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. 2020 Dec 16;13(12):468.
doi: 10.3390/ph13120468.

Eudebeiolide B Inhibits Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Regulating RANKL-Induced NF-κB, c-Fos and Calcium Signaling

Mi-Hwa Kim  1 , Hyung-Jin Lim  2 , Seon Gyeong Bak  2 , Eun-Jae Park  2 , Hyun-Jae Jang  3 , Seung Woong Lee  2 , Soyoung Lee  2 , Kang Min Lee  4 , Sun Hee Cheong  5 , Seung-Jae Lee  2 , Mun-Chual Rho  2
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V体育安卓版 - Eudebeiolide B Inhibits Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Regulating RANKL-Induced NF-κB, c-Fos and Calcium Signaling

Mi-Hwa Kim et al. Pharmaceuticals (Basel). .

Abstract

Eudebeiolide B is a eudesmane-type sesquiterpenoid compound isolated from Salvia plebeia R. Br. , and little is known about its biological activity. In this study, we investigated the effects of eudebeiolide B on osteoblast differentiation, receptor activator nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vitro and ovariectomy-induced bone loss in vivo. Eudebeiolide B induced the expression of alkaline phosphatase (ALP) and calcium accumulation during MC3T3-E1 osteoblast differentiation. In mouse bone marrow macrophages (BMMs), eudebeiolide B suppressed RANKL-induced osteoclast differentiation of BMMs and bone resorption. Eudebeiolide B downregulated the expression of nuclear factor of activated T-cells 1 (NFATc1) and c-fos, transcription factors induced by RANKL. Moreover, eudebeiolide B attenuated the RANKL-induced expression of osteoclastogenesis-related genes, including cathepsin K (Ctsk), matrix metalloproteinase 9 (MMP9) and dendrocyte expressed seven transmembrane protein (DC-STAMP). Regarding the molecular mechanism, eudebeiolide B inhibited the phosphorylation of Akt and NF-κB p65. In addition, it downregulated the expression of cAMP response element-binding protein (CREB), Bruton's tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2) in RANKL-induced calcium signaling. In an ovariectomized (OVX) mouse model, intragastric injection of eudebeiolide B prevented OVX-induced bone loss, as shown by bone mineral density and contents, microarchitecture parameters and serum levels of bone turnover markers. Eudebeiolide B not only promoted osteoblast differentiation but inhibited RANKL-induced osteoclastogenesis through calcium signaling and prevented OVX-induced bone loss. Therefore, eudebeiolide B may be a new therapeutic agent for osteoclast-related diseases, including osteoporosis, rheumatoid arthritis and periodontitis. VSports手机版.

Keywords: OVX mouse model; RANKL; calcium signal; eudebeiolide B; osteoporosis V体育安卓版. .

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Eudebeiolide B inhibits RANKL-induced osteoclast differentiation and promotes osteoblast differentiation. (A) Structure of eudebeiolide B (n = 3). (B) Cytotoxicity of eudebiolide B. Bone marrow macrophages (BMMs) were seeded and treated with indicated concentrations of eudebeiolide B in the presence of M-CSF (30 ng/mL) for 72 h. Cell viability was analyzed using the XTT assay (n = 3). (C) BMMs were treated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) after pretreatment with 1, 5, 10 or 30 μM of eudebeiolide B for 1 h. Cells were fixed and stained with TRAP staining solution. TRAP-positive multinucleated cells (TRAP + MNCs) with more than three nuclei were defined as osteoclasts and counted (n = 3). (D) BMMs were seeded and treated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) for 72 h. Cells were then treated with eudebeiolide B for 48 h. Bone resorption areas were measured using ImageJ (n = 3). (E) MC3T3-E1 cells were seeded and incubated with differentiation media and eudebeiolide B for 7 days. Osteoblast differentiation was determined by ALP staining, and ALP activity was measured using the cell lysate (n = 3). (F) MC3T3-E1 cells were seeded and incubated with differentiation media and eudebeiolide B for 21 days. Osteoblast differentiation was assessed by Alizarin red staining, and calcium accumulation was quantified with cetylpyridinium chloride solution (n = 3). (G) MC3T3-E1 cells were treated with or without eudebeiolide B (10 μM) and cultured for 24, 48 and 72 h. The mRNA expression of Runx2, Osterix, OPG and RANKL was assessed by quantitative PCR (n = 3). Values are expressed as the means ± S.D. of three individual experiments. * p < 0.05 and ** p < 0.01 versus control (C–E) obtained through one-way ANOVA followed by Dunnett’s test; * p < 0.05 and ** p < 0.01 versus the only-DM-treated group (G) obtained through the unpaired Student’s t-test.
Figure 2
Figure 2
Attenuation of RANKL-induced osteoclastogenesis-related transcription factor and gene expression by eudebeiolide B. (A–C) BMMs were pretreated with or without eudebeiolide B and stimulated with RANKL (100 ng/mL) for the indicated time. (A) Western blot analysis of c-Fos and NFATc1 protein was performed, and band densities of (B) c-Fos and (C) NFATc1 were quantified by ImageJ (n = 3). (D–G) BMMs were cultured in the presence of RANKL (100 ng/mL) with or without eudebeiolide B for 48 h. (D) NFATc1, (E) cathepsin K, (F) MMP9 and (G) DC-STAMP mRNAs were analyzed by quantitative RT-PCR (n = 3). All data are expressed as the means ± S.D. of three individual experiments. * p < 0.05 and ** p < 0.01 versus the only-RANKL-treated group (B,C) obtained through the unpaired Student’s t-test; * p < 0.05 and ** p < 0.01 versus the only-RANKL-treated group (D–G) obtained through one-way ANOVA followed by Dunnett’s test.
Figure 3
Figure 3
Effect of eudebeiolide B on RANKL-induced cellular signaling. BMM cells were pretreated with or without eudebeiolide B (10 μM) for 1 h and then stimulated with RANKL (100 ng/mL) for the indicated times. (A) Expression of signaling molecules determined by Western blot analysis. Band optical densities of (B) ERK, (C) P38, (D) JNK, I AKT and (F) NF-κB p65 were evaluated by ImageJ software (n = 3). All data are expressed as the means ± S.D. * p < 0.05 and ** p < 0.01 versus the only-RANKL-treated group obtained through the unpaired Student’s t-test.
Figure 4
Figure 4
Eudebeiolide B inhibits RANKL-induced calcium signaling. (A) Phosphorylation was evaluated by Western blot analysis. Eudebeiolide B inhibits the phosphorylation of (B) Btk, (C) PLCγ2, (D) CaMKIV and (E) CREB. BMMs were pretreated with or without eudebeiolide B (10 μM) for 1 h and then stimulated with RANKL (100 ng/mL) for the indicated times. Western blot analyses were performed with the indicated antibodies (n = 3). All data are expressed as the means ± S.D. * p < 0.05 versus the only-RANKL-treated group obtained through the unpaired Student’s t-test.
Figure 5
Figure 5
Bone mineral density BMD, bone mineral content BMC and biochemical serum analysis of the sham-operated, OVX + vehicle, OVX + eudebeiolide B-treated and OVX + alendronate-treated mice. (A) BMD and (B) BMC of the whole body were analyzed using DEXA (n = 6). The serum levels of (C) ALP, (D) c-terminal telopeptide of type 1 collagen (CTX), (E) osteoprotegerin (OPG) and (F) RANKL were determined by ELISA (n = 6). (G) RANKL/OPG ratio was calculated (n = 6). Data are expressed as the means ± S.D. # p < 0.05 versus the sham group obtained through the unpaired Student’s t-test; * p < 0.05, ** p < 0.01 versus the only-OVX-treated group obtained through one-way ANOVA followed by Dunnett’s test.
Figure 6
Figure 6
Micro-CT analysis of the proximal femurs from the eudebeiolide B- or alendronate-treated OVX mice. (A) The micro-CT images of longitudinal and transverse of the proximal femurs from the sham-operated, OVX + vehicle, OVX + eudebeiolide B-treated and OVX + alendronate-treated mice were obtained. (B) The bone volume/total volume (BV/TV), (C) trabecular thickness (Tb. Th.) and (D) trabecular number (Tb. N.) of the femurs were analyzed using micro-CT (n = 6). Data are expressed as the means ± S.D. # p < 0.05 versus the sham group obtained through the unpaired Student’s t-test; * p < 0.05, ** p < 0.01 versus the only-OVX-treated group obtained through one-way ANOVA followed by Dunnett’s test.
Figure 7
Figure 7
Histological analysis of the proximal tibia tissue sections from the eudebeiolide B- or alendronate-treated OVX mice. (A) H&E stains, (B) histochemical stains for TRAP and (C) immunohistochemical stains for ALP of the sham-operated, OVX + vehicle, OVX + eudebeiolide B-treated and OVX + alendronate-treated mice were obtained (n = 6). (D) Osteoclast surface/bone surface (Oc.S/BS) and (E) Osteoblast surface/bone surface (Ob.S/BS) were evaluated by HistoMorph software (n = 6). Data are expressed as the means ± S.D. # p < 0.05 versus the sham group obtained through the unpaired Student’s t-test; * p < 0.05 versus the only-OVX-treated group obtained through one-way ANOVA followed by Dunnett’s test.
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