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

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.

Fig. 1
Effect of SRE on cell viability.
A : RANKL(100 ng/m) only
B : RANKL(100 ng/m) + 18.7 μg/m of SRE
C : RANKL(100 ng/m) + 37.5 μg/m of SRE
D : RANKL(100 ng/m) + 75 μg/m of SRE
E : RANKL(100 ng/m) + 150 μg/m of SRE
F : RANKL(100 ng/m) + 300 μg/m of SRE
Each bar represents Mean±SD of 5 tests.
** : p<0.01 vs RANKL only
jkm-38-3-59f1.gif
Fig. 2
Effects of SRE on the formation of TRAP(+) MNCs in RANKL stimulated osteoclast.
0 : RANKL(100 ng/m) only
30 : RANKL(100 ng/m) + 30 μg/m of SRE
60 : RANKL(100 ng/m) + 60 μg/m of SRE
120 : RANKL(100 ng/m) + 120 μg/m of SRE
Each bar represents Mean±SD of 5 tests.
**: p<0.01 vs RANKL only
jkm-38-3-59f2.gif
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/m) only
30 : RANKL(100 ng/m) + 30 μg/m of SRE
60 : RANKL(100 ng/m) + 60 μg/m of SRE
120 : RANKL(100 ng/m) + 120 μg/m of SRE
Each bar represents Mean±SD of 5 tests.
* : p<0.05 vs NC
# : p<0.05 vs RANKL only
jkm-38-3-59f3.gif
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/m) only
30 : RANKL(100 ng/m) + 30 μg/m of SRE
60 : RANKL(100 ng/m) + 60 μg/m of SRE
120 : RANKL(100 ng/m) + 120 μg/m of SRE
Each bar represents Mean±SD of 5 tests.
* : p<0.05 vs NC
# : p<0.05 vs RANKL only
jkm-38-3-59f4.gif
Fig. 5
Effects of SRE on iNOS, COX-2, TNF-a, IL-6 gene expression in RANKL-stimulated osteoclast.
0 : RANKL(100 ng/m) only
30 : RANKL(100 ng/m) + 30 μg/m of SRE
60 : RANKL(100 ng/m) + 60 μg/m of SRE
120 : RANKL(100 ng/m) + 120 μg/m of SRE
Each bar represents Mean±SD of 5 tests.
** : p<0.01 vs RANKL only
jkm-38-3-59f5.gif
Fig. 6
Effects of SRE on (A)OC-STAMP and (B)CTR gene expression in RANKL-stimulated osteoclast.
NC : Vehicle
0 : RANKL(100 ng/m) only
30 : RANKL(100 ng/m) + 30 μg/m of SRE
60 : RANKL(100 ng/m) + 60 μg/m of SRE
120 : RANKL(100 ng/m) + 120 μg/m of SRE
Each bar represents Mean±SD of 5 tests.
** : p<0.01 vs NC
## : p<0.01 vs RANKL only
jkm-38-3-59f6.gif
Fig. 7
Effect of SRE on bone pit formation by RANKL-induced Osteoclast.
NC : Vehicle
0 : RANKL(100 ng/m) only
60 : RANKL(100 ng/m) + 60 μg/m of SRE
120 : RANKL(100 ng/m) + 120 μg/m of SRE
jkm-38-3-59f7.gif
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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