TRPC expression in human periodontal ligament cells and the periodontal tissue of periodontitis mice: a preliminary study.

BACKGROUND: Transient receptor potential canonical (TRPC) channels are non-selective cationic channels with permeability to Ca2+ and Na+. Despite their importance, there are currently few studies on TRPC in the periodontal ligament (PDL) and bone cells in the dental field. To provide biological information regarding TRPC in PDL cells and periodontal tissue, we evaluated TRPC channels expression in the osteoblast differentiation of PDL cells and periodontitis-induced tissue. Human PDL cells were cultured in osteogenic differentiation media for 28 days, and the expression of Runx2, osteocalcin (OCN), and TRPC1, 3, 4, and 6 was evaluated by real-time PCR. In ligature-induced periodontitis mice, the alveolar bone and osteoid areas, the osteoclast number, and the expression of Runx2, OCN, TRPC3, and TRPC6 was evaluated by H&E staining, TRAP staining, and immunohistochemistry, respectively. RESULTS: In the PDL cell differentiation group, TRPC6 expression peaked on day 7 and TRPC3 expression generally increased during differentiation. During the 28 days of periodontitis progression, alveolar bone loss and osteoclast numbers increased compared to the control group during the experimental period and the osteoid area increased from day 14. TRPC6 expression in the periodontitis group increased in the PDL area and in the osteoblasts compared to the control group, whereas TRPC3 expression increased only in the PDL area on days 7 and 28. CONCLUSIONS: These results indicate changes of TRPC3 and TRPC6 expression in PDL cells that were differentiating into osteoblasts and in periodontitis-induced tissue, suggesting the need for research on the role of TRPC in osteoblast differentiation or periodontitis progression.

Periodontitis deteriorates renal fibrosis and macrophage infiltration in rats with chronic kidney disease.

OBJECTIVE: The objective of this study was to examine the effect of periodontitis on renal function and morphology in rats with or without nephrectomy (Nx)-induced chronic kidney disease (CKD). METHODS: Rats were divided into sham surgery (Sham), Sham with tooth ligation (ShamL), Nx, and NxL groups. Periodontitis was induced by tooth ligation at 16-week olds. Creatinine, alveolar bone area, and renal histopathology were analyzed at 20-week olds. RESULTS: Creatinine did not differ between the Sham and ShamL groups or between the Nx and NxL groups. The ShamL and NxL groups (both p = 0.002) had less alveolar bone area than the Sham group. The NxL group had fewer glomeruli than the Nx group (p < 0.000). The periodontitis groups demonstrated more tubulointerstitial fibrosis (Sham vs. ShamL p = 0.002, Nx vs. NxL p < 0.000) and macrophage infiltration (Sham vs. ShamL p = 0.002, Nx vs. NxL p = 0.006) than the groups without periodontitis. Only the NxL group had greater renal TNFα expression than the Sham group (p < 0.003). CONCLUSIONS: These suggest that periodontitis increases renal fibrosis and inflammation in the presence or absence of CKD but does not affect renal function. Periodontitis also increases TNFα expression in the presence of CKD.

Alveolar bone and tibia responses to hormonal and mineral abnormalities in rats with chronic kidney disease: A pilot study.

OBJECTIVES: As the impact of chronic kidney disease (CKD) severity on different bone types remains unclear, we induced increasing levels of CKD severity in a rat model and investigated hormone and mineral levels as well as alveolar and tibia bone histomorphology. METHODS: Rats were divided into sham operation (sham), 4/6 nephrectomy (4/6Nx), 5/6Nx, and 4/6Nx with hyperphosphorous (HP) diet (4/6NxHP). At week 20, BUN, FGF23, PTH, and P were estimated in plasma. Bone parameters were evaluated by microCT, and osteoclasts and osteoid areas were evaluated by TRAP and H&E stains, respectively. RESULTS: The 4/6NxHP and 5/6Nx groups had elevated PTH, and the 4/6NxHP group alone had elevated P. Compared to the 4/6Nx group, the 4/6NxHP group demonstrated increased FGF23 and P. In the alveolar bone, the 4/6NxHP group had reduced bone volume and BMD compared to the sham and 4/6Nx groups. In the tibia cortical bone, bone surface density was higher in the 4/6NxHP group compared to the sham group. Tibia cortical bone volume was negatively correlated with FGF23 and P. Moreover, alveolar bone volume was negatively correlated with FGF23, PTH, and P. CONCLUSIONS: Our results demonstrate that hormone and mineral levels vary with CKD severity, and alveolar bone loss strongly correlates with these hormone and mineral alterations.

Distribution of neutrophil and monocyte/macrophage populations induced by the CXCR4 inhibitor AMD3100 in blood and periodontal tissue early after periodontitis induction.

CXCR4, a CXCL12 receptor, is expressed on epithelial cells, fibroblasts, and inflammatory cells. The CXCR4-CXCL12 interaction is related to the migration of neutrophils and monocytes/macrophages. Periodontitis, an inflammatory disease mainly caused by gram-negative bacteria, is characterized by infiltration of circulating inflammatory cells and alveolar bone (AB) loss. To investigate whether CXCR4 is involved in the distribution of neutrophils and monocytes/macrophages early after periodontitis induction, we examined the effects of AMD3100 (AMD), a CXCR4 antagonist, in ligature-induced periodontitis mice and LPS-injected air pouch mice. The periodontitis study was accomplished in control (C), periodontitis (P), and P + AMD groups. Periodontitis was induced by ligation of the mandibular first molar. AMD was intraperitoneally administered daily beginning the day before ligation until sacrifice on the third day after ligation. The air pouch study was accomplished in C, lipopolysaccharide (LPS), and LPS + AMD groups. Air pouches on mice backs were formed by subcutaneous injection of sterilized air. AMD was administered and then LPS was injected into the air pouch. For the detection of neutrophils and monocytes/macrophages in blood and air pouch exudates, flow cytometry was performed with anti-Ly6G/anti-CD11b antibodies (Abs) and anti-CD115 Ab, respectively. In periodontal tissue, Ly6G+ cells and CD115+ cells were counted by immunohistological analysis. AB loss was estimated by the periodontal ligament area in the furcation. In the periodontitis study, the P group showed higher numbers of Ly6G+ cells and CD115+ cells in blood and periodontal tissue than the C group. The P + AMD group showed a greater number of Ly6G+ cells and CD115+ cells in blood, but not in periodontal tissue compared to the P group. There was no difference in AB loss between the P and P + AMD groups. In the air pouch study, the LPS group had higher levels of Ly6G+ CD11b+ cells and CD115+ cells in both blood and exudates than the C group. The number of these cells in the LPS + AMD group was higher in blood than in the LPS group, but not in the exudates. The CXCR4 antagonist further increased neutrophil and monocyte/macrophage populations in the blood, but did not alter the levels in the periodontal tissue and exudates in mice with periodontitis and LPS-injected air pouches. These results suggest that during inflammatory conditions such as periodontitis, CXCR4 is involved in the distribution of neutrophils and monocytes/macrophages in the blood, but not in inflamed peripheral tissues.

Lipopolysaccharides of Fusobacterium nucleatum and Porphyromonas gingivalis increase RANKL-expressing neutrophils in air pouches of mice.

Increases of neutrophils and osteoclasts are pathological changes of periodontitis. RANKL is an osteoclast differentiation factor. The effect of periodontopathogen LPS on RANKL-expressing neutrophils has not been clarified yet. We evaluated numerical changes of RANKL-expressing neutrophils in air pouches of mice injected with LPSs of Fusobacterium nucleatum and Porphyromonas gingivalis. Mice with air pouches were assigned into saline (C)-, E. coli LPS- (Ec LPS)-, F. nucleatum LPS (Fn LPS)-, P. gingivalis LPS (Pg LPS)-, and Fn LPS and Pg LPS (Fn + Pg LPS)-injected groups. CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils in blood and air pouch exudates were determined by flow cytometry. In blood, compared to the C group, the Fn LPS group showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils whereas the Pg LPS group showed no significant differences. These increases in the Fn LPS group were not different to those in the Ec LPS group. In exudates, Fn LPS and Pg LPS groups showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils compared to the C group. Increased levels in the Fn LPS group were not different to those in the Ec LPS group, but Pg LPS group was lower than those in the Ec LPS group. In blood and exudates, the Fn + Pg LPS group showed no difference in levels of these neutrophils compared to the Ec LPS group. LPSs of F. nucleatum and P. gingivalis increased RANKL-expressing neutrophils although the degrees of increases were different. These suggest that periodontopathogen LPS can act as a stimulant to increase RANKL-expressing neutrophils.

The presence of neutrophils causes RANKL expression in periodontal tissue, giving rise to osteoclast formation.

BACKGROUNDS AND OBJECTIVE: Increased neutrophil infiltration and osteoclast formation are key characteristics of periodontitis. The effect of these neutrophils on osteoclast formation in periodontitis remains unclear. Therefore, we investigated the effects of neutrophils on osteoclast formation in a neutrophil-deficient mouse model of periodontitis. METHODS: Anti-Ly6G antibody (Ab) was used for neutrophil depletion in two mouse models: periodontitis and air pouch. In the periodontitis experiments, mice were divided into PBS-administered control (C), control Ab-administered periodontitis (P), and anti-Ly6G Ab-administered periodontitis (P + Ly6G) groups. Periodontitis was induced by ligature of mandibular first molars. In the air pouch experiments, mice were divided into PBS-administered (C), LPS and control Ab-administered (LPS), and LPS and anti-Ly6G Ab-administered (LPS + Ly6G) groups. Neutrophil migration into air pouches was induced by LPS injection. Flow cytometry was used to examine CD11b+ Ly6G+ neutrophils in the blood of periodontitis mice and CD11b+ Ly6G+ RANKL+ neutrophils in exudates of air pouch mice. In periodontal tissue, Ly6G+ neutrophil and RANKL+ cell numbers in periodontal ligament and alveolar bone areas were estimated using immunohistochemistry, osteoclast numbers were measured using TRAP assay, and alveolar bone loss was determined by H&E staining. RESULTS: In blood, CD11b+ Ly6G+ neutrophils were found in greater percentage in the P group than in the C group on days 3 and 7. However, the percentage of neutrophils was lower in the P + Ly6G group than in the C and P groups. In periodontal tissue, the numbers of Ly6G+ neutrophils and RANKL+ cells were lower in the P + Ly6G group than in the P group on day 3. Ly6G+ neutrophil numbers decreased more in the P + Ly6G group than in the P group on day 7, but RANKL+ cell numbers did not decrease in the P + Ly6G group. In exudates, the number of CD11b+ Ly6G+ RANKL+ neutrophils was greater in the LPS group than in the C and LPS + Ly6G groups. On days 3 and 7, the numbers of osteoclasts and alveolar bone loss were greater in periodontal tissue in the P and P + Ly6G groups than in the C group. Interestingly, there were fewer osteoclasts in the P + Ly6G group than in the P group on day 3. CONCLUSION: Neutrophil deficiency caused a reduction in numbers of both RANKL+ cells and osteoclasts in periodontitis-induced tissues only on day 3. Furthermore, in the LPS-injected air pouch model, neutrophil deficiency reduced the influx of RANKL+ neutrophils. These findings suggest that the presence of neutrophils induces RANKL expression and could induce osteoclast formation in the early stages of periodontitis.

A pilot study of occupational exposure to pathogenic microorganisms through lip cosmetics among dental hygienists.

OBJECTIVES: In dental clinics, dental hygienists are exposed to aerosolized pathologic bacteria, which can be transmitted to the oral cavity via lip cosmetics. Accordingly, such contamination poses a consistent health risk among staffs. Our study examined the bacterial contamination of lip cosmetics used by dental hygienists while in a clinic setting. METHODS: Sixteen dental hygienists were surveyed regarding their job assignments and habits associated with lip cosmetic. Subsequently, microorganisms were analyzed in collected samples of the hygienists' lip cosmetics using colony-forming unit (CFU) assays, 16s-rDNA polymerase chain reaction, and DNA sequencing. RESULTS: Notably, 81.3% of the submitted lip cosmetic samples were contaminated, with bacterial CFUs ranging from undetectable to innumerable. Many samples (43.8%) exceeded the microbial limits of cosmetic contamination. Of the lip cosmetic used for more than 6 months, 60% exceeded the microbial limit. When wearing a mask every time, only one of the six samples exceeded the microbial limit. More frequent dental mask changing was associated with a lower likelihood that the cosmetic sample would exceed the microbial limit. No samples from hygienists who changed their masks four times a day exceeded the microbial limit, compared to 33.3% from hygienists who only changed the mask when it became wet. Most isolated bacteria were gram-positive, facultative anaerobic, asporogenic, and opportunistically pathogenic, and the most prevalent species were Staphylococcus aureus, Streptococcus salivarius, and Staphylococcus epidermidis. CONCLUSION: Our findings indicate that dental staff, including dental hygienists, should exercise more careful workplace habits, particularly with regard to infection control and cosmetic use.

Simvastatin attenuates tibial bone loss in rats with type 1 diabetes and periodontitis.

BACKGROUND: Diabetes induces long bone loss and aggravation of periodontitis-induced alveolar bone loss. Simvastatin (SIM), which is a lipid-lowering agent is known to have an anabolic effect on bone. Therefore, we investigated effect of SIM on tibial and alveolar bone loss in type 1 diabetic rats with periodontitis. METHODS: Rats were divided into control (C), diabetes with periodontitis (DP), and diabetes with periodontitis treated with SIM (DPS) groups. DP and DPS groups were intravenously injected with streptozotocin (50 mg/kg), and C group was injected with citrate buffer. Seven days later (day 0), periodontitis was induced by ligatures of mandibular first molars. DP and DPS groups were orally administered vehicle or SIM (30 mg/kg) from day 0 to days 3, 10, or 20. Alveolar and tibial bone loss was measured using histological and m-CT analysis alone or in combination. Osteoclast number and sclerostin-positive osteocytes in tibiae were evaluated by tartrate-resistant acid phosphatase and immunohistochemical staining, respectively. Glucose, triglyceride (TG), cholesterol (CHO), and low-density lipoprotein (LDL) were evaluated. RESULTS: Consistent with diabetes induction, the DP group showed higher glucose and TG levels at all timepoints and higher CHO levels on day 20 than C group. Compared to the DP group, the DPS group exhibited reduced levels of glucose (day 3), TG (days 10 and 20), CHO, and LDL levels (day 20). Bone loss analysis revealed that the DP group had lower bone volume fraction, bone mineral density, bone surface density, and trabecular number in tibiae than C group at all timepoints. Interestingly, the DPS group exhibited elevation of these indices at early stages compared to the DP group. The DPS group showed reduction of osteoclasts (day 3) and sclerostin-positive osteocytes (days 3 and 20) compared with the DP group. There was no difference in alveolar bone loss between DP and DPS groups. CONCLUSIONS: These results suggest that SIM attenuates tibial, but not alveolar bone loss in type 1 diabetic rats with periodontitis. Moreover, attenuation of tibial bone loss by SIM may be related to inhibition of osteoclast formation and reduction of sclerostin expression.

Intermittent PTH administration improves alveolar bone formation in type 1 diabetic rats with periodontitis.

BACKGROUND: Periodontitis is an infectious disease that manifests as alveolar bone loss surrounding the roots of teeth. Diabetes aggravates periodontitis-induced alveolar bone loss via suppression of bone formation. Intermittent parathyroid hormone (PTH) administration displays an anabolic effect on bone. In this study, we investigated the effect of intermittent PTH administration on alveolar bone loss in type 1 diabetic rats with periodontitis. METHODS: Rats were divided into control (C), periodontitis (P), periodontitis treated with PTH (P + PTH), diabetes with periodontitis (DP), and diabetes with periodontitis treated with PTH (DP + PTH) groups. To induce type 1 diabetes, rats were injected with streptozotocin and periodontitis was induced bilaterally by applying ligatures to the mandibular first molars for 30 days. During the experimental period, the P + PTH and DP + PTH groups were subcutaneously injected with PTH (40 µg/kg) three times per week, whereas the C, P, and DP groups were injected with citrate buffer. To observe the mineralization of the alveolar bone, the DP and DP + PTH groups were injected with calcein on days 10 and 27, and with alizarin red on day 20. Thirty days after ligation, histological findings and fluorescence labeling were analyzed in the furcations of the mandibular first molars. Sclerostin-positive osteocytes were assessed by immunohistochemical analyses. RESULTS: The DP groups had smaller areas of alveolar bone than the other groups, and the DP + PTH group had a larger alveolar bone area than the DP group. The DP group had less osteoid formation than the C group, whereas the DP + PTH had greater osteoid formation than the DP group. Fluorescence labeling results revealed that the DP + PTH group had more mineral deposition on the alveolar bone than the DP group. The DP + PTH group exhibited lower percentage of sclerostin-positive osteocytes in alveolar bone than the DP group. CONCLUSIONS: Intermittent PTH administration diminishes alveolar bone loss and sclerostin expression in osteocytes, but increases osteoid formation and mineralization, suggesting that intermittent PTH administration attenuates diabetes-aggravated alveolar bone loss by the induction of bone formation. PTH-induced bone formation may be related to the regulation of osteocytic sclerostin expression in type 1 diabetic rats with periodontitis.

Tumor necrosis factor-α antagonist diminishes osteocytic RANKL and sclerostin expression in diabetes rats with periodontitis.

Type 1 diabetes with periodontitis shows elevated TNF-α expression. Tumor necrosis factor (TNF)-α stimulates the expression of receptor activator of nuclear factor-κB ligand (RANKL) and sclerostin. The objective of this study was to determine the effect of TNF-α expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis using infliximab (IFX), a TNF-α antagonist. Rats were divided into two timepoint groups: day 3 and day 20. Each timepoint group was then divided into four subgroups: 1) control (C, n = 6 for each time point); 2) periodontitis (P, n = 6 for each time point); 3) diabetes with periodontitis (DP, n = 8 for each time point); and 4) diabetes with periodontitis treated with IFX (DP+IFX, n = 8 for each time point). To induce type 1 diabetes, rats were injected with streptozotocin (50 mg/kg dissolved in 0.1 M citrate buffer). Periodontitis was then induced by ligature of the mandibular first molars at day 7 after STZ injection (day 0). IFX was administered once for the 3 day group (on day 0) and twice for the 20 day group (on days 7 and 14). The DP group showed greater alveolar bone loss than the P group on day 20 (P = 0.020). On day 3, higher osteoclast formation and RANKL-positive osteocytes in P group (P = 0.000 and P = 0.011, respectively) and DP group (P = 0.006 and P = 0.017, respectively) than those in C group were observed. However, there was no significant difference in osteoclast formation or RANKL-positive osteocytes between P and DP groups. The DP+IFX group exhibited lower alveolar bone loss (P = 0.041), osteoclast formation (P = 0.019), and RANKL-positive osteocytes (P = 0.009) than that of the DP group. On day 20, DP group showed a lower osteoid area (P = 0.001) and more sclerostin-positive osteocytes (P = 0.000) than P group. On days 3 and 20, the DP+IFX group showed more osteoid area (P = 0.048 and 0.040, respectively) but lower sclerostin-positive osteocytes (both P = 0.000) than DP group. Taken together, these results suggest that TNF-α antagonist can diminish osteocytic RANKL/sclerostin expression and osteoclast formation, eventually recovering osteoid formation. Therefore, TNF-α might mediate alveolar bone loss via inducing expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis.

Dynamic Expansion and Contraction of cagA Copy Number in Helicobacter pylori Impact Development of Gastric Disease.

Infection with Helicobacter pylori is a major risk factor for development of gastric disease, including gastric cancer. Patients infected with H. pylori strains that express CagA are at even greater risk of gastric carcinoma. Given the importance of CagA, this report describes a new molecular mechanism by which the cagA copy number dynamically expands and contracts in H. pylori Analysis of strain PMSS1 revealed a heterogeneous population in terms of numbers of cagA copies; strains carried from zero to four copies of cagA that were arranged as direct repeats within the chromosome. Each of the multiple copies of cagA was expressed and encoded functional CagA; strains with more cagA repeats exhibited higher levels of CagA expression and increased levels of delivery and phosphorylation of CagA within host cells. This concomitantly resulted in more virulent phenotypes as measured by cell elongation and interleukin-8 (IL-8) induction. Sequence analysis of the repeat region revealed three cagA homologous areas (CHAs) within the cagA repeats. Of these, CHA-ud flanked each of the cagA copies and is likely important for the dynamic variation of cagA copy numbers. Analysis of a large panel of clinical isolates showed that 7.5% of H. pylori strains isolated in the United States harbored multiple cagA repeats, while none of the tested Korean isolates carried more than one copy of cagA Finally, H. pylori strains carrying multiple cagA copies were differentially associated with gastric disease. Thus, the dynamic expansion and contraction of cagA copy numbers may serve as a novel mechanism by which H. pylori modulates gastric disease development.IMPORTANCE Severity of H. pylori-associated disease is directly associated with carriage of the CagA toxin. Though the sequences of the CagA protein can differ across strains, previous analyses showed that virtually all H. pylori strains carry one or no copies of cagA This study showed that H. pylori can carry multiple tandem copies of cagA that can change dynamically. Isolates harboring more cagA copies produced more CagA, thus enhancing toxicity to host cells. Analysis of 314 H. pylori clinical strains isolated from patients in South Korea and the United States showed that 7.5% of clinical strains in the United States carried multiple cagA copies whereas none of the South Korean strains did. This study demonstrated a novel molecular mechanism by which H. pylori dynamically modulates cagA copy number, which affects CagA expression and activity and may impact downstream development of gastric disease.

Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway.

Helicobacter pylori is associated with hypergastrinemia, which has been linked to the development of gastric diseases. Although the molecular mechanism is not fully understood, H. pylori is known to modulate the Erk pathway for induction of gastrin expression. Herein we found that an epidermal growth factor (EGF) receptor kinase inhibitor significantly blocked H. pylori-induced gastrin promoter activity, suggesting involvement of EGF receptor ligands. Indeed, H. pylori induced mRNA expression of EGF family members such as amphiregulin, EGF, heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α. Of these, specific siRNA targeting of HB-EGF significantly blocked H. pylori-induced gastrin expression. Moreover, H. pylori induced HB-EGF ectodomain shedding, which we found to be a critical process for H. pylori-induced gastrin expression. Thus, we demonstrate a novel role for human mature HB-EGF in stimulating gastrin promoter activity during H. pylori infection. Further investigation using specific siRNAs targeting each isoform of Raf, Mek, and Erk elucidated that the mechanism underlying H. pylori-induced gastrin expression can be delineated as the sequential activation of HB-EGF, the EGF receptor, C-Raf, Mek1, and the Erk2 molecules in the MAPK pathway. Surprisingly, whereas Erk2 acts as a potent activator of gastrin expression, siRNA knockdown of Erk1 induced gastrin promoter activity, suggesting that Erk1 typically acts as a repressor of gastrin expression. Elucidation of the mechanism of gastrin modulation by HB-EGF-mediated EGF receptor transactivation should facilitate the development of therapeutic strategies against H. pylori-related hypergastrinemia and consequently gastric disease development, including gastric cancers.

Endothelin Regulates Porphyromonas gingivalis-Induced Production of Inflammatory Cytokines.

Periodontitis is a very common oral inflammatory disease that results in the destruction of supporting connective and osseous tissues of the teeth. Although the exact etiology is still unclear, Gram-negative bacteria, especially Porphyromonas gingivalis in subgingival pockets are thought to be one of the major etiologic agents of periodontitis. Endothelin (ET) is a family of three 21-amino acid peptides, ET-1, -2, and -3, that activate G protein-coupled receptors, ETA and ETB. Endothelin is involved in the occurrence and progression of various inflammatory diseases. Previous reports have shown that ET-1 and its receptors, ETA and ETB are expressed in the periodontal tissues and, that ET-1 levels in gingival crevicular fluid are increased in periodontitis patients. Moreover, P. gingivalis infection has been shown to induce the production of ET-1 along with other inflammatory cytokines. Despite these studies, however, the functional significance of endothelin in periodontitis is still largely unknown. In this study, we explored the cellular and molecular mechanisms of ET-1 action in periodontitis using human gingival epithelial cells (HGECs). ET-1 and ETA, but not ETB, were abundantly expressed in HGECs. Stimulation of HGECs with P. gingivalis or P. gingivalis lipopolysaccharide increased the expression of ET-1 and ETA suggesting the activation of the endothelin signaling pathway. Production of inflammatory cytokines, IL-1ß, TNFα, and IL-6, was significantly enhanced by exogenous ET-1 treatment, and this effect depended on the mitogen-activated protein kinases via intracellular Ca2+ increase, which resulted from the activation of the phospholipase C/inositol 1,4,5-trisphosphate pathway. The inhibition of the endothelin receptor-mediated signaling pathway with the dual receptor inhibitor, bosentan, partially ameliorated alveolar bone loss and immune cell infiltration. These results suggest that endothelin plays an important role in P. gingivalis-mediated periodontitis. Thus, endothelin antagonism may be a potential therapeutic approach for periodontitis treatment.

Helicobacter pylori outer membrane protein, HomC, shows geographic dependent polymorphism that is influenced by the Bab family.

The array of outer membrane proteins (OMPs) found in Helicobacter pylori provides a crucial component for persistent colonization within the gastric niche. Not only does H. pylori harbor a wide number of OMPs, but these OMPs often vary across strains; this likely contributes to immune evasion, adaptation during long term colonization, and potentially differential disease progression. Previous work from our group described OMP differences among the Bab family (babA, babB, and babC) and Hom family (homA and homB) from 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). In the current study, we expanded our investigation to include the less well characterized Hom family member, HomC.Overall, we identified and genotyped three homC variants: homC S , homC L , and homC M , in both populations. Similar to other polymorphic genes, the KH group showed less overall diversity, with 97.5% of strains harboring homC L . In contrast, a more heterogeneous profile was observed in strains derived from an American population; we found nearly equal distribution of homC S and homC L . Further analysis of the AH group identified associations between homC polymorphism and bab genotype; in AH strains, there was a significant association between homC L and carriage of babA at locus A. Since babA is an important virulence factor for the development of severe gastric disease, these data may suggest that homC polymorphism plays a role in H. pylori pathogenesis.

Licochalcone F alleviates glucose tolerance and chronic inflammation in diet-induced obese mice through Akt and p38 MAPK.

BACKGROUND & AIMS: Licochalcone (lico) F is a novel synthetic retrochalcone. In this study, we investigated the anti-inflammatory effects of lico F in vitro, and its effects on obesity-induced chronic inflammation, glucose intolerance, and fatty liver in vivo. METHODS: The inhibitory effects of lico F on TNFα-induced inflammation were investigated using NF-κB luciferase reporter assay and RT-PCR. Diet-induced obese mice were treated orally, once per day, with vehicle or lico F (10 mg/kg/day), for 3 weeks, and blood, liver, and adipose tissues were analyzed. RESULTS: Lico F inhibited TNFα-induced NF-κB activation and mRNA expression of TNFα, COX-2, IL-6, IL-1ß, and NOS2. In obese mice, lico F administration significantly alleviated glucose tolerance without changes in body weight gain and food intake. Lico F reduced adipocyte size and macrophage infiltration into white adipose tissue and improved hepatic lesions, by decreasing fat droplets and glycogen deposition. The mRNA expression levels of TNFα, MCP-1, and CD68 in white adipose tissue also decreased markedly. Moreover, lico F enhanced Akt signaling, but reduced p38 MAPK signaling in white adipose tissue. CONCLUSIONS: Lico F had anti-inflammatory effects and showed beneficial effects on glucose metabolism, which could be partially caused by activation of the Akt signal pathway and obesity-induced chronic inflammation, probably by downregulating p38 signal pathway. Moreover, lico F could be used as a potential novel therapeutic compound against type 2 diabetes and obesity-induced chronic inflammation without the deleterious effects of body weight gain and fatty liver.

Osteocytic Sclerostin Expression in Alveolar Bone in Rats With Diabetes Mellitus and Ligature-Induced Periodontitis.

BACKGROUND: Osteocytic sclerostin inhibits bone formation, and its expression is stimulated by tumor necrosis factor (TNF)-α. This study investigates sclerostin and TNF-α expression in rats with diabetes mellitus (DM) and periodontitis. METHODS: Rats were divided into control (C), periodontitis (P), and DM + periodontitis (DP) groups. After induction of DM by streptozotocin, periodontitis was induced by ligature. At day 0 (control) and at days 3 and 20 after induction of periodontitis, alveolar bone, osteoclasts, osteoid area, and TNF-α and sclerostin expression were evaluated. RESULTS: The distance between the cemento-enamel junction and the alveolar bone crest of the DP group was longer than that of the P group at day 20 after induction of periodontitis, but the number of osteoclasts was not different. Osteoid area decreased in both the P and DP groups by day 3, but whereas sustained osteoid suppression was observed in the DP group at day 20, osteoid formation was increased in the P group. The number of sclerostin-positive osteocytes increased in both groups at day 3, but the increased number of sclerostin-positive osteocytes was maintained only in the DP group through day 20. The number of TNF-α-positive cells increased more in the DP group than in the P group. CONCLUSIONS: Enhanced alveolar bone loss, suppressed bone formation, and prevalent TNF-α expression were characteristic of the DP group compared with the P group. Suppressed bone formation in the DP group was observed simultaneously with increased sclerostin and TNF-α expression. These results suggest that upregulated osteocytic sclerostin expression in periodontitis accompanied by DM may play a role in suppressed bone formation.

Receptor activator of nuclear factor-κB ligand and sclerostin expression in osteocytes of alveolar bone in rats with ligature-induced periodontitis.

BACKGROUND: Osteocytes are increasingly recognized as significant sources of osteoclast differentiation factor, receptor activator of nuclear factor-κB ligand (RANKL), and osteoblast differentiation inhibitory factor, sclerostin. In this study, RANKL and sclerostin expression of osteocytes is investigated in rats with ligature-induced periodontitis. METHODS: Rats were divided into control and periodontitis groups, and periodontitis was induced by ligature on the mandibular first molars. At 1, 3, 10, and 20 days after ligature, histologic analyses of alveolar bone (AB) and osteoid areas in the molar furcation were performed. The numbers of osteoclasts and RANKL- and sclerostin-positive osteocytes were estimated by tartrate-resistant acid phosphatase staining and immunohistochemistry, respectively. RESULTS: The AB area gradually decreased at day 10 after ligature and increased at day 20. The number of osteoclasts markedly increased at day 3 and then decreased. Conversely, osteoid formation was suppressed up to day 3 and then showed a remarkable increase above control level at day 20. The number of RANKL-positive osteocytes increased at days 1 and 3 and then decreased. Sclerostin-positive osteocytes markedly increased at days 3 and 10 but decreased below control level at day 20. CONCLUSIONS: These results show that AB loss is accompanied by enhanced osteoclast formation and suppressed osteoid formation. Osteocytes express RANKL when osteoclast formation increases, and they express sclerostin when osteoid formation is suppressed. Conversely, osteocytic sclerostin expression decreases when osteoid formation increases. These findings suggest that osteocytes may be important in AB loss via RANKL and sclerostin expression in periodontitis.

Wogonin ameliorates hyperglycemia and dyslipidemia via PPARα activation in db/db mice.

BACKGROUND & AIMS: Wogonin is a flavonoid extracted from the root of Scutellaria baicalensis Gerogi. We evaluated the therapeutic effects of wogonin using db/db mice. METHODS: Mice received wogonin or vehicle by oral gavage for 2 weeks. Blood glucose, insulin, and cholesterol levels were measured, and liver morphology was observed with histopathological analysis. The mRNA expression levels of PPARα, PPARγ, and adiponectin in the liver and white adipose tissue (WAT) were determined by real-time PCR. Immunoblotting for AMPK and PPARγ, and adipocyte differentiation were investigated in vitro using 3T3-L1 cells. A luciferase assay was used to measure PPARα and PPARγ binding activity. RESULTS: The wogonin group showed decreased weight gain without a change in food intake and improved glucose tolerance. Serum insulin and cholesterol levels in the wogonin group were significantly decreased compared to those in the control group. The wogonin group also showed less accumulation of lipid droplets and glycogen in the liver. PPARα and PPARγ expression levels in the liver and WAT and adiponectin expression level in WAT in the wogonin group were higher than those in the control group. In 3T3-L1 cells, wogonin was shown to stimulate AMPK activation in a dose-dependent manner. The presence of wogonin did not affect adipocyte differentiation or PPARγ protein level during adipogenesis. Notably, wogonin enhanced PPARα but not PPARγ transactivation. CONCLUSIONS: These indicate that wogonin may have beneficial effects on glucose and lipid metabolism related to enhanced PPARα and adiponectin expression via AMPK activation. Importantly, wogonin did not cause deleterious effects, such as weight gain and fatty liver. Wogonin might be a useful therapeutic agent to treat type 2 diabetes.

Effect of the interaction between periodontitis and type 1 diabetes mellitus on alveolar bone, mandibular condyle and tibia.

OBJECTIVE: This study examined the effect of the interaction between periodontitis and type 1 diabetes mellitus on alveolar bone, mandibular condyle and tibia in animal models. MATERIALS AND METHODS: Rats were divided into normal, periodontitis, diabetic and diabetic with periodontitis groups. After injection of streptozotocin to induce diabetes, periodontitis was induced by ligation of both lower-side first molars for 30 days. Alveolar bone loss and trabecular bone volume fraction (BVF) of the mandibular condyle and tibia were estimated via hematoxylin and eosin staining and micro-computed tomography, respectively. Osteoclastogenesis of bone marrow cells isolated from tibia and femur was assayed using tartrate-resistant acid phosphatase staining. RESULTS: The cemento-enamel junction to the alveolar bone crest distance and ratio of periodontal ligament area in the diabetic with periodontitis group were significantly increased compared to those of the periodontitis group. Mandibular condyle BVF did not differ among groups. The BVF of tibia in the diabetic and diabetic with periodontitis groups was lower than that of the normal and periodontitis groups. Osteoclastogenesis of bone marrow cells in the diabetic groups was higher than that in the non-diabetic groups. However, the BVF of tibia and osteoclastogenesis in the diabetic with periodontitis group were not significantly different than those in the diabetic group. CONCLUSIONS: Type 1 diabetes mellitus aggravates alveolar bone loss induced by periodontitis, but periodontitis does not alter the mandibular condyle and tibia bone loss induced by diabetes. Alveolar bone, mandibular condyle and tibia may have different responses to bone loss stimuli in the diabetic environment.