Introduction
Osteoarthritis (OA) is the most common degenerative
joint disease affecting the articular cartilage worldwide, and
causes pain, stiffness and decreased mobility, thus resulting in a
reduced quality of life (1,2). It has been demonstrated that 10–18% of
adults >60 years of age suffer from OA, and the number of
patients with OA is estimated to increase to >130 million
worldwide by the year 2050 (3).
Factors, such as genetics, age, sex, body mass index, nutrition and
joint leptin levels are related to OA (3–5).
Initially, OA was considered to be a disease of the cartilage;
however, it was subsequently demonstrated to be a complex disease
mediated by inflammation (6).
Additionally, high cholesterol levels have been found to be closely
related to OA (7). Currently, an
increasing number of studies are focusing on cholesterol metabolism
and inflammation in OA.
The phosphoinositide 3-kinase (PI3K)/AKT/mammalian
target of rapamycin (mTOR) signaling pathway is a common signaling
pathway that plays a role in the progression of a number of
diseases, and therefore may also be involved in the development of
OA. As previously reported, the inhibition of the PI3K/AKT/mTOR
signaling pathway can attenuate the inflammatory response and
promote autophagy in articular chondrocytes in rats (8). The increment of PI3K/AKT signaling
activation has been demonstrated to be associated with the
degeneration of articular cartilage (9). Thus, the inhibition of the
PI3K/AKT/mTOR signaling pathway has been viewed as a treatment
strategy for OA (10).
Aside from the inflammatory response, high
cholesterol levels are another important risk factor of OA
(11). High levels of low-density
lipoprotein cholesterol (LDL-C) accelerate the incidence of OA in
humans and mice (12,13). The molecular mechanisms are
associated with the cholesterol 25-hydroxylase (CH25H)/cytochrome
P450 family 7 subfamily B polypeptide 1 (CYP7B1)/RAR-related orphan
receptor α axis. In psoriasis, it has been reported that the
activation of the PI3K/AKT/mTOR signaling pathway plays a vital
role in increasing cholesterol levels (14). Another study demonstrated that
insulin-induced cholesterol uptake, lipid droplet content and
apolipoprotein B secretion in CaCo-2 cells were associated with the
PI3K/AKT/mTOR signaling pathway (15). For this reason, the expression
levels of cholesterol-related proteins and proteins in the
PI3K/AKT/mTOR signaling pathway were measured in the present
study.
Chinese herbal medicine can regulate the
PI3K/AKT/mTOR signaling pathways by attenuating interleukin
(IL)-1β-induced apoptosis and extracellular matrix (ECM) catabolism
in OA (16). Chinese herbal
medicine is frequently used as a therapeutic strategy for OA.
Herbs, such as Clematis, Nigella sativa, Radix Angelica
sinensis, Salvia miltiorrhiza and the components purified from
these herbs have been reported to attenuate the development of OA
or even to function as therapies for OA (17–20).
Scutellarin is an active flavonoid isolated from the Chinese
traditional herb Erigeron breviscapus, which has been found
to possess several functions, such as anti-inflammatory and
antioxidant activities (21), and
it has also been reported to exert effects on various types of
tumors, such as malignant melanoma and bladder cancer (22,23).
The effects of scutellarin in OA have been partially
studied; however, its mechanism of action is complex. The present
study investigated the function of scutellarin in regulating the
release of inflammatory cytokines, the expression of collagen- and
cholesterol-related proteins, as well as its effects on the
PI3K/AKT/mTOR signaling pathway. IL-1β was used to stimulate human
osteosarcoma cells (SW1353) to induce OA in vitro, and the
effects of scutellarin with or without a PI3K inhibitor (LY294002)
on IL-1β-induced SW1353 cells were then observed. The results
revealed that scutellarin inhibited the expression of IL-6, and
regulated the expression levels of collagen-related proteins,
collagen II (Col2), SRY-box 9 (SOX9) and matrix metalloproteinase
(MMP)13, and cholesterol-related proteins, CH25H, CYP7B1,
apolipoprotein A-1 (APOA-1) and adenosine triphosphate-binding
cassette transporter A1 (ABCA1). As the expression levels of AKT,
phosphorylated (p)-AKT, mTOR and p-mTOR were suppressed by
scutellarin treatment, it was thus suggested that scutellarin
regulates OA in vitro by inhibiting the PI3K/AKT/mTOR
signaling pathway.
Materials and methods
Reagents
Scutellarin was purchased from Shanghai Yuanye
Bio-Technology Co., Ltd. The Cell Counting Kit-8 (CCK-8) was
obtained from Vazyme Biotech Co., Ltd. LY294002 was purchased from
Sigma-Aldrich (Merck KGaA). IL-1β was obtained from Novoprotein
Scientific, Inc. RIPA lysate was purchased from Beyotime Institute
of Biotechnology. Primary antibodies against Col2 (1:2,000; cat.
no. ab34712), SOX9 (1:1,000; cat. no. ab185966), MMP13 (1:1,000;
cat. no. ab51072), CH25H (1:500; cat. no. ab133933), CYP7B1
(1:1,000; cat. no. ab138497), ABCA1 (1:200; cat. no. ab18180),
APOA-1 (1:500; cat. no. ab75114), PI3K (1:1,000; cat. no. ab32089),
AKT (1:500; cat. no. ab8805), p-AKT (1:500; cat. no. ab38449), mTOR
(1:1,000; cat. no. ab32028) and p-mTOR (1:500; cat. no. ab84400),
and a goat anti-rabbit secondary antibody (1:5,000; cat. no.
ab6721) were purchased from Abcam. The primary antibodies and
secondary antibodies were used according to the manufacturers
instructions.
Cell culture and treatment
SW1353 cells (The Cell Bank of Type Culture
Collection of the Chinese Academy of Sciences) were cultured in
Dulbeccos modified Eagles medium (DMEM; Gibco; Thermo Fisher
Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher
Scientific, Inc.), 100 units/ml penicillin and 100 µg/ml
streptomycin. The cells were maintained at 37°C in 5%
CO2. To assess cell viability, SW1353 cells
(7×103 cells/well) were cultured in 96-well plates
overnight and then treated with increasing concentrations of
scutellarin (0, 5, 10, 20, 40, 80 and 100 µmol/l) for 48 h at 37°C.
For the second part of the experiment, SW1353 cells were
pre-treated with 80 µmol/l scutellarin for 2 h, following which
IL-1β (10 ng/ml) was added to the cells for 48 h at 37°C. The dose
of IL-1β was used as reported by Xue et al (8) and Liu et al (24). Subsequently, 10 µl CCK-8 reagent was
added to each well for a further 2 h of incubation, according to
the manufacturers instructions. The absorbance value was assessed
using a microplate reader (BioTek Epoch; BioTek Instruments, Inc.)
at 450 nm. To inhibit the PI3K/AKT signaling pathway, serum-starved
(0.5% FBS) cells were pre-treated with scutellarin (80 µmol/l) for
2 h and LY294002 (25 µg/ml) for 1 h, and then exposed to IL-1β (10
ng/ml) for 48 h. The test was repeated three times.
ELISA for conditioned medium
The expression levels of C-reactive protein (CRP),
tumor necrosis factor (TNF)-α and IL-6 in conditioned medium
(collected from culture supernatant) were measured using CRP (cat.
no. ZC-31853; ZCi Bio), TNF-α (cat. no. ZC-35733; ZCi Bio) and IL-6
(cat. no. ZC-32446; ZCi Bio) ELISA kits following the manufacturers
instructions.
Western blot analysis
RIPA buffer containing 0.1% protease inhibitor was
used to homogenize the cell samples, and cell lysates were
centrifuged at 13,680 × g for 15 min at 4°C, and the supernatants
were then collected for protein detection. Total protein
concentrations were measured using a BCA kit (Thermo Fisher
Scientific, Inc.). Equal amounts of protein (20 µg per lane for
cell samples) were separated via 10% SDS-PAGE and then transferred
onto PVDF membranes. The membranes were then blocked in 5% skimmed
milk at room temperature for 2 h. After washing in TBST three
times, the immunoblots were incubated with the following primary
antibodies (3% BSA dilution): Col2, SOX9, MMP13, CH25H, CYP7B1,
ABCA1, APOA-1, PI3K, AKT, p-AKT, mTOR and p-mTOR (details of
antibody dilutions were described in the ‘Reagents’ section)
overnight at 4°C. The membranes were then incubated with goat
anti-rabbit IgG secondary antibody (1:5,000, 1% BSA dilution) for 2
h at room temperature. The membranes were washed with TBST again
and then detected with ECL (EMD Millipore). The band sizes were
quantified using Scion Image 4.0 software (Scion Corporation).
Protein expression was normalized relative to β-actin as a loading
control. The final results are expressed as ‘fold changes’ in
comparison with the control group.
Statistical analyses
GraphPad Prism v.6.0 (GraphPad Software, Inc.) was
used to analyze the data generated in the charts in this
experiment. All data are presented as the mean ± SEM, and all
experiments were performed three times. Differences between two
groups were statistically analyzed using an unpaired, two-tailed
Students t-test. Differences among three groups were analyzed by
one-way analysis of variance followed by Tukeys post hoc test.
P<0.05 was considered to indicate a statistically significant
difference.
Results
Effects of scutellarin on SW1353 cell
viability
To analyze the effects of scutellarin on human
cartilage cells, the SW1353 cell line was used in the present
study. A CCK-8 assay was used to measure the viability of SW1353
cells. As shown in Fig. 1A, no cell
cytotoxicity was observed when the concentration of scutellarin was
<80 µmol/l; however, cell viability was reduced when the
concentration of scutellarin was >80 µmol/l. IL-1β was used to
establish the model of OA using SW1353 cells; treatment of
IL-1β-induced SW1353 cells with scutellarin significantly increased
cell viability (Fig. 1B).
Scutellarin affects the release of
inflammatory cytokines, and the expression of collagen- and
cholesterol-related proteins
Western blot analysis was performed to investigate
whether scutellarin inhibits the release of inflammatory cytokines,
attenuates the degradation of collagen by regulating
collagen-related proteins, and reduces the level of cholesterol by
regulating cholesterol-related proteins. In the present study
(Fig. 2), the protein expression
levels of CRP, TNF-α and IL-6 were measured. The results revealed
that CRP, TNF-α and IL-6 expression was increased by IL-1β;
however, only the expression of IL-6 was significantly inhibited by
scutellarin treatment (Fig.
2C).
The degradation of collagen is another feature of
OA. As demonstrated in Fig. 3, the
expression levels of Col2 and SOX9 were significantly downregulated
by IL-1β, whereas MMP13 expression was upregulated. Scutellarin
combined with IL-1β significantly increased the expression of Col2
and SOX9 (Fig. 3A-C), and
significantly decreased the expression of MMP13 compared with the
IL-1β group (Fig. 3D).
As aforementioned, high cholesterol level is another
risk factor for OA. The present study measured the expression of
CH25H, CYP7B1, ABCA1 and APOA-1 in SW1535 cells (Fig. 4). In the IL-1β-induced group, the
expression levels of CH25H and CYP7B1 significantly increased, and
those of ABCA1 and APOA-1 significantly decreased compared with the
control group. Following treatment with scutellarin in the
IL-1β-induced cell model, CH25H and CYP7B1 expression levels were
significantly downregulated (Fig. 4B
and C). By contrast, ABCA1 and APOA-1 expression levels
significantly increased (Fig. 4D and
E).
Scutellarin suppresses the
PI3K/AKT/mTOR signaling pathway
In this experiment, the expression levels of AKT,
p-AKT, mTOR and p-mTOR, and the ratio of p-mTOR/mTOR were increased
by IL-1β (Fig. 5). Following
treatment with scutellarin however, the levels of these proteins
were significantly inhibited. Of note however, the expression of
PI3K and the ratio of p-AKT/AKT remained unaltered among the groups
(Fig. 5B and G). These results
suggested that scutellarin inhibited the PI3K/AKT/mTOR signaling
pathway.
 | Figure 5.PI3K/AKT/mTOR signaling pathway in
response to Scu treatment in IL-1β-induced SW1535 cells. (A)
Protein expression levels in the PI3K/AKT/mTOR signaling pathway
were determined by western blotting. (B) The expression of PI3K
exhibited no change following treatment with Scu and IL-1β. Scu
suppressed the IL-1β-induced upregulation of (C) AKT, (D) p-AKT,
(E) mTOR, (F) p-mTOR, (G) p-AKT/AKT and (H) p-mTOR/mTOR. Data are
presented as the mean ± SEM (n=3 in each group). *P<0.05,
***P<0.001 vs. control group; #P<0.05,
##P<0.01, ###P<0.001 vs. IL-1β group.
PI3K, phosphatidylinositol 3 kinase; p-, phosphorylated; mTOR,
mammalian target of rapamycin; Scu, scutellarin; IL-1β, interleukin
1β. |
Scutellarin and LY294002 treatment
affect the release of inflammatory cytokines, and the expression of
collagen- and cholesterol-related proteins
LY294002, as an inhibitor of PI3K, was used to
examine the effects of PI3K on the release of inflammatory
cytokines, and the regulation of collagen- and cholesterol-related
proteins following treatment with scutellarin. Firstly, the
expression levels of CRP, TNF-α and IL-6 were examined (Fig. 6), and only the level of IL-6 was
significantly reduced by scutellarin and LY294002 (Fig. 6C). When compared with the IL-1β +
scutellarin group, the addition of LY294002 significantly decreased
the expression of IL-6.
In regard to the collagen-related proteins (Fig. 7), the IL-1β-induced increase in
MMP13 expression was decreased by treatment with scutellarin and
LY294002, and the greatest decrease was observed in the group
treated with scutellarin and LY294002 in combination (Fig. 7D). By contrast, compared with the
IL-1β-induced model group, the expression of Col2 (Fig. 7B) and SOX9 (Fig. 7C) increased following treatment with
scutellarin and LY294002. When compared with the IL-1β + LY294002
group, the addition of scutellarin significantly increased the
expression of Col2 and SOX9. Thus, scutellarin and LY294002 can
partially restore the loss of Col2 and SOX9 caused by IL-1β.
The expression levels of cholesterol-related
proteins were also regulated by scutellarin and LY294002 (Fig. 8). The IL-1β-induced upregulation of
CH25H and CYP7B1 expression was reversed by scutellarin and
LY294002 (Fig. 8B and C), whereas
the IL-1β-induced downregulation of ABCA1 and APOA-1 expression was
reversed by a combination of scutellarin and LY294002 (Fig. 8D and E).
Inhibition of the PI3K/AKT/mTOR
signaling pathway by scutellarin and LY294002
To directly examine the effect of scutellarin and
LY294002 on the PI3K/AKT/mTOR signaling pathway, the expression
levels of PI3K, AKT, p-AKT, mTOR and p-mTOR were determined
(Fig. 9). The results of the
western blot analysis revealed that scutellarin and LY294002
affected the expression of AKT, mTOR, and the ratio of p-mTOR/mTOR,
but not that of PI3K and the ratio of p-AKT/AKT. In combination,
LY294002 and scutellarin decreased the IL-1β-induced upregulation
of AKT, mTOR and the ratio of p-mTOR/mTOR (Fig. 9C, E and F). Thus, this indicated
that scutellarin and LY294002 are able to inactivate the
PI3K/AKT/mTOR signaling pathway.
 | Figure 9.PI3K/AKT/mTOR signaling pathway in
response to Scu and LY294002 in IL-1β-induced SW1535 cells. (A)
Protein expression bands of PI3K, AKT, p-AKT, mTOR and p-mTOR. (B)
The protein expression of PI3K was not significantly different
among the groups. (C) Scu and LY294002 treatment inhibited the
expression of AKT. (D) The ratio of p-AKT/AKT was not significantly
different among the groups. (E) Scu and LY294002 treatment
inhibited the expression of mTOR. (F) Scu and LY294002 treatment
inhibited the ratio of p-mTOR/mTOR. ***P<0.001 vs. control
group; #P<0.05, ##P<0.01,
###P<0.001 vs. IL-1β group;
&&P<0.01. PI3K, phosphatidylinositol 3
kinase; p-, phosphorylated; mTOR, mammalian target of rapamycin;
Scu, scutellarin; IL-1β, interleukin 1β. |
Discussion
OA, as a chronic disease that results in disability
and a reduced quality of life, results from several different
etiologies (25). A number of
studies have been performed to elucidate the mechanisms responsible
and to identify novel treatment strategies for OA (2). Acetaminophen, a non-steroidal
anti-inflammatory drug, and corticosteroids as oral drugs, are
common pharmacological therapies for the management of OA. However,
they may cause toxicity when at high doses and are associated with
adverse reactions, such as gastric ulceration, kidney dysfunction
and increased bleeding under particular conditions (3,26).
Thus, the development of effective drugs with limited side effects
is of utmost importance. Recently, Chinese Traditional Medicine has
gained increasing attention. Effective components of Erigeron
breviscapus, including scutellarin, have been reported to
possess anti-inflammatory properties (27) and to restrain the development of
tumors (22). In the present study,
the effects of scutellarin on the PI3K/AKT/mTOR signaling pathway
were examined, and it was determined whether it can inhibit the
inflammatory response, and the expression of collagen- and
cholesterol-related proteins in the SW1353 cell model of OA.
OA is regarded as an inflammatory disease (6). Several inflammatory cytokines are
secreted during OA, and biomarkers of inflammation, including CRP
and TNF-α are elevated. IL-6 is an inflammatory cytokine that is
associated with systemic low-grade inflammation, and its expression
is also altered in OA (28).
Scutellarin has been reported to inhibit the expression of
inflammatory factors, such as IL-1β, IL-6 and TNF-α in
collagen-induced arthritis (29).
Another study reported that treatment with scutellarin
downregulated the expression levels of IL-1β, TNF-α and IL-6 in the
serum of osteoarthritis mice (30).
In the present study, it was found that only the expression of IL-6
was influenced by scutellarin. The levels of CRP and TNF-α were not
altered. Naderi et al (31)
measured the effects of Zingiber officinale on patients with
knee osteoarthritis, and no changes in the levels of CRP were found
within 3 months. Another study demonstrated that the expression of
CRP had no association with the incidence or progression of OA
(32). Besides, in the present
study, scutellarin failed to inhibit the IL-1β-induced increase in
TNF-α expression. In fact, unlike the chondroid cells isolated from
patients and in vivo animal models, the expression levels of
CRP, TNF-α and IL-6 were all only slightly increased by IL-1β in
SW1353 cells. This was a limitation of the present research.
Although the SW1353 cell line used to establish the cell model of
OA has been reported on by some previous research (33–35)
because the cell lines could provide enough and steady cells for
the research, the IL-1β-induced OA cell model used in the present
study cannot completely mimic the in vivo environment. This
may partly explain why there were no changes in the expression
levels of CRP and TNF-α in this study.
In OA, the expression of Col2 and SOX9 are
influenced by MMP13. As reported by Otero et al (36), the expression of ETS-related
transcription factor Elf-3 protein driven by MMP13 was increased in
OA-affected cartilage, and it inhibited SOX9-driven COL2A1 promoter
activity. Ouyang et al (37)
also found decreased expression of Col2 and SOX9, but increased
expression of MMP13 in OA. Thus, indicating that the expression of
MMP13 is enhanced, whereas that of Col2 and SOX9 is inhibited in
OA. Fisetin is a polyphenol extracted from fruit and vegetables,
and it can inhibit the IL-1β-induced upregulation of MMP13
expression and decrease the degradation of Col2 and SOX9 in OA
(38). Scutellarin has been
demonstrated to have similar functions; the mRNA expression of
MMP13 was found to be decreased, whereas Col2 expression was
increased by treatment with scutellarin in OA mouse models
(30). These findings were
supported by those of another study in human primary chondrocytes
(24). In the present study,
similar results were obtained. The expression of Col2 and SOX9
increased and the expression of MMP13 decreased by treatment with
scutellarin. These results suggested that scutellarin can attenuate
the degradation of cartilage in OA.
The function of scutellarin in regulating
cholesterol-related protein expression in the OA cell model was
determined in the present study. The results revealed that
scutellarin downregulated the expression of CH25H and CYP7B1 and
upregulated the expression of ABCA1 and APOA-1. As a metabolic
disorder disease (39), it has been
demonstrated that cholesterol metabolism is abnormal in OA with
increased levels of LDL (40).
Tsezou et al (41) reported
that APOA-1 and ABCA1 expression decreased in OA-affected
cartilage. On the other hand, the expression levels of CH25H and
CYP7B1 have been found to be increased, leading to high levels of
cholesterol (42). The findings of
the present study were consistent with these previous findings,
therefore demonstrating that scutellarin can decrease total
cholesterol levels and improve lipid metabolism (43,44).
The PI3K/AKT/mTOR signaling pathway has been found
to be associated with OA (22,45),
and the inhibition of this pathway can attenuate inflammation and
articular cartilage degeneration in OA (8–10). In
the present study, no significant changes were identified in the
protein expression of PI3K between the groups, which indicated that
IL-1β, scutellarin and LY294002 may not affect the expression of
PI3K. The present study also found that scutellarin inhibited the
expression of AKT, mTOR and p-mTOR, and the ratio of p-mTOR/mTOR in
the IL-1β-induced cells. The ratio of p-AKT/AKT may not have been
affected because the decrease in p-AKT expression may be caused by
the decrease in AKT expression and the decrease in AKT expression
essentially leads to the decrease in mTOR activity. It has been
revealed that the activation of PI3K/AKT signaling can degrade
chondrocytes and accelerate the progression of OA (8–10).
Thus, these findings suggested that scutellarin can attenuate OA by
regulating the PI3K/AKT/mTOR signaling pathway.
In conclusion, the primary finding of the present
study was that scutellarin inhibited the activation of the
PI3K/AKT/mTOR signaling pathway, which alleviated cartilage
degradation by regulating relevant protein levels, and decreased
cholesterol levels by regulating cholesterol-related protein
expression in the OA cell model; however, it only affected the
release of the inflammatory cytokine, IL-6. These findings are
important for future studies investigating the function of
scutellarin in OA and they provide evidence that scutellarin could
be used as a latent medicine for OA. However, further studies are
required to confirm the pharmacological functions of scutellarin
in vivo and to explore the precise underlying
mechanisms.
Acknowledgements
Not applicable.
Funding
The present study was financially supported by the
Application Foundation Fund of Science and Technology Department of
Sichuan Province (grant no. 2018JY0264), Innovative topics of
Chengdu Sport University (grant no. CX19C01) and Innovation team of
Chengdu Sport University (grant no. CXTD201805).
Availability of data and materials
The datasets used and/or analyzed during the current
study are available from the corresponding author upon reasonable
request.
Authors contributions
SHJ, BXH and LRT designed the study. SHJ, PWL and
YLT performed the experiments. YTZ, XHL and MJW analyzed the data.
SHJ wrote the manuscript. All authors critically reviewed the
manuscript, and read and approved the final manuscript.
Ethics approval and consent to
participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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