Page 13 - N. Metabolism and metabolic diseases

Anti-osteoclastogenic activity of hibiscus syriacus
L. twig extract via inhibition of ERK/Akt-c-Fos-NFATc1 signaling axis

Shin-Hye Kim , Sik-Won Choi 1,*
1,2
1 Forest Biomaterials Research Center, National Institute of Forest Science, jinju 52817, korea
2 Department of Biological Sciences, College of Natural Science, Chonbuk National University, Jeonbuk 54896, Korea
ABSTRACT
A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal
medicines to prevent and/or treat bone disorders including osteoporosis. Hibiscus syriacus has several biological activities
including anti-osteoclasts, but its anti-osteoporotic activity has not been fully studied yet. The effect of hibiscus syriacus L.
cultivars stem with color of flowers on the differentiation of bone marrow-derived macrophages into osteoclasts was examined by
phenotype assay and confirmed by real-time PCR and immunoblotting. Interestingly, the branch extracts of red flowers with red
eyes strongly inhibited the RANKL-stimulated osteoclast differentiation compared with other varieties. In mode of action, the red
flower cultivar, ‘Nanpa’ extracts attenuated the RANKL-induced osteoclast differentiation and induction of c-Fos/NFATc1 such as
master regulator of osteoclastogenesis. Moreover, ‘Nanpa’ extracts blocked the RANKL-mediated activation of AKT/ERK
phosphorylation in BMMs. Taken together, our results suggest that the cultivar of red flowers with red eyes, ‘Nanpa’ branch
extracts could exhibit its anti-osteoclastogenic activity by inhibiting AKT/ERK-c-Fos-NFATc1 signaling molecules.
RESULTS

A B A

B

Figure 1. Effect of branch extracts according to Figure 2. Nanpa extracts inhibits RANKL-induced osteoclast differentiation.
flower color of hibiscus syriacus on RANKL- (A) Multinucleated osteoclasts were visualised using TRAP staining.
induced osteoclast differentiation. (B) TRAP+ MNCs were counted (left panel) and TRAP activity was measured (right
(A) TRAP acitivity was measured at the indicated panel). ### p<0.001(versus the negative control);*p<0.05, **p<0.01 and
concentration. Inhibition rate was shown in red ***p<0.001(versus the RANKL-treated group)
color (C) Cell viability was evaluated using CCK-8 assay.
(B) Multinucleated osteoclasts were stained with
TRAP solution. TRAP-positive MNCs were
photographed under a light microscope.
C A
Figure 3. Nanpa
Figure 4. Nanpa extracts attenuates
extracts RANKL-induced
contributes to expression of c-Fos
RANKL-mediated
AKT/ERK signaling and NFATc1 during
pathways. osteoclastogenesis.
BMMs
The
(A)
were
BMMs were pretreated stimulated with RANKL (10
with vehicle or Nanpa ng/ml) and M-CSF (30
extracts (100 ug/ml)
for 1h prior to RANKL ng/ml) in the presence or
stimulation (30 ng/ml) B absence of Nanpa branch
at indicated time extracts (100 μg/ml) for the
times.
The
indicated
periods. Then, protein indicated mRNA
expression levels were expression levels were
evluated by immuno evaluated by real-time
blot analysis. Actin PCR. *p<0.05 and
was used as the
internal control. **p<0.01(versus the
RANKL-treated group). (B)
the translational induction
was confimed by Western
blot analysis.
CONCLUSION
To our knowledge, this is the first study to report that red flowers with red eyes, Nanpa branch extracts has the potential to
inhibit RANKL-induced osteoclast differentiation. The ability of nanpa branch extracts to attenuate the RANKL-induced
activiations of AKT and ERK signaling molecules could block the inductions of c-Fos and NFATc1 that down regulate the
expressions of NFATc1-controllded osteoclast-specific genes such as DC-STAMP and cathepsin K. therefore, the anti-
osteoclastogenic property of nanpa branch extracts could provide benefits for bone health, and we believe it should be
considered as a potential treatment for osteoclast-related disorders.