Magnolol ameliorates ligature-induced periodontitis in rats and osteoclastogenesis: in vivo and in vitro study

Abstract

Periodontal disease characterized by alveolar bone resorption and bacterial pathogen-evoked inflammatory response has been believed to have an important impact on human oral health. The aim of this study was to evaluate whether magnolol, a main constituent of Magnolia officinalis, could inhibit the pathological features in ligature-induced periodontitis in rats and osteoclastogenesis. The sterile, 3-0 (diameter; 0.2 mm) black braided silk thread, was placed around the cervix of the upper second molars bilaterally and knotted medially to induce periodontitis. The morphological changes around the ligated molars and alveolar bone were examined by micro-CT. The distances between the amelocemental junction and the alveolar crest of the upper second molars bilaterally were measured to evaluate the alveolar bone loss. Administration of magnolol (100 mg/kg, p.o.) significantly inhibited alveolar bone resorption, the number of osteoclasts on bony surface, and protein expression of receptor activator of nuclear factor- κ B ligand (RANKL), a key mediator promoting osteoclast differentiation, in ligated rats. Moreover, the ligature-induced neutrophil infiltration, expression of inducible nitric oxide synthase, cyclooxygenase-2, matrix metalloproteinase (MMP)-1 and MMP-9, superoxide formation, and nuclear factor- κ B activation in inflamed gingival tissues were all attenuated by magnolol. In the in vitro study, magnolol also inhibited the growth of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans that are key pathogens initiating periodontal disease. Furthermore, magnolol dose dependently reduced RANKL-induced osteoclast differentiation from RAW264.7 macrophages, tartrate-resistant acid phosphatase (TRAP) activity of differentiated cells accompanied by a significant attenuation of resorption pit area caused by osteoclasts. Collectively, we demonstrated for the first time that magnolol significantly ameliorates the alveolar bone loss in ligature-induced experimental periodontitis by suppressing periodontopathic microorganism accumulation, NF- κ B-mediated inflammatory mediator synthesis, RANKL formation, and osteoclastogenesis. These activities support that magnolol is a potential agent to treat periodontal disease.

Figure 1

Effect of magnolol on alveolar bone loss and tissue damage. (a) Reconstructed three-dimensional micro-CT images (buccal and palatal view) of the maxilla second molars buccal (upper panel) and palatal (lower panel) alveolar bone level in nonligation (control), ligation, and ligation + magnolol (100 mg/kg) groups at the experimental day 8. (b) Histological observation (H&E stain) of maxillary intermolar tissue in various groups (scale bar = 100 μm). (c) The degree of alveolar bone loss was evaluated by measuring the ACJ-AC distance at the buccal furcation site of the maxilla second molar from the reconstructed micro-CT images. (d) Quantitative analysis of the total number of inflammatory infiltrated cells in maxillary intermolar gingivomucosal tissue was performed by accounting the number of polymorphonuclear cells (PMNs). Data are expressed as the mean ± SD (n = 5). ***P < 0.001 versus control; ^# P < 0.05, ^## P < 0.01 versus ligation group.

Figure 2

Effect of magnolol on the number of TRAP-positive multinucleated cells (MNCs) and the expression of RANKL and OPG in gingival tissues. (a) The micrographs (upper panel: 100x magnification; lower panel: 200x magnification) represent the histological sections around intermolar region of rats 8 days after ligature placement, and the quantitative analysis of the number of TRAP⁺ MNCs with red staining along the bony resorptive lacunae (cells/mm) was performed (scale bar = 50 μm) (c). (b) The protein expression of RANKL and OPG was determined by Western blot in dissected tissues, (d) and the relative protein RANKL/OPG ratio was measured. Data are expressed as mean ± SD (n = 5). **P < 0.01, ***P < 0.001 versus control; ^# P < 0.05, ^## P < 0.01 versus ligation group.

Figure 3

Effect of magnolol on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans growth in vitro. Calibrated suspensions of the periodontal pathogens, Porphyromonas gingivalis (a) and Aggregatibacter actinomycetemcomitans (b), were incubated with medium (control), vehicle (0.1% DMSO), and indicated concentration of magnolol (75–150 μM) for 24 h. The growth of the bacteria was measured with a spectrophotometer at 600 nm wavelength. Data are expressed as the mean ± SD (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001 versus control.

Figure 4

Effect of magnolol on MPO activity, superoxide anion production, and expression of COX-2 and iNOS in inflamed periodontal tissues. (a) The MPO activity and (b) superoxide anion production were determined in the gingivomucosal tissues of each experimental group. (c) The Western blot analysis was used to detect COX-2 and iNOS protein level in dissected tissues encircled upper second molar. Data are expressed as the mean ± SD (n = 5). ***P < 0.001 versus control; ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 versus ligation group.

Figure 5

Effect of magnolol on MMP-1, -9, and TIMP-1 expression and NF-κB activation. (a) The protein expression of MMP-1, -9, and TIMP-1 was determined by Western blot in dissected tissues encircled upper second molar, and the relative MMPs/TIMP ratio was measured. (b) The Western blot analysis was conducted to detect phospho-NF-κB p65 protein level in dissected tissues. Data are expressed as the mean ± SD (n = 4). **P < 0.01, ***P < 0.001 versus control; ^# P < 0.05, ^## P < 0.01 versus ligation group.

Figure 6

Effect of magnolol on RANKL-induced osteoclast differentiation. RAW 264.7 cells were cultured with the indicated dose of magnolol (2.5–20 μM) in the presence of RANKL (50 ng/mL) for 4 days. The photographs of TRAP-stained osteoclasts (a), the number of TRAP⁺ MNCs, and the TRAP activity (b) were examined (scale bar = 100 μm). Data are expressed as the mean ± SD (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001 versus RANKL-treated group alone.

Figure 7

Effect of magnolol on RANKL-induced bone resorption of mature osteoclasts in vitro. RAW 264.7 cells were incubated with various concentrations of magnolol (5–20 μM) in the presence of RANKL (50 ng/mL). (a) For the visualization of pits formation, the resorption area was analyzed by a microscope. (b) The percentage of resorption area caused by osteoclasts was measured. Data are expressed as the mean ± SD (n = 4). **P < 0.01, ***P < 0.001 versus RANKL-treated alone group.