Ethanolic extract of Schizonepeta tenuifolia attenuates osteoclast formation and activation in vitro and protects against lipopolysaccharide-induced bone loss in vivo

Abstract

Background: Excessive osteoclast activity is a major cause of metabolic bone disorders, such as osteopenia, rheumatoid arthritis, and osteoporosis. Thus, discovery of agents targeting osteoclast differentiation and bone resorption is important for development of novel treatments for bone diseases. It has been demonstrated that ethanolic extract of schizonepeta tenuifolia (EEST) has potent anti-oxidant and anti-inflammatory activities. However, the beneficial effects of EEST on bone metabolism have not been studied. Therefore, we intend to investigate the effects of EEST on osteoclast differentiation.

Methods: We examined the effects and mechanisms of action of the EEST on osteoclastogenesis in vitro in bone marrow macrophages (BMMs) stimulated with receptor activator of nuclear factor kappa-B ligand (RANKL) and in vivo using a mouse model of lipopolysaccharide (LPS)-induced bone destruction.

Results: We found that EEST inhibited phosphorylation of Akt and IkB at early stages of RANKL-induced osteoclastogenesis. Furthermore, EEST negatively controlled the transcription and translation levels of nuclear factor of activated T cells c1 (NFATc1) and the translation level of c-Fos at the final stage of osteoclast differentiation. Reflecting these effects, EEST blocked both filamentous actin (F-actin) ring formation and bone resorbing activity of mature osteoclasts in vitro. The inhibitory effects of EEST on osteoclast formation and activity were observed in an LPS-mediated bone erosion mouse model using micro-CT and histological analysis.

Conclusions: EEST is a potential agent that is able to treat osteoclast-related bone diseases, such as osteoporosis.

Keywords: Bone resorption; Osteoclast differentiation; Osteoporosis; Schizonepeta tenuifolia.

Fig. 1

EEST attenuates RANKL-induced osteoclast differentiation in a dose-dependent manner with no cytotoxicity. a BMMs were cultured for 4 days in the presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL) with the indicated concentrations of EEST. Cells were fixed in 3.7 % formalin, permeabilized with 0.1 % Triton X-100, and stained with TRAP solution. TRAP⁺ MNCs were photographed under a light microscope. b TRAP⁺ MNCs with more than 5 nuclei were counted. ***P < 0.001, **P < 0.01 vs. control. c BMMs were seeded into a 96-well plate and cultured for 3 days in the presence of M-CSF (30 ng/mL) and the indicated concentrations of EEST. After 3 days, the absorbance at 450 nm was determined using an ELISA reader

Fig. 2

EEST down-regulates RANKL-mediated phosphorylation of Akt and IkB. a BMMs were cultured for 1 day in the presence of M-CSF (10 ng/mL). Next, BMMs were starved for 3 h, pretreated with EEST (50 μg/mL) for 1 h and then stimulated with RANKL (50 ng/mL) for the indicated times. Cell lysates were analyzed by western blotting with antibodies against p-p38, p38, p-ERK, ERK, p-JNK, JNK, p-Akt, Akt, p-IκB, IκB, and GAPDH. b BMMs were infected with retroviruses expressing pMX-IRES-EGFP (pMX), pMX- CA-IKKβ-EGFP, and pMX-Akt-EGFP. Infected BMMs were cultured with or without EEST (50 μg/mL) in the presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 4 days. After culturing, the cells were fixed and stained for TRAP (left). The TRAP+ MNCs with more than 5 nuclei were counted (right). *P < 0.05, **P < 0.01 vs. the indicated group

Fig. 3

EEST inhibits protein expression of c-Fos and both mRNA and protein expression of NFATc1. a BMMs were pretreated with or without EEST (50 μg/mL) for 1 h and then stimulated with M-CSF (30 ng/mL) and RANKL (50 ng/mL) for the indicated times. The cell lysates were analyzed by western blotting with antibodies against c-Fos, NFATc1, and GAPDH. b BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of EEST (50 μg/mL) for the indicated times. Total RNA was isolated from cells using the QIAzol reagent, and mRNA expression of c-Fos and NFATc1 was determined using quantitative real-time RT-PCR. ***P < 0.001 vs. control in the indicated time

Fig. 4

EEST negatively controls F-actin structure, bone-resorbing activity, and expression of osteoclast-specific genes. a BMMs were cultured for 4 days in the presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL) with or without EEST (50 μg/mL). Cells were fixed with 3.7 % formalin, permeabilized with 0.1 % Triton X-100, and stained with phalloidin and DAPI. Mature osteoclasts were seeded on hydroxyapatite-coated plates for 24 h with EEST (50 μg/mL) treatment. Adherent cells were removed and photographed under a light microscope. b The relative ratio of pit areas was quantified using Image J. ***P < 0.001 vs. control. c BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of EEST (50 μg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent, and mRNA expression of β3-integrin, DC-STAMP, Atp6v0d2, and Cathepsin K was determined using quantitative real-time RT-PCR. ***P < 0.001 vs. control in the indicated time

Fig. 5

EEST recovers LPS-induced inflammatory bone loss in mice. a Mice were sacrificed 8 days after the first LPS injection and 2D or 3D radiographs of the coronal and transverse planes of the proximal femora were obtained by micro-CT. b The BV/TV, Tb.Sp, Tb.Th, and Tb.N of the femora were determined using the micro-CT data and analyzed by INFINITT-Xelis software. ***P < 0.001 vs. control; ^## P < 0.01, ^### P < 0.001 vs. LPS group. c Dissected femora were fixed, decalcified, embedded, and sectioned. Sections were stained with TRAP and H&E. d The number of osteoclasts per field of tissue was counted by histomorphometric analysis