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Secreotory Leukocyte Protease Inhibitor Regulates Bone Formation via RANKL, OPG, and Runx2 in Rat Periodontitis and MC3T3-E1 Preosteoblast
J Dent Hyg Sci 2023;23:282-95
Published online December 31, 2023;  https://doi.org/10.17135/jdhs.2023.23.4.282
© 2023 Korean Society of Dental Hygiene Science.

Seung-Yeon Lee1 ,*, Soon-Jeong Jeong2 ,*, Myoung-Hwa Lee1 , Se-Hyun Hwang3 , Do-Seon Lim4 , and Moon-Jin Jeong1,†

1Department of Oral Histology and Developmental Biology, College of Dentistry, Chosun University, Gwangju 61452, 2Department of Dental Hygiene and Institute of Basic Science for Well-Aging, College of Health Science, Youngsan University, Yangsan 50510, 3Department of Dental Hygiene, Yeungnam College, Daegu 42415, 4Department of Dental Hygiene, College of Health Science, Eulji University, Seongnam 13135, Korea
Correspondence to: Moon-Jin Jeong, https://orcid.org/0000-0002-5547-898X
Department of Oral Histology and Developmental Biology, College of Dentistry, Chosun University, 146 Chosundae-gil, Gwangju 61452, Korea
Tel: +82-62-230-6895, Fax: +82-62-608-5732, E-mail: mjjeong@chosun.ac.kr

*These authors contributed equally to this work.
Received October 31, 2023; Revised November 17, 2023; Accepted November 21, 2023.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Secretory leukocyte protease inhibitor (SLPI) protects tissues from proteases and promotes cell proliferation and healing. SLPI also reduces periodontal inflammation and alveolar bone resorption by inhibiting proinflammatory cytokine expression in rat periodontal tissues and osteoblasts. However, little is known of the role of SLPI in the expression of osteoclast regulatory factors from osteoblasts, which are crucial for the interaction between osteoblasts and osteoclasts. Therefore, we aimed to determine the effects of SLPI on the regulation of osteoclasts and osteoblasts in LPS-treated alveolar bone and osteoblasts.
Methods: Periodontitis was induced in rats using LPS. After each LPS injection, SLPI was injected into the same area. Immunohistochemical analysis was performed with antibodies against SLPI, RANKL, OPG, and Runx2 in the periodontal tissue. RT-PCR and western blotting were performed to determine the expression levels of SLPI, RANKL, OPG, and Runx2 in LPS- and SLPI/LPS-treated MC3T3-E1 cells. SLPI/LPS-treated MC3T3-E1 cells were also stained with Alizarin Red S.
Results: Immunohistochemical analysis showed that the expression levels of SLPI, OPG, and Runx2 were higher while that of RANKL was lower in the LPS/SLPI group relative to those in the LPS group. The mRNA and protein expression of SLPI, OPG, and Runx2 was higher in SLPI/LPS/MC3T3-E1 cells than in LPS/MC3T3-E1 cells, and RANKL expression was lower. During differentiation, OPG and Runx2 protein levels were higher whereas RANKL levels were lower in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 cells on days 0, 4, 7, and 10. In addition, mineralization and matrix deposition were higher in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 on days 7 and 10. SLPI decreased RANKL expression in LPS-treated alveolar bone and osteoblasts but increased the expression of OPG and Runx2.
Conclusion: SLPI can be considered as a regulatory molecule that indirectly regulates osteoclast activation via osteoblasts and promotes osteoblast differentiation.
Keywords : Bone remodeling, Osteoblasts, Osteoclasts, Periodontitis, Secretory leukocyte protease inhibitor
Introduction

1.Background

Bone remodeling occurs via bone formation and resorp-tion. The tissue surrounding the bone includes various types of cells, of which osteoblasts promote bone matrix formation and tissue mineralization, whereas osteoclasts are involved in bone resorption1). Moreover, osteoblasts stimulate osteoclast formation to facilitate bone resorption by secreting factors, such as parathyroid hormone (PTH), interleukin-1beta (IL-1b), and tumor necrosis factor-a (TNF-a)2). Lipopolysaccharide (LPS) is an endotoxin of gram-negative bacteria, which is known to stimulate oste-oclasts thereby promoting bone resorption; it also enha-nces IL-1b and TNF-a production from osteoblasts, fibro-blasts and macrophages of infected periodontal tissue3). Additionally, LPS induces the expression of TNF-a and receptor activator of nuclear factor-kB ligand (RANKL) by activating nuclear factor-kB (NF-kB) through toll-like receptor4 (TLR4) on the surface of osteoblasts4).

Macrophage-colony stimulating factor (M-CSF) and RANKL are the major cytokines involved in osteoclast differentiation5). RANKL, a member of the TNF family, is expressed in T cells, B cells, osteoblasts and stromal cells, and is crucial for osteoclast differentiation6,7). Further-more, RANKL directly induces the differentiation of hemato-poietic myeloid progenitor cells of the monocyte or macro-phage lineage into osteoclasts8). RANKL, a membrane- bound molecule expressed in osteoblasts, is an important factor that regulates osteoclasts together with M-CSF and osteoprotegerin (OPG). RANKL inhibits osteoclastogenesis via OPG, a decoy receptor of RANKL6,7,9). OPG inhibits osteoclastogenesis by blocking the binding of RANKL- RANK to ST2 cells5,8). OPG production in osteoblasts is reduced by bone-resorption stimulating hormone (PTH, prostaglandin E2) and TNF-a and IL-16). RANKL also induces osteoclast differentiation and activation, whereas OPG inhibits osteoclastogenesis5,10). Therefore, the ratio of OPG to RANKL expression in osteoblasts is an impor-tant factor that regulates osteoclasts7). Runt-related trans-cription factor 2 (Runx2), a member of the Runx family of transcription factors, is an important regulator that is expressed early in osteoblast differentiation and is essential for the differentiation of mesenchymal cells into osteo-blasts11). Runx2 regulates the expression of genes related to bone matrix formation such as type I collagen (Col I), osteopontin (OPN), and bone sialoprotein (BSP) in osteo-blasts. Moreover, Runx2 plays an important role in main-taining immature osteoblast supply11,12). Secretory leukocyte protease inhibitor (SLPI) has anti-inflammatory, anti-bac-terial, and antiviral functions, and inhibits LPS-induced NF-kB activity and TNF-a production in SLPI-treated macrophages13). SLPI expression is also temporarily increased by LPS stimulation or wounds in dental pulp odontoblasts, which suppresses the inflammatory response by decreasing NF-kB activity14). SLPI secreted from odontoblasts regulates dentine matrix formation and mine-ralization, and is known to increase the adhesion and via-bility of osteoblasts on titanium surfaces15,16). Furthermore, SLPI increases the expression of mineralization and differ-entiation-related factors during osteoblast differentiation on titanium surfaces17). Recently, SLPI was found to in-hibit inflammation in periodontal tissues and alveolar bone resorption by reducing the expression of the infla-mmatory cytokines, IL-1b and TNF-a, in periodontitis tissue and LPS-stimulated osteoblasts18).

2.Objectives

Periodontitis and alveolar bone resorption have been found to be reduced via inhibition of the inflammatory res-ponse by SLPI. We posed the question of whether alveolar bone resorption could be suppressed via some pathway unrelated to the direct regulation of SLPI during the interaction between osteoblasts and osteoclasts because of inflammation-related molecules inhibited by SLPI. However, because SLPI was found to play an important role in cell adhesion, survival, differentiation, and mineralization in osteoblasts, the relationship between the inhibition of inflammatory cytokines and reduced alveolar bone resor-ption remains unclear. We aimed to clarify the role of SLPI in regulating the molecules involved in osteoclast induc-tions, which are expressed in inflammatory periodontal tissues and osteoblasts. Therefore, this study sought to reveal the role of SLPI in molecules involved in the regulation of osteoclast differentiation and activation in LPS-stimulated alveolar bone tissue and osteoblasts.

Materials and Methods

1. Study design

1) Induction of periodontitis via LPS injection

Nine-week-old Sprague-Dawley rats (Samtako Bio, Osan, Korea) weighing 350 to 400 g were used. Eighteen rats were divided into three groups of six. Periodontitis was induced over 10 days by administering LPS (Sigma- Aldrich, St Louis, MO, USA) (1 mg/ml) to the bottom of the gingival groove at the buccal aspect of the right second molar of the maxilla every 48 hours, five times. SLPI (rhSLPI; R&D systems, Minneapolis, MN, USA) (0.1 or 1 mg/ml) was also injected into the same area after each LPS injection every 48 hours, five times. The groups were clas-sified as follows: the LPS group was injected with only LPS, the LPS/SLPI-L group was injected with SLPI (0.1 mg/ml) after LPS injection, and the LPS/SLPI-H group was injec-ted with SLPI (1 mg/ml) after LPS injection. The left maxi-llary second molar in each group was used as the control. All animal experiments were approved by the Institutional Animal Care and Use Committee of Chosun University (No. CIACUC2014 A0025).

2) Tissue preparation and immunohistochemistry

The maxilla was washed with phosphate-buffered saline (PBS) and decalcified for 3 months in a solution of 10% EDTA supplement with 1% paraformaldehyde. After deca-lcification, tissue blocks (12 mm thickness) were prepared using Histocut 820 (Leica, Wetzlar, HE, Germany). Sam-ples were deparaffinized, rehydrated, and washed in tris- buffered saline containing tween 20 (TBS-T). Hematoxylin and eosin staining was performed to observe the histolo-gical changes and bone resorption levels. For immuno-histochemistry, the samples were incubated with 1:1,000 of rabbit anti-mouse SLPI antiserum18), 1:1,000 of anti- mouse RANKL (Novus Biologicals, Littleton, CO, USA), and anti-goat OPG (Santa Cruz Biotechnology, Dallas, TX, USA), anti-rabbit Runx2 (Abcam, Cambridge, United Kingdom), and antibodies were diluted in fresh normal goat serum for 16 hours at 4°C. Biotinylated goat anti- rabbit IgG or horse anti-mouse IgG or rabbit anti-goat IgG (1:250; Vector Laboratories, Newark, CA, USA) were used as secondary antibodies for 1 hour at room temperature. For color development, the samples were incubated with 0.05% deaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories) for 3 minutes. The samples were counter-stained with Harris hematoxylin, dehydrated and mounted. Stained tissues were observed under an optical microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany). The intensity of SLPI, RANKL, OPG, and Runx2 protein expression in the bottom of the alveolar bone at the root furcation of the left and right maxillary second molars was quantified using Axiovision LE release 4.6 software (Carl Zeiss). The Runx2-positive cells in the same area were counted.

3) Cell culture and differentiation with LPS and/or SLPI treatment

MC3T3-E1 cells were cultured in alpha-modified Eagle’s medium (a-MEM) (Gibco, Billings, MT, USA) containing 10% fetal bovine serum (FBS; WelGENE, Gyeongsan, Korea) and 1% antibiotic-antimycotic solution (WelGENE). For 2, 4, 6, 12, and 24 hours, MC3T3-E1 cells (LPS/ MC3T3-E1) was treated with LPS (200 ng/ml), and the cells (SLPI/LPS/MC3T3-E1) were treated with SLPI (R&D systems) (1 mg/ml) after LPS treatment. For differen-tiation, the cells were transferred to a differentiation medium (a-MEM supplemented with 5% FBS, 10 mM b-glycerol phosphate and 50 mg/ml ascorbic acid) with or without SLPI (1 mg/ml) after LPS pretreatment for 2 hours, every 2 days. The cells were placed into a humi-dified chamber and maintained in an atmosphere contai-ning 5% CO2 at 37°C.

4) Extraction of total RNA and RT-PCR

Total RNA was extracted using the Tri reagent (MRC Inc., Cincinnati, OH, USA) according to the manufac-turer’s instructions. One microgram of total RNA was used to synthesize the complementary DNA (cDNA). cDNA synthesis was performed using the RT Premix (GeNet Bio, Daejeon, Korea). PCR reaction was carried out in a thermo cycler (Takara, Kusatsu, Shiga, Japan) after adding 1 ml of cDNA to the PCR premix (GeneAll, Seoul, Korea). The following primers (Bioneer, Daejeon, Korea) were used for RT-PCR analysis: 1) SLPI Forward 5´-TGC TTA ACC CTC CCA ATG TC-3´, Reverse: 5´-AAT GCT GAG CCA AAA GGA GA-3´; 2) RANKL Forward, 5´-TAT GAT GGA AGG CTC ATG GT-3´, Reverse: 5´-TGT CCT GAA CTT TGA AAG CC-3´; 3) OPG Forward, 5´-CAG AGA CTA ATA GAT CAA AGG CAG G-3´, Reverse: 5´-ATG AAG TCT CAC CTG AGA AGA ACC-3´; 4) Runx2 Forward, 5´-GCA GTG CCC CGA TTG AGG-3´, Reverse: 5´-CAT ACR GGG ATG AGG AAT GCG-3´; and 5) GAPDH Forward, 5´-CCA TGG AGA AGG CTG GG-3´, Reverse: 5´-CAA AGT TGT CAT GGA TGA CC-3´. GAPDH was used as an internal control for RT-PCR. The PCR condi-tions were 60°C (30 cycles), 59°C (36 cycles), 59.7°C (30 cycles), 59°C (34 cycles), and 60°C (30 cycles) for SLPI, RANKL, OPG, Runx2, and GAPDH, respectively. The pro-ducts were electrophoresed, stained with ethidium bromide (EtBr), and visualized using Gel-Doc (BioRad, Hercules, CA, USA). The intensities of the bands were calculated, and semi-quantitative analysis was performed using the ratio of each gene/GAPDH using Science Lab Image Gauge software (version 3.12; FUJI FILM, Minato, Tokyo, Japan).

5) Western blotting

Total protein was extracted from MC3T3-E1 cells using NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, [pH 7.4], 2 mM Na3VO4, 2 mM Na4P2O, 50 mM NaF, 2 mM EDTA [pH 7.4], 0.1 µg/ml of leupeptin, and 1 µg/ml of aprotinin). After protein extraction, the concen-tration was determined as 30 mg using an assay kit (BioRad) and electrophoresed in 10% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA) and blocked with either 5% non-fat dry milk or 5% bovine serum albumin (Bio-Shop, Burlington, Ontario, Canada) for 1 hour at room temperature. The membranes were blotted with 1:1,000 of rabbit anti-mouse SLPI antiserum18), anti-mouse RANKL (Novus Biologicals), anti-goat OPG (Santa Cruz bio-technology), anti-rabbit Runx2 antibody (Abcam), and 1:2,500 of anti-mouse b-actin antibody (Santa Cruz Bio-technology) for 16 hours at 4°C. After washing, the membrane was blotted with 1:5,000∼1:10,000 of HRP- conjugated goat anti-rabbit or mouse-IgG or donkey anti- goat (Santa Cruz Biotechnology) at room temperature for 1 hour. Development was performed using an X-ray film (FUJI FILM) following detection using ECL solution (Merck Millipore). The density of the expressed bands was measured using a Science Lab Image Gauge (FUJI FILM).

6) Alizarin Red S staining and quantitative analysis

To identify the formation of mineralized nodules follo-wing MC3T3-E1 cell differentiation following LPS or LPS/ SLPI treatment, the cells were stained with 2% Alizarin Red S (Sigma-Aldrich). The mineralized nodules were observed using a stereoscopic microscope (Carl Zeiss). Alizarin Red S-stained MC3T3-E1 cells were incubated with cetylpyridinium chloride (Thermo Fisher Scientific, Waltham, MA, USA) to dissolve and release calcium- bound Alizarin Red S into the solution. The absorbance of the released Alizarin Red S was measured at 562 nm using a microplate reader (BioTek, Winooski, VT, USA).

2.Statistical analysis

All experiments were performed at least in triplicate. All data are reported as means and standard deviations using SPSS (version 27.0; IBM Co., Armonk, NY, USA). Significant differences (p<0.05, p<0.01) were deter-mined using an independent samples t-test.

Results

1.Immunohistochemisty of SLPI, RANKL, OPG, and Runx2

SLPI was not expressed in the alveolar bone matrix, but was identified in several osteoblasts and cells located in the intercellular space of the periodontal ligament (PDL) in the control group. It was also expressed in the junctional region between the root and the PDL fibers. SLPI in the LPS group was strongly expressed in the alveolar bone matrix, osteo-blasts, dispersed collagen, and connective tissue cells in the PDL compared to that in the control. SLPI in the LPS/ SLPI-L group was not expressed in the alveolar bone matrix, but was expressed strongly in almost all cells in the PDL compared to that in the LPS group. SLPI expre-ssion in the LPS/SLPI-H group was similar to that in the LPS group (Fig. 1A, 1C). RANKL was weakly expressed in almost all cells in the PDL of the control group. In the LPS group, RANKL was strongly expressed in the presen-ting cells and collagen fibers located in the resorbed region of the alveolar bone. RANKL in the LPS/SLPI-L group was expressed in the cells, and collagen fibers were locali-zed in the intracellular space of the PDL attached to the marginal alveolar bone. In the LPS/SLPI-H group, it was weakly expressed in cells of the intercellular space and collagen fibers in the PDL attached to the marginal alveo-lar bone (Fig. 1B, 1D). OPG was expressed in the con-nective tissue cells in the PDL and PDL fibers in the control group. In the LPS group, it was not expressed in PDL fibers or cells of the intercellular space. OPG in the LPS/SLPI-L and -H groups was strongly expressed in cells of the intercellular space and collagen fibers in the PDL compared to that in the LPS group (Fig. 2A, 2C). Runx2 was found to be expressed in osteocytes and adjacent cells in alveolar bone. Runx2 in the LPS group was weakly expressed in several alveolar bone osteocytes. In the LPS/ SLPI-L and -H groups, it was more strongly expressed in the osteocytes of the alveolar bone and cells attached to the marginal alveolar bone than in the LPS group. Runx2 positive cells were SLPI dose-dependently higher in the LPS/SLPI group than in the LPS group (Fig. 2B, 2D).

Fig. 1. SLPI and RANKL expression in LPS or LPS/SLPI-injected periodontal tissue. (A, C) SLPI expression in alveolar bone and PDL tissue was high in the LPS/SLPI-L group compared to that in the LPS group. (B, D) RANKL expression in cells of intercellular space and collagen fiber in PDL attached to marginal alveolar bone was high in the LPS group compared to that of the control, and it was low in the LPS/SLPI group in an SLPI dose-dependent manner compared to that of the LPS group. Inset boxes of left column images represent the corresponding area at higher magnification (right panel). LPS: lipopolysaccharide, SLPI: secretory leukocyte protease inhibitor. **p<0.01.
Fig. 2. OPG and Runx2 expression in LPS or LPS/SLPI-injected periodontal tissue. (A, C) OPG expression in cells of intercellular space and collagen fiber in PDL was low in the LPS group compared to that in the control, and it was high in the LPS/SLPI group in an SLPI dose-dependent manner compared to that in the LPS group. (B, D) Runx2 expression in osteocytes of alveolar bone and cells in PDL was low in the LPS group compared to that in the control, and it was high in the LPS/SLPI group in an SLPI dose-dependent manner compared to that of the LPS group. Inset boxes of left column images represent the corresponding area at higher magnification (right panel). LPS: lipopolysaccharide, SLPI: secretory leukocyte protease inhibitor. **p<0.01.

2.mRNA and protein expression of SLPI, RANKL, OPG, and Runx2 in MC3T3-E1 cells

The mRNA and protein expression patterns of SLPI, RANKL, OPG, and Runx2 were identical in MC3T3-E1 cells (Fig. 3, 4). The expression of SLPI mRNA and protein in SLPI/LPS/MC3T3-E1 cells was higher than that in LPS/MC3T3-E1 cells until 24 hours (Fig. 3A, 3B, 4A, 4B). The mRNA and protein expression of RANKL in LPS/MC3T3-E1 cells was higher than that in the control cells until 24 hours. The expression of SLPI/LPS/MC3T3- E1 was lower than that of LPS/MC3T3-E1 until 24 hours (Fig. 3A, 3C, 4A, 4C). The expression of OPG mRNA and protein in LPS/MC3T3-E1 cells was lower than that in the control until 24 h. In SLPI/LPS/MC3T3-E1 cells, it was higher than that in LPS/MC3T3-E1 cells at 2, 4, and 6 hours (Fig. 3A, 3D, 4A, 4D). The expression of Runx2 mRNA and protein in LPS/MC3T3-E1 cells was lower than that in the control cells from 4 to 24 hours. In SLPI/ LPS/MC3T3-E1 cells, it was higher than that in LPS/ MC3T3-E1 cells from 4 to 24 hours (Fig. 3A, 3E, 4A, 4E).

Fig. 3. mRNA expression of SLPI, RANKL, OPG, and Runx2 in LPS or SLPI/LPS-treated MC3T3-E1 cells. (A∼E) RANKL expression was decreased in SLPI/LPS/MC3T3-E1 compared to that in the LPS/MC3T3-E1 group, and SLPI was increased in LPS/MC3T3-E1 compared to that in the control. OPG and Runx2 expressions were increased in SLPI/LPS/MC3T3-E1 compared to that of the LPS/MC3T3-E1. LPS: lipopolysaccharide, SLPI: secretory leukocyte protease inhibitor. *p<0.05, **p<0.01.
Fig. 4. Protein expression of SLPI, RANKL, OPG, and Runx2 in LPS or SLPI/LPS-treated MC3T3-E1 cells. (A∼E) RANKL expression was decreased in SLPI/LPS/MC3T3-E1 compared to that of the LPS/MC3T3-E1. OPG and Runx2 levels were increased in SLPI/LPS/MC3T3-E1 compared to that of the LPS/MC3T3-E1. LPS: lipopolysaccharide, SLPI: secretory leukocyte protease inhibitor. **p<0.01.

3.Protein expression of SLPI, RANKL, OPG and Runx2 in MC3T3-E1 cells during differentiation

SLPI protein expression in LPS/MC3T3-E1 cells during differentiation was higher than that in the control on all days. In SLPI/LPS/MC3T3-E1, it was lower on day 0, 4, and 7 compared to LPS/MC3T3-E1 and was similar to the control on day 10 (Fig. 5A, 5B). RANKL expression was very high on days 0, 4, and 7 in LPS/MC3T3-E1 com-pared to that in the control, and in SLPI/LPS/MC3T3-E1 its expression was lower than that in LPS/MC3T3-E1 (Fig. 5A, 5C). OPG expression was lower in LPS/MC3T3-E1 than in the control on days 4, 7, and 10, and was higher in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 on days 0 and 4 (Fig. 5A, 5D). Runx2 expression in LPS/MC3T3-E1 was lower than that in the control on days 4 and 7, and that in SLPI/LPS/MC3T3-E1 was higher on days 4 and 7 than that in LPS/MC3T3-E1 (Fig. 5A, 5E).

Fig. 5. Protein expression of SLPI, RANKL, OPG and Runx2 in LPS or SLPI/LPS-treated MC3T3-E1 cells during differentiation. (A∼E) SLPI was increased in LPS/MC3T3-E1 compared to that of the control and RANKL expression was decreased in SLPI/LPS/MC3T3-E1 compared to that of the LPS/MC3T3-E1. OPG and Runx2 expressions were increased in SLPI/LPS/MC3T3-E1 compared to that of the LPS/MC3T3-E1. LPS: lipopolysaccharide, SLPI: secretory leukocyte protease inhibitor. **p<0.01.

4.Mineralization of MC3T3-E1 cells during differentiation

Mineralized nodule formation observed with Alizarin Red S staining decreased on days 7 and 10 in LPS/MC3T3- E1 compared to that in the control and increased in SLPI/ LPS/MC3T3-E1 compared to that in LPS/MC3T3-E1 (Fig. 6A). Additionally, the absorbance for mineral depo-sition was lower in LPS/MC3T3-E1 than in the control on days 7 and 10, and higher in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 (Fig. 6B).

Fig. 6. Mineralization in LPS or SLPI/LPS-treated MC3T3-E1 cells. (A) Alizarin Red S staining showed that the SLPI/LPS/MC3T3-E1 exhibited higher mineral deposition compared to that of the LPS group. (B) Quantification of Alizarin Red S staining demonstrated an increase in SLPI/LPS/MC3T3-E1 cells compared with that of the LPS group at day 7 and 10. LPS: lipopolysaccharide, SLPI: secretory leukocyte protease inhibitor. *p<0.05.

5.SLPI plays a regulatory role between osteoblasts and osteoclasts

Fig. 7 shows the function of SLPI as a regulatory mole-cule between osteoblasts and osteoclasts in inflamed peri-odontal tissues and osteoblasts. SLPI inhibits the infla-mmation via reduced TNF-a and IL-1b18), which indi-rectly inhibits the osteoclastogenic activity. SLPI represses RANKL expression in LPS-stimulated preosteoblasts, which directly inhibits mature osteoclasts to activate osteoclasts. Moreover, increased OPG and Runx2 levels could increase osteoblastogenic activity for bone formation. The activa-ted osteoblasts inhibit the activation of mature osteoclasts, resulting in reduced bone resorption.

Fig. 7. Schematic showing regula-tion of SLPI in LPS-stimulated preo-steoblasts. RANKL, OPG, and Runx2 expressions regulated by SLPI leads to the activation of osteoblasts and inhibition of mature osteoclasts. LPS: lipopolysaccharide, SLPI: secretory leukocyte protease inhibitor.
Discussion

1.Key results, comparison with previous results, and interpretation

Periodontitis is an inflammatory and infectious disease caused by bacterial LPS and is characterized by the des-truction of periodontal tissues, including the alveolar bone, PDL, and cementum3). The secretion of inflamma-tory cytokines such as IL-1, IL-6, and TNF-a from osteo-blasts of the periodontal tissue and alveolar bone is increased by LPS19), and increased inflammatory cytokines induce destruction of periodontal tissue including loss of periodo-ntal attachment, destruction of collagen, and alveolar bone resorption by osteoclasts20). SLPI inhibits the resorption and destruction of cartilage and bone in acute and chronic inflammation via inhibiting TNF-a and nitric oxide (NO) production21,22), and especially can relieve periodontitis accompanied by alveolar bone resorption3,22). SLPI pre-vented the infiltration of inflammatory cells into the infla-med site in the PDL and alveolar bone resorbing tissues of rats induced with periodontitis via LPS injection, and it decreased the expression of TNF-a and IL-1b proteins, which are bone resorption factors of the intracellular space in the PDL and collagen fibers. In addition, SLPI inhibited the expression of TNF-a and IL-1b proteins in LPS- stimulated preostoblasts18). Therefore, SLPI could inhibit bone resorption by inhibiting the secretion of inflammation- related factors in periodontal tissue and osteoblasts, and directly acting on osteoblasts in the inflammatory respo-nse to regulate the expression of factors related to the differentiation and induction of osteoclasts.

Bone tissue including alveolar bone is a dynamic tissue that is regenerated throughout life through a balance bet-ween the activity of osteoblasts and osteoclasts on the surface of the bone1), and the imbalance between bone formation and resorption leads to bone diseases such as osteopetrosis, osteoporosis, rheumatoid arthritis and perio-dontal bone disease2,5). Osteoblasts are bone-forming cells derived from mesenchymal cells, and form new bones by forming and mineralizing an osteoid, an organic mixture1), and produce RANKL, IL-1, TNF, IL-6 and MIP-1a that induce differentiation of osteoclast by stimulating LPS and 1a,25-dihydroxyvitamin D3 (Vitamin D3)2,23). Under nor-mal and pathological conditions, bone metabolism is regulated via the RANKL/RANK/OPG pathway6,24). RANKL is one of the TNF family members produced by osteoblasts, and its secretion is increased by bone resor-ption-inducing factors such as LPS, Vitamin D3 and Ti ions; it is an important factor for osteoclastogenesis that directly induces monocytes or macrophages-type hemato-poietic bone marrow progenitor cells into osteoclasts7,18). In this study, SLPI expression in periodontal tissue was increased in both the LPS and LPS/SLPI groups compared to the control group. RANKL expression was increased in the LPS group but decreased in the LPS/SLPI group, and the decreasing trend was more prominent as the concen-tration of SLPI increased. Moreover, the mRNA and protein expression levels of SLPI increased in MC3T3-E1 cells after LPS treatment, and those of RANKL, which were increased by LPS, were significantly decreased after SLPI treatment.

OPG, an osteoclast inhibitory factor (OCIF), is expre-ssed in a various cells, including osteoblasts and heart, kidney, and lung cells, and RANKL-induced osteoclast formation is inhibited by OPG9). In addition, OPG is a decoy receptor for RANKL in ST2 cells (a stromal cell line) and inhibits osteoclast formation by blocking RANKL- RANK binding5-8). Runx2 is one of the Runx family mem-bers and an important transcription factor related to the differentiation of preosteoblastic cells into osteoblasts, expre-ssion of Runx2 indicates differentiation of osteoblasts25). Runx2 not only regulates the expression of genes related to bone matrix formation, such as Col I, OPN, and BSP, in osteoblasts25), but also plays an important role in main-taining the supply of immature osteoblasts by retaining their immature stage12). In this study, the expression of OPG in periodontal tissue decreased in the LPS group and significantly increased in the LPS/SLPI group. In contrast, Runx2 positive cells were significantly lower in the LPS group than in the control group and higher in the LPS/ SLPI group than in the control group. The mRNA and protein expression levels of OPG and Runx2 in MC3T3- E1 cells were decreased by LPS treatment compared to those in the control group but increased following treat-ment with SLPI. Therefore, SLPI inhibited RANKL and increased OPG and Runx2 levels in osteoblasts during the inflammatory response, indicating that SLPI directly regu-lates the differentiation and activity of osteoblasts and induces osteoclasts activity.

Osteoblasts and osteoclasts communicate with each other through direct cell-cell contact, cytokines and extracellular matrix interactions. Osteoblasts influence osteoclast for-mation, differentiation, and apoptosis through several signaling pathways including OPG/RANKL/RANK, LGR4/ RANKL/RANK, Ephrin2/ephB4, and Fas/FasL26). The soybean-derived isoflavone, genistein (4´,5,7-trihydroxy-isoflavone), induces bone formation by increasing the expression of Runx2 and bone morphogenetic protein-2 (BMP-2) expression in rat calvarial osteoblasts27). The overexpression of neural epidermal growth factor-like 1 (Nell-1) in rat bone marrow mesenchymal stem cells (BMSCs) increases the expression of Runx2 and osterix, which induces bone formation and promotes matrix cal-cification. The activation of Nell-1 in BMSC inhibits osteoclast differentiation by increasing the expression ratio of OPG/RANKL28). In the present study, RANKL protein levels were significantly reduced after SLPI treatment during the differentiation of LPS-treated MC3T3- E1 cells. After SLPI treatment, the expression of OPG increased on day 4, the early stage of differentiation, and that of Runx2 increased on day 4 to 7. These results indi-cate that SLPI not only increases the differentiation and activity of osteoblasts by increasing the expression of Runx2 and OPG during differentiation, but also inhibits osteo-clast formation by reducing RANKL-RANK binding by directly inhibiting RANKL expression.

During the differentiation of preosteoblasts into osteo-blasts, the expression of bone matrix-forming genes, such as alkaline phosphatase (ALP), Col I, and osteocalcin (OCN), increases, and the organic matrix secreted in the late stage of osteoblast differentiation is mineralized by calcium deposition17,24). Cordyceptin and Leonurus sibiricus L. (LS) extract increased the expression of various bone- forming genes, including ALP, osteonectin (OSN), OPN, Col I, and BSP during the differentiation of MC3T3-E1 cells. Moreover, the LS extract increased the formation of calcified nodules during the differentiation of MC3T3-E1 cells29,30). SLPI regulates not only the formation of the bone matrix and mineralization in osteoblasts attached to titanium17), but also the formation of the dentin matrix and mineralization in odontoblasts15). Alizarin Red staining is generally used to confirm the formation of calcium nodu-les (mineralization) by osteoblasts during the late stages of differentiation31). Alizarin Red staining and the quantita-tive results revealed that SLPI increased nodule formation in LPS-treated MC3T3-E1 cells on days 7 and 10 during differentiation. This result suggests that SLPI not only promotes bone matrix formation and mineral deposition during the osteoblast differentiation in an inflammatory response, but may also directly regulate mineralization in fully differentiated osteoblasts.

2.Suggestion

SLPI was found to regulate the expression of RANKL, OPG, and Runx2 in LPS-treated periodontitis tissue and MC3T3-E1 cells during differentiation, promoting bone matrix formation and mineralization by inducing the diffe-rentiation and activation of osteoblasts. Therefore, SLPI may be an important molecule that can inhibit bone resor-ption, not only by inhibiting inflammatory mediators, but also by directly acting on osteoblasts to regulate osteo-clasts. The present study revealed for the first time that SLPI can regulate the differentiation of osteoblasts and the expression of factors related to osteoclast induction by directly acting on osteoblasts during the inflammatory res-ponse of periodontal tissue. To further identify the regu-latory mechanism of SLPI in the interaction between osteoblasts and osteoclasts, future on the function of SLPI for activating osteoclasts in the co-culture of osteoblasts and osteoclasts are necessary.

Acknowledgments

None.

Conflict of Interest

Prof. Soon-Jeong Jeong has been journal editor-in-chief of the Journal of Dental Hygiene Science since January 2023. She was not involved in the review process of this study. Otherwise, there is no potential conflict of interest relevant to this article was reported.

Ethical Approval

This study was approved by the institutional review board of Chosun University (IRB No. CIACUC2014 A0025).

Author contributions

Conceptualization: Moon-Jin Jeong and Seung-Yeon Lee. Data acquisition: Seung-Yeon Lee and Moon-Jin Jeong. Formal analysis: Seung-Yeon Lee, Myoung-Hwa Lee, and Se-Hyun Hwang. Funding: Moon-Jin Jeong. Supervision: Moon-Jin Jeong. Writing-original draft: Seung-Yeon Lee, Soon-Jeong Jeong, and Moon-Jin Jeong. Writing-review & editing: Do-Seon Lim, Soon-Jeong Jeong, and Moon-Jin Jeong.

Funding

This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03028872).

Data availability

Raw data is provided at the request of the corresponding author for reasonable reason.

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