MicroRNA-126 Regulates Inflammatory Cytokine Secretion in Human Gingival Fibroblasts Under High Glucose via Targeting Tumor Necrosis Factor Receptor Associated Factor 6.

BACKGROUND: MicroRNAs (miRs) play a crucial role in inflammatory diseases, including periodontitis. Meanwhile, miRs act as biomarkers for predicting diabetes mellitus (DM). However, the regulatory mechanism of miR-126 on development of periodontitis in patients with DM still remains unclear. METHODS: Human gingival fibroblasts were cultured with low (5.5 mmol/L), medium (15 mmol/L), and high (25 mmol/L) glucose, respectively. Expressions of miR-126, tumor necrosis factor (TNF) receptor associated factor (TRAF) 6, and related cytokines were analyzed by real-time polymerase chain reaction (PCR). After transfection with miR-126 mimic, PCR and western blot were performed to detect level of TRAF6, and luciferase reporter assay confirmed if TRAF6 is the direct target of miR-126. Production of cytokines was measured using enzyme-linked immunosorbent assay. RESULTS: Increased glucose significantly suppressed miR-126 expression in human gingival fibroblasts (P <0.05). Also, high glucose increased TRAF6, interleukin (IL)-6, TNF-α, and chemical chemokine ligand (CCL) 2 levels, whereas it decreased IL-10 level. MiR-126 mimic significantly decreased TRAF6 mRNA and protein levels under high glucose (P <0.05). Also, miR-126 directly targeted TRAF6 through binding to its 3' untranslated region in human gingival fibroblasts. Overexpression of miR-126 significantly abrogated high glucose-induced secretion of proinflammatory cytokines such as IL-6, TNF-α, and CCL2 and promoted production of IL-10. CONCLUSION: These data suggest that miR-126 inhibits inflammation of human gingival fibroblasts under high glucose through targeting TRAF6, which may be a potential therapeutic target for periodontitis concomitant with DM.

The negative feedback regulation of microRNA-146a in human periodontal ligament cells after Porphyromonas gingivalis lipopolysaccharide stimulation.

OBJECTIVE: Toll-like receptors (TLRs) pathway has been demonstrated to play an important role in periodontitis. However, the regulatory mechanism of microRNAs (miRNAs) on TLRs pathway is still unclear. Hence, this study is to explore the function of miRNA-146a in inflammatory reaction induced by Porphyromonas gingivalis lipopolysaccharide (LPS) in human periodontal ligament cells (hPDLCs). METHODS: Cells were treated with 1 or 10 µg/ml P. gingivalis LPS. The expression of TLR2, TLR4 and miRNA-146a were measured by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to detect nuclear factor (NF)-κ B p65 nuclear activity, interleukin-1ß (IL-1ß), IL-6, IL-8 and tumor necrosis factor-α (TNF-α). To examine the underlying mechanisms, cells were exposed to anti-TLR2/4 mAb or miRNA-146a inhibitor/mimic and evaluated by real-time PCR and ELISA. RESULTS: 10 µg/ml P. gingivalis LPS increased the expressions of TLR2 (3.79 ± 0.31), TLR4 (2.21 ± 0.31), and miRNA-146a (4.91 ± 0.87), NF-κ B p65 nuclear activity (6.51 ± 0.77 fold) (p < 0.05). 1 µg/ml P. gingivalis LPS induced TLR2 (3.05 ± 0.23), miRNA-146a (3.66 ± 0.83) and NF-κ B p65 nuclear activity (4.06 ± 0.78 fold) (p < 0.05), except TLR4 (1.11 ± 0.30, p > 0.05). Also, cytokines production increased (p < 0.05). The up-regulation of miRNA-146a could be blocked by anti-TLR2/4 mAb (p < 0.05). After the blockage of miRNA-146a, TLR2, TLR4, NF-κ B p65 nuclear activity and proinflammatory cytokines increased. However, after application of miRNA-146a mimic, the levels of these indexes decreased obviously (p < 0.05). CONCLUSION: MiRNA-146a functions as a negative feedback regulator via down-regulating proinflammatory cytokine secretion and blocking TLRs signaling pathway in hPDLCs after P. gingivalis LPS stimulation.

High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts.

Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p<0.05). Also, high glucose increased nuclear factor kappa B (NF-κB) p65 nuclear activity, tumor necrosis factor-α (TNF-α) and interleukin-lß (IL-1ß) levels. Protein kinase C (PKC)-α and δ knockdown with siRNA significantly decreased TLR2 and NF-κB p65 expression (p<0.05), whereas inhibition of PKC-ß had no effect on TLR2 and NF-κB p65 under high glucose (p<0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-κB expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-α and IL-1ß secretion via inducing TLR2 through PKC-α and PKC-δ in human gingival fibroblasts.

[Corrosion resistance of a new Ti-12.5Zr-3Nb-2.5Sn alloy].

OBJECTIVE: to evaluate the corrosion resistance of a new titanium alloy for dental restoration in artificial saliva. METHODS: in simulated oral environment, the electrochemical behavior of a new titanium alloy (Ti-12.5Zr-3Nb-2.5Sn) for dental restoration based on the d-electron alloy design method with high elastic modulus, high mechanical and good biological safety properties was investigated together with that of Ti-6Al-4V and TA2 as control groups. The anodic polarization curve and polarization resistance of these alloys were analyzed and the element release of Ti-12.5Zr-3Nb-2.5Sn and Ti-6Al-4V alloy after immersion in artificial saliva for 1, 2, 3, 5, 7, 15 d were measured. RESULTS: polarization curve indicated that Ti-6Al-4V alloy had lower breakdown potential (0.8 V) than Ti-12.5Zr-3Nb-2.5Sn alloy did (> 2.5 V). Ti-6Al-4V alloy showed higher passivation current density (1.45 × 10(-4) - 1.09 × 10(-3) A/cm(2)) than Ti-12.5Zr-3Nb-2.5Sn alloy (3.32 × 10(-6) - 3.46 × 10(-5) A/cm(2)) and TA2(5.03 × 10(-6) - 2.65 × 10(-5) A/cm(2)) did. Polarization resistance results showed that polarization resistance volume of TA2, Ti-12.5Zr-3Nb-2.5Sn alloy and Ti-6Al-4V alloy were 371.0, 252.0, and 60.1 kΩ×cm(2) respectively. With the increasing of dipping time in artificial saliva, the ion release of Ti-12.5Zr-3Nb-2.5Sn alloy and Ti-6Al-4V alloy increased to different degrees. Ti-6Al-4V alloy always showed more ion release than Ti-12.5Zr-3Nb-2.5Sn alloy did in the experiment. CONCLUSIONS: data from this study indicated that Ti-12.5Zr-3Nb-2.5Sn alloy, as a dental restoration material, had good corrosion resistance in artificial saliva.

Study of the surface wear resistance and biological properties of the Ti-Zr-Nb-Sn alloy for dental restoration.

A new titanium alloy (Ti-12.5Zr-3Nb-2.5Sn) was developed to meet the needs of clinical requirements for medical titanium alloys and improve the properties of existing titanium alloys. The as-prepared alloy was solution treated at 500 °C for 3 h in vacuum followed by water quenching. Tensile, wear and hardness tests were carried out to examine the mechanical properties of the Ti-Zr-Nb-Sn alloy. Oral mucous membrane irritation test was performed to evaluate the surface biological properties of the Ti-Zr-Nb-Sn alloy. The results suggested that the surface hardness and wear-resistant properties of the Ti-12.5Zr-3Nb-2.5Sn alloy were superior to commercially pure Ti. The oral mucous irritation test showed that all samples had no mucous membrane irritation. It indicates that Ti-12.5Zr-3Nb-2.5Sn has large potential to be used as dental restoration material.

Role of chloride formed on anodized titanium surfaces against an oral microorganism.

A modified titanium surface, anodized after being discharged in electrolytes, provides antibacterial activity against oral bacteria as well as osteoconductivity. However, the mechanism of this antibacterial effect against oral bacteria is still unclear. This study aims to investigate whether it is the chloride or the hydrophilicity properties of the anodized titanium, which is effective against the oral bacteria. Titanium plates are anodized in various electrolytes with or without chloride and are characterized. Then the survival of Streptococcus mutans on each specimen is evaluated. The results demonstrate that the peroxidation effects of HClO generated from the TiCl(3) formed on the titanium surface anodized in various chloride solutions efficiently killed adherent S. mutans on the surface whereas the presence of hydrophilicity alone do not demonstrate antibacterial activity. This method of anodizing titanium surface in a chloride solution may provide a novel strategy for use in orthopedic or dental implant systems.