Epithelial Cells Secrete Interferon‐γ Which Suppresses Expression of Receptor Activator of Nuclear Factor Kappa‐B Ligand in Human Mandibular Osteoblast‐Like Cells

Background: Prostaglandin (PG)E₂ accumulates in inflamed periodontal tissue and induces receptor activator of nuclear factor kappa‐B ligand (RANKL)‐RANK‐osteoprotegerin (OPG) signaling associated with bone resorption. Although oral epithelial cells maintain tissue homeostasis, the role of these cells in RANKL regulation remains unknown. Methods: To mimic an inflamed condition, RANKL upregulation in human mandibular osteoblast‐like cells (HMOBs) were stimulated with PGE₂. Effect of recombinant human interferon (IFN)‐γ or epithelial‐derived IFN‐γ in constitutively released or Porphyromonas gingivalis lipopolysaccharide (PgLPS)‐stimulated epithelial supernatant was investigated in HMOBs. Some HMOBs were pretreated with an anti‐IFN‐γ antibody before PGE₂ stimulation. THP‐1 human monocytes and HMOBs were cocultured in a transwell system to investigate RANKL‐driven THP‐1 osteoclastic activity. Results: PGE₂ significantly increased RANKL messenger RNA (mRNA) and protein in HMOBs in a dose‐dependent manner, while OPG protein remained similar to baseline. Epithelial cells constitutively released IFN‐γ, which was substantially increased by PgLPS. HMOBs treated with epithelial supernatant or recombinant IFN‐γ, concurrently with PGE₂ stimulation, reduced RANKL, but not OPG, expression. In contrast, anti‐IFN‐γ antibody reversed the effect of epithelial mediators on RANKL expression. When cocultured with THP‐1, RANKL released by PGE₂‐stimulated HMOBs is adequate to drive THP‐1 differentiation as osteoclastogenic gene expression and bone resorption pit are increased. However, recombinant IFN‐γ, or IFN‐γ derived from oral epithelial cells, suppressed RANKL expression at both the mRNA and protein level, resulting in decreased THP‐1‐derived osteoclastic activity. Conclusion: Oral epithelial cells interact with HMOBs by releasing IFN‐γ to regulate RANKL expression and contribute to osteoclastogenesis.     

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.     

Gingival Crevicular Fluid,Serum Levels of Receptor Activator of Nuclear Factor‐κB Ligand,Osteoprotegerin, and Interleukin‐17 in Patients With Rheumatoid Arthritis and Osteoporosis and With Periodontal Disease

Background: This study is performed to evaluate gingival crevicular fluid (GCF) and serum levels of soluble receptor activator of nuclear factor‐κB ligand (sRANKL), interleukin (IL)‐17A, IL‐17E, IL‐17F, IL‐17A/F, and osteoprotegerin (OPG) in women with rheumatoid arthritis (RA), osteoporosis (OPR), and those who are systemically healthy (SH), all with periodontal disease. Methods: GCF and serum samples were obtained before any periodontal intervention from 17 women with RA, 19 with OPR, and 13 who were SH with periodontitis. Full‐mouth clinical periodontal measurements were recorded. sRANKL, OPG, and IL‐17 levels were determined by enzyme‐linked immunosorbent assay. Results: Clinical periodontal measurements were similar in the three study groups. Although the total amounts of GCF albumin, OPG, IL‐17A, and IL‐17A/F were similar in the study groups, there were statistically significant differences in GCF concentrations of sRANKL, OPG, IL‐17A, IL‐17E, IL‐17F, and IL‐17A/F. The sRANKL/OPG ratios were significantly higher in the RA group than in the OPR and SH groups (P <0.05). Serum sRANKL, sRANKL/OPG, and IL‐17A/IL‐17E ratios were significantly higher, whereas OPG concentrations were significantly lower in the RA group compared to other groups (P <0.05). Serum IL‐17A concentrations were significantly higher in the RA and OPR groups than in the SH group (P <0.05). Conclusion: Increased inflammatory mediator levels in patients with RA, despite the long‐term use of various anti‐inflammatory drugs, suggest that these patients may have a propensity to overproduce these inflammatory mediators.     

Response of Human Periodontal Ligament Cells to Baicalin

Background: Periodontitis is the most common cause of tooth loss in adults. Periodontal ligament cell (PLC)–based therapy is considered one of the most promising methods in periodontal tissue regeneration. The traditional Chinese medicine baicalin has been shown to possess antimicrobial and anti‐inflammatory activities and enhance cell proliferation and alkaline phosphatase activity. The aim of this study is to investigate the response of human PLCs (HPLCs) to baicalin. Methods: The effect of baicalin on cultured HPLC proliferation was measured with a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. The effect of baicalin on the expression of osteoprotegerin (OPG), receptor activator of nuclear factor‐κB ligand (RANKL), core binding factor α1 (Cbfα1), and osteocalcin (OC) was determined by quantitative real‐time polymerase chain reaction and immunodetection. Results: Baicalin at a concentration of 0.01 μg/mL promoted HPLC proliferation, upregulated OPG messenger RNA (mRNA) and protein expression, and downregulated RANKL mRNA and protein expression. In addition to reducing the RANKL/OPG expression ratio significantly, it also increased Cbfα1 and OC mRNA and protein expression. Conclusion: Baicalin showed multifaceted regulation of genes with important roles in tissue growth and differentiation, and thus it has the potential to be a promising candidate for HPLC‐based periodontal regeneration therapy.     

Activation of Receptor Activator of Nuclear Factor‐κB Ligand and Matrix Metalloproteinase Production in Periodontal Fibroblasts by Endothelin Signaling

Background: Periodontitis is a group of inflammatory diseases affecting the tissues supporting the teeth that will progressively cause the loss of alveolar bone and periodontal ligaments and eventually the dentition. Activation of osteoclast activity by receptor activator of nuclear factor‐κB ligand (RANKL) and released enzymes such as matrix metalloproteinases (MMPs) are among the factors involved in the breakdown of the periodontium. However, the mechanisms regulating their production in periodontitis are poorly understood. Endothelin signaling via the activation of the endothelin‐A receptor (EDNRA) by endothelin‐1 may play a role in the disease because the expression of the receptor and ligand is elevated in the periodontal tissues of patients with periodontitis. Methods: Cultured primary human periodontal fibroblasts were treated with 20 and 100 nM endothelin‐1 for 6 and 24 hours and then collected to assess MMP and RANKL production by immunoblotting. Inhibitors were used to identify the molecular pathways activated by EDNRA in these cells. Results: Endothelin‐1 stimulated the production of MMP1, MMP8, and RANKL in a dose‐ and time‐dependent manner; blocking EDNRA function with the antagonist TBC3214 inhibited the response, although EDNRA activation had no effects on osteoprotegerin production. These mechanistic studies indicate that EDNRA activates phospholipase C, which then 1) increases the MMP1 protein levels through activation of the extracellular signal‐regulated kinase mitogen‐activated protein kinase‐dependent pathway and 2) upregulates RANKL by a different pathway. Conclusion: These results suggest that EDNRA may function in the breakdown of the periodontal tissues associated with periodontitis by promoting the protein expression of MMPs and RANKL via the phospholipase C pathway.     

Gingival Crevicular Fluid Levels of Sclerostin,Osteoprotegerin, and Receptor Activator of Nuclear Factor‐κB Ligand in Periodontitis

Background: To investigate changes in the levels and relative ratios of sclerostin, osteoprotegerin (OPG), and receptor activator of nuclear factor‐κB ligand (RANKL) in the gingival crevicular fluid (GCF) of patients with periodontitis after non‐surgical periodontal treatment. Methods: Fifty‐four individuals (27 healthy controls and 27 patients with chronic periodontitis [CP]) were enrolled in the study. Periodontitis patients received non‐surgical periodontal therapy. GCF sampling and clinical periodontal parameters were assessed before and 6 weeks after therapy. Sclerostin, OPG, and RANKL levels were measured by enzyme‐linked immunosorbent assay, and their relative ratios were calculated. Results: Total amounts and concentrations of sclerostin were significantly higher in patients with CP than in healthy individuals (P <0.025) and decreased after treatment (P <0.05). The RANKL/OPG ratio was significantly lower in healthy individuals than in patients with periodontitis before and after treatment (P <0.025), but no significant difference was observed in patients with periodontitis after treatment (P >0.05). The sclerostin/OPG and sclerostin/RANKL ratios were significantly lower in healthy individuals than in patients with periodontitis before and after treatment (P <0.025) and decreased in patients with periodontitis after treatment (P <0.05). Conclusions: The GCF sclerostin level may be more reliable than the RANKL/OPG ratio as a diagnostic and prognostic marker of periodontal disease and treatment outcome. Regulation of sclerostin levels may aid the development of new therapeutic strategies for the treatment of periodontal disease.     

The Influences of Periodontal Status and Periodontal Pathogen Quantity on Salivary 8‐Hydroxydeoxyguanosine and Interleukin‐17 Levels

Background: Periodontitis is a biofilm‐initiated disease that is characterized by elevated inflammatory status. 8‐Hydroxydeoxyguanosine (8‐OHdG) and interleukin (IL)‐17 are highly associated with inflammation and bone resorption and therefore are regarded as potential biomarkers for periodontitis. In this study, the associations between salivary 8‐OHdG and IL‐17 levels and clinical and microbial parameters before and after non‐surgical treatment are investigated. Methods: Forty‐five patients with chronic periodontitis (CP) and 47 periodontally healthy volunteers were recruited for the study. Clinical parameters, including the probing depth (PD), clinical attachment level (CAL), sulcular bleeding index, and simplified oral hygiene index (OHI‐S), were examined for each participant. Microbial parameters including the quantities of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola in the subgingival plaque and saliva were determined by real‐time polymerase chain reaction at baseline and 1 and 3 months after the non‐surgical treatment. Salivary 8‐OHdG and IL‐17 levels were detected by enzyme‐linked immunosorbent assays. Results: Compared with healthy volunteers, CP group patients had significantly higher salivary 8‐OHdG and IL‐17 levels at baseline. Baseline salivary 8‐OHdG and IL‐17 levels were positively correlated with all clinical parameters as well as the quantities of T. forsythia and T. denticola. After non‐surgical treatment, baseline levels of salivary 8‐OHdG and IL‐17 were reduced significantly at both the 1‐ and 3‐month follow‐ups. The hierarchical linear model revealed that variations in the PD, CAL, and OHI‐S had significant positive effects on variation in the salivary 8‐OHdG level. However, variations in the PD; quantity of T. forsythia in the subgingival plaque; and quantities of P. gingivalis, T. forsythia, and T. denticola in saliva were associated significantly with variation in the salivary IL‐17 levels. Conclusions: There was a strong association between salivary 8‐OHdG and IL‐17 levels and periodontitis. Variation in the salivary 8‐OHdG level was correlated with variations in the clinical parameters, whereas variation in the IL‐17 level was correlated with variation in the microbial parameters.     

Endothelin‐1 Stimulates Proinflammatory Cytokine Expression in Human Periodontal Ligament Cells via Mitogen‐Activated Protein Kinase Pathway

Background: Endothelin‐1 (ET‐1) is a 21–amino acid peptide with multifunctional regulation. Initial research indicated that ET‐1 is related to the inflammatory pathogenesis of periodontitis and involved in the regulation of cytokines, but the mechanisms involved remain unclear. The primary aim of this study is to investigate how ET‐1 affects proinflammatory cytokine expression in human periodontal ligament (hPDL) cells. Methods: hPDL cells were obtained from both healthy (H)‐ and periodontitis (P)‐affected periodontal tissues. H‐hPDL and P‐hPDL cells were treated with ET‐1 (1, 10, and 100 nM) for 12, 24, and 48 hours. The untreated cells served as a control. To confirm the specificity of the ET‐1 effects, 100 nM of the specific endothelin A (ET_A) receptor antagonist BQ123 and 100 nM of the specific ET_B receptor antagonist BQ788, as negative control, were used. To examine the signaling pathways and molecular mechanisms involved in ET‐1–mediated cytokine expression, H‐hPDL and P‐hPDL cells were pretreated with specific inhibitors for extracellular signal–regulated kinase (ERK1/2) (PD98059), c‐Jun N‐terminal kinase (SP600125), and p38 kinase (SB203580) for 1 hour before 100 nM ET‐1 stimulation. Tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, and IL‐6 messenger RNA (mRNA) and protein levels were evaluated by quantitative real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: ET‐1 dose‐ and time‐dependently induced the production of proinflammatory cytokines TNF‐α, IL‐1β, and IL‐6 by H‐hPDL and P‐hPDL cells at both mRNA and protein levels. However, ET_A and ET_B receptor antagonists inhibited the stimulatory effects of ET‐1 on inflammatory cytokine expression in H‐hPDL and P‐hPDL cells. Furthermore, inhibitors of the mitogen‐activated protein kinases (MAPKs) significantly reduced ET‐1–stimulated TNF‐α, IL‐1β, and IL‐6 expression in H‐hPDL and P‐hPDL cells. Conclusion: ET‐1 may be involved in the inflammatory process of periodontitis, at least in part, by stimulating proinflammatory cytokine production via the MAPK pathway in hPDL cells.     

Calcium Phosphate Particles Induce Interleukin‐8 Expression in a Human Gingival Epithelial Cell Line via the Nuclear Factor‐κB Signaling Pathway

Background: Dental calculus is calcified plaque composed primarily of calcium phosphate mineral salts, and there is a clear association between the presence of calculus and the initiation/progression of periodontitis. However, it is still inconclusive whether dental calculus can be a direct causative factor. The authors examined the effect of nano/microsized calcium phosphate particles, which may be generated in the process of early precipitation and/or dissolution of calcium phosphate mineral, on the expression of interleukin (IL)‐8 in human gingival epithelial cells. Methods: Primary human gingival epithelial cells and/or a human gingival carcinoma cell line (Ca9‐22) were stimulated with calcium phosphate particles. Gene and protein levels were assessed by real‐time polymerase chain reaction analysis and enzyme‐linked immunosorbent assay, respectively. The activity of nuclear factor (NF)‐κB signaling was measured by an immunofluorescence assay to evaluate NF‐κB p65 nuclear translocation. Results: The results show that nano/microsized particles stimulate IL‐8 expression in human gingival epithelial cells at gene and protein levels. The activity to induce IL‐8 expression depends on the particle size: particles with a diameter of 200 nm are more effective than those of 40‐nm and 5‐μm diameters. Calcium phosphate particles (diameter 200 nm) stimulated NF‐κB activity. Pretreatment with BMS‐345541, an NF‐κB signaling inhibitor, inhibited the particle‐mediated IL‐8 gene induction, suggesting a requirement for the NF‐κB signaling pathway. Conclusion: These findings suggest that calcium phosphate particles, which may be related to calculus development, may act as a direct causative factor in the pathogenesis of gingival epithelium.     

Assessment of Interleukin‐6 Receptor Inhibition Therapy on Periodontal Condition in Patients With Rheumatoid Arthritis and Chronic Periodontitis

Background: Overproduction of interleukin (IL)‐6 may play a pathologic role in rheumatoid arthritis (RA) and chronic periodontitis (CP). The present study assesses IL‐6 receptor (IL‐6R) inhibition therapy on the periodontal condition of patients with RA and CP. Methods: The study participants were 28 patients with RA and CP during treatment with IL‐6R inhibitor, and 27 patients with RA and CP during treatment without IL‐6R inhibitor. Periodontal and rheumatologic parameters and serum levels of cytokine and inflammatory markers and immunoglobulin G against periodontopathic bacteria were examined after medication with IL‐6R inhibitor for 20.3 months on average (T1) and again 8 weeks later (T2). Results: No differences were observed between the groups in any parameter values at T1, except for serum IL‐6 levels. The anti–IL‐6R group showed a significantly greater decrease in gingival index, bleeding on probing (BOP), probing depth (PD), clinical attachment level (CAL), and serum levels of IL‐6 and matrix metalloproteinase (MMP)‐3 from T1 to T2 than the control group (P <0.05). A significant correlation was found between changes in serum anticyclic citrullinated peptide levels and those in PD and CAL in the anti–IL‐6R group (P <0.05), whereas both groups exhibited a significant association between changes in serum MMP‐3 levels and those in BOP (P <0.05). Conclusion: Changes in periodontal and serum parameter values were different between the patients with RA and CP during treatment with and without IL‐6R inhibitor.     

Evaluation of Receptor Activator of Nuclear Factor Kappa B Ligand,Osteoprotegerin, Osteopontin,and Tumor Necrosis Factor Alpha on Chronic Apical Periodontitis in Smokers

IntroductionSmoking can be considered a risk factor for chronic apical periodontitis (CAP). This study compared the immunoexpression of biomarkers receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), osteopontin (OPN), and tumor necrosis factor alpha (TNF-α) in CAP in smokers and nonsmokers.MethodsTwelve smokers and 12 nonsmokers diagnosed with CAP and indicated for tooth extraction were selected. Exclusion factors were teeth with a diagnosis of root fracture, previous endodontic treatment, or endoperiodontal injury, in addition to individuals with systemic diseases, under 18 years of age, users of anti-inflammatory and/or antibiotics in the last 3 months, and drug users. Specimens were processed for histopathologic and immunohistochemical analysis.ResultsQualitative analysis of RANKL expression showed 66.66% weak/moderate and 33.33% strong in smokers and 100% weak/moderate in nonsmokers. OPG and OPN expressions were 100% negative to focal in the smoker group and 50% negative to focal and 50% weak/moderate in the nonsmoker group. TNF-α was 25% negative to focal and 75% weak/moderate in the smoker group and 33.33% negative to focal and 66.66% weak/moderate in the nonsmoker group. Quantitative analysis of the data using the Mann-Whitney U test showed that there was a significant difference in the immunoexpression of RANKL (P < .05), OPG (P < .05), and OPN (P < .05), but there was no statistical difference in the immunoexpression of TNF-α (P > .05) between the 2 groups.ConclusionsThese findings suggest that smoking is capable of altering the inflammatory response, influencing the evolution of CAP.     

Tumor Necrosis Factor‐α Induces Matrix Metalloproteinases‐3, ‐10, and ‐13 in Human Periodontal Ligament Cells

Background: Various biologic mediators, including matrix metalloproteinases (MMPs), that are implicated in periodontal tissue breakdown can be induced by cytokines. MMPs are known to degrade periodontal ligament attachment, and bone matrix proteins and tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of MMPs. The aim of this study is to investigate the effect of tumor necrosis factor (TNF)‐α on the expression of MMPs in human periodontal ligament (PDL) cells in vitro and establish which MMPs are expressed specifically in response to that stimulus. Methods: Cultured PDL cells were stimulated with TNF‐α and analyzed with an MMP antibody array. Real‐time polymerase chain reaction (PCR), enzyme‐linked immunosorbent assay (ELISA), and western blot with cell lysate and zymography were used to measure messenger RNA (mRNA) and protein levels of MMP‐3, ‐10, and ‐13. To examine TNF receptor (TNFR) expression, PDL cells were examined by flow cytometry, and expression of MMP‐3, ‐10, and ‐13 was observed after blocking the TNFR with an antagonist. Results from real‐time PCR, ELISA, and western blot were analyzed by paired t test. Results: The antibody array showed that the protein most strongly upregulated by TNF‐α stimulation was MMP‐3, followed by MMP‐13 and MMP‐10. The TNF‐α receptor blocker specifically inhibited expression of MMP‐3 and ‐13. In addition, TNF‐α increased levels of MMP mRNAs in MMP‐3, ‐13, and ‐10 (in decreasing order). However, ELISAs showed that MMP‐13 was the most upregulated protein, followed by MMP‐10 and MMP‐3. Western blotting indicated that TNF‐α increased MMP‐3 and ‐13 levels but had no significant effect on the level of MMP‐10, and zymography showed that TNF‐α increased the activities of all forms of MMP‐3 and ‐13, but MMP‐10 was not detected. Flow cytometry demonstrated that the majority of PDL cells expressed TNFR1. Conclusions: TNF‐α (10 ng/mL) upregulates levels of MMP‐3, ‐10, and ‐13 in human PDL cells. These results suggest that these proteins play an important role in the inflammation of PDLs.     

The immunopathogenic and immunomodulatory effects of interleukin‐12 in periodontal disease

Interleukin 12 (IL‐12) is an inflammatory cytokine that promotes the response of the immune system. This cytokine has been implicated as a potent stimulator of several diseases characterized by inflammatory‐induced bone destruction, such as rheumatoid arthritis and periodontitis. Yet, the exact role of IL‐12 in the development and progress of periodontitis has not been clarified. Several studies have demonstrated a positive correlation between the level of IL‐12 and the severity of periodontal destruction. Deletion of IL‐12 in mice with periodontitis significantly suppressed the level of bone destruction. Interestingly, next to a role in modulating the pathogenesis, IL‐12 also has immunological‐regulatory properties. This cytokine induces expression of immunosuppressive molecules, such as indoleamine‐pyrrole 2,3‐dioxygenase (IDO). Thus, these findings suggest both negative and positive influences of IL‐12 in periodontal disease. It is currently proposed that the diversity of action of cytokines is a molecular key which regulates biological development and homeostasis. Accordingly, the actions of IL‐12 might be one of the mechanisms that regulate homeostasis of periodontal tissue during and following inflammation. Therefore, this article aims to review both destructive and protective functionalities of IL‐12 with an emphasis on periodontal disease.     

The Effects of Initial Periodontal Therapy on the Serum Receptor Activator of Nuclear Factor‐κβ Ligand/Osteoprotegerin System in Patients With Type 2 Diabetes Mellitus and Periodontitis

Background: The aim of the present study is to evaluate the serum receptor activator of nuclear factor‐κβ ligand (RANKL)/osteoprotegerin (OPG) system in patients with chronic periodontitis (CP) and type 2 diabetes mellitus (T2DM) and its changes after periodontal intervention. Methods: Thirty‐five patients with CP + T2DM, 35 systemically healthy patients with CP, and 35 healthy controls were enrolled, and serum levels of RANKL and OPG were measured at baseline. Then the CP + T2DM group was divided into a well‐controlled subgroup and a poorly controlled subgroup according to their hemoglobin A1c (HbA1c), and initial periodontal therapy was performed. After 3 months, patients in both subgroups were recalled, and serum RANKL and OPG levels were tested again and compared with the baseline. Results: At baseline, serum levels of OPG in the T2DM + CP group were much lower than in the CP group and healthy controls (197.41 ± 57.05 pg/mL versus 232.60 ± 70.85 pg/mL [CP group] or 244.96 ± 85.13 pg/mL [healthy controls], P <0.05), whereas their RANKL levels were much higher than in the other two groups (324.35 ± 87.62 pg/mL versus 284.52 ± 90.35 pg/mL [CP group] or 163.01 ± 45.24 pg/mL [healthy control], P <0.05), as was the RANKL/OPG (R/O) ratio (1.68 ± 0.33 versus 1.26 ± 0.35 [CP group] or 0.72 ± 0.25 [healthy control], P <0.001). Serum levels of OPG in both disease groups had significant negative correlations with HbA1C, and serum levels of RANKL in all participants had significant positive correlations with periodontal parameters. After periodontal intervention, both the well‐controlled and poorly controlled subgroups exhibited significant increases in OPG and decreases in RANKL in serum, and the R/O ratio was also notably reduced. Additionally, the poorly controlled subgroup exhibited a greater reduction in HbA1c and a greater increase in OPG than the well‐controlled subgroup. Conclusions: The changing trend in the serum RANKL/OPG system in patients with T2DM + CP was similar to that seen in CP patients and may be even more pronounced. Periodontal intervention effectively improved glucose metabolism and changed the serum RANKL/OPG system regardless of whether patients’ HbA1c was well‐controlled or poorly controlled over the 3‐month observation period.     

The Proteasome Inhibitor Bortezomib Inhibits Inflammatory Response of Periodontal Ligament Cells and Ameliorates Experimental Periodontitis in Rats

Background: Periodontitis is a chronic inflammatory disease initiated by bacteria and their virulence factors. Bortezomib (BTZ) is the first proteasome inhibitor for clinical treatment of malignancies. Its anticancer activity is accompanied by an anti‐inflammatory effect. However, there are few reports about its anti‐inflammatory effect and underlying mechanism in periodontal disease, especially on human periodontal ligament cells (hPDLCs), which are considered a promising cell‐based therapy for treating periodontitis. Methods: hPDLCs were treated with lipopolysaccharide (LPS) and pretreated with BTZ. mRNA and protein levels of tumor necrosis factor (TNF)‐alpha, interleukin (IL)‐1β, IL‐6, and IL‐8 were determined. The anti‐inflammatory mechanism of BTZ was studied. Further, experimental rat periodontitis was induced with ligature and LPS injection, and simultaneously and locally treated with BTZ (three injections/week). Four weeks after treatment, microcomputed tomography, immunohistochemical, and histopathologic analyses were performed. Results: Bortezomib administration at safe concentrations (≤1 nM) inhibited production of proinflammatory cytokines in LPS‐stimulated hPDLCs via nuclear factor (NF)‐kappa B, p38/extracellular signal‐regulated kinase, and mitogen‐activated protein kinase/activator protein‐1 pathways. Moreover, in the LPS and ligature‐induced periodontitis rat model, BTZ suppressed expression of TNF‐α, IL‐1β, IL‐6, and IL‐8, reduced the ratio of receptor activator of NF‐κB ligand/osteoprotegerin, and prevented alveolar bone absorption. Conclusion: These findings demonstrate the anti‐inflammatory activity of BTZ against periodontal inflammatory response and present BTZ as a promising therapy for periodontal disease.     

Effect of Curcumin on Systemic T Helper 17 Cell Response; Gingival Expressions of Interleukin‐17 and Retinoic Acid Receptor‐Related Orphan Receptor γt; and Alveolar Bone Loss in Experimental Periodontitis

Background: Curcumin has anti‐inflammatory and antioxidant effects and is reported to have many biologic activities. The current study examines effect of curcumin on: 1) systemic T helper 17 (Th17) cell response; 2) gingival expressions of interleukin (IL)‐17 and retinoic acid receptor‐related orphan receptor (ROR) γt; and 3) alveolar bone loss (ABL) in experimental periodontitis. Methods: Thirty‐eight male albino Wistar rats were divided into four groups: 1) group 1 = periodontitis; 2) group 2 = periodontitis with curcumin treatment; 3) group 3 = periodontally healthy with curcumin treatment; and 4) group 4 = periodontally healthy. Curcumin was administered via oral gavage (30 mg/kg/d) for 15 days. After sacrifice via exsanguination, the following serum levels were determined using enzyme‐linked immunosorbent assay: 1) IL‐1β; 2) IL‐6; 3) IL‐17A; 4) IL‐23; and 5) transforming growth factor‐ β. Morphometric evaluation of ABL was conducted and expression levels of IL‐17 and RORγt in gingival tissues were evaluated immunohistochemically. Results: Group 2 had significantly lower ABL than group 1 (P <0.0125). Highest expression levels of IL‐17 and RORγt were observed in group 1 and were significantly higher than those in all other groups (P <0.0125). The only serum biochemical parameter significantly different among groups was level of IL‐23 (P <0.05). Serum IL‐23 levels were higher in groups 1 and 2 than groups 3 and 4 (P <0.0125); however, they were not significantly different for groups 1 and 2 (P >0.0125). Conclusion: Curcumin seems to be a promising host modulatory agent in periodontal disease pathogenesis regarding IL‐17/IL‐23 axis, with a decreasing effect on ABL and gingival expressions of IL‐17 and RORγt.     

Dry Extract of Matricaria recutita L. (Chamomile) Prevents Ligature‐Induced Alveolar Bone Resorption in Rats via Inhibition of Tumor Necrosis Factor‐α and Interleukin‐1β

Background: Matricaria recutita L. (chamomile) has demonstrated anti‐inflammatory activity. Accordingly, the ability of the Matricaria recutita extract (MRE) to inhibit proinflammatory cytokines and its influence on alveolar bone resorption (ABR) in rats. Methods: Wistar rats were subjected to ABR by ligature with nylon thread in the second upper‐left molar, with contralateral hemiarcade as control. Rats received polysorbate TW₈₀ (vehicle) or MRE (10, 30, and 90 mg/kg) 1 hour before ligature and daily until day 11. The periodontium was analyzed by macroscopy, histometry, histopathology, and immunohistochemistry for the receptor activator of nuclear factor‐kappa B ligand (RANKL), osteoprotegerin (OPG), and tartrate‐resistant acid phosphatase (TRAP). The gingival tissue was used to quantify the myeloperoxidase (MPO) activity and tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β levels by enzyme‐linked immunosorbent assay. Blood samples were collected to evaluate bone‐specific alkaline phosphatase (BALP), leukogram, and dosages of aspartate and alanine transaminases, urea, and creatinine. Aspects of liver, kidneys, spleen, and body mass variations were also evaluated. Results: The 11 days of ligature induced bone resorption, low levels of BALP, leukocyte infiltration; increase of MPO, TNF‐α, and IL‐1β; immunostaining increase for RANKL and TRAP; reduction of OPG and leukocytosis, which were significantly prevented by MRE, except for the low levels of BALP and the leukocytosis. Additionally, MRE did not alter organs or body weights of rats. Conclusion: MRE prevented the inflammation and ABR by reducing TNF‐α and IL‐1β, preventing the osteoclast activation via the RANKL–OPG axis, without interfering with bone anabolism.     

Cimetidine Reduces Interleukin‐6, Matrix Metalloproteinases‐1 and ‐9 Immunoexpression in the Gingival Mucosa of Rat Molars With Induced Periodontal Disease

Background: Histamine seems to act, via H₂ receptor, on inflammatory processes by stimulating interleukin (IL)‐6 and matrix metalloproteinase (MMP) release. As cimetidine is an H₂ receptor antagonist, the authors hypothesize that this antiulcer drug reduces IL‐6, MMP‐1, and MMP‐9 immunoexpression in gingiva with induced periodontal disease (PD). To confirm a possible modulatory role of IL‐6 on MMPs, the relationship between IL‐6/MMP‐1 and IL‐6/MMP‐9 immunoexpression was evaluated. Methods: Forty‐six male rats were distributed into the cimetidine group (CimG: received daily intraperitoneal injections of 100 mg/kg of body weight of cimetidine) or the saline group (SG). PD was induced by cotton ligature around the maxillary left first molars (PDSG and PDCimG). The right molars were used as controls (SG and CimG). After 7, 15, 30, and 50 days, maxillary fragments were processed for paraffin embedding or for transmission electron microscopy. Tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in the alveolar process surface and number of IL‐6, MMP‐1, and MMP‐9‐immunolabeled cells in the gingival mucosa were quantified. Statistical analyses were performed (P ≤0.05). Results: In PDSG and PDCimG, gingival mucosa exhibited few collagen fibers among numerous inflammatory cells. In PDCimG, the number of TRAP‐positive osteoclasts and IL‐6, MMP‐1, and MMP‐9‐immunolabeled cells was significantly lower than in PDSG at all periods. A positive correlation between IL‐6/MMP‐1 and IL‐6/MMP‐9 was detected in PDSG and PDCimG. Conclusion: Cimetidine decreases bone loss through reduction of osteoclast number and induces reduction of IL‐6, MMP‐1, and MMP‐9 immunoexpression, reinforcing the idea that the beneficial effect of cimetidine in PD may be due to reduction of IL‐6 immunolabeling in the inflamed gingival mucosa.