Inhibitory Effects of Ssangbohwan on Osteoclast Differentiation and Bone Resorption

Article information

Acupunct. 2015;32(3):69-81
1Department of Acupuncture & Moxibustion Medicine, College of Oriental Medicine, Wonkwang University
2Department of Oriental Medical Ophthalmology & Otolaryngology & Dermatology, College of Oriental Medicine, Wonkwang University
3Research Center of Traditional Korean Medicine, Wonkwang University
*Corresponding author: Department of Acupuncture & Moxibustion Medicine, College of Oriental Medicine, Wonkwang University, 460, Iksandae-ro, Iksan-si, Jeollabuk-do, 54538, Republic of Korea, Tel: +82-63-270-1022, E-mail: freezo@wonkwang.ac.kr
Received 2015 August 06; Revised 2015 September 07; Accepted 2015 September 09.

Abstract

Purpose:

The first purpose of this study is to find out whether the water extract of Rehmanniae Radix Preparat(RRP), Cuscutae Semen(CS) and their combination(Ssangbohwan, SBH) have the effect of suppressing Receptor activator of nuclear factor kappa-B ligand(RANKL)-induced osteoclast differentiation. The second purpose of this study is to find out whether the water extract of RRP, CS and SBH have the effect of inhibiting osteoporosis in an osteoporosis model induced by lipopolysaccharide(LPS).

Methods:

After promoting differentiation of osteoclasts by treating the RANKL, we observed the effect by the administration of RRP, CS and SBH. In addition, by means of Reverse transcription polymerase chain reaction(RT-PCR), we assayed mRNA expression levels of NFATc1, c-Fos, TRAP and GAPDHS(Glyceraldehyde-3-phosphate dehydrogenase, spermatogeni) from bone marrow macrophages(BMMs). Similarly, the protein expression levels of NFATc1 (Nuclear factor of activated T-cells, cytoplasmic1), C-Fos, MAPKs(Mitogen-activated protein kinases) and β-actin in cell lysates were analyzed by means of Western Blotting. Finally, we determined the anti-osteoporotic effects of RRP, CS and SBH, through the use of Lipopolysaccharide-induced bone-loss mouse.

Results:

RRP, CS and SBH showed remarkable inhibitive effect on RANKL-treated osteoclast differentiation without cytotoxicity. SBH inhibited the phosphorylation of p38, Jun N-terminal kinases(JNK), and I-κB and down-regulated the induction of c-Fos and NFATc1 by RANKL. RRP, CS suppressed degradation of I-κB, but it did not affect c-Fos and NFATc1 by RANKL. Lastly, in vivo data showed that RRP and SBH prevented bone erosion by LPS treatment.

Conclusions:

These results demonstrate SBH can be effective remedy for bone-loss diseases such as osteoporosis.

Fig. 1

Ssangbohwan(SBH) inhibits osteoclast differentiation

Bone marrow macrophages(BMMs) were cultured for 4 d with M-CSF(30 ng/ml) and RANKL(100 ng/ml) in the presence or absence of Ssangbohwan.

A: after 4 d, cells were fixed in 3.7 % formalin, permeabilized in 0.1 % triton x-100, and stained for TRAP.

B: TRAP-positive cells were counted as osteoclasts. Asterisks(*) indicate statistical differences from the control(*: p<0.05, **: p<0.01, ***: p<0.001).

N=4

Fig. 2

Rehmanniae Radix Preparat(RRP) and Cuscutae Semen(CS) inhibits osteoclast differentiation

Bone marrow macrophages(BMMs) were cultured for 4 d with M-CSF(30 ng/ml) and RANKL(100 ng/ml) in the presence or absence of Rehmanniae Radix Preparat and Cuscutae Semen.

A: After 4 d, cells were fixed in 3.7 % formalin, permeabilized in 0.1 % Triton x-100, and stained for TRAP.

B: TRAP-positive cells were counted as osteoclasts. Asterisks(*) indicate statistical differences from the control(*: p<0.05, **: p<0.01, ***: p<0.001).

N=4

Fig. 3

The effect of Rehmanniae Radix Preparat (RRP), Cuscutae Semen(CS) and Ssangbohwan (SBH) on cell viability

BMMs were cultured for 3 d with M-CSF(30 ng/ml) in the presence or absence of Rehmanniae Radix Preparat, Cuscutae Semen and Ssangbohwan.

After 3 d, each well were added with XTT solution and incubated for 4–6 h.

The plate was measured at 450 nm using a microplate reader.

Fig. 4

Ssangbohwan(SBH) suppresses RANKL-induced c-Fos, NFATc1 and TRAP expression

BMMs were pretreated with or without Ssangbohwan(100 μg/ml) for 1 h and then stimulated with RANKL(100 ng/ml) for the indicated times.

A: each mRNA were analyzed by RT-PCR.

B: quantitative analysis by image J program.

Significance(*) indicates significance for the difference between control and extraction treatment group(*: p<0.05, **: p<0.01, ***: p<0.001).

Error bars indicate SDs; N=4.

Fig. 5

Rehmanniae Radix Preparat(RRP) and Cuscutae Semen(CS) suppresses RANKL-induced c-Fos, NFATc1 and TRAP expression BMMs were pretreated with or without Rehmanniae Radix Preparat and Cuscutae Semen(50 μg/ml) for 1 h and then stimulated with RANKL(100 ng/ml) for the indicated times.

A: each mRNA were analyzed by RT-PCR.

B: quantitative analysis by image J program. Significance(*) indicates significance for the difference between control and extraction treatment group(*: p<0.05, **: p<0.01, ***: p<0.001).

Error bars indicate SDs; N=4.

Fig. 6

Ssangbohwan(SBH) suppresses RANKL-induced c-Fos and NFATc1 expression

BMMs were pretreated with or without Ssangbohwan(100 μg/ml) for 1 hour and then stimulated with RANKL(100 ng/ml) for the indicated times.

The cell lysates were analyzed by western blotting with antibodies for c-Fos, NFATc1, and β-actin.

Fig. 7

Rehmanniae Radix Preparat(RRP) and Cuscutae Semen(CS) suppresses RANKL-induced c-Fos and NFATc1 expression

BMMs were pretreated with or without Rehmanniae Radix Preparat and Cuscutae Semen(50 μg/ml) for 1 hour and then stimulated with RANKL(100 ng/ml) for the indicated times.

The cell lysates were analyzed by Western blotting with antibodies for c-Fos, NFATc1, and β-actin.

Fig. 8

Ssangbohwan(SBH) inhibits RANKL signaling pathways

BMMs were pretreated with or without Ssangbohwan(100 μg/ml) and further stimulated with RANKL(100 ng/ml) for the indicated time.

The cells were lysed and proteins were revolved by 10 % SDS-PAGE and subjected to western blot analysis.

Fig. 9

Rehmanniae Radix Preparat(RRP) and Cuscutae Semen(CS) inhibits RANKL signaling pathways

BMMs were pretreated with or without Rehmanniae Radix Preparat and Cuscutae Semen(50 μg/ml) and further stimulated with RANKL(100 ng/ml) for the indicated time.

The cells were lysed and proteins were revolved by 10 % SDS-PAGE and subjected to western blot analysis.

Fig. 10

Skyscan analysis of LPS-induced bone loss in mice

A: Mice were oral-treated Rehmanniae Radix Preparat(RRP), Cuscutae Semen(CS) and Ssangbohwan(SBH) extracts(200 or 400 mg/kg/day) or 1×PBS for every 10 days. LPS(5 μm/g of body weight) or 1×PBS was administered intraperitoneally on day 4 and 7, and the femurs were collected on day 10 after the first of PPR, CS and SBH extracts. Radiographs were taken with a Skyscan.

B: Bone volume/tissue volume(BV/TV), trabecular thickness, trabecular separation and trabecular number was analyzd using the histomorphometric results. Asterisks indicate statistical difference from the Lipopolysaccharide(LPS) group. *: p<0.001. **: p <0.05, ***:p: <0.01.

Extraction Yield of Medicinal Herbs Used in This Study

Primers for PCR Amplification

Skyscan Analysis of LPS-induced Bone Loss in Mice

Notes

This study was supported by academic research grant of Wonkwang University in 2013

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Article information Continued

Fig. 1

Ssangbohwan(SBH) inhibits osteoclast differentiation

Bone marrow macrophages(BMMs) were cultured for 4 d with M-CSF(30 ng/ml) and RANKL(100 ng/ml) in the presence or absence of Ssangbohwan.

A: after 4 d, cells were fixed in 3.7 % formalin, permeabilized in 0.1 % triton x-100, and stained for TRAP.

B: TRAP-positive cells were counted as osteoclasts. Asterisks(*) indicate statistical differences from the control(*: p<0.05, **: p<0.01, ***: p<0.001).

N=4

Fig. 2

Rehmanniae Radix Preparat(RRP) and Cuscutae Semen(CS) inhibits osteoclast differentiation

Bone marrow macrophages(BMMs) were cultured for 4 d with M-CSF(30 ng/ml) and RANKL(100 ng/ml) in the presence or absence of Rehmanniae Radix Preparat and Cuscutae Semen.

A: After 4 d, cells were fixed in 3.7 % formalin, permeabilized in 0.1 % Triton x-100, and stained for TRAP.

B: TRAP-positive cells were counted as osteoclasts. Asterisks(*) indicate statistical differences from the control(*: p<0.05, **: p<0.01, ***: p<0.001).

N=4

Fig. 3

The effect of Rehmanniae Radix Preparat (RRP), Cuscutae Semen(CS) and Ssangbohwan (SBH) on cell viability

BMMs were cultured for 3 d with M-CSF(30 ng/ml) in the presence or absence of Rehmanniae Radix Preparat, Cuscutae Semen and Ssangbohwan.

After 3 d, each well were added with XTT solution and incubated for 4–6 h.

The plate was measured at 450 nm using a microplate reader.

Fig. 4

Ssangbohwan(SBH) suppresses RANKL-induced c-Fos, NFATc1 and TRAP expression

BMMs were pretreated with or without Ssangbohwan(100 μg/ml) for 1 h and then stimulated with RANKL(100 ng/ml) for the indicated times.

A: each mRNA were analyzed by RT-PCR.

B: quantitative analysis by image J program.

Significance(*) indicates significance for the difference between control and extraction treatment group(*: p<0.05, **: p<0.01, ***: p<0.001).

Error bars indicate SDs; N=4.

Fig. 5

Rehmanniae Radix Preparat(RRP) and Cuscutae Semen(CS) suppresses RANKL-induced c-Fos, NFATc1 and TRAP expression BMMs were pretreated with or without Rehmanniae Radix Preparat and Cuscutae Semen(50 μg/ml) for 1 h and then stimulated with RANKL(100 ng/ml) for the indicated times.

A: each mRNA were analyzed by RT-PCR.

B: quantitative analysis by image J program. Significance(*) indicates significance for the difference between control and extraction treatment group(*: p<0.05, **: p<0.01, ***: p<0.001).

Error bars indicate SDs; N=4.

Fig. 6

Ssangbohwan(SBH) suppresses RANKL-induced c-Fos and NFATc1 expression

BMMs were pretreated with or without Ssangbohwan(100 μg/ml) for 1 hour and then stimulated with RANKL(100 ng/ml) for the indicated times.

The cell lysates were analyzed by western blotting with antibodies for c-Fos, NFATc1, and β-actin.

Fig. 7

Rehmanniae Radix Preparat(RRP) and Cuscutae Semen(CS) suppresses RANKL-induced c-Fos and NFATc1 expression

BMMs were pretreated with or without Rehmanniae Radix Preparat and Cuscutae Semen(50 μg/ml) for 1 hour and then stimulated with RANKL(100 ng/ml) for the indicated times.

The cell lysates were analyzed by Western blotting with antibodies for c-Fos, NFATc1, and β-actin.

Fig. 8

Ssangbohwan(SBH) inhibits RANKL signaling pathways

BMMs were pretreated with or without Ssangbohwan(100 μg/ml) and further stimulated with RANKL(100 ng/ml) for the indicated time.

The cells were lysed and proteins were revolved by 10 % SDS-PAGE and subjected to western blot analysis.

Fig. 9

Rehmanniae Radix Preparat(RRP) and Cuscutae Semen(CS) inhibits RANKL signaling pathways

BMMs were pretreated with or without Rehmanniae Radix Preparat and Cuscutae Semen(50 μg/ml) and further stimulated with RANKL(100 ng/ml) for the indicated time.

The cells were lysed and proteins were revolved by 10 % SDS-PAGE and subjected to western blot analysis.

Fig. 10

Skyscan analysis of LPS-induced bone loss in mice

A: Mice were oral-treated Rehmanniae Radix Preparat(RRP), Cuscutae Semen(CS) and Ssangbohwan(SBH) extracts(200 or 400 mg/kg/day) or 1×PBS for every 10 days. LPS(5 μm/g of body weight) or 1×PBS was administered intraperitoneally on day 4 and 7, and the femurs were collected on day 10 after the first of PPR, CS and SBH extracts. Radiographs were taken with a Skyscan.

B: Bone volume/tissue volume(BV/TV), trabecular thickness, trabecular separation and trabecular number was analyzd using the histomorphometric results. Asterisks indicate statistical difference from the Lipopolysaccharide(LPS) group. *: p<0.001. **: p <0.05, ***:p: <0.01.

Table 1

Extraction Yield of Medicinal Herbs Used in This Study

Korean name Pharmaceutical name Abbreviation Yield
Sukjihwang Rehmanniae Radix Preparat RRP 49.6 %
Tosaja Cuscutae Semen CS 11.9 %
Ssangbohwan Ssangbohwan SBH 15.1%

Table 2

Primers for PCR Amplification

Primer Sequence
c-Fos Forward
Reverse
5′- CTGGTGCAGCCCACTCTGGTC-3′
5′- CTTTCAGCAGATTGGCAATCTC-3′
NFATc1 Forward
Reverse
5′- CAACGCCCTGACCACCGATAG-3′
5′- GGCTGCCTTCCGTCTCATAGT-3′
TRAP Forward
Reverse
5′-ACTTCCCCAGCCCTTACTAC-3′
5′-TCAGCACATAGCCCACACCG-3′
GAPDH Forward
Reverse
5′-ACCACAGTCCATGCCATCAC-3′
5′-TCCACCACCCTGTTGCTGTA-3′

Table 3

Skyscan Analysis of LPS-induced Bone Loss in Mice

CTR RRP200 CS200 LPS +
RRP200 CS200 SBH200 SBH400
BV/TV(%) 32.3±3.1 29.5±3.4 28.0±1.6 26.5±0.6 29.4±0.6 21.6±1.9 29.7±2.4 26.1±1.0
Trabecular thickness(μm) 16.0±1.1 13.9±0.6 13.8±0.2 14.7±0.5 15.8±0.1 13.0±0.4 2.1±16.3 14.6±1.4
Trabecular separation(μm) 33.5±2.7 33.4±3.9 35.6±2.3 41.0±2.0 37.9±0.8 50.3±0.8 38.4±0.6 41.4±2.2
Trabecular number(1/μm) 0.021±0.0 0.021±0.0 0.020±0.0 0.018±0.0 0.019±0.0 0.016±0.0 0.018±0.0 0.018±0.0

CTR: control group. RRP: Rehmanniae Radix Preparat. CS: Cuscutae Semen. SBH: Ssangbohwan. LPS: lipopolysaccharide.