Periodontal disease is an infective disease of the periodontal tissues caused by periopathogenic bacteria accumulated on the tooth surface. Once periodontitis occurs, severe inflammation and alveolar bone resorption progress, causing the destruction of tooth-supporting tissues [1]. Consequently, the loss of teeth limits dental function and the quality of the personal diet might be dramatically depressed. Furthermore, several reports have revealed that periodontitis is highly associated with systemic diseases, including cardiovascular disease, diabetes, and Alzheimer’s disease [2-4]. Considering that oral health status contributes significantly to the quality of life and systemic health, the treatment and prevention of periodontitis is an important strategy for maintaining health.

Research on the effects of anti-periodontitis can be approached in three methodological aspects: (i) antibacterial activity against periopathogens, (ii) anti-inflammatory activity, and (iii) anti-osteoclastic activity. Periodontitis is initiated by infection with gram-negative microorganisms such as Porphyromonas gingivalis and caused by plaque formation of Streptococcus mutans on the gingival sulcus. Hosts activate their immune systems in response to microbial endotoxins (lipopolysaccharide, LPS) secreted from the periopathogens. Consequently, the expression of inflammatory factors interleukin (IL)-1β and nuclear factor kappa B (NF-κB) from gingival fibroblasts and periodontal ligament fibroblasts increases in gingival tissues. The inflammatory response elevates matrix metalloproteinases (MMPs), which are proteases that degrade matrix components such as collagens and gelatins, finally collapsing the gingival tissues [5].

These inflammatory responses are mediated by the mitogen-activated protein kinase (MAPK) signaling and activator protein-1 (AP-1) complex formation pathways. MAPK signaling proteins, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, are phosphorylated by the binding of LPS to the Toll-like receptor (TLR), resulting in activation of c-Jun and c-Fos. Activated c-Jun and c-Fos form the AP-1 complex, which acts as a major transcription factor for the expression of MMP-2 and MMP-8 [6].

Inflammatory responses increase the secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) from gingival fibroblasts or osteoblasts, which leads to the differentiation of osteoclasts [7]. The osteoclasts are a type of bone cell that resorbs the bone matrix, whereas osteoblasts produce new bone. These cell types continually maintain bone resorption and remodeling in balance. In the case of periodontitis, osteoclasts are more predominant than osteoblasts, which causes the destruction of alveolar bone due to the imbalance of bone cells [8]. When RANKL binds to the RANK receptor, osteoclastogenesis occurs via the MAPK signaling pathways initiated by the promotion of tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins [9]. Similarly, phosphorylated ERK (p-ERK), p-JNK, and p-p38 activate the osteoclastogenesis-related key transcription factors nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and c-Fos [10]. NFATc1 is self-stimulating, so the differentiation signal is highly amplified. These serial responses increase the expression of enzymes involved in bone resorption, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K [11].

Panduratin A (Fig. 1) is a natural chalcone derivative isolated from Boesenbergia pandurata Roxb. (B. pandurata), which has bioactivities, including anti-obesity, anticancer, and antioxidant effects [12]. Previous results reported that panduratin A effectively inhibited the growth of periopathogens, including P. gingivalis [13, 14]. Moreover, panduratin A markedly suppressed the activation of MMP-9 in human oral epithelial (KB) cells [15]. However, the inhibitory effect of panduratin A on gingival inflammation caused by inflammatory cytokines and transcription factors in periodontal tissues has not been reported. Thus, we evaluated the anti-inflammatory effect of panduratin A using LPS-stimulated human gingival fibroblast-1 (HGF-1). Since periodontal inflammation accelerates osteoclastogenesis, we also investigated whether panduratin A prevents osteoclast differentiation in RANKL-induced RAW 264.7 cells.

Figure 1. Chemical structure of panduratin A.

Preparation of Panduratin A

The dried rhizomes of B. pandurata were ground and extracted with 95% (v/v) ethanol for 3 days at room temperature. B. pandurata extract (BPE) was subsequently obtained by evaporating the solvent (yield 12.0%). The standardized BPE contained 8%panduratin A, which was isolated as previously described [16]. The purity of panduratin A was measured to be ≥98% using high-performance liquid chromatography analysis.

Cell Culture

HGF-1 and murine monocytes RAW 264.7 were purchased from the American Type Culture Collection (USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, USA) supplemented with 10% fetal bovine serum (FBS; HyClone), streptomycin (75 μg/ml) and penicillin (120 units/ml) (both from Invitrogen, USA) under condition of 5% CO₂ at 37°C. To induce inflammation in HGF-1, LPS (Sigma-Aldrich, USA) from Escherichia coli was used and the cells were cultured in medium containing 1 μg/ml LPS with panduratin A. RAW 264.7 cells are characterized as monocytes that differentiate into multinucleated osteoclast-like cells in the presence of RANKL. To differentiate RAW 264.7 into osteoclasts, alpha-minimum essential Eagle’s Medium (alpha-MEM; Gibco, USA) containing 10% FBS, streptomycin (75 μg/ml), and penicillin (120 units/ml) was used. The differentiation medium containing 25 ng/ml RANKL (Peprotech Inc., USA) with panduratin A was changed every 2 days during the 4-day incubation.

Cell Viability

To determine the cytotoxicity of panduratin A, cell viability was measured by employing the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich). Each cell line was cultured in plates (2 × 10⁵ cells/well) for 24 h with panduratin A at various concentrations [7]. Then, the cells were additionally incubated in fresh medium containing 200 μl of MTT (0.5 mg/ml) for 4 h. After removing the medium, the formed insoluble product was dissolved with 200 μl of dimethyl sulfoxide and the absorbance of the resultant solution was detected with a VERSAmax tunable microplate reader (Molecular Devices Inc., USA) at a wavelength of 540 nm. There was no significant cytotoxicity at concentrations of up to 1 μM panduratin A in both HGF-1 and RAW 264.7 cells (data not shown). Further experiments were implemented at concentrations below 1 μM panduratin A in HGF-1 and RAW 264.7 cells.

TRAP Staining and Activity

The RAW 264.7 cells were seeded in plates (8 × 10⁴ cells/well) with DMEM for 12 h. The cells were serially incubated in RANKL-containing alpha-MEM with panduratin A for 4 days. The differentiation medium was replaced every 2 days.

After culture, differentiated osteoclasts were observed and then the cells were fixed with citrate-acetone-formaldehyde for 30 min. The expression of TRAP was observed by employing a staining kit (Sigma-Aldrich, USA) following the manufacturer’s instructions. Briefly, osteoclasts were incubated with TRAP solution consisting of Fast Garnet GBC base, acetate, naphthol AS-Bi phosphate, sodium nitrite, and tartrate solutions for 1 h at 37°C. Stained multinucleated osteoclasts were observed using an Eclipse TE2000U inverted microscope with twin CCD cameras (magnification ×100; Nikon, Japan). For the TRAP activity assay, differentiated osteoclasts were lysed with 0.1% Triton-X (Oriental Chemical Industries, Japan) and the lysates were centrifuged at 16,600 ×g at 4°C for 10 min. Then, the supernatant was co-incubated with TRAP solution at a 1:1 ratio for 1 h at 37°C, and the absorbance was measured at 540 nm with a VERSAmax tunable microplate reader to determine the TRAP activity.

Western Blotting

The cells were lysed with NP40 lysis buffer (Elpis Biotech, Korea) containing protease inhibitor cocktail (Sigma-Aldrich, USA). The protein concentration of the lysate was assessed by the Bradford assay (Bio-Rad Laboratories Inc., USA) and then equal amounts of protein (20 μg) were seperated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto nitrocellulose membranes (Whatman GmBH, Germany) and the membranes were incubated with primary antibodies against NF-κB, IL-1β, MMP-2, MMP-8, TRAP, NFATc1, cathepsin K, c-Fos, p-ERK, p-JNK, p-p38 (1:1,000 dilution; Santa Cruz Biotechnology Inc., USA), p-c-Jun, total (t)-c-Jun, t-ERK, t-JNK, t-p38, and α-tubulin (1:1,000 dilution; Cell Signaling, USA) overnight at 4°C. For the secondary reaction, the membranes were incubated with secondary antibody (1:5,000 dilution; Bethyl Laboratories, Inc., USA) for 1 h at room temperature. The protein intensity was measured by the enhanced chemiluminescence detection system (Amersham Biosciences, UK) and visualized with the G:BOX EF imaging system (Syngene, UK) and the GeneSys program.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated by Trizol reagent (Takara, Japan), and then 2 μg of total RNA was quantified by NanoDrop 1000 spectrophotometry (Thermo Fisher Scientific Inc., USA). The RNA was mixed with reverse transcriptase premix (Elpis Biotech, Korea) and oligo (dT) to synthesize cDNA (20 μl). Reverse transcription was carried out by the following process: initiation at 70°C for 5 min, incubation at 42°C for 55 min, and termination at 70°C for 15 min. The amplification of cDNA by polymerase chain reaction with specific primer pairs (Bioneer, Korea) (Table 1) was implemented by the following process: denaturation at 94°C for 30 sec, annealing for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 5 min. The intensity of the products was detected using 1.5-2.0% agarose gel electrophoresis and stained with loading star (Dyne Bioinc, Korea). The bands were visualized using the G:BOX EF imaging system (Syngene, UK) and the Genesys program.

Table 1 . Primer sequences used in the reverse transcription-polymerase chain reaction (RT-PCR) analysis..

Origin	Gene	Direction	Sequence (5ʹ-3ʹ)
Mouse	TRAP	Forward	AAA TCA CTC TTT AAG ACC AG
		Reverse	TTA TTG AAT AGC AGT GAC AG
	cathepsin K	Forward	ATC TCT CTG TAC CCT CTG CA
		Reverse	CCT CTC TTG GTG TCC ATA CA
	NFATc1	Forward	CCT GGA GAT CCC GTT GCT TC
		Reverse	TCC CGG TCA GTC TTT GCT TC
	c-Fos	Forward	CAG CTC AGT AAC AGT CCG CC
		Reverse	TCA CTA TTG GCA ACG AGC GG
	β-Actin	Forward	CAG CTC AGT AAC AGT CCG CC
		Reverse	TCA CTA TTG GCA ACG AGC GG
Human	IL-1β	Forward	CCT GAG CTC GCC AGT GAA AT
		Reverse	GCA TCG TGC ACA TAA GCC TC
	NF-κB	Forward	GGG CCA TCT GCT GTT GGC AG
		Reverse	CGC GCC GCT TAG GAG GGA GA
	MMP-2	Forward	CAT ACA AAG GGA TTC CCA GG
		Reverse	ATC GCT CCA GAC TTG GAA GG
	MMP-8	Forward	AGA AAG CCA GGA GGG GTA GA
		Reverse	TCC ACA GCG AGG CTT TTT CA
	GAPDH	Forward	CTC CTG TTC GAC AGT CAG CC
		Reverse	TCG CCC CAC TTG ATT TTG GA

Statistical Analysis

All the experiments were repeated at least three times in triplicate. Each value is reported as the mean ± standard deviation (SD). The statistical analysis was performed by the Statistical Package for the Social Sciences ver. 23.0 (SPSS Inc., USA). Group differences were estimated using a one-way analysis of variance followed by the Scheffe’s test and ^## p < 0.01 (untreated control vs. LPS or RANKL control) and **p < 0.01 (LPS or RANKL control vs. panduratin A-treated group) were considered statistically significant.

Panduratin A Inhibits Gingival Inflammation in LPS-Stimulated HGF-1 Cells

To determine whether panduratin A suppresses gingival inflammation, the activation of inflammatory factors was measured in LPS-stimulated HGF-1 cells. In the group induced with LPS, the expression levels of inflammatory cytokine IL-1β and transcription factor NF-κB were significantly enhanced, whereas the groups treated with panduratin A (0.1 and 1 μM) showed a significant decrease (Fig. 2). Similarly, the levels of MMP-2 and MMP-8 were diminished by panduratin A, compared with the LPS group. The results imply that panduratin A exerts an anti-inflammatory effect by controlling inflammatory factors and matrix-degrading enzymes.

Figure 2. Effects of panduratin A on inflammatory factor expression in LPS-stimulated human gingival fibroblast-1 (HGF-1) cells. The cells were seeded in plates (5 × 10⁵ cells/well) and cultured in medium containing 1 μg/ml LPS with panduratin A overnight. (A) The mRNA levels of IL-1β, NF-κB, MMP-2, and MMP-8 were measured using RT-PCR, with GAPDH as the housekeeping gene. (B) The protein levels were evaluated by western blotting with α-tubulin as the loading control. The relative mRNA levels are presented as the mean ± standard deviation (SD, % control) of three independent experiments. ^## p < 0.01 (untreated control vs. LPS control) and **p < 0.01 (LPS control vs. sample-treated cells).

Panduratin A Regulates Gingival Inflammation by Inactivating MAPK Signaling Pathways and AP-1 Complex Formation Pathways in LPS-Induced HGF-1 Cells

The anti-inflammatory effect of panduratin A was estimated by examining the MAPK signaling and AP-1 complex formation pathways at the protein level. Panduratin A (1 μM) markedly suppressed the expression of p-ERK, p-JNK, and p-p38 and attenuated the expression of p-c-Jun and c-Fos in LPS-stimulated HGF-1 cells (Fig. 3). These results indicate that the anti-inflammatory effect of panduratin A are regulated via phosphorylated MAPK signaling proteins, thereby blocking the formation of the AP-1 complex.

Figure 3. Effects of panduratin A on (A) MAPK signaling pathways and (B) the AP-1 complex formation pathway in LPS-stimulated human gingival fibroblast-1 cells. Cells were seeded in plates (5 × 10⁵ cells/well) and cultured in medium containing 1 μg/ml LPS for 30 min after 2 h of panduratin A pretreatment. The protein expression was detected using western blotting with α-tubulin as the loading control.

Panduratin A Inhibits Osteoclastogenesis in RANKL-Stimulated RAW 264.7 Cells

RANKL-stimulated RAW 264.7 cells have been employed as a cell model for osteoclastogenesis studies [7, 17]. The effect of panduratin A on osteoclastogenesis was examined in RANKL-treated RAW 264.7 cells. When compared with the RANKL-induced group, panduratin A (1 μM) significantly decreased the mRNA and protein activation of osteoclastic transcription factors, including NFATc1 and c-Fos, and bone resorptive enzymes such as cathepsin K and TRAP (Fig. 4). These results indicate that panduratin A exerts the anti-osteoclastic effect by controlling differentiation-related transcription factors and enzymes.

Figure 4. Effects of panduratin A on osteoclastic factor expression in RANKL-induced RAW 264.7 cells. The cells were seeded in plates (8 × 10⁴ cells/well) and cultured in alpha-MEM with 25 ng/ml RANKL containing panduratin A for 4 days. (A) The mRNA levels of NFATc1, c-Fos, TRAP, and cathepsin K were measured by RT-PCR, with β-actin as the housekeeping gene. (B) The protein levels were evaluated by western blotting, with α-tubulin as the loading control. The relative mRNA levels are presented as the mean ± standard deviation (SD, % control) of three independent experiments. ^## p < 0.01 (untreated control vs. RANKL control) and **p < 0.01 (RANKL control vs. sample-treated cells).

Panduratin A Regulates Osteoclastogenesis by Inactivating MAPK Signaling Pathways in RANKL-Treated RAW 264.7 Cells

The differentiation of pre-osteoclast cells into mature osteoclasts is mediated by the MAPK signaling pathways, which are stimulated by RANKL binding [7]. In this study, the MAPK signaling proteins, p-ERK, p-JNK, and p-p38, were highly expressed following the induction of differentiation in RANKL-treated RAW 264.7 cells (Fig. 5). However, panduratin A significantly blunted the phosphorylation of ERK, JNK, and p38 in RANKL-induced RAW 264.7 cells. These results imply that panduratin A downregulates MAPK signaling transduction by suppressing the phosphorylated signaling proteins, resulting in the inhibition of osteoclasto-genesis.

Figure 5. Effects of panduratin A on MAPK signaling pathways in RANKL-treated RAW 264.7 cells. The cells were seeded in plates (5 × 10⁵ cells/well) and incubated with 25 ng/ml RANKL for 30 min after 2 h panduratin A pretreatment. The protein expression was detected using western blotting, with α-tubulin as the loading control.

Panduratin A Decreases TRAP Activity in RANKL-Treated RAW 264.7 Cells

To assess the inhibitory effect of panduratin A on TRAP activity in osteoclasts, a TRAP activity assay was carried out in RANKL-induced RAW 264.7 cells. At 1 μM panduratin A, the activity of TRAP was significantly suppressed by 61.8% compared with that of the RANKL-treated group (Fig. 6). Furthermore, a decrease in mature osteoclast formation was observed through TRAP staining. The cells in the RANKL-treated group were differentiated into large, multinucleated, and fused osteoclasts, whereas the cells treated with 1 μM panduratin A retained the normal RAW 264.7 morphology despite RANKL stimulation. Therefore, panduratin A might efficiently prevent alveolar bone destruction by deactivating osteoclasts and TRAP, which absorb the bone matrix.

Figure 6. Effect of panduratin A on TRAP activity in RANKL-treated RAW 264.7 cells. The cells were seeded in plates (8 × 10⁴ cells/well) and grown in alpha-MEM containing 25 ng/ml RANKL with panduratin A for 4 days. (A) TRAP activity was evaluated by measuring the absorbance at 540 nm wavelength. The relative TRAP activity is given as the mean ± standard deviation (SD, % control) of three independent experiments. ^## p < 0.01 (untreated control vs. RANKL control) and **p < 0.01 (RANKL control vs. sample-treated cells). (B) Morphological changes of osteoclastic transformation, measured with TRAP staining. For osteoclastogenesis, RANKL induces TRAP activity and the formation of TRAP-positive multinucleated osteoclasts.

Periodontitis is an inflammatory disease initiated by the accumulation of oral pathogens, leading to the destruction of gingival tissues and alveolar bone [1]. Therefore, periodontitis can be prevented by inhibiting periopathogenic microbial growth, inflammatory responses, and bone loss in the periodontium. Previous studies showed that panduratin A possessed a remarkable antibacterial effect against several pathogens causing periodontal disease. Panduratin A inhibited the growth of P. gingivalis and S. mutans at 4 μg/ml, which was comparable to several antibiotics [13, 14]. These results indicate that panduratin A might be a good candidate for the prevention of periodontitis. In the current study, we evaluated the inhibitory effects of panduratin A on inflammation and osteoclastogenesis using LPS-stimulated HGF-1 and RANKL-treated RAW 264.7 cells.

In the LPS-stimulated HGF-1 cells, panduratin A significantly alleviated the expression of MMP-2, MMP-8, and inflammatory factors such as IL-1β and NF-κB (Fig. 2). Once LPS infection occurs, cytokines such as IL-1β enhance the proinflammatory response and stimulate periodontal inflammatory mediators such as NF-κB, IL-6, and IL-8, resulting in the overexpression of MMPs [18]. These MMPs are enzymes that degrade the proteins composing the extracellular matrix of periodontal tissues, such as elastin, collagen, and fibronectin [6]. Specifically, MMP-2 is a gelatinase targeting collagen type IV, which is distributed in the base membranes of gingival connective tissues [19]. In addition, MMP-8 is a collagenase degrading collagen type I and III, which are the principal organic constituents found in the periodontal tissues [20]. Therefore, MMP-2 and MMP-8 might be responsible for the destruction of the free gingival tissues as well as the loss of periodontal ligaments, resulting in the deepening of the gingival sulcus. Accordingly, panduratin A should efficiently attenuate the MMP-induced destruction of periodontal tissues by inhibiting inflammatory factors IL-1β and NF-κB.

These inflammatory responses are mediated by MAPK signaling pathways [6]. The phosphorylated MAPK signaling proteins (ERK, JNK, and p38) activate AP-1 and NF-κB, which act as key transcription factors for the expression of MMPs [21]. In fact, our previous study reported that panduratin A markedly diminished MMP-9 expression by regulating the MAPK signaling pathways [22]. Overall, these results indicate that panduratin A inhibits inflammatory factors, including NF-κB and AP-1, by regulating MAPK signaling proteins and ultimately protects MMP expression (Fig. 3).

LPS-induced inflammatory responses overexpress RANKL in osteoblasts, resulting in its binding to the RANK receptor on osteoclasts, causing osteoclastogenesis [7]. Thus, we investigated whether panduratin A prevented osteoclast differentiation, using RANKL-stimulated RAW 264.7 cells. The levels of TRAP and cathepsin K were markedly reduced by treatment with panduratin A in RANKL-induced RAW 264.7 cells (Fig. 4). The effects might be derived from the downregulation of two important transcription factors, c-Fos and NFATc1. When RANKL binds to RANK, these transcription factors play a role in translocating themselves to target genes in the nucleus, which are essential for osteoclastogenesis [23]. The results showed that the expression of osteoclastic proteins TRAP, cathepsin K, calcitonin receptor, and osteoclast-associated receptor was highly increased in response to the transcription signals. However, panduratin A blocked the serial signaling transduction starting from the RANKL/RANK axis by interrupting c-Fos and NFATc1. In particular, although the NFATc1 signal was highly amplified due to self-stimulation, panduratin A remarkably suppressed the expression of NFATc1 and ultimately prevented the amplification. The effects imply that panduratin A could be an important factor in preventing osteoclastic enzyme activation. The transcription factors in osteoclast differentiation, NFATc1 and c-Fos, are activated by the phosphorylated MAPK signaling proteins [17, 24]. Therefore, panduratin A could suppress the expression of osteoclastic transcription factors via control of MAPK signaling pathway proteins, ultimately blocking TRAP and cathepsin K activation. Collectively, panduratin A suppresses gingival inflammation and osteoclastogenesis by inhibiting at least one pathway, MAPK, in LPS-stimulated HGF-1 and RANKL-treated RAW 264.7 cells. Previous studies reported several natural MAPK pathway inhibitors on periodontal inflammation from medicinal plants [25, 26]. Although myricetin, luteolin, morin, and fisetin have been shown to reduce MAPK protein expression in periodontal cell lines, these natural compounds were used at a higher concentration than that of panduratin A [25, 26].

LPS from oral bacteria is a main inducer of the inflammatory response in gingival tissues, subsequently stimulating RANKL from gingival fibroblasts or osteoblasts [7]. The RANKL increased by the inflammatory response enhances bone resorption in alveolar bone. Thus, since panduratin A reduced the inflammatory response in LPS-induced HGF-1 cells (Fig. 2), panduratin A might prevent RANKL-induced osteoclast differentiation in animals or humans.

Panduratin A is one of a few natural sources to confer antimicrobial, anti-inflammatory, and anti-osteoclastic functions. This indicates that panduratin A can effectively prevent periodontitis by simultaneously blocking various factors, leading to periodontal destruction. In addition to a preventive effect on periodontitis, the therapeutic effect of panduratin A can be achieved by further enhancing the bone regeneration ability initiated by osteoblast differentiation. If the inhibitory effect of panduratin A on periodontitis is verified through in vivo and clinical studies, panduratin A could be used as a target agent for treating or preventing periodontitis.

This research is supported by “The Project of Conversion by the Past R&D Results” through the Ministry of Trade, Industry and Energy (MOTIE) (N0002221, 2016).