J Bone Metab.  2019 May;26(2):113-121. 10.11005/jbm.2019.26.2.113.

Extracts of Flavoparmelia sp. Inhibit Receptor Activator of Nuclear Factor-κB Ligand-Mediated Osteoclast Differentiation

Affiliations
  • 1Department of Pharmacy, Sunchon National University, Suncheon, Korea. sony@sunchon.ac.kr
  • 2Korean Lichen Research Institute, Sunchon National University, Suncheon, Korea. jshur1@sunchon.ac.kr

Abstract

BACKGROUND
Osteoporosis is a geriatric disease with diminished bone density. The increase in the number of patients and medical expenses due to a global aging society are recognized as problems. Bone loss is the most common symptom of bone disease, not only osteoporosis but Paget's disease, rheumatoid arthritis, multiple myeloma, and other diseases. The main cause of this symptoms is excessive increase in the number and activity of osteoclasts. Osteoclasts are multinucleated giant cells that can resorb bone. They are differentiated and activation from monocytes/macrophages in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL).
METHODS
The effect of extract of Flavoparmelia sp. (EFV), a genus of lichenized fungi within the Parmeliaceae, on the differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts was examined by phenotype assay and the cell cytotoxicity was evaluated by cell counting kit-8. The osteoclast differentiation-related genes and proteins were investigated by real-time polymerase chain reaction and immunoblotting. The functional activity of osteoclast in response to EFV treatment was evaluated by an Osteo Assay plate.
RESULTS
In this study, we found that EFV, a genus of lichenized fungi within the Parmeliaceae, inhibited osteoclast formation. And we investigated its inhibitory mechanism. EFV reduced RANKL-mediated osteoclast formation and activation by inhibiting expression of nuclear factor of activated T cells 1, a key factor of osteoclastogenesis.
CONCLUSIONS
Taken together, our results show that EFV is a promising candidate for health functional foods or therapeutic agents that can help treat bone diseases such as osteoporosis.

Keyword

Flavoparmelia; Lichens; NFATC transcription factors; Osteoclasts; Osteoporosis

MeSH Terms

Aging
Arthritis, Rheumatoid
Bone Density
Bone Diseases
Cell Count
Functional Food
Fungi
Giant Cells
Humans
Immunoblotting
Lichens
Macrophage Colony-Stimulating Factor
Macrophages
Multiple Myeloma
NFATC Transcription Factors
Osteoclasts*
Osteoporosis
Parmeliaceae
Phenotype
Real-Time Polymerase Chain Reaction
T-Lymphocytes
Macrophage Colony-Stimulating Factor
NFATC Transcription Factors

Figure

  • Fig. 1 High performance liquid chromatography (HPLC) profiling of methanol extract from Flavoparmelia sp. methanol extract of Flavoparmelia sp. was analyzed by HPLC with YMC-Pack ODS-A reversed-phase C18 column. The ultraviolet–visible-spectra of the maximum absorption wavelengths (λmax) of galbinic acid are present in the insert. By comparing both the retention time and λmax with the standard, the main compound of the extract was identified to be galbinic acid. The molecular structure of galbinic acid is also presented in the inset.

  • Fig. 2 The extracts of Flavoparmelia sp. (EFV) inhibited osteoclast differentiation. (A) Bone marrow-derived macrophages (BMMs) were cultured with 10 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) and 30 ng/mL macrophage colony-stimulating factor (M-CSF) for 4 days in the presence of the vehicle (0.1% dimethyl sulfoxide [DMSO]) or the indicated concentrations of EFV. The cells were fixed in 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained with tartrate-resistant acid phosphatase (TRAP) solution. (B) TRAP-positive multinucleated cells (3 or more nuclei) were counted as osteoclasts. (C) BMMs were cultured with 30 ng/mL M-CSF for 3 days in the presence of the vehicle (0.1% DMSO) or the indicated concentrations of EFV. The effects of EFV on the BMMs viability were assessed using a cell counting kit-8. *P<0.001 (n=3).

  • Fig. 3 Effects of the extracts of Flavoparmelia sp. (EFV) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated mRNA expressions of nuclear factor of activated T cells 1 (NFATc1). The bone marrow-derived macrophages were treated with the vehicle (0.1% DW) or EFV (10 µg/mL) and macrophage colony-stimulating factor (30 ng/mL) for 1 hr and then RANKL (10 ng/mL) were treated at the indicated times. The total RNA was then isolated using the TRIzol reagent, and the mRNA expression levels were evaluated by performing real-time polymerase chain reaction. (A) NFATc1, (B) dendritic cell-specific transmembrane protein (DC-STAMP), (C) tartrate-resistant acid phosphatase (TRAP), and (D) cathepsin K. Glyceraldehyde-3-phosphate dehydrogenase was used as the internal control. *P<0.05. **P<0.01. ***P<0.001.

  • Fig. 4 The extracts of Flavoparmelia sp. (EFV) abolishes receptor activator of nuclear factor-κB ligand (RANKL)-mediated protein expression of nuclear factor of activated T cells 1 (NFATc1). Bone marrow-derived macrophages were pretreated with the vehicle (0.1% dimethyl sulfoxide) or EFV (10 µg/mL) and macrophage colony-stimulating factor (30 ng/mL) for 1 hr prior to RANKL (10 ng/mL) stimulation for the indicated times. The cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting was performed with anti-NFATc1, and anti-actin antibodies as indicated.

  • Fig. 5 The extracts of Flavoparmelia sp. (EFV) inhibited bone resorption by receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclasts. (A) Bone marrow-derived macrophages were plated on an Osteo Assay Plate and treated with 30 ng/mL macrophage colony-stimulating factor and 10 ng/mL RANKL in the presence of 10 µg/mL of EFV. After 4 days of culture, the cells attached to the Osteo Assay plate were removed and photographed under an optical microscope. (B) Pit areas were quantified using the ImageJ program. *P<0.001 (n=3).


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