Antagonism of GPR4 with NE 52-QQ57 and the Suppression of AGE- Induced Degradation of Type II Collagen in Human Chondrocytes
Haochuan Liu, Yulong Liu, and Bing Chen*
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ABSTRACT: Osteoarthritis (OA) is a common degenerative joint disease for
which an effective therapeutic strategy has not yet been established. AGEs are
widely recognized as a contributor to OA pathogenesis. GPR4, a recently
discovered proton-sensing transmembrane receptor, has been shown to possess a
wide range of physiological functions. However, the potential role of this receptor
in chondrocytes and the pathogenesis of OA is unclear. In the present study, we
investigated the potential of GPR4 to modulate the effects of advanced glycation
end products (AGEs) in SW1353 human chondrocytes. First, we demonstrate
that GPR4 is fairly expressed in SW1353 chondrocytes and that exposure to AGEs
increases the expression of this transmembrane receptor. Second, we found that
antagonism of GPR4 with NE 52-QQ57 significantly inhibited the AGE-induced
increased expression of several key inflammatory cytokines and signaling
molecules, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-
6
, inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase 2
(COX2), and prostaglandin E (PGE ). We also found that antagonisn of GPR4 had a remarkable ability to rescue type II collagen
2
2
from AGE-induced degradation by inhibiting the expression of matrix metalloproteinase (MMP)-3 and MMP-13. As a key pro-
inflammatory signaling pathway, we further tested the effect of GPR4 antagonism on the activation of nuclear factor-κB (NF-κB) and
found that NF-κB activation was indeed suppressed, thereby indicating that the NF-κB signaling pathway may mediate the effects of
GPR4 antagonism described above. These findings provide a basis for further research into the role of GPR4 -mediated signaling in
OA.
1
. INTRODUCTION
In recent years, the pivotal role of inflammation in OA has
received increasing attention. Tumor necrosis factor-α (TNF-
α) is a major cytokine involved in AGE-mediated inflamma-
tion. AGE-induced dysfunctional chondrocytes produce high
levels of TNF-α and interleukin (IL)-1β, which triggers the
release of IL-6 and ECM-degrading proteoglycans, such as
matrix metalloproteinase (MMP)-3 and MMP-13, while
Osteoarthritis (OA) is an increasingly common joint disease
characterized by excessive destruction of the articular
extracellular matrix (ECM). Chondrocytes are the main cell
type found in articular cartilage and serve a dual regulatory role
by synthesizing new cartilage tissue and destroying old or
injured tissue. However, in OA, chondrocyte dysfunction leads
to excessive degradation of the ECM and irreversible joint
damage. Factors involved in the initiation of OA include
obesity, gender, genetics, mechanical injury, diet, and most
significantly, age. Advanced glycation end-products (AGEs)
inhibiting the synthesis of aggrecan and type II collagen, the
1
,2
11,12
main structural components of the articular ECM.
Inhibiting the degradation of type II collagen, which has a
particularly slow rate of turnover, is an attractive treatment
3
,4
result from the process of nonenzymatic glycation involving
reducing sugars, amino acids, lipids, and DNA, and accumulate
in tissues over time. The accumulation of AGEs has been
associated with a wide range of intractable and degenerative
diseases, including diabetes, chronic kidney disease, Alz-
target. TNF-α and IL-6 are associated with age-related
systemic inflammation, which is recognized as a major
contributor to OA and other age-related pathologies. IL-6 is
strongly correlated with reduced physical function and the
heimer’s disease, and OA, among others.5 In chondrocytes,
AGEs induce the expression of proinflammatory cytokines,
secretion of degradative enzymes, and activation cellular
−8
Received: March 24, 2020
Published: May 6, 2020
signaling pathways, including the so-called “master switch” of
9
inflammation, nuclear factor-κB (NF-κB). Recent research has
demonstrated that inhibiting the effects of AGEs may be a
method to slow or halt the progression of OA.10
©
XXXX American Chemical Society
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development of degenerative diseases, and increased serum
levels of IL-6 are viewed as a biomarker for the pathogenesis of
OA. Additionally, IL-1β induces the expression of inducible
2.3. Western Blot Analysis. After the indicated treatment, RIPA
buffer supplemented with protease inhibitor cocktail was used to lyse
the chondrocytes. A total of 20 μg of cell lysates was loaded onto 4−
1
3
2
0% precasted poly acrylamide gel electrophoresis (PAGE) gel and
nitric oxide synthase (iNOS), which mediates nitric oxide
(NO) production. NO is an important proinflammatory
mediator that contributes to the pathogenesis of OA by
driving the destruction of type II collagen.14 Prostaglandin E2
(PGE2) is a product of arachidonic acid metabolism that has
been shown to promote cartilage degradation, bone remodel-
ing, and cartilage turnover in OA.15 Numerous studies have
focused on reducing the expression of these cytokines and
signaling molecules to hinder cartilage destruction as a
potential treatment method for OA. However, safe and
effective treatment targets remain to be identified.
the proteins were separated according to size. The separated protein
mixture was then transferred onto polyvinylidene fluoride (PVDF)
membranes. The membranes were blotted against the primary
°
antibodies overnight at 4 C and, after washing 3 times, were
incubated with corresponding HRP-conjugated secondary antibodies
for 1 h at room temperature. HRP substrate and ImageJ software were
used to detect and visualize the resulting protein signals.
2
.4. ELISA. The protein secretions of the target genes were
measured using enzyme-linked immunosorbent assay (ELISA).
Briefly, the supernatants were collected from the culture medium by
centrifugation at 1000 rpm for 10 min. Commercial ELISA kits for
TNF-α, IL-1β, IL-6, PGE , MMP-3, and MMP-13 were purchased
2
G protein-coupled receptors (GPCRs) have been receiving
increasing attention for their diverse range of physiological
roles. GPR4 is a proton-sensing transmembrane receptor that
is activated in response to low pH levels. Antagonism of this
receptor has been shown to reduce intestinal inflammation in
from R&D Systems and used in accordance with the manufacturer’s
instructions. The results are presented as fold-changes.
2.5. Determination of NO. We performed a cellular staining
experiment using the cell-permeable fluorescent probe 4-amino-5-
methylamino-2′,7′-difluorofluorescein (DAF-FM DA; Thermo Fisher
Scientific, U.S.A.) to determine the intracellular production of NO.
Briefly, SW1353 chondrocytes were grown to full confluence on a 96-
well plate and then incubated with 5 μM DAF-FM DA for 10 min.
The fluorescent signals were visualized with excitation at 495 nm and
emission detection at 515 nm via fluorescence microscopy.
1
6
17
mice and modulate nociception and angiogenesis, which
may be of use in the treatment of OA. Interestingly, recent
research has shown that activation of GPR4 by acidosis can
alter cytoskeletal phenotype and cell migration.18 However,
there is little known regarding the role of GPR4 in cartilage
and particularly, in the pathogenesis of OA. In the present
study, we identified that GPR4 is expressed in chondrocytes
and increased upon exposure to AGEs. Antagonism of GPR4
with its antagonist NE 52-QQ57 (chemical name 2-(4-((2-
ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)-
phenyl)-5-(piperidin-4-yl)-1,3,4-oxadiazole) significantly re-
duced the AGE-induced expression of various inflammatory
mediators and degradative enzymes, thereby demonstrating a
potential role for GPR4 antagonism in the prevention and
2.6. NF-κB Luciferase Reporter Assay.
Promoter luciferase
activity was measured to evaluate the transcriptional activity of NF-
κB. Briefly, the cells were cotransfected with NF-κB promoter
(Beyotime Biotechnology, China) and pRL-TK firefly promoter
(Promega, U.S.A.) plasmids using Lipofectamine 2000 reagent
(Thermo Fisher Scientific, U.S.A.). After the indicated treatment,
the total cell lysates were collected and a dual luciferase reporter assay
(Promega, U.S.A.) was used to measure the dual luciferase activity of
renilla and NF-κB. The relative luciferase activity was calculated by
normalizing the firefly to the renilla luciferase activity.
2.7. Statistical Analysis. The experimental data from all
procedures are presented as means ±
SEM. One-way analysis of
variance (ANOVA) followed by Bonferroni’s posthoc test were used
to determine the statistical significance of differences. It was calculated
that a P value of <0.05 represented a statistically significant difference.
1
9
treatment of OA.
2
. MATERIALS AND METHODS
2
.1. Cell Culture and Treatment. Human chondrosarcoma cell
line SW1353 chondrocytes purchased from the American Type
Culture Collection (ATCC) were used in all experiments. Human
umbilical vein endothelial cells (HUVECs) from ATCC were used as
a positive control in the GPR4 expression experiment. The cells were
stored in a humidified incubator with 95% oxygen and 5% CO at 37
3. RESULTS
3
.1. GPR4 as Expressed in Human Chondrocytes and
Increased by AGEs. We began by confirming that GPR4 is
expressed in SW1353 chondrocytes. HUVECs have been
shown to express GPR420 and were used as a positive control.
As shown in Figure 1A,B, GPR4 is indeed expressed at both
the mRNA and protein levels. Next, we determined whether
the expression of GPR4 is responsive to AGE treatment. The
results show that, upon stimulation with 50, 100, and 200 μg/
mL AGEs, the expression of GPR4 increased 1.9-, 2.6-, and
2
°
C and maintained in Leibovitz’s L-15 medium supplemented with
1
6
0% FBS and 1% penicillin/streptomycin. The cells were seeded into
-well plates and grown to full confluence. For AGE treatment
experiments, the AGE treatment reagent was freshly prepared in 150
mg/mL stock solution. The confluent SW1353 cells were then treated
with 50, 100, and 150 μg/mL AGEs for 24 h to determine the AGE-
induced expression of GPR4. For subsequent experiments, the cells
were stimulated with 100 μg/mL AGEs for 24 h in the presence or
absence of the GPR4 antagonist NE 52-QQ57 (0.5 and 1 μM;
Novartis, Basal, Switzerland). To measure the activity of NF-κB, the
cells were stimulated with AGEs for 2 h in the presence or absence of
NE 52-QQ57.
3
.1-fold at the mRNA level (Figure 2A) and 1.8-, 2.4-, and 2.9-
2
.2. Reverse-Transcription Polymerase Chain Reaction (RT-
PCR) Analysis. For analysis of the mRNA expression of the target
genes, RNA was extracted from SW1353 cells using a commercial
RNA MiniPrep Purification Kit (Qiagen) in accordance with the
procedures in the manufacturer’s manual. Briefly, cDNA was
synthesized using 1 μg of isolated RNA with an RT-PCR One-Step
Kit (Bio-Rad, U.S.A.). The SYBR Green-based real-time PCR method
was used to measure the mRNA transcripts of the target genes on the
ABI 7500 Real-Time PCR platform. The expression levels of each
target gene were normalized to GAPDH and calculated using the
Figure 1. GPR4 is expressed in human SW1353 chondrocytes. The
expression of GPR4 was detected with human umbilical vein
endothelial cells (HUVECs) as a positive control. (A) mRNA of
GPR4 was measured by reverse-transcription PCR; (B) protein of
GPR4 was measured by Western blot analysis.
−
ΔΔCT
2
method.
B
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Figure 2. AGEs increased the expression of GPR4 in human SW1353 chondrocytes. Cells were stimulated with 50, 100, and 200 μg/mL AGEs for
2
4 h. (A) mRNA of GPR4; (B) protein of GPR4 (**, ****, P < 0.01, 0.0001 vs vehicle group).
fold at the protein level (Figure 2B), respectively. Thus, the
expression of GPR4 is considerably increased upon exposure to
AGEs.
antagonism of GPR4 dose-dependently ameliorated this effect
to only 2-fold.
3.4. Antagonism of GPR4 and the Inhibition of the
Degradation of Type II Collagen. Next, we determined the
effect of GPR4 antagonism on the degradation of type II
collagen mediated by MMP-3 and MMP-13. The expression of
these two enzymes was measured at the mRNA and protein
levels using real-time PCR and ELISA. Exposure to AGEs
increased the mRNA expression of MMP-3 and MMP-13 to
3.6- and 4.2-fold. However, the addition of 0.5 and 1 μM NE
3
.2. Antagonism of GPR4 and the Reduction of the
Expression of Proinflammatory Cytokines Induced by
AGEs. Next, we investigated the effects of antagonism of
GPR4 using NE 52-QQ57 on the expression of several key
inflammatory cytokines by SW1353 chondrocytes treated with
AGEs. The molecular structure of NE 52-QQ57 is shown in
Figure 3. We measured the mRNA expression of TNF-α, IL-
5
2
2-QQ57 dose-dependently reduced this increase to less than
-fold (Figure 7A,B). At the protein level, antagonism of GPR4
dose-dependently reduced the protein secretion of MMP-3 and
MMP-13 to only roughly 1.5-fold (Figure 7C,D). To
determine whether this decrease in degradative enzymes
inhibited the degradation of type II collagen, we employed
Western blot analysis. The results in Figure 8 show that
stimulation with AGEs induced a decrease in type II collagen
of 53%, while antagonism of GPR4 dose-dependently
mitigated this decrease to only 28% and 7%, thereby suggesting
a potent ability of GPR4 antagonism to prevent MMP-
mediated cartilage destruction.
Figure 3. Molecular structure of NE 52-QQ57.
1
β, and IL-6 via real-time PCR and found that 0.5 and 1 μM
NE 52-QQ57 dose-dependently reduced the AGE-induced
increase in TNF-α from 4.5-fold to only 3.2- and 2.1-fold, IL-
3
.5. Antagonism of GPR4 and the Inhibition of the
1β from 3.6-fold to 2.4- and 1.6-fold, and IL-6 from 5.5-fold to
3
.6- and 2.3-fold, respectively (Figures 4A−C). At the protein
Activation of NF-κB. NF-κB is widely regarded as a master
regulator of inflammation. Here, we determined whether
antagonism of GPR4 could suppress the activation of NF-κB.
As shown in Figure 9A, there was an increase of 3.5-fold in the
nuclear translocation of p65 protein, which was reduced to 2.4-
and 1.7-fold by NE 52-QQ57 (Figure 9A). The results of
luciferase reporter assay demonstrate that the increase in the
luciferase activity of NF-κB of 3-fold induced by AGEs was
only 2- and 1.2-fold in the presence of the two respective doses
of GPR4 antagonist (Figure 9B).
level, a similar effect of GPR4 antagonism was observed, dose-
dependently reducing the increased secretion of these three
cytokines (Figure 4D−F).
3
.3. Antagonism of GPR4 and the Reduction of the
Expression of Inflammatory Mediators Induced by
AGEs. Here, we assessed the effect of GPR4 antagonism on
the AGE-induced expression of several important inflammatory
mediators. First, we determined the effect on iNOS and NO
production via real-time PCR, Western blot analysis, and DAF-
FM DA staining. As shown in Figure 5A,B, AGEs increased the
mRNA and protein expression of iNOS 3.7- and 2.9-fold, while
the two doses of GPR4 antagonist dose-dependently reduced
these increases to only 2.4- and 1.7-fold at the mRNA level and
4
. DISCUSSION
In the present study, we examined the role of the pH-sensing
transmembrane receptor GPR4 in human chondrocytes
stimulated with AGEs. Researchers have postulated a
connection between acidity and osteoarthritis since as far
back as 1935,20 and “matrix acidosis” has been cited as a
contributor to chondrocyte apoptosis.21 More recent studies
have shown, that while many degradative enzymes operate at
relatively neutral pH, others cleave type II collagen more
efficiently at a lower pH. For example, MMP-3 is most active at
a pH of 5.5, while cathepsin K, an acidic cystine
endoproteinase, is activated by acidic conditions and operates
at a pH of 4.5−6.22 As a newly discovered GPCR, there is
2
.1- and 1.6-fold at the protein level. Consistently, we found
that GPR4 antagonism dose-dependently reduced the increase
in NO production from 2.9-fold to only 2.2- and 1.7-fold
(Figure 5C). Next, we determined the effect on AGE-induced
COX2 and PGE2 expression. The mRNA and protein
expression of COX2 increased 3.3- and 2.8-fold, while the
two doses of GPR4 antagonist reduced these levels to 2.3- and
1
.6-fold at the mRNA level and 2.2- and 1.5-fold at the protein
level (Figure 6A,B). The results of ELISA in Figure 6C show
that AGEs increased the secretion of PGE2 nearly 4-fold, while
C
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Figure 4. Treatment with the GPR4 antagonist NE 52-QQ57 reduced AGE-induced expression and secretion of pro-inflammatory cytokines TNF-
α, IL-1β, and IL-6 in human SW1353 chondrocytes. Cells were stimulated with 100 μg/mL AGEs in the presence or absence of NE 52-QQ57 (0.5
and 1 μM) for 24 h. (A) mRNA of TNF-α; (B) mRNA of IL-1β; (C) mRNA of IL-6; (D) secretions of TNF-α; (E) secretions of IL-1β; (F)
secretions of IL-6 (****, P < 0.0001 vs vehicle group; #, ##, ###, ####, P < 0.05, 0.01, 0.001, 0.0001 vs AGEs treatment group).
Figure 5. Treatment with the GPR4 antagonist NE 52-QQ57 reducedAGE-induced expression of inducible nitric oxide synthase (iNOS) and
production of nitric oxide (NO). Cells were stimulated with 100 μg/mL AGEs in the presence or absence of NE 52-QQ57 (0.5 and 1 μM) for 24
h. (A) mRNA of iNOS as measured by real-time PCR; (B) protein of iNOS as measured by Western blot; (C) production of NO as measured by
DAF-FM DA staining (****, P < 0.0001 vs vehicle group; #, ##, ###, ####, P < 0.05, 0.01, 0.001, 0.0001 vs AGEs treatment group).
D
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Figure 6. Treatment with the GPR4 antagonist NE 52-QQ57 prevented AGE-induced expression of cyclooxygenase 2 (COX2) and production of
prostaglandin E (PGE ). Cells were stimulated with 100 μg/mL AGEs in the presence or absence of NE 52-QQ57 (0.5, 1 μM) for 24 h. (A)
2
2
mRNA of COX2 as measured by real-time PCR; (B) protein of COX2 as measured by Western blot; (C) production of PGE2 as measured by
ELISA (****, P < 0.0001 vs vehicle group; #, ###, ####, P < 0.05, 0.001, 0.0001 vs AGEs treatment group).
Figure 7. Treatment with the GPR4 antagonist NE 52-QQ57 inhibited AGE-induced expression of MMP-3 and MMP-13. Cells were stimulated
with 100 μg/mL AGEs in the presence or absence of NE 52-QQ57 (0.5 and 1 μM) for 24 h. (A) mRNA of MMP-3; (B) mRNA of MMP-13; (C)
protein of MMP-3 as measured by ELISA; (D) protein of MMP-13 as measured by ELISA (****, P < 0.0001 vs vehicle group; ###, ####, P <
0
.001, 0.0001 vs AGEs treatment group).
Figure 8. Treatment with the GPR4 antagonist NE 52-QQ57 prevented AGE-induced degradation of type II collagen. Cells were stimulated with
00 μg/mL AGEs in the presence or absence of NE 52-QQ57 (0.5 and 1 μM) for 24 h. Protein level of type II collagen was measured by Western
blot analysis (****, P < 0.0001 vs vehicle group; ##, ####, P < 0.01, 0.0001 vs AGEs treatment group).
1
limited research regarding the physiological effects of GPR4.
GPR4 acts by altering adenylate cyclase activity and has been
shown to be moderately expressed in bone.23 Interestingly,
GPR4 has been shown to be expressed in rheumatoid arthritis-
E
https://dx.doi.org/10.1021/acs.chemrestox.0c00111
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Figure 9. Treatment with the GPR4 antagonist NE 52-QQ57 prevented AGE-induced activation of NF-κB. Cells were stimulated with 100 μg/mL
AGEs in the presence or absence of NE 52-QQ57 (0.5 and 1 μM) for 2 h. (A) Nuclear translocation of NF-κB p65; (B) luciferase activity of NF-κB
(****, P < 0.0001 vs vehicle group; ##, ####, P < 0.01, 0.0001 vs AGEs treatment group).
affected synoviocytes and synovial tissues, and the associated
acidic environment increases the expression of degradative
enzymes, including MMPs.24 However, there is little
information regarding GPR4 expression in cartilage tissue.
Here, we confirmed that GPR4 is expressed in human
chondrocytes at both the mRNA and protein levels and
AGEs increased the expression of this receptor. However, the
possible connection between AGEs and acidity is not yet
tered in the cytoplasm in its inactive form. Upon activation,
p65 protein, the precursor to NF-κB, is translocated to the
nucleus where it triggers the transcription of NF- B and a
wide-reaching inflammatory cascade.35 To better elucidate the
mechanism behind GPR4 antagonist-mediated improved levels
of type II collagen and inflammatory cytokines, we measured
the effect on the activation of NF-κB. Indeed, antagonism of
GPR4 significantly reduced the level of nuclear p65 and the
activity of NF-κB. Taken together, our findings indicate a
potential role for the proton-sensing receptor GPR4 in
mediating the effects of AGEs in OA. As a newly discovered
receptor, little is known about the extent of the effects of
GPR4. Further research will provide valuable information
regarding the possibilities of GPR4 agonism/antagonism as a
potential treatment for a wide range of diseases. The promising
results of the present study provide a basis for further research
on the type II collagen-rescuing properties of GPR4
antagonism.
κ
2
5
clear.
Reducing the expression of pro-inflammatory cytokines and
other mediators is an important aspect of treatment for
countless diseases, including OA. In recent years, OA has come
to be recognized as a chronic inflammatory disease, with TNF-
α, IL-1β, and IL-6 being of great significance to the
pathogenesis of the disease.26 IL-1β induces an inflammatory
cascade by triggering the expression of TNF-α, NO, COX2,
2
7
and PGE . Previous research has shown that acidosis
2
increases the expression of TNF-α, PGE , and RNA for bone
cell receptor activator of NF-κB ligand (RANKL), a precursor
2
■
AUTHOR INFORMATION
to NF-κB activation.28 It has also been shown that TNF-α and
IL-1β promote acidosis-induced chondrocyte apoptosis and
reduced type II collagen synthesis.29 Here, we found that,
while AGEs increased the expression of these cytokines,
antagonism of GPR4 suppressed this effect. GPR4 antagonism
also decreased the expression of iNOS, which likely mediated
the reduction in NO production.
Corresponding Author
Bing Chen − Department of Anesthesiology, China-Japan Union
Hospital of Jilin University, Changchun City, Jilin Province
130033, China; orcid.org/0000-0002-9638-0161;
Phone: +86-431-8499513; Email: [email protected]
Authors
The progressive degradation of type II collagen is the main
hallmark of OA. Type II collagen is the most abundant
structural component in articular cartilage and the concen-
tration of type II collagen degradative byproducts in urine
correlates with the severity of the disease.30 Due to its
extremely slow rate of turnover, type II collagen is considered
to be essentially irreplaceable.31 Thus, it is of utmost
importance to slow or halt the deterioration of type II collagen
in patients with OA. MMP-3 (stromelysin-1) and MMP-13
(collagenase-3) are the primary zinc-dependent catabolic
Haochuan Liu − Department of Orthopaedics, China-Japan
Union Hospital of Jilin University, Changchun City, Jilin
Province 130033, China
Yulong Liu − Department of Orthopaedics, China-Japan Union
Hospital of Jilin University, Changchun City, Jilin Province
130033, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrestox.0c00111
Notes
The authors declare no competing financial interest.
enzymes responsible for type II collagen degradation via
cleavage of the collagen triple helix at the P4−P11′ site.3
Reducing the expression of these enzymes is an important
aspect of a therapeutic approach to slow the progression of
OA. In the present study, we found that antagonism of GPR4
significantly reduced the AGE-induced increase in MMP-3 and
MMP-13 expression. We further confirmed that this action
resulted in a remarkable amelioration of AGE-induced type II
collagen degradation. NF-κB is well-recognized as a promoter
of MMP-3 and MMP-13 expression and type II collagen
2,33
■
ACKNOWLEDGMENTS
This study is Supported by Young Scholar Research Grant of
Chinese Anesthesiologist Association(21900004).
■
REFERENCES
(1) Glyn-Jones, S., Palmer, A. J., Agricola, R., Price, A. J., Vincent, T.
L., Weinans, H., and Carr, A. J. (2015) Osteoarthritis. Lancet 386
(9991), 376−87.
3
4
degradation. In healthy conditions, NF-κB remains seques-
F
https://dx.doi.org/10.1021/acs.chemrestox.0c00111
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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(2) Ma, F., Li, G., Yu, Y., Xu, J., and Wu, X. (2019) MiR-33b-3p
promotes chondrocyte proliferation and inhibits chondrocyte
apoptosis and cartilage ECM degradation by targeting DNMT3A in
osteoarthritis. Biochem. Biophys. Res. Commun. 519 (2), 430−7.
(3) Palazzo, C., Nguyen, C., Lefevre-Colau, M. M., Rannou, F., and
Poiraudeau, S. (2016) Risk factors and burden of osteoarthritis.
Annals of physical and rehabilitation medicine. 59 (3), 134−8.
(4) Silverwood, V., Blagojevic-Bucknall, M., Jinks, C., Jordan, J. L.,
Protheroe, J., and Jordan, K. P. (2015) Current evidence on risk
factors for knee osteoarthritis in older adults: a systematic review and
meta-analysis. Osteoarthritis and cartilage. 23 (4), 507−15.
endothelial cell adhesion through the cAMP/Epac pathway. PLoS One
6 (11), No. e27586.
(20) Hartung, E. F., and Steinbrocker, O. (1935) Gastric acidity in
chronic arthritis. Ann. Intern. Med. 9 (3), 252
−
7.
(21) Mobasheri, A. (2002) Role of chondrocyte death and
hypocellularity in ageing human articular cartilage and the patho-
genesis of osteoarthritis. Med. Hypotheses 58 (3), 193
(22) Konttinen, Y. T., Mandelin, J., Li, T. F., Salo, J., Lassus, J.,
m, M., Hukkanen, M., Takagi, M., Virtanen, I., and Santavirta,
−
7.
Liljestro
̈
S. (2002) Acidic cysteine endoproteinase cathepsin K in the
degeneration of the superficial articular hyaline cartilage in osteo-
arthritis. Arthritis Rheum. 46 (4), 953−
60.
(5) Nowotny, K., Jung, T., Ho
(2015) Advanced glycation end products and oxidative stress in type
diabetes mellitus. Biomolecules 5 (1), 194−222.
̈
hn, A., Weber, D., and Grune, T.
(23) Frick, K. K., Krieger, N. S., Nehrke, K., and Bushinsky, D. A.
(2009) Metabolic acidosis increases intracellular calcium in bone cells
through activation of the proton receptor OGR1. J. Bone Miner. Res.
2
(6) Stinghen, A. E., Massy, Z. A., Vlassara, H., Striker, G. E., and
Boullier, A. (2016) Uremic toxicity of advanced glycation end
products in CKD. J. Am. Soc. Nephrol. 27 (2), 354−70.
(7) Byun, K., Yoo, Y., Son, M., Lee, J., Jeong, G. B., Park, Y. M.,
Salekdeh, G. H., and Lee, B. (2017) Advanced glycation end-products
produced systemically and by macrophages: A common contributor
to inflammation and degenerative diseases. Pharmacol. Ther. 177, 44−
24 (2), 305
−
13.
(24) Albloui, F. S. (2014) FRI0343 Acidification Enhances Acid
Sensing Protein and Matrix Metalloproteinase Expression in Human
Rheumatoid Fibroblast like Synoviocytes. Ann. Rheum. Dis. 73, 511.
(25) bae Park, J., Yoo, Y., Ong, B. X., Kim, J., and Cho, H. S. (2017)
Purification, crystallization and X-ray diffraction of heparan sulfate
bounded human RAGE. Biodesign 5, 122−5.
5
5.
(26) Feng, Z., Zheng, W., Li, X., Lin, J., Xie, C., Li, H., Cheng, L.,
Wu, A., and Ni, W. (2017) Cryptotanshinone protects against IL-1β-
induced inflammation in human osteoarthritis chondrocytes and
ameliorates the progression of osteoarthritis in mice. Int. Immuno-
pharmacol. 50, 161−7.
(27) Zheng, W., Zhang, H., Jin, Y., Wang, Q., Chen, L., Feng, Z.,
Chen, H., and Wu, Y. (2017) Butein inhibits IL-1β-induced
inflammatory response in human osteoarthritis chondrocytes and
slows the progression of osteoarthritis in mice. Int. Immunopharmacol.
(8) Li, Y., Zhang, Y., Chen, C., Zhang, H., Ma, C., and Xia, Y. (2016)
Establishment of a rabbit model to study the influence of advanced
glycation end products accumulation on osteoarthritis and the
protective effect of pioglitazone. Osteoarthritis and cartilage. 24 (2),
3
07−14.
(9) Lei, C., Wu, S., Wen, C., Li, Y., Liu, N., Huang, J., Li, L., Fu, M.,
and Liu, J. (2019) Zafirlukast attenuates advanced glycation end-
products (AGEs)-induced degradation of articular extracellular matrix
(ECM). Int. Immunopharmacol. 68, 68−73.
(10) Zhang, H. B., Zhang, Y., Chen, C., Li, Y. Q., Ma, C., and Wang,
Z. J. (2016) Pioglitazone inhibits advanced glycation end product-
induced matrix metalloproteinases and apoptosis by suppressing the
activation of MAPK and NF-κB. Apoptosis 21 (10), 1082−93.
(11) Wang, Y., Xu, J., Zhang, X., Wang, C., Huang, Y., Dai, K., and
Zhang, X. (2017) TNF-α-induced LRG1 promotes angiogenesis and
mesenchymal stem cell migration in the subchondral bone during
osteoarthritis. Cell Death Dis. 8 (3), No. e2715.
(12) Han, P. F., Wei, L., Duan, Z. Q., Zhang, Z. L., Chen, T. Y., Lu,
J. G., Zhao, R. P., Cao, X. M., Li, P. C., Lv, Z., and Wei, X. C. (2018)
Contribution of IL-1β, 6 and TNF-α to the form of post-traumatic
osteoarthritis induced by “idealized” anterior cruciate ligament
reconstruction in a porcine model. Int. Immunopharmacol. 65, 212−
4
2, 1−0.
(28) Frick, K. K., LaPlante, K., and Bushinsky, D. A. (2005) RANK
ligand and TNF-α mediate acid-induced bone calcium efflux in vitro.
American Journal of Physiology-Renal Physiology. 289 (5), F1005−11.
(29) Zhou, R. P., Dai, B. B., Xie, Y. Y., Wu, X. S., Wang, Z. S., Li, Y.,
Wang, Z. Q., Zu, S. Q., Ge, J. F., and Chen, F. H. (2018) Interleukin-
1
β and tumor necrosis factor-α augment acidosis-induced rat articular
chondrocyte apoptosis via nuclear factor-kappa B-dependent
upregulation of ASIC1a channel. Biochim. Biophys. Acta, Mol. Basis
Dis. 1864 (1), 162−77.
(30) Bakilan, F., Armagan, O., Ozgen, M., Tascioglu, F., Bolluk, O.,
and Alatas, O. (2016) Effects of native type II collagen treatment on
knee osteoarthritis: A Randomized Controlled Trial. Eurasian J. Med.
4
8 (2), 95.
2
0.
(31) Heinemeier, K. M. (2017) Type II Collagen; Designed to Last
(13) Greene, M. A., and Loeser, R. F. (2015) Aging-related
a Lifetime? Osteoarthritis and Cartilage. 25, S5.
inflammation in osteoarthritis. Osteoarthritis and cartilage. 23 (11),
(32) Ma, J.-D., Zhou, J.-J., Zheng, D.-H., Chen, L.-F., Mo, Y.-Q.,
Wei, X.-n., Yang, L.-J., and Dai, L. (2014) Serum matrix metal-
loproteinase-3 as a noninvasive biomarker of histological synovitis for
diagnosis of rheumatoid arthritis. Mediators Inflammation 2014, 1.
(33) Gudmann, N. S., and Karsdal, M. A. (2016) Type II Collagen.
In Biochemistry of Collagens pp 13−20, Laminins and Elastin.
(34) Li, Y., Wang, L. M., Xu, J. Z., Tian, K., Gu, C. X., and Li, Z. F.
(2017) Gastrodia elata attenuates inflammatory response by
inhibiting the NF-κB pathway in rheumatoid arthritis fibroblast-like
synoviocytes. Biomed. Pharmacother. 85, 177−81.
(35) Liu, W., Wang, X., Zheng, Y., Shang, G., Huang, J., Tao, J., and
Chen, L. (2016) Electroacupuncture inhibits inflammatory injury by
targeting the miR 9 mediated NF κB signaling pathway following
ischemic stroke. Mol. Med. Rep. 13 (2), 1618−26.
1
966−71.
(14) Ma, Z., Wang, Y., Piao, T., and Liu, J. (2016) Echinocystic acid
inhibits IL-1β-induced COX-2 and iNOS expression in human
osteoarthritis chondrocytes. Inflammation 39 (2), 543−9.
(15) Akhtar, N., Khan, N. M., Ashruf, O. S., and Haqqi, T. M.
(2017) Inhibition of cartilage degradation and suppression of PGE2
and MMPs expression by pomegranate fruit extract in a model of
posttraumatic osteoarthritis. Nutrition 33, 1−3.
(16) Sanderlin, E. J., Marie, M., Velcicky, J., Loetscher, P., and Yang,
L. V. (2019) Pharmacological inhibition of GPR4 remediates
intestinal inflammation in a mouse colitis model. Eur. J. Pharmacol.
8
52, 218−30.
(17) Velcicky, J., Miltz, W., Oberhauser, B., Orain, D., Vaupel, A.,
Weigand, K., Dawson King, J., Littlewood-Evans, A., Nash, M., Feifel,
R., and Loetscher, P. (2017) Development of selective, orally active
GPR4 antagonists with modulatory effects on nociception, inflam-
mation, and angiogenesis. J. Med. Chem. 60 (9), 3672−3683.
(18) Krewson, E. A., Yang, L. V., and Dong, L. (2017) Acidic tumor
microenvironment stimulation of GPR4 alters cytoskeletal dynamics
and migration of vascular endothelial cells. Tumor Biol., 1993.
(19) Chen, A., Dong, L., Leffler, N. R., Asch, A. S., Witte, O. N., and
Yang, L. V. (2011) Activation of GPR4 by acidosis increases
G
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