(S)-Equol Is More Effective than (R)-Equol in Inhibiting Osteoclast Formation and Enhancing Osteoclast Apoptosis, and Reduces Estrogen Deficiency–Induced Bone Loss in Mice

doi:10.1093/jn/nxac130

The Journal of Nutrition

Volume 152, Issue 8, August 2022, Pages 1831-1842

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Abstract

Background

Equol, a metabolite of daidzein, binds to the estrogen receptor with greater affinity than daidzein and exhibits various biological properties. It exists as an enantiomer, either (S)-equol or (R)-equol.

Objectives

We have previously shown that the inhibitory effect of (S)-equol on bone fragility is stronger than that of racemic equol in ovariectomized (OVX) mice; however, the effect of (R)-equol has not been elucidated. The aim of this study was to compare the activities of equol enantiomers on bone metabolism in vitro and in vivo.

Methods

Bone marrow cells (BMCs) and RAW 264.7 cells were treated with equol enantiomers. The number of osteoclasts and caspase-3/7 activity were measured. We examined the effect of equol enantiomers on osteoblast differentiation in MC3T3-E1 cells. In vivo, 8-wk-old female ddY mice were assigned to 4 groups: sham-operated (sham), OVX, OVX + 0.5 mg/d of (S)-equol (S-eq), and OVX + 0.5 mg/d of (R)-equol (R-eq). Four weeks after the intervention, femoral bone mineral density (BMD) and osteoclastic gene expression were analyzed, along with concentrations of equol enantiomers in the serum and tissues.

Results

(S)-equol and (R)-equol inhibited osteoclast differentiation in BMCs (97% and 60%, P < 0.05) and RAW 264.7 cells (83% and 68%, P < 0.05). (S)-equol promoted apoptosis of mature osteoclasts by inducing caspase-3/7 activity (29%, P < 0.05) and enhanced osteoblast differentiation (29%, P < 0.05). In OVX mice, BMD was ameliorated in (S)-equol-treated mice (11%, P < 0.05), but not in (R)-equol-treated mice. The concentrations of (S)-equol were greater than those of (R)-equol in the serum, tibia, liver, and kidney (by 148%, 80%, 22%, and 139%, respectively).

Conclusions

These results suggest that (S)-equol is more effective than (R)-equol in inhibiting osteoclast formation and enhancing osteoclast apoptosis in vitro, supporting the beneficial effect of (S)-equol to reduce estrogen deficiency–induced bone loss in OVX mice.

Key words

equol enantiomers

osteoclastogenesis

osteoporosis

ovariectomized mice

bone mineral density

Abbreviations

AP-1

activator protein-1

BMC

bone marrow cell

BMD

bone mineral density

CCK-8

Cell Counting Kit-8

Ctsk

cathepsin K

DC-STAMP

dendritic cell-specific transmembrane protein

estrogen receptor

ERK

extracellular signal-regulated kinase

JNK

c-jun N-terminal kinase

MAPK

mitogen-activated protein kinase

NFATc1

nuclear factor of activated T cells c1

OC-STAMP

osteoclast stimulatory transmembrane protein

OSCAR

osteoclast-associated receptor

OVX

ovariectomized

PPAR

peroxisome proliferator– activated receptor

RANKL

receptor activator of nuclear factor-κB ligand

R-eq

ovariectomized + 0.5 mg/d of (R)-equol group

S-eq

ovariectomized + 0.5 mg/d of (S)-equol group

SERM

selective estrogen receptor modulator

TRAP

tartrate-resistant acid phosphatase

1α,25-(OH)₂D₃

1α,25-dihydroxycholecalciferol

Cited by (0)

: Supported by Japan Society for the Promotion of Science KAKENHI grants JP15H01767 (to MU), JP19K24321 (to MT), JP20H02938 (to MU), and JP21K17646 (to MT).

: Author disclosures: the authors report no conflicts of interest.

: Supplemental Table 1 is available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/.