Anti-Inflammatory and Protective Effects of Juncus effusus L. Water Extract on Oral Keratinocytes.

Periodontitis is a chronic inflammatory disease caused by periodontopathogenic bacteria that form biofilms in periodontal pockets. The gingival epithelium acts as the first physical barrier in fighting attacks by periodontopathogenic pathogens, such as the primary etiological agent Porphyromonas gingivalis, and various exogenous chemicals, as well as regulates the local innate immune responses. Therefore, the development of novel oral care products to inhibit inflammatory reactions caused by bacterial infection and protect the gingival epithelium is necessary. Juncus effusus L. has generally been used as an indigenous medicine, such as a diuretic, an antipyretic, and an analgesic, in ancient practice. In this study, we examined the effects of a water extract from J. effusus L. on the inhibition of the inflammatory reaction elicited by bacterial infection and protection of the oral epithelium by chemical irritation. Pretreatment of oral epithelial cells with the water extract from J. effusus L. significantly reduced P. gingivalis or its lipopolysaccharide- (LPS-) mediated production of chemokines (interleukin-8 and C-C-chemokine ligand20) in a concentration-dependent manner with comparable to or greater effects than epigallocatechin gallate and protected oral epithelial cells from injury by chemical irritants, cetylpyridinium chloride, and benzethonium chloride. Moreover, the water extract from J. effusus L. in the presence of antimicrobial agents or antifibrinolytics already used as ingredients in mouthwash could significantly reduce the production of chemokines from P. gingivalis LPS-stimulated oral epithelial cells in a concentration-dependent manner. These findings suggest that the water extract from J. effusus L. is potentially useful for oral care to prevent oral infections, such as periodontal infections, and maintain oral epithelial function.

The Pathogenic Factors from Oral Streptococci for Systemic Diseases.

The oral cavity is suggested as the reservoir of bacterial infection, and the oral and pharyngeal biofilms formed by oral bacterial flora, which is comprised of over 700 microbial species, have been found to be associated with systemic conditions. Almost all oral microorganisms are non-pathogenic opportunistic commensals to maintain oral health condition and defend against pathogenic microorganisms. However, oral Streptococci, the first microorganisms to colonize oral surfaces and the dominant microorganisms in the human mouth, has recently gained attention as the pathogens of various systemic diseases, such as infective endocarditis, purulent infections, brain hemorrhage, intestinal inflammation, and autoimmune diseases, as well as bacteremia. As pathogenic factors from oral Streptococci, extracellular polymeric substances, toxins, proteins and nucleic acids as well as vesicles, which secrete these components outside of bacterial cells in biofilm, have been reported. Therefore, it is necessary to consider that the relevance of these pathogenic factors to systemic diseases and also vaccine candidates to protect infectious diseases caused by Streptococci. This review article focuses on the mechanistic links among pathogenic factors from oral Streptococci, inflammation, and systemic diseases to provide the current understanding of oral biofilm infections based on biofilm and widespread systemic diseases.

Caffeic Acid Phenethyl Ester (CAPE) Induces VEGF Expression and Production in Rat Odontoblastic Cells.

Caffeic acid phenethyl ester (CAPE), the main component of propolis, has various biological activities including anti-inflammatory effect and wound healing promotion. Odontoblasts located in the outermost layer of dental pulp play crucial roles such as production of growth factors and formation of hard tissue termed reparative dentin in host defense against dental caries. In this study, we investigated the effects of CAPE on the upregulation of vascular endothelial growth factor (VEGF) and calcification activities of odontoblasts, leading to development of novel therapy for dental pulp inflammation caused by dental caries. CAPE significantly induced mRNA expression and production of VEGF in rat clonal odontoblast-like KN-3 cells cultured in normal medium or osteogenic induction medium. CAPE treatment enhanced nuclear factor-kappa B (NF-κB) transcription factor activation, and furthermore, the specific inhibitor of NF-κB significantly reduced VEGF production. The expression of VEGF receptor- (VEGFR-) 2, not VEGFR-1, was up regulated in KN-3 cells treated with CAPE. In addition, VEGF significantly increased mineralization activity in KN-3 cells. These findings suggest that CAPE might be useful as a novel biological material for the dental pulp conservative therapy.

The roles of odontoblasts in dental pulp innate immunity.

Odontoblasts located in the outermost layer of dental pulp form a natural barrier between mineralized tissues, dentin, and soft tissues, dental pulp, of the vital tooth, and they first recognize caries-related pathogens and sense external irritations. Therefore, odontoblasts possess a specialized innate immune system to fight oral pathogens invading into dentin. Generally, the rapid initial sensing of microbial pathogens, especially pathogen-associated molecular patterns (PAMPs) shared by microorganisms, are mediated by pattern recognition receptors (PRRs), such as Toll-like receptor and the nucleotide-binding oligomerization domain (NOD). The innate immune responses in odontoblasts initiated by sensing oral pathogens provide host protective events, such as inflammatory reactions, to produce a variety of pro-inflammatory mediators, including chemokines and cytokines. These attract various inflammatory cells and cause antibacterial reactions, such as the production of defensins, to kill microorganisms in the proximal region of the odontoblast layer. This review focuses on innate immunity, especially cellular and molecular mechanisms regarding the sensing of PAMPs from oral pathogens by PRRs, in odontoblasts and provides information for future studies for the development of novel therapeutic strategies, including diagnosis and treatment, to prevent exceeding dental pulp inflammation and preserve the dental pulp tissues.

Antibiofilm and Anti-Inflammatory Activities of Houttuynia cordata Decoction for Oral Care.

Dental biofilms that form in the oral cavity play a critical role in the pathogenesis of several infectious oral diseases, including dental caries, periodontal disease, and oral candidiasis. Houttuynia cordata (HC, Saururaceae) is a widely used traditional medicine, for both internal and external application. A decoction of dried HC leaves (dHC) has long been consumed as a health-promoting herbal tea in Japan. We have recently reported that a water solution of HC poultice ethanol extract (wHCP) exerts antimicrobial and antibiofilm effects against several important oral pathogens. It also exhibits anti-inflammatory effects on human keratinocytes. In our current study, we examined the effects of dHC on infectious oral pathogens and inflammation. Our results demonstrated that dHC exerts moderate antimicrobial effects against methicillin-resistant Staphylococcus aureus (MRSA) and other oral microorganisms. dHC also exhibited antibiofilm effects against MRSA, Fusobacterium nucleatum (involved in dental plaque formation), and Candida albicans and inhibitory effects on interleukin-8, CCL20, IP-10, and GROα productions by human oral keratinocytes stimulated by Porphyromonas gingivalis lipopolysaccharide (a cause of periodontal disease), without cytotoxic effects. This suggests that dHC exhibits multiple activities in microorganisms and host cells. dHC can be easily prepared and may be effective in preventing infectious oral diseases.

Royal Jelly Inhibits Pseudomonas aeruginosa Adherence and Reduces Excessive Inflammatory Responses in Human Epithelial Cells.

Pseudomonas aeruginosa is a Gram-negative bacterium and causes respiratory infection especially in elderly patients. Royal jelly has been used worldwide as a traditional remedy and as a nutrient; however, the effect against P. aeruginosa is unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly against P. aeruginosa. Wild-type strain PAO1 and clinical isolates of P. aeruginosa were used for antibacterial assay and antiadherent assay to abiotic surface and epithelial cells, which are pharynx (Detroit 562) and lung (NCI-H292) epithelial cells. In anti-inflammatory assay, epithelial cells were pretreated with royal jelly before bacterial exposure to investigate its inhibitory effect on interleukin (IL-8) and macrophage inflammatory protein-3α/CCL20 overproduction. Although royal jelly did not have antibacterial activity at concentration of 50% w/v, antiadherent activity was confirmed on the abiotic surface and epithelial cells under concentration of 25%. Pretreatment with royal jelly significantly inhibited overproduction of IL-8 and CCL20 from both cells. These results demonstrated that royal jelly inhibits P. aeruginosa adherence and protects epithelial cells from excessive inflammatory responses against P. aeruginosa infection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused by P. aeruginosa.

Functional Roles of NOD1 in Odontoblasts on Dental Pulp Innate Immunity.

Caries-related pathogens are first recognized by odontoblasts and induce inflammatory events that develop to pulpitis. Generally, initial sensing of microbial pathogens is mediated by pattern recognition receptors, such as Toll-like receptor and nucleotide-binding oligomerization domain (NOD); however, little is known about NODs in odontoblasts. In this study, the levels of NODs expressed in rat odontoblastic cell line, KN-3, were assessed by flow cytometry and the levels of chemokines in NOD-specific ligand-stimulated KN-3 cells were analyzed by real-time PCR and ELISA. The signal transduction pathway activated with NOD-specific ligand was assessed by blocking assay with specific inhibitors and reporter assay. In KN-3 cells, the expression level of NOD1 was stronger than that of NOD2 and the production of chemokines, such as CINC-1, CINC-2, CCL20, and MCP-1, was upregulated by stimulation with NOD1-specific ligand, but not with NOD2-specific ligand. CINC-2 and CCL20 production by stimulation with NOD1-specific ligand was reduced by p38 MAPK and AP-1 signaling inhibitors. Furthermore, the reporter assay demonstrated AP-1 activation in NOD1-specific ligand-stimulated KN-3 cells. These findings indicated that NOD1 expressed in odontoblasts functions to upregulate the chemokines expression via p38-AP-1 signaling pathway and suggested that NOD1 may play important roles in the initiation and progression of pulpitis.

Electromagnetic wave irradiation promotes osteoblastic cell proliferation and up-regulates growth factors via activation of the ERK1/2 and p38 MAPK pathways.

BACKGROUND/AIMS: Periodontitis with bone resorption is caused by inflammatory reactions to bacterial infection. We recently reported that electromagnetic wave irradiation (EMWI) has bactericidal effects. However, the effects of EMWI on periodontal tissues remain unclear. This study was aimed to investigate the effects of EMWI on osteoblasts. METHODS: Osteoblastic cells MC3T3-E1 were treated with EMWI (500-1,000 kHz, 5 times, 1 sec/time). Cell growth and cytotoxicity were determined by cell proliferation assays and measurement of lactate dehydrogenase release, respectively. Gene expression and protein production of growth factors were analyzed using real-time PCR and ELISA, respectively. EMWI-activated cellular signal transduction pathways were investigated by immunoblotting and blocking assay with specific inhibitors. RESULTS: Osteoblasts proliferation was significantly enhanced 3 days after EMWI and no cytotoxicity was observed. EMWI up-regulated various growth factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). EMWI induced ERK1/2, p38 MAPK and SAPK/JNK phosphorylation within 5 min, and the production of PDGF-ΑΑ and VEGF was partially reduced by MAPK-specific inhibitor. CONCLUSION: These findings demonstrated that EMWI increases osteoblastic cell activity and the expression of growth factors via ERK1/2 and p38 MAPK pathways and suggested that EMWI may be beneficial to bone tissue repair such as periodontitis.

Anti-inflammatory and protective effects of 2-methacryloyloxyethyl phosphorylcholine polymer on oral epithelial cells.

Periodontitis is a chronic inflammatory disease initiated by a microbial biofilm formed in the periodontal pocket. Gingival epithelium plays important roles as the first physical barrier to bacterial invasion and in orchestrating the innate immune reaction via toll-like receptors (TLRs), which recognize various bacterial products, and maintaining its function. Newly developed oral care products to inhibit bacterial adherence, subsequent inflammatory reaction and protect the gingival epithelium are expected. We previously reported that 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer coating decreased bacterial adhesion to human oral keratinocytes, RT-7, and mouth-rinsing with MPC-polymer inhibited the increase of oral bacteria. In this study, regarding the possibility of MPC-polymer application for preventing the adherence of periodontal pathogen, subsequent inflammatory reaction and protection of gingival epithelium, we examined the effects of MPC-polymer on the adherence of Porphyromonas gingivalis, major periodontitis-related pathogen, and TLR2 ligand to RT-7 and subsequent interleukin (IL)-8 production. MPC-polymer treatment significantly reduced P. gingivalis adherence by 44% and TLR2-mediated IL-8 production by blocking the binding of its specific-ligand in a concentration-dependent manner. Furthermore, MPC-polymer pretreatment protected RT-7 from injury by chemical irritants, cetylpyridinium chloride. These findings suggest that MPC-polymer is potentially useful for oral care to prevent oral infection and to maintain oral epithelial function.

Modulatory roles of interferon-γ through indoleamine 2, 3-dioxygenase induction in innate immune response of dental pulp cells.

INTRODUCTION: Marked infiltration of inflammatory cells such as activated T cells producing interferon-γ (IFN-γ) is observed in severe pulpitis. However, the roles of IFN-γ in the innate immune response of dental pulp have not been reported. Indoleamine 2, 3-dioxygenase (IDO) is a regulator of immune responses, and the IDO expression is induced by IFN-γ in many cells whose expression in dental pulp is unknown. The purpose of this study was to determine the role of IFN-γ in the immune response through microbial pattern recognition receptors (PRRs) such as Toll-like receptors or nucleotide-binding oligomerization domain-like receptors on the production of proinflammatory cytokines such as CXCL10 and interleukin (IL)-6 and the expression of IDO in cultured human dental pulp cells (HDPCs). METHODS: HDPCs were established from explant cultures of healthy pulp tissues. CXCL10 and IL-6 production was determined using enzyme-linked immunosorbent assay. Confirmation of IDO localization in dental pulp tissues was examined using immunohistochemistry. IDO expression in HDPCs was analyzed by immunoblot. RESULTS: IFN-γ significantly up-regulated CXCL10 and IL-6 production in the HDPCs stimulated with ligands for PRRs in a concentration-dependent manner. The expression of IDO was detected in inflamed pulp tissue. In addition, IFN-γ in combination with the PRR ligands enhanced IDO expression in HDPCs compared with IFN-γ alone. Moreover, CXCL10 production in IFN-γ-stimulated HDPCs was inhibited by an IDO inhibitor. CONCLUSIONS: This study showed the synergistic effects by IFN-γ on cytokine production and IDO expression in HDPCs, suggesting that IFN-γ may modulate the innate immune response of dental pulp.

Anti-inflammatory effect of catechin on cultured human dental pulp cells affected by bacteria-derived factors.

Catechins (bioactive polyphenols in green tea) are known to exhibit potent anti-inflammatory properties. However, the anti-inflammatory effects of catechins on inflamed dental pulp tissue are not known. In this study, we investigated the effect of epigallocatechin-3-gallate (EGCG) and epicatechin gallate (ECG), the major components of green tea catechins, on the expression of pro-inflammatory cytokines and adhesion molecules in human dental pulp cells stimulated with bacteria-derived factors such as lipopolysaccharide (LPS) and peptidoglycan (PG). The expression of interleukin (IL)-6 and of IL-8 was examined using the reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assays. The expression of intercellular adhesion molecule-1 (ICAM-1) and of vascular cell adhesion molecule-1 (VCAM-1) on dental pulp cells was analyzed using flow cytometry. The presence of EGCG and ECG significantly reduced, in a concentration-dependent manner, the expression of IL-6 and IL-8 in dental pulp cells exposed to LPS or PG. Increased expression of ICAM-1 and VCAM-1 on the dental pulp cells in response to bacterial components was also decreased by treatment with EGCG and ECG. These findings suggest that green tea catechins may prevent the exacerbation of pulpitis.

Tea catechins reduce inflammatory reactions via mitogen-activated protein kinase pathways in toll-like receptor 2 ligand-stimulated dental pulp cells.

AIMS: In this study, we evaluated whether catechins could inhibit the expression of pro-inflammatory mediators induced by dental caries-related bacteria, Streptococci, or pathogen-associated molecular patterns (PAMPs) stimulation in human dental pulp fibroblasts (HDPF). We further determined the mechanisms of the anti-inflammatory activity of catechins. MAIN METHODS: Streptococci or PAMP-stimulated HDPF were treated with catechin, and then the expression and production of pro-inflammatory mediators were determined by RT-PCR and ELISA. Furthermore, the signal transduction pathways activated with toll-like receptor (TLR)2 ligand were assessed by Immunoblot and ELISA using blocking assay with specific inhibitors. KEY FINDINGS: Increased expressions of pro-inflammatory mediators are found in inflamed dental pulp, especially in HDPF. We recently reported that dental pulpal innate immune responses may mainly result from the predominantly-expressed TLR2 signaling. Catechins, polyphenolic compounds in green tea, exert protective and healing effects through multiple mechanisms, including antioxidative and anti-inflammatory effects. However, there are no reports concerning the effects of catechins on dental pulp. In this study, we demonstrated that the up-regulated expressions of IL-8 or PGE(2) in Streptococci or PAMP-stimulated HDPF were inhibited by catechins, (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG). In TLR2 ligand-stimulated HDPF, specific inhibitors of extracellular signal regulated kinase (ERK)1/2, p38, c-jun NH(2)-terminal kinase (SAP/JNK), NF-kappaB or catechins markedly reduced the level of pro-inflammatory mediators and the phosphorylation of these signal transduction molecules was suppressed by catechins. SIGNIFICANCE: These findings suggest that catechins might be useful therapeutically as an anti-inflammatory modulator of dental pulpal inflammation.

Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways.

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (rSi-HLP). rSi-HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1 beta and TNF-alpha) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of rSi-HLP was heat-unstable. In subsequent studies, rSi-HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that rSi-HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si-HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.