Iris Koreana NAKAI Inhibits Osteoclast Formation via p38-Mediated Nuclear Factor of Activated T Cells 1 Signaling Pathway

Abstract

Background: Iris Koreana NAKAI (IKN) is a flowering perennial plant that belongs to the Iridaceae family. In this study, we aimed to demonstrate the effects of IKN on osteoclast differentiation in vitro and in vivo. We also sought to verify the molecular mechanisms underlying its anti-osteoclastogenic effects.

Methods: Osteoclasts were formed by culturing mouse bone marrow macrophage (BMM) cells with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). Bone resorption assays were performed on dentin slices. mRNA expression levels were analyzed by quantitative polymerase chain reaction. Western blotting was performed to detect protein expression or activation. Lipopolysaccharide (LPS)-induced osteoclast formation was performed using a mouse calvarial model.

Results: In BMM cultures, an ethanol extract of the root part of IKN suppressed RANKL-induced osteoclast formation and bone resorptive activity. In contrast, an ethanol extract of the aerial parts of IKN had a minor effect on RANKL-induced osteoclast formation. Mechanistically, the root part of IKN suppressed RANKL-induced p38 mitogen-activated protein kinase (MAPK) activation, effectively abrogating the induction of c-Fos and nuclear factor of activated T cells 1 (NFATc1) expression. IKN administration decreased LPS-induced osteoclast formation in a calvarial osteolysis model in vivo.

Conclusions: Our study suggested that the ethanol extract of the root part of IKN suppressed osteoclast differentiation and function partly by downregulating the p38 MAPK/c-Fos/NFATc1 signaling pathways. Thus, the root part.

Keywords: Bone diseases, metabolic; NFATc1 transcription factors; Osteoclasts; p38 mitogen-activated protein kinases.

Fig. 1

Root part of Iris Koreana NAKAI (IKN) decreases osteoclast formation in a co-culture system. (A) Mouse bone marrow cells and primary osteoblasts were co-cultured with 1,25-dihydroxy-vitamin D₃ (1,25[OH]₂D₃) (10 nM) in the presence or absence of an ethanol extract of the root part of IKN (10 μg/mL) for 6 days. Tartrate-resistant acid phosphatase-positive (TRAP⁺) multinucleated osteoclasts (MNCs) containing 3 or more nuclei were counted as osteoclasts. Scale bar= 200 μm. (B, C) Mouse primary osteoblasts were pretreated with an ethanol extract of the root part of IKN (10 μg/mL) for 30 min and then cultured with 1,25(OH)₂D₃ for 24 hr. The mRNA expression levels were determined using real-time polymerase chain reaction. Data are expressed as mean±standard deviation of at least 3 independent experiments. Scale bar=200 μm. *P<0.05. Veh, vehicle; RANKL, receptor activator of nuclear factor-κB ligand; OPG, osteoprotegerin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 2

Root part of Iris Koreana NAKAI (IKN) inhibits receptor activator of nuclear factor-κB ligand (RANKL)–induced osteoclast differentiation. (A, B) Bone marrow macrophages (BMMs) were incubated with RANKL (100 ng/mL) and macrophage colony-stimulating factor (M-CSF; 30 ng/mL) in the presence of the indicated concentrations of an ethanol extract of the root of IKN for 4 days. Tartrate-resistant acid phosphatase-positive (TRAP⁺) multinucleated osteoclasts (MNCs) (A) or actin-rings (B) were counted. (C) BMMs were cultured with M-CSF (30 ng/mL) with or without the ethanol extract of the root part of IKN (10 μg/mL) for 48 hr. Cell viability was measured using an MTT assay. (D) BMMs were incubated on dentine slices with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days followed by the ethanol extract of the root part of IKN (10 μg/mL) for an additional 2 days. The number of resorption pits were counted. Data are expressed as mean±standard deviation of at least 3 independent experiments. Scale bar=200 μm. *P<0.05. Veh, vehicle.

Fig. 3

Root part of Iris Koreana NAKAI (IKN) inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced expression of c-Fos and nuclear factor of activated T cells 1 (NFATc1) via p38 mitogen-activated protein kinase activation. (A–C) Bone marrow macrophages were preincubated with or without an ethanol extract of the root part of IKN (10 μg/mL) for 30 min, and then treated with 100 ng/mL of RANKL for 24 hr (for NFATc1 and c-Fos) or 15 min (for p-p38). Cell lysates were then subjected to Western blot analysis using (A) anti-NFATc1, (B) anti-c-Fos, (C) anti-p-p38 antibodies. Data are expressed as mean±standard deviation of at least 3 independent experiments. *P<0.05. Veh, vehicle.

Fig. 4

Anti-osteoclastogenic effect is unique to the root part of Iris Koreana NAKAI (IKN). (A–C) Bone marrow macrophages (BMMs) were incubated with receptor activator of nuclear factor-κB ligand (RANKL) (100 ng/mL) and macrophage colony-stimulating factor (M-CSF; 30 ng/mL) in the presence of the indicated concentrations of an ethanol extract of (A) the aerial part of IKN, (B) the root part of Iris germanica L. (IGL), or (C) the aerial part of IGL for 4 days. Tartrate-resistant acid phosphatase-positive (TRAP⁺) multinucleated osteoclasts were counted. Data are expressed as mean±standard deviation of at least 3 independent experiments. *P<0.05. Veh, vehicle.

Fig. 5

Root part of Iris Koreana NAKAI (IKN) inhibits lipopolysaccharide (LPS)-induced osteoclast formation in vivo. Mice were subcutaneously injected with LPS (0.5 mg) into the calvarial bone. Mice were also administered daily intraperitoneal injections of the ethanol extract of IKN root (50 mg/kg) or vehicle for 7 days. Calvariae of mice treated with vehicle, LPS, or LPS combined with IKN were subjected to tartrate-resistant acid phosphatase (TRAP) staining. TRAP-stained areas were measured using ImageJ software. Data are expressed as mean±standard deviation of at least 3 independent experiments. Scale bar=200 μm. *P<0.05. Veh, vehicle.