Yasumasa Endo

Involvement of toll-like receptor 2 and 4 in association between dyslipidemia and osteoclast differentiation in apolipoprotein E deficient rat periodontium

BackgroundDyslipidemia increases circulating levels of oxidized low-density lipoprotein (OxLDL) and this may induce alveolar bone loss through toll-like receptor (TLR) 2 and 4. The purpose of this study was to investigate the effects of dyslipidemia on osteoclast differentiation associated with TLR2 and TLR4 in periodontal tissues using a rat dyslipidemia (apolipoprotein E deficient) model.MethodsLevels of plasma OxLDL, and the cholesterol and phospholipid profiles in plasma lipoproteins were compared between apolipoprotein E-deficient rats (16-week-old males) and wild-type (control) rats. In the periodontal tissue, we evaluated the changes in TLR2, TLR4, receptor activator of nuclear factor kappa B ligand (RANKL) and tartrate resistant acid phosphatase (TRAP) expression.ResultsApolipoprotein E-deficient rats showed higher plasma levels of OxLDL than control rats (p<0.05), with higher plasma levels of total cholesterol (p<0.05) and LDL-cholesterol (p<0.05) and lower plasma levels of high-density lipoprotein cholesterol (p<0.05). Their periodontal tissue also exhibited a higher ratio of RANKL-positive cells and a higher number of TRAP-positive osteoclasts than control rats (p<0.05). Furthermore, periodontal gene expression of TLR2, TLR4 and RANKL was higher in apolipoprotein E-deficient rats than in control rats (p<0.05).ConclusionThese findings underscore the important role for TLR2 and TLR4 in mediating the osteoclast differentiation on alveolar bone response to dyslipidemia.

Supplementation of green tea catechins in dentifrices suppresses gingival oxidative stress and periodontal inflammation

OBJECTIVE this study examined the effects of a dentifrice containing green tea catechins on gingival oxidative stress and periodontal inflammation using a rat model. DESIGN twenty-four male Wister rats were randomly divided into four groups. The first group (Control group) received no treatment for 8 weeks. Periodontal inflammation was induced in the second group for 8 weeks. Periodontal inflammation was induced in the last two groups for 8 weeks and dentifrices with or without green tea catechins were topically applied to the gingival sulcus daily for 4 weeks prior to the end of the experimental period. RESULTS rats that had experimental periodontal inflammation showed apical migration of the junctional epithelium, alveolar bone loss and inflammatory cell infiltration in the connective tissue subjacent to the junctional epithelium at 8 weeks, whilst the control group showed no pathologic changes. Topical application of a green tea catechin-containing dentifrice reduced inflammatory cell infiltration in the periodontal lesions to a greater degree than the control dentifrice at 8 weeks. The gingiva in which green tea catechin-containing dentifrice was applied also showed a lower level of expression of hexanoyl-lysine (a marker of lipid peroxidation), nitrotyrosine (a marker of oxidative protein damage), and tumour necrosis factor-α (an indicator of pro-inflammatory cytokines) at 8 weeks compared to gingiva in which the control dentifrice was applied. CONCLUSIONS adding green tea catechins to a dentifrice may contribute to prevention of periodontal inflammation by decreasing gingival oxidative stress and expression of pro-inflammatory cytokines.

Preventive effects of a cocoa-enriched diet on gingival oxidative stress in experimental periodontitis.

BACKGROUND Oxidative stress affects the progression of periodontitis. Cocoa is a rich source of flavonoids with antioxidant properties, which could suppress gingival oxidative stress in periodontal lesions. The purpose of the present study was to investigate the effects of a cocoa-enriched diet on gingival oxidative stress in a rat-periodontitis model. METHODS In this 4-week study, rats were divided into three groups (n = 8/group): a control group (fed a regular diet) and two periodontitis groups (fed a regular diet or cocoa-enriched diet [10% of food intake]). Periodontitis was induced by ligature placement around the mandibular first molars. Serum levels for reactive oxygen metabolites were measured at baseline and 2 and 4 weeks. At 4 weeks, the levels of 8-hydroxydeoxyguanosine and reduced/oxidized glutathione ratio were determined to evaluate gingival oxidative damage and antioxidant status, respectively. RESULTS Rats with experimental periodontitis that were fed a regular diet showed an increase in the level of serum reactive oxygen metabolites in a time-dependent manner. These rats also had an increased 8-hydroxydeoxyguanosine level and decreased reduced/oxidized glutathione ratio in the gingival tissue, inducing alveolar bone loss and polymorphonuclear leukocyte infiltration. Although experimental periodontitis was induced in the rats fed a cocoa-enriched diet, they did not show impairments in serum reactive oxygen metabolite level and gingival levels for 8-hydroxydeoxyguanosine and reduced/oxidized glutathione ratio. Alveolar bone loss and polymorphonuclear leukocyte infiltration after ligature placement were also inhibited by cocoa intake. CONCLUSION Consuming a cocoa-enriched diet could diminish periodontitis-induced oxidative stress, which, in turn, might suppress the progression of periodontitis.

Experimental Periodontitis Induces Gene Expression of Proinflammatory Cytokines in Liver and White Adipose Tissues in Obesity

BACKGROUND Recent studies indicated that periodontitis induces systemic low-grade inflammation. The increase in systemic low-grade inflammation induced by periodontitis may alter the effects of obesity on the production of inflammatory molecules, including C-reactive protein (CRP), interleukin (IL)-6, and tumor necrosis factor-alpha (TNF-alpha), in the liver and white adipose tissue (WAT). The purpose of the present study is to investigate the effects of periodontitis on the expression of proinflammatory cytokines in the liver and WAT in obese Zucker rats. METHODS Obese Zucker rats and their lean litter mates were divided into four groups of six rats each: lean Zucker rats without periodontitis (control group), lean Zucker rats with periodontitis (periodontitis group), obese Zucker rats without periodontitis (obesity group), and obese Zucker rats with periodontitis (combination group). Periodontitis was ligature induced for 4 weeks in the periodontitis and combination groups, whereas the other groups were left unligated. RESULTS At 4 weeks, the gene expression for CRP, IL-6, and TNF-alpha in the liver and CRP and IL-6 in the WAT of combination groups was significantly higher than in each of the three groups. Serum TNF-alpha in the periodontitis and obesity groups was significantly higher than in the control group. Serum CRP and TNF-alpha in the combination group was significantly higher than in each of the three groups. CONCLUSION Systemic low-grade inflammation after experimental periodontitis was associated with increased gene expression for hepatic levels of TNF-alpha and CRP and adipose tissue levels of IL-6 and CRP in the obese-rat model.

Imbalance of oxidative/anti-oxidative status induced by periodontitis is involved in apoptosis of rat submandibular glands

OBJECTIVE Epidemiologic studies suggest a relationship between periodontitis and salivary gland dysfunction. A rat periodontitis model was used to investigate whether a causal relationship exists between periodontitis and pathological changes of submandibular glands. DESIGN Fourteen male Wistar rats (8 weeks old) were divided into two groups (n=7/group): a control group and periodontitis group. Periodontitis was induced by ligature placement around the mandibular first molars. Serum levels for reactive oxygen metabolites, anti-oxidant and tumour necrosis factor (TNF)-alpha were measured at baseline, 2 and 4 weeks. At 4 weeks, the levels of 8-hydroxydeoxyguanosine were determined to evaluate oxidative damage of submandibular glands. Expression of TNF-alpha mRNA and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) as well as histological findings were also evaluated in the submandibular glands. RESULTS The rats with experimental periodontitis showed increase in the levels of serum reactive oxygen metabolites and TNF-alpha, and a decrease of anti-oxidant power in a time-dependent manner. At 4 weeks, these rats also had significantly increased levels of 8-hydroxydeoxyguanosine and TNF-alpha, and increased number of TUNEL-positive cells and vacuolisation in the submandibular glands compared to the control rats. CONCLUSIONS Imbalance of circulating oxidative/anti-oxidative status may be involved in vacuolisation and apoptosis of submandibular glands in the rat periodontitis model.

Effects of periodontitis on aortic insulin resistance in an obese rat model

The combination of obesity and its associated risk factors, such as insulin resistance and inflammation, results in the development of atherosclerosis. However, the effects of periodontitis on atherosclerosis in an obese body remain unclear. The aim of the study was to investigate the effects of ligature-induced periodontitis in Zucker fatty rats on initiation of atherosclerosis by evaluating aortic insulin resistance. Zucker fatty rats (n=24) were divided into two groups. In the periodontitis group, periodontitis was ligature-induced for 4 weeks, whereas the control group was left unligated. After the 4-week experimental period, descending aorta was used for measuring the levels of lipid deposits, immunohistochemical analysis, and evaluation of gene expression. Levels of serum C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and insulin were also measured. Rats in the periodontitis group had significantly enhanced lipid deposits in the aorta, but not in the control group. Expression of suppressor of cytokine signaling 3, vascular cell adhesion molecule 1, reactive oxygen species, nitrotyrosine, and endothelin-1 in the periodontitis group was more intense than that in the control group. Significantly decreased levels of phosphatidylinositol 3-kinase (Pi3k) catalytic β-polypeptide (Pi3kcb), Pi3kp85, and insulin receptor substrate 1 and 2 were observed in the periodontitis group. Levels of serum CRP and TNF-α were significantly increased in the periodontitis group. Under insulin-stimulated conditions, aorta in the periodontitis group altered the Akt phosphorylation. Periodontitis in obesity induced the initial stage of atherosclerosis and disturbed aortic insulin signaling.

Combined effects of hydrogen sulfide and lipopolysaccharide on osteoclast differentiation in rats.

BACKGROUND Lipopolysaccharide (LPS) stimulates osteoclast differentiation through toll-like receptors (TLRs) 2 and 4, and hydrogen sulfide (H(2)S) induces osteoclast differentiation. If H(2)S activates TLRs, H(2)S may enhance the effects of LPS on osteoclast differentiation. The purpose of the present study is to examine the combined effects of sodium hydrogen sulfide (NaHS, an H(2)S donor drug) and LPS on osteoclast differentiation and TLR expression in rat periodontal tissue. METHODS Twenty-eight male Wistar rats (8 weeks old) were divided into four groups (n = 7 per group): a control (no treatment) group and three experimental groups (NaHS group, LPS group, and a combination [NaHS + LPS] group). At 1 day after topical application of NaHS and/or Porphyromonas gingivalis LPS into the gingival sulcus of first molars, the number of tartrate-resistant acid phosphate (TRAP)-positive osteoclasts in the periodontal tissue was counted. Expression of TLR2 and TLR4 mRNAs and proteins in the gingival was also assessed. RESULTS The number of TRAP-positive osteoclasts was significantly higher in the combination group than in any other group (P <0.01). The combination group had 11.0-fold higher TLR4 mRNA levels than the control group. TLR4 protein levels were also higher in the combination group than in the NaHS or LPS group. However, the TLR2 mRNA and protein levels were not significantly different in the combination group and the LPS group. CONCLUSION In rat periodontal tissue, NaHS and LPS had an additive effect on osteoclast differentiation through activation of the TLR4 pathway but not the TLR2 pathway.

Supplementation of broccoli or Bifidobacterium longum–fermented broccoli suppresses serum lipid peroxidation and osteoclast differentiation on alveolar bone surface in rats fed a high-cholesterol diet

High-cholesterol diet enhances osteoclastic activity on alveolar bone by increasing serum lipid peroxidation. We hypothesized that supplementation with dietary antioxidants, such as found in broccoli and its fermented products, might suppress increases in serum lipid peroxidation, contributing to the inhibition of osteoclastic activity after high-cholesterol diet intake. The purpose of the present study was to investigate the effects of broccoli and fermented broccoli consumption on serum lipid peroxidation and osteoclast differentiation in alveolar bone of rats fed a high-cholesterol diet. In this 12-week study, rats were divided into 4 groups (n = 6/group): a control group (fed regular diet) and 3 experimental groups (fed a high-cholesterol [1% wt/wt] diet, or a high-cholesterol diet supplemented with either broccoli powder [5% wt/wt] or Bifidobacterium longum-fermented broccoli powder [5% wt/wt]). Serum hexanoyl-lysine (HEL) levels were measured as a parameter of lipid peroxidation. The number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in alveolar bone was enumerated to evaluate osteoclast differentiation. When compared with regular diet, the high-cholesterol diet increased serum HEL levels and resulted in a higher number of TRAP-positive osteoclasts at 12 weeks. The high-cholesterol diet supplemented with broccoli or B. longum-fermented broccoli showed lower levels of serum HEL and fewer TRAP-positive osteoclasts than the high-cholesterol diet at 12 weeks. In conclusion, consumption of broccoli, or its fermented product, inhibited the effects of a high-cholesterol diet on osteoclast differentiation in rat alveolar bone by suppressing serum lipid peroxidation.

Effects of exercise training on gingival oxidative stress in obese rats

OBJECTIVE The purpose of the present study was to investigate the effects of exercise training on serum reactive oxygen species (ROS) level and gingival oxidative stress in obese rats fed a high-fat diet. DESIGN Rats were divided into three groups (n = 14/group): one control group (fed a regular diet) and two experimental groups (fed a high-fat diet with and without exercise training [treadmill: 5 days/week]). The rats were sacrificed at 4 or 8 weeks. The level of serum reactive oxidative metabolites (ROM) was measured as an indicator of circulating ROS. The level of 8-hydroxydeoxyguanosine (8-OHdG) and reduced-form glutathione (GSH)/oxidised-form glutathione (GSSG) ratio were determined to evaluate gingival oxidative stress. RESULTS The obese rats fed a high-fat diet without exercise training showed higher serum ROM levels [Carratelli Units (CARR U)] (mean ± SD; 413 ± 64) than the control (333 ± 12) at 4 weeks (p = 0.023). Such a condition resulted in higher 8-OHdG levels (ng/mg mtDNA) (0.97 ± 0.18) (p < 0.05) and a lower GSH/GSSG ratio (17.0 ± 3.1) (p < 0.05) in gingival tissues, compared to the control (0.55 ± 0.13 for 8-OHdG and 23.6 ± 5.8 for GSH/GSSG ratio) at 8 weeks. In addition, the obese rats fed a high-fat diet with exercise training showed lower serum ROM (623 ± 103) (p < 0.001) and gingival 8-OHdG levels (0.69 ± 0.17) (p = 0.012) than those without exercise training (1105 ± 95 for ROM and 0.55 ± 0.13 for 8-OHdG) at 8 weeks. CONCLUSIONS Obesity prevention by exercise training may effectively suppress gingival oxidative stress by decreasing serum ROS in rats.

Hydrogen-rich water prevents lipid deposition in the descending aorta in a rat periodontitis model

OBJECTIVE Periodontitis has been causally linked to atherosclerosis, which is mediated by the oxidative stress. As hydrogen-rich water (HW) scavenges reactive oxygen species (ROS), we hypothesized that HW could prevent lipid deposition induced by periodontitis in the aorta. The aim of this study was to investigate the effects of HW on the initiation of atherosclerosis in a rat periodontitis model. DESIGN Eighteen 8-wk-old male Wistar rats were divided into three groups of six rats; the periodontitis group, periodontitis+HW group and the no treatment (control) group. In the periodontitis and periodontitis+HW groups, periodontitis was induced using a ligature for 4 wk, while the periodontitis+HW group was given water containing 800-1000 μg/L hydrogen during the 4-wk experimental period. RESULTS In the periodontitis group, lipid deposition in the descending aorta was observed. The periodontitis group also showed significant higher serum levels for ROS and oxidised low-density lipoprotein-cholesterol (ox-LDL) (1.7 and 1.4 times, respectively), and higher aortic expression levels of nitrotyrosine and hexanoyl-lysine (HEL) (7.9 and 16.0 times, respectively), as compared to the control group (p<0.05). In the periodontitis+HW group, lipid deposition was lower. Lower serum levels of ROS and ox-LDL (0.46 and 0.82 times, respectively) and lower aortic levels of nitrotyrosine and HEL (0.27 and 0.19 times, respectively) were observed in the periodontitis+HW group than in the periodontitis group (p<0.05). CONCLUSIONS HW intake may prevent lipid deposition in the rat aorta induced by periodontitis by decreasing serum ox-LDL levels and aortic oxidative stress.