Sophoraflavanone G prevents Streptococcus mutans surface antigen I/II-induced production of NO and PGE2 by inhibiting MAPK-mediated pathways in RAW 264.7 macrophages

BackgroundSophora flavescens AITON (Leguminosae) is a typical traditional Korean medical herb considered to exhibit antibacterial, anti-inflammatory, and antipyretic effects, and is also used for the treatment of skin and mucosal ulcers, sores, diarrhea, gastrointestinal hemorrhage, arrhythmia, and eczema.Objective and designThis study examined the inhibitory effects of sophoraflavanone G (SF) of S. flavescens on the bacterial fibrillar protein, Antigen I/II (AgI/II)-N recombinant protein isolated from Streptococcus mutans(rAg I/II)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE₂). The investigation was focused on whether SF could inhibit the production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin (PG) E2 as well as the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-a, interleukin (IL)-6, nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) in rAgI/II-stimulated RAW 264.7 cells using Griess reagent, Enzyme linked immunosorbent assay (ELISA), and Western blotting analysis.ResultsSG significantly inhibited the production of NO and PGE₂ and pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor α in Ag I/II-N-stimulated RAW264.7 cells, which were mediated by the down-regulation of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. The SF inhibited the phosphorylation of IκB-α, nuclear translocation of p65, and subsequent activation of NF- κB in the rAgI/II-stimulated cells. In addition, the SF suppressed the rAgI/II-stimulated activation of ERK MAPK as well as the MAPK inhibitor significantly reduced the rAgI/II-induced production of NO and PGE₂.ConclusionCollectively, we suggest that the SF inhibits the expression and production of inflammatory mediators by blocking the ERK MAPK mediated pathway and inhibiting the activation of NF-κB.     

Effect of PGE₂ induced by compressive and tensile stresses on cementoblast differentiation in vitro

Objective

The aim of the study was to clarify the mechanisms underlying orthodontically induced root resorption by characterizing the role of PGE₂ induced by compressive stress (CS) and tensile stress (TS) on cementoblast metabolism in vitro.

Design

Mouse cementoblast cell line OCCM-30 was continuously stimulated with 0.2 KPa CS or 5.0 KPa TS. COX-2 mRNA expression and PGE₂ production were thus quantified. In addition, cells were treated with COX-2 inhibitor and the role of PGE₂ induced by CS or TS on the expression of genes related to cementoblast differentiation was examined. PGE₂ receptors mRNA expression induced by CS or TS was also evaluated. Moreover, cells were treated with exogenous PGE₂ and the role of PGE₂ concentration on matrix mineralization was verified.

Results

CS and TS enhanced COX-2 mRNA expression and PGE₂ production. PGE₂ synthesis, however, was markedly induced by CS. Gene expression of bone morphogenetic protein 2 (BMP-2), osteocalcin (OCN) and receptor activator for nuclear factor kappaB ligand (RANKL) was enhanced by CS on an endogenous PGE₂-mediated manner. Osteoprotegerin (OPG) expression was not affected by CS. Meanwhile, TS up-regulated the expression of BMP-2 and alkaline phosphatase (ALP) on an endogenous PGE₂-mediated manner. TS down-regulated RANKL mRNA expression, whilst OPG expression was not affected. Moreover, EP4 mRNA expression was considerably enhanced by TS. Regarding PGE₂ concentration, only cells treated with low concentration presented anabolic response.

Conclusions

Gene expression was differentially regulated according to the type of mechanical stimulation applied to cementoblasts. In addition, it is shown that PGE₂ plays an important role on mediating cementoblast mechanosensitivity.     

Involvement of oxidative stress in mutagenicity and apoptosis caused by dental resin monomers in cell cultures

OBJECTIVE: This investigation studied the possibility that apoptosis as well as mutagenicity induced by resin monomers are mediated by oxidative stress. METHODS: A range of dilutions of three resin monomers (GMA, TEGDMA, and HEMA) was added to culture medium (DMEM/10% FBS), of V79-4 fibroblasts and RPC-C2A pulp cells for 24 h. Their cytotoxic effects were measured by a colorimetric functional assay (MTT). Chromosomal aberration induced by the resin monomers was investigated by counting micronuclei in V79-4 cells. The effects of the resin monomers on DNA fragmentation were viewed by agarose gel electrophoresis of DNA, isolated from RPC-C2A pulp cells that were treated by resin compounds. Resin monomer-induced apoptosis was further confirmed by flow cytometry (staining with both annexin V-FITC and PI). RESULTS: All monomers exhibited a dose-dependent cytotoxic effect, and the ranking of the cytotoxicity based on TC50 was GMA > TEGDMA > HEMA. The resin monomer-induced cytotoxicity was significantly decreased by co-treatment with N-acetylcystein (NAC), an antioxidant. The authors also confirmed a dose-dependent genotoxicity of the resin monomers that had induced micronucleated cells in V79-4 fibroblasts. Similar to the effects on cytotoxicity, NAC reduced the numbers of micronuclei in comparison with those generated by the resin monomers. The preventive effects of NAC were also observed in monomer-induced apoptosis in RPC-C2A cells. A DNA ladder pattern, characteristic of apoptosis, was shown at cytotoxic concentrations, but NAC blocked the resin monomer-mediated DNA fragmentation. The preventive effects of NAC on apoptosis were confirmed by Annexin V staining. Cells exposed to 300 microM GMA, 7 mM TEGDMA, or 14 mM HEMA for 24 h showed a significant increase in apoptotic cells, while NAC co-treatment caused a reduction in apoptotic cells compared to controls. SIGNIFICANCE: These findings suggest that glutathione depletion and oxidative stress are responsible for GMA, TEGDMA, and HEMA-induced mutagenicity and apoptosis.     

Epigallocatechin-3-gallate Blocks Triethylene Glycol Dimethacrylate–induced Cyclooxygenase-2 Expression by Suppressing Extracellular Signal-regulated Kinase in Human Dental Pulp and Embryonic Palatal Mesenchymal Cells

IntroductionMethacrylate resin–based materials could release components into adjacent environment even after polymerization. The major components leached include triethylene glycol dimethacrylate (TEGDMA). TEGDMA has been shown to induce the expression of cyclooxygenase-2 (COX-2). However, the mechanisms are not completely understood. The aims of this study were to investigate the molecular mechanism underlying TEGDMA-induced COX-2 in 2 oral cell types, the primary culture of human dental pulp (HDP) cells and the human embryonic palatal mesenchymal (HEPM) pre-osteoblasts, and to propose potential strategy to prevent or ameliorate the TEGDMA-induced inflammation in oral tissues.MethodsTEGDMA-induced COX-2 expression and its signaling pathways were assessed by Western blot analyses in HDP and HEPM cells. The inhibition of TEGDMA-induced COX-2 protein expression using various dietary phytochemicals was investigated.ResultsCOX-2 protein expression was increased after exposure to TEGDMA at concentrations as low as 5 μmol/L. TEGDMA-induced COX-2 expression was associated with reaction oxygen species, the extracellular signal-regulated kinase 1/2, and the p38 mitogen-activated protein kinase signaling pathways in HDP and HEPM cells. The activation of p38 mitogen-activated protein kinase was directly associated with reactive oxygen species. Epigallocatechin-3-gallate suppressed TEGDMA-induced COX-2 expression by inhibiting phosphorylation of extracellular signal-regulated kinase 1/2.ConclusionsCells exposed to low concentrations of TEGDMA may induce inflammatory responses of the adjacent tissues, and this should be taken into consideration during common dental practice. Green tea, which has a long history of safe beverage consumption, may be a useful agent for the prevention or treatment of TEGDMA-induced inflammation in oral tissues.     

Interleukin-1 beta affects cyclooxygenase-2 expression and cartilage metabolism in mandibular condyle

Extracellular matrix degradation in mandibular condylar cartilage is mediated by various cytokines in the temporomandibular joint (TMJ). Interleukin-1 beta (IL-1β) is detected in joint structures with pathologic status, and participates in catabolic action in the extracellular matrix. The purpose of this study was to investigate the effects of IL-1β on cyclooxygenase-2 (COX-2) expression and cartilage metabolism using cultured chondrocytes from mandibular condyle. Articular chondrocytes from the porcine mandibular condylar cartilage around the surface were cultured and treated with 0–10 ng/ml IL-1β or 0–1000 ng/ml prostaglandin (PGE₂) for 0–24 h. The mRNA levels of COX-2, MMP-1, -3, and -13 were evaluated by real-time PCR analysis. The protein levels of PGE₂ and MMPs were examined by ELISA and Western blot analysis, respectively. The expression levels of COX-2 and PGE₂ were enhanced by exogenous IL-1β in chondrocytes. The mRNA levels of MMP-1, -3, and -13 were up-regulated by PGE₂ treatment dose-dependently. It is shown that the expression of COX-2/PGE₂ was enhanced by IL-1β in articular chondrocytes from mandibular condyle, and that MMP-1, -3, and -13 were induced by PGE₂, suggesting that IL-1β-induced COX-2/PGE₂ play a crucial role in catabolic processes of mandibular condylar cartilage under inflammatory conditions.     

Sodium butyrate induces the production of cyclooxygenases and prostaglandin E₂ in ROS 17/2.8 osteoblastic cells

ObjectiveSodium butyrate (butyric acid; BA) is a major metabolic by-product of the anaerobic periodontopathic bacteria present in subgingival plaque. We examined the effects of BA and/or indomethacin on cell proliferation, the expression of cyclooxygenases (COXs), prostaglandin (PG) receptors (EP1-4), extracellular matrix proteins, such as type I collagen and osteopontin, and PGE₂ production, using ROS17/2.8 cells as osteoblasts.MethodsThe rat clonal cell line ROS 17/2.8 was cultured with 0, 10^?5, 10^?4, and 10^?3 M BA in the presence or absence of 0.5 μM indomethacin, for up to 7 days. The expression of COX-1, COX-2, EP1, EP2, EP3, EP4, type I collagen, and osteopontin was examined at the mRNA and protein levels using real-time PCR and Western blotting, respectively. The amount of PGE₂ in the culture medium was measured by ELISA.ResultsProliferation of ROS 17/2.8 cells was not affected by the addition of BA. However, PGE₂ production and the expression of COX-1 and COX-2 increased with the addition of BA. In contrast, indomethacin, an inhibitor of COX, blocked the stimulatory effect of BA. Furthermore, EP2 expression increased with BA treatment, whereas EP1 expression was not affected and the expression of EP3 and EP4 was not detected. The addition of BA also increased the expression of type I collagen and osteopontin. Indomethacin blocked about 50% of the stimulatory effect of BA on type I collagen, whereas it did not block the effect on osteopontin.ConclusionsThese results suggest that BA induces PGE₂ production by increasing the expression of COX-1 and COX-2 in osteoblasts, and that an autocrine action of the produced PGE₂, via EP1 or BA-induced EP2, is related to an increase in type I collagen expression by BA.     

Prostaglandin E2 Affects Interleukin 6 and Monocyte Chemoattractant Protein 1/CCL2 Production by Cultured Stem Cells of Apical Papilla

IntroductionRoot canal treatment of immature necrotic teeth is a major challenge in current endodontics. The effect of inflammatory mediators, such as prostaglandin, on the modulation of stem cells of the apical papilla (SCAP) is not completely understood. The aim of this study was to investigate the role of prostaglandin E₂ (PGE₂) on SCAP activation by Escherichia coli lipopolysaccharide (LPS) in vitro.MethodsSCAP cultures were established and characterized. Increasing concentrations of lipopolysaccharide (0.1–10 μg/mL) were used to investigate cyclooxygenase-2 (COX-2/PTGS2) and PGE₂ receptors (EP1–4) gene expression. Then, SCAP were treated with a COX-2 inhibitor (indomethacin) before treatment with different concentrations of LPS. The levels of the chemokine CCL2/monocyte chemoattractant protein 1 and interleukin (IL)-6 were detected in cell supernatants (24 hours) by enzyme-linked immunosorbent assay. Data analysis was performed using analysis of variance followed by the Tukey post test.ResultsThe expression of COX-2 was up-regulated in the group treated with LPS at 1μg/mL compared with that in the control group. EP1–4 were detected in all experimental conditions at similar levels. SCAP treated with indomethacin presented a down-regulation in the production of LPS-induced CCL2 and the secretion of IL-6.ConclusionsSCAP showed increased COX-2 (PTGS2) gene expression induced by LPS and a PGE₂-dependent production of IL-6 and CCL2.     

Inhibition of TEGDMA and HEMA-induced genotoxicity and cell cycle arrest by N-acetylcysteine.

OBJECTIVES: Dental resin monomers like triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) are able to cause an imbalance of the redox state in mammalian cells. The resulting oxidative stress originating from reactive oxygen species (ROS) has been associated with cytotoxicity. We hypothesized that ROS might contribute to the generation of genotoxicity by TEGDMA and HEMA as well. Therefore, we examined the formation of micronuclei in V79 cells by both resin monomers in the presence of the antioxidant N-acetylcysteine (NAC), which scavenges ROS. In addition, we analyzed the effects of TEGDMA and HEMA on the normal cell cycle in the presence of NAC. METHODS: V79 fibroblasts were exposed to increasing concentrations of TEGDMA and HEMA in the presence and absence of NAC for 24h. Genotoxicity was indicated by the formation of micronuclei. The modification of the normal cell cycle was analyzed by flow cytometry (FACS). RESULTS: A dose-related increase in the number of micronuclei in V79 cells-induced by TEGDMA and HEMA indicated genotoxicity of both chemicals. However, the formation of micronuclei was reduced in the presence of 10 mmol/L NAC, indicating its protective role. A cell cycle delay in G2 phase caused by TEGDMA was absent when cells were co-treated with NAC. Similarly, the presence of NAC led to a reversion of the cell cycle delay in HEMA-treated cell cultures. SIGNIFICANCE: Our results suggest that genotoxic effects and the modification of the cell cycle caused by TEGDMA and HEMA are mediated, at least in part, by oxidative stress.     

Vasodilatory effect of hydroxyethyl methacrylate and triethylene glycol dimethacrylate in rat aorta through calcium antagonistic action

Introduction

Resin-based dental materials contain various diluent monomers that can interfere with vascular function by causing vasodilation. In this study, we evaluated the vasoactive potential of hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) and the possible mechanism of their vascular action on isolated rat aorta.

Methods

Responses of thoracic aorta rings were recorded isometrically by using force displacement transducers. After precontracting aorta rings with phenylephrine, relaxations to HEMA and TEGDMA were recorded in the absence and presence of nitric oxide synthase inhibitor N^ω-nitro-L-arginine methyl ester, cyclooxygenase inhibitor indomethacin, and K⁺ channel inhibitors tetraethylammonium, glibenclamide, and 4-aminopyridine. To investigate the Ca²⁺-channel antagonistic effect of HEMA and TEGDMA in different aorta rings, concentration-response curves to CaCl₂ were obtained in the absence and presence of the test monomers.

Results

Both HEMA and TEGDMA elicited concentration-dependent relaxations. The vasorelaxant effect of HEMA and TEGDMA was not mediated via endothelium-dependent nitric oxide and prostanoid-dependent mechanisms or by K⁺ efflux through K⁺ channels. Both monomers significantly inhibited the contractions induced by CaCl₂.

Conclusions

Our results showed that HEMA and TEGDMA induce vasodilation via Ca²⁺-antagonistic action, whereas nitric oxide and cyclooxgenase pathway and K⁺ channels were not responsible for this vasoactive effect.     

Evaluation of polymerization characteristics and penetration into enamel caries lesions of experimental infiltrants

ObjectivesTo evaluate the properties of experimental infiltrant blends by comparing them with the commercial infiltrant Icon^® and penetration homogeneity into enamel caries lesions.MethodsGroups were set up as follows: G1 (TEGDMA 100%); G2 (TEGDMA 80%, Ethanol 20%); G3 (TEGDMA 80%, HEMA 20%); G4 (TEGDMA 75%, BisEMA 25%); G5 (TEGDMA 60%, BisEMA 20%, Ethanol 20%); G6 (TEGDMA 60%, BisEMA 20%, HEMA 20%); G7 (TEGDMA 75%, UDMA 25%); G8 (TEGDMA 60%, UDMA 20%, Ethanol 20%); G9 (TEGDMA 60%, UDMA 20%, HEMA 20%) and Icon^®. Ten specimens were comprised by each group for the following tests (n = 10): degree of conversion (DC), elastic modulus (EM), Knoop hardness (KH), and softening ratio (SR). Infiltrant penetration was evaluated using confocal microscopy (CLSM). Data were subjected to two-way ANOVA and a Tukey's test (5%). Data comparing experimental materials and Icon^® were analysed using ANOVA and Dunnett's test (5%).ResultsThe highest DC values were found in G1, G7, G8, and G9. The lowest DC values were found in G2, G4, G5, and G6. EM and KHN were significantly lower in HEMA and with ethanol addition for all blends, except for G9. There was no significant difference among the groups regarding SR, and it was not possible to take KHN readings of G2, G5, and G8 after storage. There was no significant difference among groups for infiltrant penetration into enamel lesions.ConclusionsThe addition of hydrophobic monomers and solvents into TEGDMA blends affected DC, EM, and KHN. UDMA added to TEGDMA resulted in an increase in DC, EM, and KHN. Overall, solvents added to monomer blends resulted in decreased properties. The addition of hydrophobic monomers and solvents into TEGDMA blends does not improve the penetration depth of the infiltrants.     

n-Butanol extracts of Panax notoginseng suppress LPS-induced MMP-2 expression in periodontal ligament fibroblasts and inhibit osteoclastogenesis by suppressing MAPK in LPS-activated RAW264.7 cells

Objective

Periodontitis is a group of inflammatory diseases that affect connective tissue attachments and the supporting bone that surround the teeth. Osteoclasts are responsible for skeletal modeling and remodeling but may also destroy bone in several bone diseases, including osteoporosis and periodontitis. This study examined the anti-inflammatory effects of Panax notoginseng (PN) on periodontal ligament fibroblasts (PDLFs) and RAW264.7 cells under lipopolysaccharide (LPS) induced inflammatory conditions.

Design

The effects of PN on PDLFs were determined by measuring the cell viability and mRNA expression of tissue-destructive proteins. The effects of PN on osteoclasts were examined by measuring the following: (1) the cell viability, (2) the formation of Tartrate-resistant acid phosphatase (TRAP)(+) multinucleated cells, (3) MAPK signaling pathways, (4) mRNA expression of inflammatory-related proteins and (5) nitric oxide (NO) production.

Results

The n-butanol extracts of PN (bPN) increased the cell proliferation of the PDLFs and decreased the mRNA expression of matrix metalloproteinase (MMP)-2 in the PDLFs. bPN inhibited the formation of LPS-stimulated TRAP(+) multinucleated cells. bPN also inhibited the LPS-stimulated activation of JNK and ERK signaling, and inhibited the LPS-stimulated degradation of I_KB in the RAW264.7 cells. In addition, bPN decreased the mRNA expression of MMP-9 and iNOS, which are involved in the range of pathophysiological processes, such as inflammation in the RAW264.7 cells. NO production was also decreased via the inhibition of iNOS.

Conclusions

These findings suggest that bPN has therapeutic effects on bone-destructive processes, such as those that occur in periodontal diseases.     

Curcumin abrogates LPS-induced pro-inflammatory cytokines in RAW 264.7 macrophages. Evidence for novel mechanisms involving SOCS-1, -3 and p38 MAPK

Curcumin is the active compound in the extract of Curcuma longa rhizomes with anti-inflammatory properties mediated by inhibition of intracellular signalling. SOCS and MAPKinases are involved in the signalling events controlling the expression of IL-6, TNF-α and PGE₂, which have important roles on chronic inflammatory diseases. The aim was to assess if these pathways are involved in curcumin-mediated effects on LPS-induced expression of these cytokines in macrophages. RAW 264.7 murine macrophages were stimulated with Escherichia coli LPS in the presence and absence of non-cytotoxic concentrations of curcumin. Curcumin potently inhibited LPS-induced expression of IL-6, TNF-α and COX-2 mRNA and prevented LPS-induced inhibition of SOCS-1 and -3 expression and the inhibition of the activation of p38 MAPKinase by modulation of its nuclear translocation. In conclusion, curcumin potently inhibits expression of LPS-induced inflammatory cytokines in macrophages via mechanisms that involve modulation of expression and activity of SOCS-1 and SOCS-3 and of p38 MAPK.     

Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stem/progenitor cells derived from deciduous teeth

Objectives

The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells.

Methods

Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH₂PO₄,β-glycerophosphate and l-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5 mM) and TEGDMA (0.05-0.25 mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were also evaluated.

Results

DTSCs cultures were positive for STRO-1 (7.53 ± 2.5%), CD146 (91.79 ± 5.41%), CD34 (11.87 ± 3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated.

Significance

These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.     

Kaempferol Inhibits P. intermedia Lipopolysaccharide‐Induced Production of Nitric Oxide Through Translational Regulation in Murine Macrophages: Critical Role of Heme Oxygenase‐1‐Mediated ROS Reduction

Background: Nitric oxide (NO) could be a potential target for the development of new therapeutic approaches to the treatment of periodontal disease because this molecule plays a significant role in the tissue destruction observed in periodontitis. In this study, the authors investigate the effect of kaempferol on the production of NO by murine macrophage‐like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and try to determine the underlying mechanisms of action. Methods: NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Real‐time polymerase chain reaction was performed to quantify inducible NO synthase (iNOS) and heme oxygenase‐1 (HO‐1) mRNA expression. iNOS and HO‐1 protein expression and phosphorylation of c‐Jun N‐terminal kinase and p38 were characterized via immunoblot analysis. Reactive oxygen species (ROS) production was measured using the redox‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate. Results: Kaempferol significantly inhibited NO production and expression of iNOS protein in P. intermedia LPS‐stimulated RAW246.7 cells without affecting iNOS mRNA expression. Kaempferol upregulated HO‐1 expression in LPS‐activated cells. Inhibition of HO‐1 activity by tin protoporphyrin IX (SnPP) abolished the suppressive effect of kaempferol on NO production. In addition, kaempferol significantly attenuated P. intermedia LPS‐induced increase of intracellular ROS, and SnPP blocked this reduction. Treatment with antioxidants downregulated the production of LPS‐induced NO. Conclusions: Kaempferol inhibits NO production and iNOS protein expression in P. intermedia LPS‐stimulated RAW264.7 cells at the translational level via HO‐1‐mediated ROS reduction and could be an efficient modulator of host response in the treatment of periodontal disease.     

Cyclic Mechanical Strain Induces Interleukin-6 Expression via Prostaglandin E2 Production by Cyclooxygenase-2 in MC3T3-E1 Osteoblast-like Cells

Interleukin (IL)-6, produced by osteoblasts, is one of the key cytokines that promote bone resorption. This study examined the effect of cyclic mechanical strain on the expression of IL-6 mRNA and protein, and the induction mechanism of IL-6 in MC3T3-E1 osteoblast-like cells using RT-PCR and ELISA. MC3T3-E1 cells were cultured in α-MEM with 10% FBS on flexible, collagen I-coated membranes and subjected to cyclic mechanical strain at 6 cycles/min. The production of IL-6 protein was increased at 3 h, reached a peak at 6 h, and was maintained up to 24 h by the cyclic mechanical strain. The strain increased the production of IL-6 protein in a force-dependent manner. The expression of IL-6 mRNA was increased by the cyclic mechanical strain. Prostaglandin(PG)E₂ production was induced at 1 h, dramatically increased to 3 h, and was gradually increased from 3 to 24 h by the cyclic mechanical strain. The strain increased PGE₂ production in a force-dependent manner. Treatment with PGE₂ increased the production of IL-6 protein from MC3T3-E1 cells. The cyclic mechanical strain promoted cyclooxygenase (COX)-2 but not COX-1 mRNA expression. Pretreatment with indomethacin and NS-398 prevented PGE₂ production and partly inhibited the production of IL-6 protein induced by the cyclic mechanical strain. These findings suggest that cyclic mechanical strain increases IL-6 expression in osteoblasts, the induction of which is in part mediated via PGE₂ production by COX-2.     

低强度脉冲式超声波在脂多糖诱导的RAW264.7巨噬细胞分化中的抗炎和抗氧化作用

目的研究低强度脉冲式超声波（LIPUS）对RAW264.7巨噬细胞极化的影响及相关分子机制。 方法100 ng/mL脂多糖（LPS）和10 ng/mL白细胞介素（IL）-4诱导巨噬细胞RAW264.7分别向M1和M2型极化，45 mW/cm²强度LIPUS对巨噬细胞处理25 min。采用流式细胞术检测巨噬细胞氧化应激活性氧（ROS）水平、M1分化标志物CD80和CD11b，以及M2分化标志物CD163的表达水平。采用反转录聚合酶链反应（RT-PCR）技术检测CD80、CD11b、核转录因子κB（NF-κB）p65、肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-1β和IL-6的mRNA水平。采用Western blot技术检测细胞p65、p-p65、TNF-α、IL-1β和IL-6表达水平。采用流式细胞术检测细胞培养上清TNF和IL-6表达水平。 结果LIPUS可明显减少LPS诱导的RAW264.7细胞ROS水平。LPS诱导后，RAW264.7细胞M1分化标志物CD80和CD11b表达和转录水平均上调；LIPUS可抑制LPS对RAW264.7细胞向M1的诱导，差异具有统计学意义。并且，LIPUS可促进IL-4诱导的巨噬细胞M2分化标志物CD163表达。LPS诱导的RAW264.7细胞炎症因子NF-κB p65、TNF-α、IL-1β和IL-6 mRNA水平上调，LIPUS可下调这些炎症因子的mRNA水平，差异均具有统计学意义。LIPUS抑制了LPS对RAW264.7细胞因子蛋白p65和p-p65、IL-1β、TNF-α和IL-6蛋白表达上调的作用。胰酶可以通过激活ROS-NF-κB通路，回复LIPUS促进巨噬细胞M1分化的作用。 结论LIPUS可通过ROS-NF-κB抑制RAW264.7向巨噬细胞M1型分化，促进RAW264.7向巨噬细胞M2型分化。氧化应激和炎症因子表达水平被抑制。LIPUS可能在牙周疾病中起到抑制氧化和炎症的作用，从而发挥对牙周疾病的治疗功能。     

In vitro stability of methylmethacrylic acid,TEGDMA and HEMA exposed to esterases

ObjectivesComonomers used in dental restorative materials, e.g. triethylenglycoledimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA) and methylmethacylic acid (MMA) are methacrylic acid esters. Because of the chemical structure of these esters in earlier studies two different in vitro pathways were suggested. The present study was performed in order to analyze which of these chemical pathways is preferred in vitro: (a) saponification of TEGDMA, HEMA, and MMA leading to free methacrylic acid (MA) as an ionic intermediate or (b) enzymatically epoxidation of mother compounds leading to lipophilic intermediates.MethodsExperiments have been performed in an isolated system using MMA, TEGDMA or HEMA, respectively exposed to pig liver esterase (PLE) in phosphate buffer. The reaction of tested comonomers was terminated by the use of sodium hydroxide solution, sulfuric acid or saturated NaCl solution, respectively. The samples were analyzed by the use of headspace-gas chromatography–mass spectrometry.ResultsIn all samples a decomposition of comonomers by the use of PLE was observed. Due to the high rate constant k of MMA (e.g. k_MMA > 1.3 mg/(l s)) epoxidation of non-cleaved molecules of this substance can be excluded. Compared to MMA the decomposition of TEGDMA and HEMA is significantly slower (e.g. k_{(TEGDMA, PLE = 7 units/ml)} = 0.004 mg/(l s) and k_{(HEMA, PLE = 7 units/ml)} = 0.00013 mg/(l s)). Thus in case of a low liver enzyme concentration the epoxidation of non-cleaved molecules of TEGDMA and HEMA cannot be excluded.SignificanceIn the metabolic pathway of TEGDMA and HEMA the probability of an auxiliary chemical pathway was demonstrated. In case of MMA the formation of epoxidated metabolites can be excluded. In contrast to this the chemical pathway for TEGDMA and HEMA might lead to lipophilic intermediates which can be accumulated in fatty tissue.     

The induction of micronuclei in vitro by unpolymerized resin monomers

Components of resin materials may damage DNA, leading to genetic alterations in mammalian cells. Here, monomers were analyzed for the induction of chromosomal aberrations indicated by micronuclei induced in V79 cells. A dose-related increase in the numbers of micronuclei was observed with triethyleneglycol dimethacrylate (TEGDMA), 2-hydroxyethyl methacrylate (HEMA), and glycidyl methacrylate (GMA). These effects were reduced, however, by a metabolically active microsomal fraction from rat liver. The very low activity of Bis-GMA and UDMA and the elevated numbers of micronuclei caused by high concentrations of methyl methacrylate and bisphenol A were associated with cytotoxicity. Our findings provide evidence for the induction of micronuclei by TEGDMA, HEMA, and GMA under physiological conditions, indicating clastogenic activity of these chemicals in vitro. Since it has been shown that TEGDMA also caused gene mutations and DNA sequence deletions in mammalian cells, the activity of this substance should be analyzed in vivo.