Effect of  Extract on Osteoclast Differentiation and Bone-pit Formation

Min-Youn Ahn; Hyung-Ho Lim; Ahn, Min-Youn; Lim, Hyung-Ho

doi:10.13048/jkm.17025

JKM > Volume 38(3); 2017 > Article

Ahn and Lim: Effect of Sochungryong-tang Extract on Osteoclast Differentiation and Bone-pit Formation

Original Article

The Journal of Korean Medicine 2017; 38(3): 59-72.

Published online: September 30, 2017

DOI: https://doi.org/10.13048/jkm.17025

Effect of Sochungryong-tang Extract on Osteoclast Differentiation and Bone-pit Formation

Min-Youn Ahn, Hyung-Ho Lim

Colledge of Korean Medicine, Gachon University

Correspondence to：임형호(Hyung-Ho Lim) 경기도 성남시 수정구 성남대로 1342 가천대학교 Tel：+8-31-750-8599, E-mail : omdlimhh@gachon.ac.kr

Received August 7, 2017 Revised September 19, 2017 Accepted September 20, 2017

Abstract

Objectives

This study was performed to evaluate effects of Sochungryong-tang Extract(SRE) on osteoclast differentiation and bone resorptionin order to find out the possibility for clinical use in preventing and treating osteoporosis.

Methods

To evaluate the effect of SRE on osteoclast differentiation, we induced RAW 264. 7 cells to be differentiated to osteoclasts by RANKL (receptor activator of nuclear factor-κB ligand). We measured effect on TRAP (Tartrate-resistant acid phosphatase), NFATc, cathepsin K, MMP-9, inflammation related factors, histogenesis factors and bone resorption.

Results

SRE decreased osteoclast differentiation, and also decreased expression of bone resorbing factors such as MMP-9, cathepsin K, TRAP, NFATc1, MITF, c-Fos, osteoclast stimulatory transmembrane protein, calcitonin receptor in RANKL-induced osteoclast. SRE also decreased Cyclooxygenase-2, indusible nitric oxide synthase, TNF-α, which are thought to be related with the inflammatory bone destruction.

Conclusion

SRE inhibits osteoclast differentiation and bone resorption. The results indicate that the BHT extract can potentially be applied for preventing and treating osteoporosis.

Keywords: Sochungryong-tang, Osteoclast, Bone resorption, osteoporosis

Fig. 1

Effect of SRE on cell viability.

A : RANKL(100 ng/mℓ) only

B : RANKL(100 ng/mℓ) + 18.7 μg/mℓ of SRE

C : RANKL(100 ng/mℓ) + 37.5 μg/mℓ of SRE

D : RANKL(100 ng/mℓ) + 75 μg/mℓ of SRE

E : RANKL(100 ng/mℓ) + 150 μg/mℓ of SRE

F : RANKL(100 ng/mℓ) + 300 μg/mℓ of SRE

Each bar represents Mean±SD of 5 tests.

** : p<0.01 vs RANKL only

Fig. 2

Effects of SRE on the formation of TRAP(+) MNCs in RANKL stimulated osteoclast.

0 : RANKL(100 ng/mℓ) only

30 : RANKL(100 ng/mℓ) + 30 μg/mℓ of SRE

60 : RANKL(100 ng/mℓ) + 60 μg/mℓ of SRE

120 : RANKL(100 ng/mℓ) + 120 μg/mℓ of SRE

Each bar represents Mean±SD of 5 tests.

**: p<0.01 vs RANKL only

Fig. 3

Effects of SRE on (A)TRAP, (B) MMP-9 and (C)cathepsin K gene expression in RANKL-stimulated osteoclast.

NC : Vehicle

0 : RANKL(100 ng/mℓ) only

30 : RANKL(100 ng/mℓ) + 30 μg/mℓ of SRE

60 : RANKL(100 ng/mℓ) + 60 μg/mℓ of SRE

120 : RANKL(100 ng/mℓ) + 120 μg/mℓ of SRE

Each bar represents Mean±SD of 5 tests.

* : p<0.05 vs NC

# : p<0.05 vs RANKL only

Fig. 4

Effects of SRE on (A)NFATc1, (B)c-Fos and (C)MITF gene expression in RANKL-stimulated osteoclast.

NC : Vehicle

0 : RANKL(100 ng/mℓ) only

30 : RANKL(100 ng/mℓ) + 30 μg/mℓ of SRE

60 : RANKL(100 ng/mℓ) + 60 μg/mℓ of SRE

120 : RANKL(100 ng/mℓ) + 120 μg/mℓ of SRE

Each bar represents Mean±SD of 5 tests.

* : p<0.05 vs NC

# : p<0.05 vs RANKL only

Fig. 5

Effects of SRE on iNOS, COX-2, TNF-a, IL-6 gene expression in RANKL-stimulated osteoclast.

0 : RANKL(100 ng/mℓ) only

30 : RANKL(100 ng/mℓ) + 30 μg/mℓ of SRE

60 : RANKL(100 ng/mℓ) + 60 μg/mℓ of SRE

120 : RANKL(100 ng/mℓ) + 120 μg/mℓ of SRE

Each bar represents Mean±SD of 5 tests.

** : p<0.01 vs RANKL only

Fig. 6

Effects of SRE on (A)OC-STAMP and (B)CTR gene expression in RANKL-stimulated osteoclast.

NC : Vehicle

0 : RANKL(100 ng/mℓ) only

30 : RANKL(100 ng/mℓ) + 30 μg/mℓ of SRE

60 : RANKL(100 ng/mℓ) + 60 μg/mℓ of SRE

120 : RANKL(100 ng/mℓ) + 120 μg/mℓ of SRE

Each bar represents Mean±SD of 5 tests.

** : p<0.01 vs NC

## : p<0.01 vs RANKL only

Fig. 7

Effect of SRE on bone pit formation by RANKL-induced Osteoclast.

NC : Vehicle

0 : RANKL(100 ng/mℓ) only

60 : RANKL(100 ng/mℓ) + 60 μg/mℓ of SRE

120 : RANKL(100 ng/mℓ) + 120 μg/mℓ of SRE

Table 1

Composition of Sochungryong-tang

Herbal name	Pharmacognostic Name	Dose amount(g)
麻黃	Ephedrae Herba	6
桂枝	Cinnamomi Ramulus	6
白芍藥	Paeoniae Radix	6
半夏	Pinelliae Tuber	6
乾薑	Zingiberis Rhizoma	4
五味子	Schizandra Fructus	2
細辛	Asari Herba	2
甘草	Glycyrrhizae Radix et Rhizoma	2

Table 2

Primers

Target gene	Forward (5′-3′)	Reverse (5′-3′)
TRAP	ACACAGTGATGCTGTGTGGCAACTC	CCAGAGGCTTCCACATATATGATGG
Cathepsin K	AGGCGGCTATATGACCACTG	CCGAGCCAAGAGAGCATATC
MMP-9	CGTCGTGATCCCCACTTACT	AGAGTACTGCTTGCCCAGGA
NFATc1	GGGTCAGTGTGACCGAAGAT	GGAAGTCAGAAGTGGGTGGA
iNOS	CCTTGTTCAGCTACGCCTTC	AAGGCCAAACACAGCATACC
MITF	GGAACAGCAACGAGCTAAGG	TGATGATCCGATTCACCAGA
b-actin	TCACCCACACTCTGCCCAT	TCCTTAATGTCACGCACCATTT

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