α‐Mangostin promotes apoptosis of human rheumatoid
arthritis fibroblast‐like synoviocytes by reactive oxygen
species‐dependent activation of ERK1/2 mitogen‐activated
Xiaoyun Sheng1 | Jun Li2 | Chao Zhang3 | Lianggong Zhao1 | Laiwei Guo1 |
Tianen Xu1 | Jiaxin Jin1 | Meng Wu1 | Yayi Xia1,4
Department of Orthopaedics, Lanzhou
University Second Hospital, Lanzhou,
Gansu, PR China
The Anesthesia Surgery Clinical Medical
Center, Lanzhou University Second
Hospital, Lanzhou, Gansu, PR China
Department of Spine Surgery, The
Second Xiangya Hospital of Central South
University, Changsha, Hunan, PR China
Gansu Key Laboratory of Orthopaedics,
lanzhou, Gansu, China
Yayi Xia, Department of Orthopaedics,
Lanzhou University Second Hospital,
No. 82 Cuiyingmen, Lanzhou, 730000
Gansu, PR China; Gansu Key Laboratory
of Orthopaedics, Lanzhou, Gansu, PR
Email: [email protected];
α‐Mangostin (α‐M) is a commonly used traditional medicine with various
biological and pharmacological activities. Our study aimed to explore the effects
and mechanism of α‐M in regulating apoptosis of rheumatoid arthritis
fibroblast‐like synoviocytes (RA‐FLS). α‐M of 10 to 100 μM was used to treat
RA‐FLS for 24 hours, followed by measuring cell viability and apoptosis. The
involvement of reactive oxygen species (ROS) and mitogen‐activated protein
kinases was detected. Treatment of α‐M promoted apoptosis and reduced
viability of RA‐FLS in a dose‐dependent manner. The mitochondrial membrane
potential in RA‐FLS was remarkably reduced by α‐M treatment, accompanied
by the cytochrome c accumulation in the cytosol and increased activities of
caspase‐3 and caspase‐9. Moreover, we found that α‐M treatment promoted
ROS production and extracellular signal‐regulated kinase 1/2 (ERK1/2)
phosphorylation. The proapoptotic activity of α‐M in RA‐FLS was markedly
reversed by the co‐induction with the ERK1/2 inhibitor LY3214996 or ROS
scavenger N‐acetyl‐L‐cysteine. In conclusion, our studies found that α‐M had
remarkable proapoptotic activities in RA‐FLS, which is regulated by the
induction of ROS accumulation and ERK1/2 phosphorylation. α‐M may thus
have potential therapeutic effects for rheumatoid arthritis.
apoptosis, extracellular signal‐regulated kinase 1/2, rheumatoid arthritis, rheumatoid arthritis
fibroblast‐like synoviocytes, α‐mangostin
1 | INTRODUCTION
Rheumatoid arthritis (RA) is a systemic inflammatory
disease that primarily affects the joints. In RA, the immune
system damages the lining of the joints and synovium,
which leads to the destruction of bone and articular
cartilage. Mechanism of the disease is highly complex and
the underlying mechanisms are still being elucidated.
However, it is known that fibroblast‐like synoviocytes
(FLS) play a critical role in the pathogenesis of RA.1 As
the disease progresses, the activation of FLS leads to the
production of various proinflammatory mediators that
J Cell Biochem. 2019;1-9. wileyonlinelibrary.com/journal/jcb © 2019 Wiley Periodicals, Inc. | 1
signal to recruit, retain, and activate local immune cells.
These cells, along with resident tissue cells, cause tissue
destruction via an unregulated immune response.2 The
synovium, usually consisting of an inner lining and three
layers of adjacent cells, is expanded to form multiple layers
of proliferative and destructive tissue in RA. Known as
pannus, this is a direct result of increased numbers of FLS,
which actively and aggressively remodel the tissue structure
of the joint via inflammatory processes. These cells can
contribute to local cartilage degradation and promote
synovial inflammation by releasing chemokines and
cytokines, and metalloproteases that break down the
extracellular cellular matrix.3 In addition, the release of
pro‐inflammatory molecules and growth factors can
activate FLS to secrete interleukins and prostanoids to
further propagate the unhealthy immune response.4
Furthermore, additional studies have indicated that FLS
directly interact with infiltrating T and B cells via LFA‐1/
ICAM‐1 to secrete inflammatory cytokines.5
Inactivation or removal of FLS presents a therapeutic
approach for treating RA, based upon the pivotal role
played by these cells in disease initiation and progression
via inflammatory processes. α‐Mangostin (α‐M) is a
naturally occurring xanthone with a range of biological
activities and the potential to be used as a treatment for
many diseases, including cancers6,7 and obesity.8 α‐M is
isolated from the mangosteen tree and displays activity as
an antineoplastic agent, an antioxidant, an antiproliferative,
and an inducer of apoptosis.9 α‐M was shown to induce
cervical cancer cell apoptosis by activating ASK1/p38
signaling and promoting the production of reactive oxygen
species (ROS).10 Many studies have demonstrated the anti‐
inflammatory properties of the compound and it has been
shown to reduce edema through anti‐inflammatory
effects.11 Regarding arthritis, the compound reduced
immunocytoadherence in animal studies and inhibited
primary and secondary responses to adjuvant‐induced
disease.12 Other studies have also shown that xanthones
can ease experimental arthritis in mice by inhibiting
proliferation of FLS through modulation of mitogen‐
activated protein kinase (MAPK) signaling.13,14
Previous studies have shown that the therapeutic
effect of α‐M in RA is mainly via immunosuppressive
effects, cytokine regulation, and antioxidant activity.
However, more recent studies have indicated that α‐M
is also able to induce the apoptosis of human rheumatoid
arthritis fibroblast‐like synoviocytes (RA‐FLS) by increasing ROS accumulation and the ratio of Bax/Bcl‐2,15
which suggests that α‐M may act via additional,
previously unknown pathways. The current study aims
to uncover additional mechanisms related to the apoptosis‐inducing abilities of α‐M by studying the effects of
α‐M on apoptosis of RA‐FLS. The results obtained show
that α‐M is able to significantly inhibit the viability of
RA‐FLS and increase the proportion of apoptotic cells in
a concentration‐dependent manner. We also show that
these remarkable proapoptotic activities are regulated by
the accumulation of ROS and extracellular signal‐
regulated kinase 1/2 (ERK1/2) phosphorylation. Data
provide new insights into the mechanisms of RA
progression and indicate that α‐M may be a promising
treatment for RA by reducing FLS survival.
2 | MATERIALS AND METHODS
2.1 | Cell treatment
The medical ethics committee of our hospital (LUSH‐
2016‐0042; approval date: 23 May 2016) approved this
study, and all patients provided informed consent. Five
female patients with RA (aged 48‐69 years) who were
treated with synovectomy or joint replacement donated
synovial tissues. Healthy control synovial tissue samples
were obtained from five emergent trauma amputation
patients consists of three males and two females. All
healthy control specimens were free from RA or
osteoarthritis. To isolate synoviocytes, synovial tissues
were cut and subjected to collagenase digestion in
Dulbeccoʼs modified Eagleʼs medium (DMEM)/F12
medium at 37°C for 2 hours. DMEM/F12 medium
supplemented with 10% fetal bovine serum was used as
a cell culture medium and cell culture conditions were
37°C and 5% CO2. α‐M (Sigma‐Aldrich, MO) of different
concentrations (10‐100 μM) were used to treat the cells
for 24 hours. In inhibition experiments, ERK1/2 inhibitor
N‐acetylcysteine (NAC) (5 mM) or LY3214996 (1 mM)
was used to treat cells for 1 or 2 hours before exposure to
2.2 | Cell viability measurement
Cells were transferred to a 96‐well plate with 104 cells in
each well. Cells were cultivated under conditions of 37°C
and 5% CO2 overnight and 0.1% dimethyl sulfoxide
(DMSO) and α‐M (various dosages) were added to treat
cells for 24 hours. After that, MTT was added and the
final concentration was 5 mg/mL. Cells were cultivated
for additional 4 hours, and DMSO was added. Finally,
optical density (OD) values were measured at 570 nm.
2.3 | Flow cytometry
Cells were transferred to a six‐well plate with 105 cells per
well. After cell culture for 24 hours, incubation with 0.1%
DMSO or α‐M was performed for 72 hours. After that,
cells were harvested and mixed with binding buffer,
2 | SHENG ET AL.
followed by treatment with FITC‐conjugated PI and
annexin V (Becton Dickinson Biosciences, San Jose, CA)
for 20 minutes in the dark. Finally, FACSCalibur flow
cytometer (Becton Dickinson Biosciences) was used to
detect apoptotic cells.
2.4 | Mitochondrial membrane
Rhodamine‐123 (Rho‐123) dye was used to measure
mitochondrial membrane potential. When 60% confluence
was reached, cells were treated with 100 μM α‐M or 0.1%
DMSO in a six‐well plate with 106 cells per well. After
24 hours, cells were washed and stained with Rho‐123
(1 mL/L) at 37°C for 30 minutes in the dark. Rho‐123
fluorescence was quenched by mitochondrial energization
and the fluorescence emissions were analyzed by flow
cytometry. The rate of fluorescence decay was represented as
the loss of Δψm.
2.5 | Western blot analysis
) were mixed with 1 mL radioimmunoprecipitation assay buffer to extract total protein and cells
concentrations were measured using BCA protein assay
kit (Pierce Chemical Co, Rockford, IL). After that, 10%
sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was performed, followed by gel transfer to nitrocellulose membranes. After incubation in 5% nonfat milk for
1.5 hours, incubation with primary antibodies was
performed overnight at 4°C as follows: rabbit monoclonal
to p38 MAPK, 1:1000; rabbit monoclonal to p‐p38 MAPK,
1:500; mouse monoclonal to p‐ERK1/2, 1:500; mouse
monoclonal to ERK1/2, 1:1000; mouse monoclonal to
p‐JNK, 1:500; mouse monoclonal to JNK, 1:1000; rabbit
monoclonal to cytochrome c, 1:500; rabbit monoclonal to
β‐actin, 1:2000. After washing, horseradish peroxidase‐
conjugated secondary antibodies were added to the
membrane. ECL (GE Healthcare, Madison, WI) was used
to develop signals, which were processed using the
Quantity One image software (Pierce Biotechnology Inc.,
2.6 | Caspase activity
After treatments aforementioned, cells were mixed with
caspase assay buffer, followed by incubation on ice for
30 minutes. The mixtures were then centrifuged for
10 minutes at 1000g to collect the supernatant. Colorimetric substrates of c caspase‐3 (Ac‐DEVD‐pNA) or
caspase‐9 (Ac‐LEHD‐pNA) was then used to incubate
with the supernatant for 90 minutes. OD values were
measured to reflect caspase activity.
2.7 | ROS measurement
After treatments aforementioned, oxidative conversion of
2′,7′‐dichlorofluorescin diacetate (DCFH‐DA) to fluorescent
dichlorofluorescein in the cells was measure to quantify
intracellular ROS levels. Following this, cells were treated
with DCFH‐DA for 30 minutes and the ROS levels were
quantified by flow cytometry using the excitation wavelength of 488 nm and the emission wavelength of 525 nm.
2.8 | Statistical analysis
Mean values were from three biological replicates. One‐
way analysis of variance and Tukeyʼs multiple comparison test were used for statistical analyses. P < 0.05 was
3 | RESULTS
3.1 | α‐M inhibits the cell viability and
promotes apoptosis in RA‐FLS
To examine the effects of α‐M (Figure 1A) treatment on
RA‐FLS, cell viability and apoptosis were measured. As
shown in Figure 1B, α‐M of 10 to 100 μM significantly
attenuated the cell viability of RA‐FLS in a concentration‐
dependent manner compared to untreated cells (P < 0.05
or P < 0.01). Compared to untreated cells, RA‐FLS treated
with α‐M also demonstrated increased apoptosis (at
100 μM, P < 0.01) (Figure 1C and 1D). α‐M of 100 μM
was used for the further experiments.
Next, we measured the effect of α‐M on cell viability
and apoptosis in normal FLS from healthy individuals. It
was shown that α‐M at the concentrations from 10 to
100 μM did not affect the cell viability (Figure 1E) and
cell apoptosis (Figure 1F and 1G) in normal FLS.
3.2 | α‐M triggers mitochondrial
apoptotic cascade in RA‐FLS
To investigate the effects of α‐M on the mitochondria‐
dependent apoptosis, the changes of Δψm and cytochrome
c in RA‐FLS were examined. As shown in Figure 2A and
2B, α‐M treatment resulted in a significant reduction of
Δψm in RA‐FLS (P < 0.05). The accumulation of cytochrome c in the cytosol was significantly increased by α‐M
treatment as revealed by western blot analysis (P < 0.05,
Figure 2C and 2D). To elucidate the involvement of
caspase‐3 and caspase‐9 in α‐M‐induced apoptosis, the
activities of caspase‐3 and caspase‐9 in RA‐FLS were
measured. We show that the activities of caspase‐3 and
caspase‐9 were significantly increased by α‐M treatment
(P < 0.05, Figure 2E).
SHENG ET AL. | 3
FIGURE 1 Continued.
4 | SHENG ET AL.
3.3 | α‐M treatment leads to activation
of ERK1/2 in RA‐FLS
Western blot analysis revealed that α‐M significantly
increased the phosphorylation of ERK1/2 in RA‐FLS
(P < 0.05, Figure 3A and 3B). Meanwhile, no remarkable changes were observed on the phosphorylation
status of p38 MAPK or JNK. To determine the role of
ERK1/2 signaling in α‐M‐induced apoptosis in
RA‐FLS, the ERK1/2 inhibitor LY3214996 was used
FIGURE 1 α‐M inhibits the cell viability and promotes apoptosis of RA‐FLS. A, Chemical structure of α‐M. B, The cell viability of
RA‐FLS treated with indicated concentrations of α‐M for 24 hours was measured by MTT assay. C, D, The proportion of apoptosis in RA‐FLS
treated with indicated concentrations of α‐M for 24 hours was measured and quantified by flow cytometry. E‐G, The effects of α‐M on the
cell viability and apoptosis of normal FLS from healthy individuals were measured by MTT assay and flow cytometry. *P < 0.05 compared
with the control group and **P < 0.01 compared with the control group. FLS, fibroblast‐like synoviocytes; RA‐FLS, rheumatoid arthritis
fibroblast‐like synoviocytes; α‐M, α‐mangostin
FIGURE 2 α‐M induces the mitochondrial apoptotic cascade in RA‐FLS. After treated with 100 μM α‐M for 24 hours, the activation of
the mitochondrial apoptotic cascade in RA‐FLS was measured by assay kit. A, B, The changes of Δψm in RA‐FLS were examined by
Rho‐123. Representative figure of three independent experiments was shown. Data were expressed as percentage of control cells. C,D, The
relative expression of cytochrome c protein in the cytoplasmic extracts of RA‐FLS was measured and quantified by Western blot analysis.
E, The activities of caspases in RA‐FLS were measured by assay kit. Data were expressed as fold change compared to untreated cells.
*P < 0.05 compared with the control group. RA‐FLS, rheumatoid arthritis fibroblast‐like synoviocytes; α‐M, α‐mangostin
SHENG ET AL. | 5
FIGURE 3 ERK1/2 is involved in α‐M‐induced apoptosis of RA‐FLS. A,B, After treated with 100 μM α‐M for 24 hours, the indicated
proteins in RA‐FLS were examined and quantified by Western blot analysis. C,D, RA‐FLS was pretreated with or without ERK1/2 inhibitor
LY3214996 before exposure to 100 μM α‐M. The apoptosis percentage of RA‐FLS was measured and quantified by flow cytometry. *P < 0.05
compared with the control group and #
P < 0.05 compared with α‐M treatment alone. RA‐FLS, rheumatoid arthritis fibroblast‐like
synoviocytes; α‐M, α‐mangostin
6 | SHENG ET AL.
to block the phosphorylation of ERK1/2. As shown in
Figure 3C and 3D, pretreatment with LY3214996
significantly reversed the α‐M‐induced apoptosis
in RA‐FLS (P < 0.05, compared to α‐M treatment
3.4 | ROS generation is involved in
α‐M‐induced ERK1/2 activation and
apoptosis in RA‐FLS
Next, we investigated the effects of α‐M on cellular ROS
level in RA‐FLS. α‐M treatment resulted in a significant
FIGURE 4 ROS plays an important role in α‐M‐induced ERK1/2 activation and apoptosis of RA‐FLS. A, Detection of cellular ROS
levels in RA‐FLS treated with or without NAC exposure to 100 μM α‐M. B, C, The proportion of apoptosis in RA‐FLS was determined and
quantified by flow cytometry. D, E, The phosphorylated of ERK1/2 was measured and quantified by Western blot analysis. *P < 0.05
compared with the control group and #
P < 0.05 compared with α‐M treatment alone. NAC, N‐acetylcysteine; RA‐FLS, rheumatoid arthritis
fibroblast‐like synoviocytes; ROS, reactive oxygen species; α‐M, α‐mangostin
SHENG ET AL. | 7
increase in cellular ROS level compared to untreated cells
(P < 0.05, Figure 4A). Finally, we examined the involvement of ROS in the α‐M‐induced ERK1/2 activation. We
found that pretreatment with ROS scavenger NAC
significantly reversed the upregulation of ROS production induced by α‐M (Figure 4A, P < 0.05), compared to
α‐M treatment alone. Moreover, NAC significantly
reversed the apoptosis (Figure 4B and 4C) and ERK1/2
phosphorylation (Figure 4D and 4E) induced by α‐M
(P < 0.05), compared to α‐M treatment alone.
4 | DISCUSSIONS
Many studies have highlighted a role for FLS in the
initiation and progression of RA and it was known that
RAFLS promotes joint destruction via proinflammatory
processes.16 However, most therapies approved for
treating RA do not target FLS themselves and therapies
that are capable of eliminating these destructive cells may
provide better clinical outcomes.17 α‐M has the potential
to fulfill this unmet clinical need since it has previously
been shown to induce apoptosis of FLS via ROS
accumulation and modulation of Bax:Bcl‐2.15 The pharmacokinetics, bioavailability, and clinical efficacy of the
compound have also been improved in recent years via
new lipid‐soluble formulations and micro emulsions.18,19
In this study, we show that α‐M treatment induces
significant apoptosis of RA‐FLS in a dose‐dependent
manner and that it does not affect healthy FLS. This is
in line with earlier studies, which have demonstrated
that α‐M can induce apoptosis via several molecular
mechanisms.20 However, α‐M at the same concentration range did not affect the viability and apoptosis of
normal FLS. Similarly, previous study also revealed
little cytotoxicity of α‐M in normal cells and normal
animals.21 Here we demonstrate that the process
involves caspase‐3 and caspase‐9 because these were
significantly increased by the induction of α‐M. Both of
these caspases are known to either initiate or execute
programmed cell death.22,23 ERK phosphorylation can
promote proapoptotic functions under certain circumstances24 and α‐M was also shown to significantly
increase the phosphorylation of ERK1/2 solely in RA‐
FLS. ERK activity can stimulate either intrinsic or
extrinsic apoptotic pathways by inducing the release of
mitochondrial cytochrome c or via caspase‐8 activation.25 Our finding that levels of cytochrome c were
increased in the cytoplasm of treated cells indicates
that apoptosis was stimulated by cytochrome c release
from the respiratory chain. This process is dependent
on the presence of ROS26 and we observed significant
increases in ROS in RA‐FLS following treatment. The
ROS‐dependency of the process was confirmed following the addition of the ROS scavenger NAC, which was
able to significantly reverse α‐M‐mediated apoptosis.
Taken together, these results indicate that α‐M induces
ROS‐dependent apoptosis via the ERK1/2 signaling
pathway in RA‐FLS, in a comparable mechanism to
that observed in human osteosarcoma cells.27 ROS
accumulation and cytochrome c release has been
previously observed in α‐M‐treated HL60 cells, which
indicates that α‐M most likely targets mitochondria in
the early phase, resulting in the initiation of apoptosis
via cytochrome c release.28
Significant RH‐FLS death was observed following
treatment with α‐M, whereas no effect was seen in
non‐RA cells. This result demonstrates that the compound could offer a new therapy for RA because these
cells promote synovial proliferation and hyperplasia that
forms the driving force for the chronic inflammation and
joint destruction observed in RA patients. These destructive synovial cells produce excessive proinflammatory
cytokines and matrix metalloproteinases that directly
damage the tissue and extracellular matrix. These
changes are supported by a cycle of remodeling of the
rheumatoid pannus,26 which is driven by synovial
angiogenesis. Inhibition of synovial angiogenesis and
synovial hyperplasia is potentially possible through
control of FLS by α‐M, which could prevent the
development and progression of RA.
5 | CONCLUSIONS
In summary, we show that α‐M induces apoptosis via the
ROS/ERK1/2 signaling pathway in RA‐FLS. Since these
cells drive RA progression then α‐M could offer a new
treatment for RA through the abolition of these damaging
cells. Recent improvements in drug delivery methods
leading to increased bioavailability of α‐M support this
development; however, future studies in animal models
and humans are still required to determine the safety and
clinical efficacy of the compound for treating RA.
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
XS, JL, CZ, and YX designed and carried out the study.
XS, LZ, LG, TX, JJ, and MW participated in experiments
and statistical analysis. XS and LG wrote the manuscript.
YX revised the manuscript. All authors read and
approved the final manuscript.
8 | SHENG ET AL.
AVAILABILITY OF DATA AND
The analyzed data sets generated during the study are
available from the corresponding author on reasonable
Yayi Xia http://orcid.org/0000-0002-1692-2937
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How to cite this article: Sheng X, Li J, Zhang C,
et al. α‐Mangostin promotes apoptosis of human
rheumatoid arthritis fibroblast‐like synoviocytes by
reactive oxygen species‐dependent activation of
ERK1/2 mitogen‐activated protein kinase. J Cell
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