down-regulates IL-1b–mediated
catabolism in chondrocytes
To determine the role of ghrelin in catabolism of degenerative chondrocytes, human articular chondrocytes
were isolated and stimulated with IL-1b (10 ng/ml) for
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Figure 2. Effect of ghrelin on
cartilage degeneration in the
ACLT model. A) Ghrelin protected against destruction of
bone structure in the ACTL
mouse model as detected by
micro-CT (n = 4/group). B)
Ghrelin protected the structure
of articular cartilage as detected
by Safranin-O staining (n = 4/
group). Scale bars, 100 mm. C)
OARSI score of OA based on the
result of Safranin-O staining.
After ACLT operation, PBS or
ghrelin was intraperitoneally injected into the mice, and the
samples were collected 4 wk
later. Significant differences are
indicated (2-tailed Student’s
t test; n = 4/group; means 6
SEM). *P , 0.05 vs. control
group. D–F ) Comparison of
OA severity between the PBS
and ghrelin treatment groups
in the ACLT model as assessed
by proteoglycan loss, chondrocyte number, and articular cartilage thickness in the tibia. Significant differences are indicated (2-tailed Student’s t test;
n = 4/group; means 6 SEM). *P , 0.05 vs. control group.
12 h with or without simultaneous stimulation of ghrelin
(50 ng/ml). Ghrelin remarkably suppressed IL-1b–induced
increases of MMP-13, ADAMTS-5, IL-6, and TNF-a, as
assayed by real-time PCR (Fig. 5A–D).
Chondrocytes were stimulated by IL-1b for 72 h. Protein extracts were collected from chondrocytes of each
group, and iNOS expression was tested using Western
blot. iNOS was markedly induced by IL-1b, which was
abolished by additional treatment of ghrelin (Fig. 5E, F).
Conditioned medium of each group was collected, and
ELISA was performed. The release of IL-6 and TNF-a
in the culture medium was greatly reduced after treatment with ghrelin (Fig. 5G). To further study the role of
ghrelin alone in chondrocyte catabolis, primary human
chondrocytes were cultured in the presence or absence
of 50 ng/ml ghrelin for 12 h, and real-time PCR was performed for degeneration-associated biomarkers, including
IL-1b, ADAMTS-5, and MMP-13. Ghrelin alone slightly
reduced the levels of the catabolic and degenerative biomarkers in chondrocytes, but no significant change was
observed (Supplemental Fig. S4A–C).
Ghrelin reverses IL-1b repression of
chondrocyte anabolism
To investigate the role of ghrelin in impaired anabolism in
chondrocyte degeneration, human articular chondrocytes
were isolated and stimulated with IL-1b (10 ng/ml) for
12 h with or without simultaneous stimulation of ghrelin
(50 ng/ml). Ghrelin remarkably suppressed IL-1b–
induced reduction in the mRNA levels of aggrecan, Col
2, and Sox-9, as assayed by real-time PCR (Fig. 6A–C).
Chondrocyte was stimulated by IL-1b for 72 h. Conditioned medium was collected from each group, and
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GAG synthesis was tested. GAG content was markedly
diminished by IL-1b, which was attenuated by ghrelin
(Fig. 6D). Cell extract of each group was collected, and
Western blot was performed. Col 2 level was remarkably
maintained after treatment with ghrelin (Fig. 6E, F). To
further demonstrate the role of ghrelin alone in chondrocyte anabolism, primary human chondrocytes were
cultured in the presence or absence of 50 ng/ml ghrelin for
12 h, and real-time PCR was performed for anabolic biomarkers, including Sox-9, aggrecan, and Col 2. Ghrelin
alone significantly promoted anabolism in chondrocytes
(Supplemental Fig. S4D–F). To investigate whether the
anabolic effect of ghrelin is dependent on the Akt signaling
pathway, primary chondrocytes were stimulated by IL-1b
in the presence or absence of ghrelin. IL-1b diminished
activity of the Akt signaling pathway, whereas ghrelin
largely maintained Akt signaling in chondrocytes
(Fig. 6G–I). Moreover, MK-2206, a typical Akt inhibitor,
markedly suppressed the role of ghrelin in anabolism of
IL-1b–stimulated chondrocytes. Real-time PCR showed
that ghrelin induction of aggrecan, Col 2, and Sox-9 was
remarkably repressed by MK-2206 (Fig. 6J).
Total protein was collected, and Western blot for Col 2
was performed. Maintenance of Col 2 by ghrelin was antagonized by MK-2206 (Fig. 6K, L). Additionally, GAG
synthesis assay (Fig. 6M) showed that ghrelin-mediated
GAG content in conditioned medium was dramatically
inhibited by MK-2206. To study whether activation of the
Akt signaling pathway alone can play a role similar to that
of ghrelin in the attenuation of impaired anabolism during
degeneration, SC-79, a novel Akt activator (42), was used
to treat the IL-1b–stimulated chondrocytes for 12 h, and
real-time PCR for anabolic biomarkers, including aggrecan, Col 2, and Sox-9, was performed. Activation of the
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Figure 3. Effect of ghrelin on catabolic and inflammatory markers in ACLT model. A–F ) Ghrelin antagonized expression of
MMP-13, ADAMTS-5, TNF-a, IL-6, iNOS, and COX-2 in cartilage of the mouse model as assayed by real-time PCR. Total RNA
was isolated, followed by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM).
*P , 0.05; **P , 0.01; ***P , 0.005 vs. indicated control group. G) Ghrelin inhibited expression of MMP-13 and ADAMTS-5 as
detected by immunohistochemistry (n = 4/group). Scale bar, 100 mm. H ) Administration of ghrelin attenuated circulating levels
of IL-6 and TNF-a as measured by ELISA. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6
SEM). *P , 0.05 vs. indicated control group. I–K ) Levels of iNOS and COX-2 in cartilage of the ACLT mouse model
were suppressed by ghrelin. Total protein from each group was extracted, followed by Western blot (n = 3/group). *P , 0.05,
**P , 0.01, ***P , 0.005 vs. indicated control group.
Akt signaling pathway through SC-79 greatly abolished
IL-1b mediation of impaired anabolism (Supplemental
Fig. S5).
Ghrelin represses degeneration of cartilage
explants induced by IL-1b ex vivo
It is well established that IL-1b mediates cartilage destruction in OA (43). To demonstrate the function of
ghrelin in cartilage destruction, cartilage samples from
patients with OA were isolated and cultured with 10
ng/ml IL-1b in the presence or absence of 50 ng/ml
ghrelin for 48 h, followed by Safranin-O staining. IL-1b
enhanced the loss of proteoglycan, whereas the additional
treatment of ghrelin largely rescued this effect of IL-1b
(Fig. 7A).
Total mRNA was collected from all the groups,
followed by real-time PCR. Production of Col2 and
aggrecan in cartilage was markedly diminished by
IL-1b, whereas this effect was antagonized by ghrelin
(Fig. 7B, C). Levels of catabolism-associated molecules,
including MMP-13 and ADAMTS-5, were significantly higher in the IL-1b–treated group, and additional ghrelin administration greatly reduced the levels
ROLE OF GHRELIN IN OSTEOARTHRITIS
of these molecules (Fig. 7D, E). Total protein was collected
from all the groups, and Western blot was performed
for iNOS. IL-1b induction of iNOS was dramatically
inhibited by ghrelin (Fig. 7F, G).
Culture medium was collected from each group for
testing the TNF-a level. Ghrelin significantly reduced
expression of TNF-a in the conditioned medium (Fig.
7H). Culture medium was also collected to analyze the
release of GAG using DMMB assay. Ghrelin treatment
markedly reduced the release of GAG into the culture
medium from IL-1b–treated human cartilage samples
(Fig. 7I).
Ghrelin attenuates TNF-a–induced
disorganization of metabolism
in chondrocytes
Because TNF-a plays a detrimental role in OA development and chondrocyte degeneration (44) and ghrelin antagonized TNF-a in several conditions (45), we determined
the role of ghrelin in TNF-a–mediated chondrocyte catabolism. Human articular chondrocytes were isolated and
stimulated with TNF-a (10 ng/ml) for 12 h with or without simultaneous administration of ghrelin (50 ng/ml).
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7
Figure 4. Function of ghrelin on destruction of cartilage structure in the DMM model. A) Ghrelin protected the structure of
articular cartilage in the DMM model as detected by Safranin-O staining (n = 4/group). Scale bars, 100 mm. B) OARSI score of
OA based on the result of Safranin-O staining. After the DMM operation, PBS or ghrelin was intraperitoneally injected into
the mice, and the samples were collected 4 wk later. Significant differences are indicated (2-tailed Student’s t test; n = 4–6/
group; means 6 SEM). *P , 0.05 vs. control group. C–E ) Comparison of OA severity between PBS and ghrelin treatment
groups in the DMM model as assessed by proteoglycan loss, chondrocyte number, and articular cartilage thickness in the
tibia. Significant differences are indicated (2-tailed Student’s t test; n = 4–6/group; means 6 SEM). *P , 0.05, **P , 0.01 vs.
control group.
Ghrelin markedly suppressed the TNF-a–mediated increase in the mRNA levels of MMP-13, ADAMTS-5, and
IL-1b, as assayed by real-time PCR (Fig. 8A).
IL-6 has been reported to be closely associated with
cartilage degeneration (46). In the present study,
chondrocytes were stimulated by TNF-a for 72 h with
PBS or ghrelin. Conditioned medium was collected
from each group, and IL-6 expression was tested using ELISA. IL-6 was markedly induced by TNF-a in
chondrocytes, which was rescued by additional treatment of ghrelin (Fig. 8B). Protein extracts were collected,
and Western blot was performed for iNOS. iNOS was
Figure 5. Effect of ghrelin on catabolic and inflammatory markers in IL-1b–treated human articular chondrocyte. A–D) Human
articular chondrocyte was treated with ghrelin, followed by treatment with IL-1b. Total RNA was isolated, and levels of mRNA for
MMP-13, ADAMTS-5, IL-6, and TNF-a were detected by real-time PCR. Significant differences are indicated (2-tailed Student’s
t test; n = 4–6/group; means 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005 vs. control group. E, F ) Treatment of ghrelin attenuated
IL-1b–mediated expression of iNOS as assayed by Western blot. Significant differences are indicated (2-tailed Student’s t test;
n = 3/group). *P , 0.05; **P , 0.01 vs. control group. G) Levels of IL-6 as well as TNF-a were suppressed by ghrelin. Conditioned
medium from each group was collected, and ELISA was performed. Significant differences are indicated (2-tailed Student’s t test;
n = 4–6/group; means 6 SEM). *P , 0.05 vs. indicated control group.
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QU ET AL.
Figure 6. Ghrelin reverses IL-1b repression of anabolism in chondrocyte through activation of the Akt signaling pathway. A–C )
Ghrelin remarkably suppressed the IL-1b–induced reduction of aggrecan, Col 2, and Sox-9 as assayed by real-time PCR.
Significant differences are indicated (2-tailed Student’s t test; n = 4–6/group; means 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005
vs. control group. D) GAG content was markedly diminished by IL-1b, which was attenuated by ghrelin. Chondrocyte was
stimulated by IL-1b for 72 h. Conditioned medium was collected from chondrocyte of each group, and GAG synthesis was tested.
Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. control
group. E, F ) Ghrelin remarkably maintained Col 2 level suppressed by IL-1b. Chondrocyte was stimulated by IL-1b for 72 h, and
cell extract of each group was collected, followed by Western blot. Significant differences are indicated (2-tailed Student’s t test;
n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. control group. G–I ) IL-1b diminished activity of the Akt signaling pathway,
whereas ghrelin largely maintained Akt signaling in chondrocyte as measured by Western blot. Primary chondrocyte was
stimulated by IL-1b in the presence or absence of ghrelin. Significant differences are indicated (2-tailed Student’s t test; n = 3/
group; means 6 SEM). ***P , 0.005 vs. indicated control group. NS, no significant difference. J ) Ghrelin induction of aggrecan,
Col 2, and Sox-9 was remarkably repressed by MK-2206 as indicated by real time PCR. Significant differences are indicated
(2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005 vs. indicated control group. K, L)
Maintenance of Col 2 by ghrelin was antagonized by MK-2206. Total protein was collected, and Western blot for Col 2 was
performed. *P , 0.05, **P , 0.01 vs. indicated control group. M ) Ghrelin-mediated GAG content in conditioned medium was
dramatically inhibited by MK-2206 as detected by GAG synthesis assay. Significant differences are indicated (2-tailed Student’s
t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group.
greatly reduced after treatment with ghrelin in a dosedependent manner (Fig. 8C).
To investigate the role of ghrelin in disorganized anabolism mediated by TNF-a, human articular chondrocytes
were isolated and stimulated with TNF-a (10 ng/ml)
for 12 h with or without simultaneous stimulation of ghrelin (50 ng/ml). Real-time PCR indicated
that ghrelin inhibited impairment of anabolism biomarkers induced by TNF-a, including aggrecan, Sox9, and Col 2 (Fig. 8D). Chondrocytes were stimulated
by TNF-a for 72 h with PBS or ghrelin, and protein
extracts were collected from chondrocytes of each
group. Ghrelin attenuated the reduction of Col 2 mediated by TNF-a (Fig. 8E). In addition, chondrocytes
were stimulated by TNF-a for 72 h, conditioned
ROLE OF GHRELIN IN OSTEOARTHRITIS
medium was collected from chondrocytes of each group,
and GAG synthesis was tested. GAG content was
markedly diminished by TNF-a but was greatly improved by ghrelin (Fig. 8F).
Ghrelin antagonizes exaggerated catabolism
in degenerative chondrocytes by inhibiting
the NF-kB signaling pathway
Disorganization of metabolism in OA involves impaired
anabolism and abnormally enhanced catabolism, and activation of NF-kB signaling is known to play a predominant role in cartilage degradation of OA (47). It is
reported that NF-kB signaling represses anabolism of
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9
Figure 7. Protective role of
ghrelin in IL-1b–induced degeneration of cartilage explants. A) IL-1b–mediated loss
of proteoglycan in cartilage
explants was rescued by ghrelin
as detected by Safranin-O staining (n = 4–6/group). Scale bar,
100 mm. B, C ) Production of
Col 2 and aggrecan in cartilage
were markedly diminished by
IL-1b, but this effect was antagonized by ghrelin as measured
by real-time PCR. Total mRNA
was isolated from each group of
cartilage as assessed by realtime PCR. Significant differences are indicated (2-tailed
Student’s t test; n = 3/group;
means 6 SEM). *P , 0.05,
**P , 0.01 vs. indicated control
group. D, E ) Levels of MMP-13
and ADAMTS-5 induced by IL1b. Ghrelin treatment abolished this effect as assayed by
real-time PCR. Significant differences are indicated (2-tailed
Student’s t test; n = 3/group;
means 6 SEM). *P , 0.05,
**P , 0.01 vs. indicated control
group. F, G) IL-1b induction of
iNOS was dramatically inhibited by ghrelin as detected by Western blot. Total protein was extracted from cartilage of all groups,
and Western blot was performed for iNOS. Culture medium was collected for testing TNF-a. Significant differences are indicated
(2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. indicated control group. H ) Ghrelin reduced
expression of TNF-a in culture medium as measured by ELISA. Culture medium was collected, and ELISA was performed.
Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group.
I ) Ghrelin treatment markedly decreased the release of GAG into the culture medium from IL-1b–treated human cartilage
samples. Culture medium was collected to analyze the release of GAG using DMMB assay. Significant differences are indicated (2tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group, ***P , 0.005 vs. indicated control
group.
chondrocytes (48) and that the NF-kB signaling pathway
triggers production of catabolic and inflammatory biomarkers in chondrocytes (49). In the current study, the
results suggest that ghrelin activated Akt signaling and
promoted anabolism of chondrocytes while suppressing
the expression of catabolic and inflammatory biomarkers.
Based on these data, we determined whether treatment of
ghrelin affected the signaling activity of NF-kB in surgically induced OA models. Total protein extracts were
collected from cartilage of an ACLT mouse model. Ghrelin
treatment greatly impaired phosphorylation of IkBa in the
ACLT OA model (Fig. 9A, B). Immunohistochemistry of
p-IkBa was also performed for cartilage of the ACLT
model, and ghrelin markedly suppressed p-IkBa expression (Fig. 9C).
mRNA and protein were collected from cartilage samples of the DMM model. Real-time PCR
for NF-kB2 (Supplemental Fig. S6A) and Western blot
for p-IkBa (Supplemental Fig. S6B) indicated that
ghrelin treatment suppressed activation of the NF-kB
signaling pathway. Furthermore, immunohistochemistry
of p-IkBa revealed that ghrelin inhibited phosphorylation of IkBa in the DMM model (Supplemental
Fig. S6C).
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To further investigate whether ghrelin represses enhanced activity of the NF-kB signaling pathway in primary chondrocytes, human chondrocytes were isolated
and treated with IL-1b in the presence or absence of
ghrelin, and the NF-kB signaling pathway was assayed.
The NF-kB2 level was measured with real-time PCR, and
IL-1b–mediated NF-kB2 was antagonized with ghrelin
treatment (Fig. 9D). Total protein was collected from
chondrocytes. Ghrelin attenuated p-IkBa expression as
compared with the PBS group (Fig. 9E, F). Nuclear translocation of NF-kB p65 is a parameter for activity of NF-kB
signaling. In this study, cell immunostaining was performed for chondrocytes of each treatment group, and
ghrelin greatly antagonized nuclear translocation of
NF-kB p65 (Fig. 9G). These findings reveal that ghrelin
treatment reduces the degeneration-associated activity of the NF-kB signaling pathway in chondrocytes.
DISCUSSION
OA is a common degenerative disease worldwide
and is mainly characterized by destruction of articular cartilage (50). Proinflammatory cytokines, including
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QU ET AL.
Figure 8. Protective role of ghrelin on TNF-a–mediated disorganization of metabolism in human articular chondrocytes. A)
Human articular chondrocytes were treated with ghrelin, followed by treatment with TNF-a. Total RNA was isolated, and levels of
mRNA for MMP-13, ADAMTS-5, and IL-1b were detected by real-time PCR. Significant differences are indicated (2-tailed
Student’s t test; n = 4–6/group; mean 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005 vs. indicated control group. B) The TNFa–mediated elevation of IL-6 level was suppressed by ghrelin. Conditioned medium from each group was collected, and ELISA
for IL-6 was performed. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; mean 6 SEM). *P , 0.05,
**P , 0.01 vs. indicated control group. C ) Treatment of ghrelin attenuated IL-1b–mediated expression of iNOS as assayed by
Western blot. Each experiment was repeated 3 times. D) TNF-a–induced impairment of Aggrecan, Col 2, and Sox-9 expression
was remarkably repressed by ghrelin as indicated by real-time PCR. Significant differences are indicated (2-tailed Student’s t test;
n = 4–6/group; mean 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005 vs. indicated control group. E ) TNF-a–induced reduction of
Col 2 level in chondrocyte was antagonized by ghrelin. Total protein was collected, and Western blot for Col 2 was performed.
Each experiment was repeated 3 times. F ) TNF-a inhibited GAG content in conditioned medium, which was antagonized by
additional ghrelin as detected by GAG synthesis assay. Significant differences are indicated (2-tailed Student’s t test; n = 3/group;
means 6 SEM). *P , 0.05 vs. indicated control group.
TNF-a and IL-1b, are well known to induce OA, and
previous data have shown the potential antagonization
between these cytokines and ghrelin in several conditions (30, 51, 52). In the current study, we demonstrated
that ghrelin was detected in human articular cartilage
and that the ghrelin expression level was remarkably
diminished in degenerative cartilage, inflammatory
cytokine–stimulated chondrocytes, and cartilage in a
surgically induced arthritis model, which implies the
potential involvement of ghrelin in the development
of OA.
Previous studies have reported the potential role of
ghrelin in patients who underwent hip joint replacement
(53). However, the function of ghrelin in the development
of OA is unknown. Mouse models of surgically induced
OA play a key role in studies of OA (54, 55). Among them,
the DMM model and the ACLT model are widely used for
identification of OA-related molecules (55). In the current
study, both models were established, and systemic as well
as local delivery methods of ghrelin were used. The halflife of ghrelin is short, and repeated intraperitoneal injection of ghrelin was required to achieve a therapeutic
result in several cases (56–58). In this study, we performed
ROLE OF GHRELIN IN OSTEOARTHRITIS
repeated intraperitoneal injection and tested the expression level of ghrelin. An elevated ghrelin level was observed in articular cartilage tissue of the ACLT model,
suggesting that systemic ghrelin administration reached
the cartilage tissue. Development of OA was assayed
through histology as well as other experiments. As a result,
systemic and local delivery of ghrelin markedly alleviated
the severity of OA in both surgically induced OA models,
which implied that ghrelin might play a protective role in
degeneration of articular cartilage.
Exaggerated catabolism and inflammatory reaction has
been extensively studied and widely reported during development of OA (59). It is known that inflammatory cytokines TNF-a and IL-1b induce expression of molecules
associated with OA in human chondrocyte, including
ADAMTS-5 and MMP-13, which degrade the critical
matrix components aggrecan and Col 2, respectively (60).
Furthermore, oxidative stress reaction and inflammatory
response are observed in cartilage degeneration, during
which process COX-2, IL-6, iNOS, and TNF-a levels are
usually enhanced (61). Herein, we assessed all the mentioned biomarkers in IL-1b as well as TNF-a–induced
chondrocytes and found that the additional use of ghrelin
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Figure 9. Ghrelin antagonizes IL-1b induction of catabolism by inhibiting the NF-kB signaling pathway. A, B) Ghrelin treatment
impaired phosphorylation of IkBa in OA models as detected by Western blot. Protein was collected from cartilage of ACLT
models in the PBS and ghrelin treatment groups, and Western blot was performed. Significant differences are indicated (2-tailed
Student’s t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group. C ) Ghrelin markedly suppressed p-IkBa
expression in cartilage in the ACLT model as shown by immunohistochemistry of p-IkBa (n = 3/group). Scale bar, 100 mm. D)
IL-1b–mediated NF-kB2 expression in primary chondrocyte was antagonized by ghrelin treatment. Human chondrocytes were
isolated and treated with IL-1b for 12 h in the presence or absence of ghrelin. Significant differences are indicated (2-tailed
Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. indicated control group. E, F ) Ghrelin attenuated pIkB-a
expression as compared with the PBS group with stimulation of IL-1b. Human chondrocyte was treated with IL-1b for 12 h in the
presence or absence of ghrelin, followed by Western blot. Significant differences are indicated (2-tailed Student’s t test; n = 3/
group; means 6 SEM). **P , 0.01 vs. indicated control group. G) Ghrelin suppressed IL-1b induction of NF-kB p65 nuclear
translocation as detected by cell immunostaining. Human chondrocytes were treated with IL-1b for 12 h in the presence or
absence of ghrelin, and cell immunostaining was performed (n = 3/group).
greatly reduced the mentioned molecules associated with
OA.
Exaggerated apoptosis of chondrocyte is commonly
observed with cartilage degeneration, and how to antagonize apoptosis has become a therapeutic target in OA
(62). In this study, ghrelin treatment suppressed chondrocyte apoptosis in the DMM OA mouse model. Chondrocyte hypertrophy is another parameter to detect the
development of OA (63), and in the current study, exogenous ghrelin inhibited chondrocyte hypertrophy in the
DMM model, supporting the therapeutic potential of
ghrelin in OA.
Diminished production of extracellular matrix components, including Col 2 and aggrecan, is a feature in the
aging process of cartilage (64). A critical aspect of the
therapeutic strategy of OA is to attenuate the loss of Col
2 and aggrecan (65). It is known that IL-1b and TNF-a
lead to reduced matrix production and elevated loss of
these extracellular matrix components in cartilage (16,
66). In the current study, to imitate degeneration, primary human chondrocyte and cartilage explants were
cultured and stimulated with IL-1b or TNF-a in the
presence or absence of ghrelin. As a result, Col 2 and
aggrecan expression levels were protected, which suggested the protective role of ghrelin in disorganized
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anabolism of cartilage. Moreover, activation of the Akt
signaling pathway is reported to promote chondrocyte
anabolism, and impaired activity of Akt signaling in
chondrocyte suppresses chondrocyte anabolism in OA
development (67). Ghrelin is reported to activate Akt
signaling and to promote the regeneration procedure in
several conditions (42, 68, 69). In this study, ghrelin
maintained phosphorylation of the Akt signaling pathway, and a specific Akt inhibitor, MK-2206, repressed
the protective role of ghrelin. Moreover, it is known that
activation of Akt signaling maintains the impaired
anabolism of chondrocyte during degeneration (70).
In this study, a commercial Akt activator, SC-79 (54),
was used to antagonize the detrimental role of IL-1b
in anabolism, which had similar effect compared
with ghrelin. This finding further suggested that ghrelin
might suppress the disorganized anabolism through Akt
signaling.
The NF-kB signaling pathway is a key mediator of
age-dependent cartilage degeneration (71). Studies have
shown that activation of NF-kB signaling accelerates—
whereas suppression of this signaling attenuates—
cartilage-degenerative diseases associated with aging
(15, 72). Phosphorylation of IkBa is a commonly tested
parameter for the activation of the NF-kB signaling
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QU ET AL.
pathway (21). In our research, we found that the level
of p-IkBa was markedly elevated in inflammatory
cytokine–treated chondrocytes and degenerative cartilage, although this was largely abolished by exogenous ghrelin. Moreover, nuclear transaction of p65 is a
well-established feature for activation of the NF-kB
signaling pathway (73, 74), which was inhibited by
ghrelin in the current study. Taken together, these data
suggest that excessive activation of the NF-kB signaling pathway in chondrocytes induced by inflammatory cytokines or OA was repressed by ghrelin, which
may lead to the attenuation of cartilage degeneration.
Our results show that ghrelin inhibits cartilage degeneration through maintaining chondrocyte homeostasis, which may have implications for the treatment
of OA.
ACKNOWLEDGMENTS
This work was supported by Key Research and Development Projects of Shandong Province (Grant 2015GSF118115),
the Natural Science Foundation of Shandong Province
(Grants BS2014YY048 and BS2015SW028), and the National
Natural Science Foundation of China (Grants 81501880 and
26010105131643). The authors declare no conflicts of interest.
AUTHOR CONTRIBUTIONS
K. Vasilev, W. Li, and Y. Zhao, conceived and designed the
experiments; R. Qu, W. Wang, C. Qiu, and L. Guo performed
the experiments; R. Qu, M. Ban, and J. Chen, analyzed the
data; and W. Li and Y. Zhao contributed reagents, materials,
and analysis tools.
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Received for publication March 26, 2017.
Accepted for publication October 16, 2017.
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15
Ghrelin protects against osteoarthritis through interplay with Akt
and NF- κB signaling pathways
Ruize Qu, Xiaomin Chen, Wenhan Wang, et al.
FASEB J published online October 25, 2017
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