Screening and detection of antimicrobials from Dai pharmaceuticals against clinical diseases by methicillin-resistant Staphylococcus aureus.

Oroxylum indicum is one of the valuable Dai pharmaceuticals; the dry seeds and bark of O. indicum were used to treat acute cough, sore throat and so on. Of the seven compounds from O. indicum were determined and obtained using the bioassay-guided method. Among them, compound 7 was obtained from the plant for the first time. Eight bacterial strains and one yeast fungi were exposed to the compounds. Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) or minimum fungicidal concentrations were determined according to the standard broth microdilution method. Baicalein (2) exhibited relative strong antibacterial activities with MIC of 8 µg ml-1 and MBC of 16 µg ml-1 against three MRSA strains of Staphylococcus aureus of SCCmec III type, whereas flavonoids 3, 5 and 7 showed some degree of activities against methicillin-susceptible S. aureus (MSSA, ATCC 25923). The findings may offer new evidence that why O. indicum was used widely in Dai peoples' life.

The regulation of cytotoxicity and cyclooxygenase-2 expression by 2-hydroxy-ethyl methacrylate in human osteoblasts are related to intracellular glutathione levels.

AIM: To investigate the effects of 2-hydroxy-ethyl methacrylate (HEMA) on cytotoxicity and cyclooxygenase-2 (COX-2) protein expression in human osteoblasts. METHODOLOGY: Cytotoxicity was judged using an Alamar Blue reduction assay on human osteoblast cell line U2OS. Western blot was used to evaluate the expression of COX-2 protein by HEMA. To determine whether glutathione (GSH) levels were important in cytotoxicity and COX-2 expression of HEMA, cells were pre-treated with the GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. Paired Student's t-tests were applied for the statistical analysis of the results. RESULTS: HEMA demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P < 0.05). The 50% inhibition concentration of HEMA was approximately 3 mmol L(-1) . HEMA was found to induce COX-2 protein expression in U2OS cells (P < 0.05). The addition of OTZ acted as a protective effect on HEMA-induced cytotoxicity and COX-2 expression (P < 0.05). In contrast, the addition of BSO enhanced HEMA-induced cytotoxicity and COX-2 expression (P < 0.05). CONCLUSION: Taken together, the levels of HEMA that were tested inhibited cell growth on U2OS cells. HEMA has a significant potential for periapical toxicity. The activation of COX-2 protein expression may be one of the mechanisms of HEMA-induced periapical inflammation. These inhibitory effects were associated with intracellular GSH levels.

The role of DNA damage and caspase activation in cytotoxicity and genotoxicity of macrophages induced by bisphenol-A-glycidyldimethacrylate.

AIM: To evaluate the potential toxicological implications of BisGMA on murine macrophage cell line RAW264.7. METHODOLOGY: Lactate dehydrogenase release, flow cytometry, Western blot and fluorometric assays were used to detect cell viability, mode of cell death and caspase activities, respectively. In addition, alkaline single-cell gel electrophoresis and cytokinesis-block micronucleus assays were applied to detect genotoxicity. Statistical analyses were performed using anova followed by the Bonferroni's t-test for multi-group comparisons test. RESULTS: BisGMA demonstrated a cytotoxic effect on RAW264.7 cells in a dose-dependent and a time-dependent manner (P < 0.05). BisGMA was found to induce two modes of cell death. The mode of cell death changed from apoptosis to necrosis as the concentrations of BisGMA elevated. Caspase-3, caspase-8 and caspase-9 activities were significantly induced by BisGMA in a dose-dependent manner (P < 0.05). Moreover, BisGMA exhibited genotoxicity via a dose-related increase in the numbers of micronucleus and DNA strand breaks (P < 0.05). CONCLUSIONS: Cytotoxicity and genotoxicity induced by BisGMA are mediated by DNA damage and caspase activation.

The upregulation of tumour necrosis factor-α and surface antigens expression on macrophages by bisphenol A-glycidyl-methacrylate.

AIM: To evaluate the expression of tumour necrosis factor-α and surface antigens by bisphenol A-glycidyl-methacrylate (BisGMA) on murine macrophage cell line RAW264.7. METHODOLOGY: Cytotoxicity was measured by tetrazolium bromide reduction assay. Tumour necrosis factor (TNF)-α was analysed by enzyme-linked immunosorbent assay. Cell surface antigens were investigated by flowcytometry. Statistical analyses were performed using anova followed by the Bonferroni's t-test for multigroup comparisons. RESULTS: BisGMA exhibited cytotoxicity to RAW264.7 in a dose-dependent manner (P < 0.05) during 2-h incubation period. BisGMA was found to increase TNF-α secretion in a dose-dependent manner (P < 0.05). In addition, CD11, CD14, CD45, CD54, CD40, CD80, and MHC II were significantly stimulated by BisGMA in a dose-dependent manner (P < 0.05). However, MHC I expression was not affected by BisGMA (P > 0.05). CONCLUSIONS: Taken together, the ability of macrophages to induce an appropriate immune response when exposed to BisGMA has the potential to upregulate TNF-α production and expression of surface antigens.

Cytotoxicity of dentine bonding agents on human pulp cells is related to intracellular glutathione levels.

AIM: To evaluate ex vivo the mechanisms of cytotoxicity of dentine bonding agents in human pulp cells in vitro. METHODOLOGY: Human pulp cells were obtained from impacted third molars with informed consent and then cultured using an explant technique. Set specimens from Clearfil SE Bond (CB), Prime & Bond 2.1 (PB), and Single Bond (SB) were eluted with culture medium. Cytotoxicity was judged using an assay of tetrazolium bromide reduction. To determine whether glutathione (GSH) levels were important in the cytotoxicity of dentine bonding agents, cells were pretreated with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or buthionine sulfoximine (BSO) to deplete GSH. Three replicates of each dentine bonding agents were performed in each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was by one-way analysis of variance (anova). Tests of differences of the treatments were analysed by Duncan's test. RESULTS: Clearfil SE Bond, PB, and SB were cytotoxic to pulp cells in a concentration-dependent manner (P<0.05). The cytotoxicity was upregulated by dentine bonding agents in the following order: PB>SB>CB. Addition of OTZ extracellularly protected the pulp cells from dentine bonding agents-induced cytotoxicity (P<0.05). Addition of BSO enhanced pulp cell death on dentine bonding agents-induced cytotoxicity (P<0.05). CONCLUSIONS: Dentine bonding agents have significant potential for pulpal toxicity. GSH depletion could be the mechanism for dentine bonding agents-induced cytotoxicity.

Up-regulation of osteolytic mediators in human osteosarcoma cells stimulated with nicotine.

BACKGROUND AND OBJECTIVE: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. However, little is known about how nicotine influences the expression of osteolytic mediators in cigarette smoking-associated periodontal diseases. The aim of this study was to investigate the expression of interleukin-1, interleukin-8, receptor activator of nuclear factor-kappaB ligand (RANKL), gelatinases and tissue-type plasminogen activator in U2OS cells (from the human osteosarcoma cell line) stimulated with nicotine. MATERIAL AND METHODS: Differences in the expression of interleukin-1, interleukin-8 and RANKL mRNAs, in response to exposure to various concentrations of nicotine (0, 0.125, 0.25, 0.5 and 1 mm) were evaluated in U2OS cells using the reverse transcription-polymerase chain reaction.In addition, the levels of interleukin-1, interleukin-8 and RANKL proteins were determined using enzyme-linked immunosorbent assays. The gelatinolytic and caseinolytic activities in nicotine treated-U2OS cells were demonstrated using gelatin and casein zymography, respectively. RESULTS: Nicotine was found to increase the expression of interleukin-1, interleukin-8 and RANKL mRNA and protein in U2OS cells (p < 0.05). The gelatin zymograms revealed that matrix metalloproteinase (MMP)-2 and MMP-9 were secreted by U2OS cells. The secretion of MMP-2 and MMP-9 occurred in a dose-dependent manner that was dependent on the concentration of nicotine (p < 0.05). Casein zymography exhibited a caseinolytic band with a molecular weight of 70 kDa, indicative of the presence of tissue-type plasminogen activator. Tissue-type plasminogen activator was also found to be up-regulated by nicotine in a dose-dependent manner (p < 0.05). CONCLUSION: Taken together, the results of the present study indicated that smoking modulation of bone destruction in periodontal disease may involve various osteolytic mediators, such as interleukin-1, interleukin-8, RANKL, MMP-2, MMP-9, and tissue-type plasminogen activator.

The upregulation of oncostatin M in inflamed human dental pulps.

AIM: To compare oncostatin M (OSM) expression in clinically healthy and inflamed specimened human pulp tissue. METHODOLOGY: Thirty pulpal tissue specimens (15 clinically healthy and 15 inflamed) were obtained from extracted third molars with informed consent from patients. The levels of OSM were compared between clinically healthy pulp and inflamed pulp tissues using the semi-quantitative reverse-transcriptase polymerase chain reaction. In addition, immunohistochemistry was used to identify the in situ localization of OSM expression in pulp specimens. For testing of differences in the OSM between the clinically healthy and inflamed human dental pulps, the Wilcoxon-Mann-Whitney rank sum test was applied. Differences in OSM expression between tissue with low and high levels of inflammation were subsequently analysed by Fisher's exact test. RESULTS: Inflamed pulps exhibited significantly higher OSM mRNA gene expression than clinically healthy pulps (P < 0.05). Immunohistochemistry demonstrated that OSM expression was significantly higher in inflamed than clinically healthy pulps (P < 0.05). OSM staining was detected in odontoblasts, fibroblasts, inflammatory infiltrates and endothelial cells. CONCLUSIONS: Oncostatin M expression was elevated in inflamed pulp tissue. OSM is potentially involved in the disease process of pulpal inflammation.

The upregulation of receptor activator NF-kappaB ligand expression by interleukin-1alpha and Porphyromonas endodontalis in human osteoblastic cells.

AIM: To investigate the receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. METHODOLOGY: The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1alpha and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t-test. RESULTS: IL-1alpha was found to upregulate RANKL production in U2OS cells (P < 0.05). Investigations of the time dependence of RANKL expression in IL-1alpha-treated cells revealed a rapid accumulation of RANKL protein after 1 h of exposure; it remained elevated throughout the 24-h incubation period shown by Western blot and ELISA. In addition, P. endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA (P < 0.05). CONCLUSIONS: IL-1alpha and P. endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.

The upregulation of cystatin C in human gingival fibroblasts stimulated with cyclosporine A.

BACKGROUND AND OBJECTIVE: Cystatin C is a 13 kDa non-glycosylated, basic protein belonging to the cystatin family. It is consistently and dramatically upregulated in a variety of fibrotic diseases. However, little is known about the correlation between cystatin C and cyclosporine A-induced gingival overgrowth. The aim of this study was to compare cystatin C expression in normal, healthy gingival tissues and cyclosporine A-induced gingival overgrowth specimens and further explore the potential mechanism that may result in cystatin C expression. MATERIAL AND METHODS: Fifteen cyclosporine A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Three human gingival fibroblast (HGF) strains were established from crown-lengthening surgery. The reverse transcriptase-polymerase chain reaction was used to investigate the effects on HGFs exposed to cyclosporine A. In addition, predominant periodontal pathogens (Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis) and proinflammatory cytokines (interleukin-1alpha and tumor necrosis factor-alpha) were added to seek the possible regulatory mechanisms of cystatin C expression. RESULTS: The cystatin C staining in gingival tissue was stronger in the cyclosporine A-induced gingival overgrowth group than in the normal gingival group (p < 0.05). Intensive staining for cystatin C expression was observed mainly in the cytoplasm of fibroblasts, epithelial cells and inflammatory cells. Moreover, cystatin C expression was significantly higher in cyclosporine A-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). A concentration of 200 ng/mL cyclosporine A was found to increase cystatin C expression in HGFs in a time-dependent manner (p < 0.05). The addition of periodontal pathogens and proinflammatory cytokines significantly increased the expression of cystatin C compared with cyclosporine A alone (p < 0.05). CONCLUSION: The increased ability of protein accumulation by cystatin C is one of several factors mediating cyclosporine A-induced gingival overgrowth. In addition, cyclosporine A may predispose to gingival overgrowth in inflammatory environments.

Immunohistochemical localization of oncostatin M in epithelialized apical periodontitis lesions.

AIM: To investigate the in situ location of oncostatin M (OSM) in epithelialized apical periodontitis lesions. METHODOLOGY: Thirty periapical lesions of pulpal origin were collected with informed consent from patients at the time of apical surgery. Tissue specimens were fixed with 10% buffered formalin overnight, dehydrated in an ascending series of graded alcohol, and embedded in paraffin. Five micron sections from formalin-fixed, paraffin-embedded specimens were examined by immunohistochemistry. In addition, another section from each specimen was stained with haematoxylin and eosin to assess the presence of inflammatory infiltrates. Differences in OSM expression between tissue with low and high levels of inflammation were subsequently analyzed by Fisher's exact test. RESULTS: Based on histological examination of haematoxylin and eosin stained sections, all specimens revealed the morphology of epithelialized apical periodontitis lesions. The results from immunohistochemistry demonstrated that OSM stain was detected in the inflammatory infiltrates, epithelium, connective tissue, and endothelium. The OSM signal was mainly expressed in endothelial cells (100%) followed by inflammatory cells (93.33%), epithelial cells (53.33%), and fibroblasts (16.67%). In addition, OSM expression was significantly higher in epithelialized apical periodontitis with higher levels of inflammatory infiltrates (P < 0.001). CONCLUSIONS: Oncostatin M was found to be expressed in epithelialized apical periodontitis lesions and would form part of the cytokine network involved in the disease process of apical periodontitis.

The up-regulation of type I plasminogen activator inhibitor in human gingival fibroblasts stimulated with cyclosporin A.

BACKGROUND AND OBJECTIVE: Cyclosporin A is used as an immunosuppressive agent and its prominent side-effect is the induction of fibrous gingival overgrowth. The progression of fibrous gingival overgrowth results from the accumulation of extracellular matrix. Type I plasminogen activator inhibitor (PAI-1) acts as the inhibitor of extracellular matrix degradation and is involved in some fibrotic diseases. However, little is known about the correlation between PAI-1 and cyclosporin A-induced gingival overgrowth. The aim of this study was to investigate the effects of cyclosporin A on the expression of PAI-1 mRNA and protein in human gingival fibroblasts human gingival fibroblasts in vitro and to compare PAI-1 expression in normal healthy gingival tissues and cyclosporin A-induced gingival overgrowth specimens in vivo. MATERIAL AND METHODS: Quantitative reverse transcription-polymerase chain reaction and western blot assay were used to investigate the effects on human gingival fibroblasts exposed to cyclosporin A. In addition, 10 cyclosporin A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. RESULTS: Investigations of the time dependence of PAI-1 mRNA expression in human gingival fibroblasts treated with 200 ng/ml of cyclosporin A revealed a rapid accumulation of the transcript: a significant signal was first detectable after 1 h of exposure and the signal remained elevated throughout the 24-h incubation period (p < 0.05). Cyclosporin A was also found to up-regulate PAI-1 protein in a time-dependent manner (p < 0.05). The PAI-1 staining in gingival tissue was stronger in the cyclosporin A-induced gingival overgrowth group than in the normal gingival group (p < 0.05). In the cyclosporin A-induced gingival overgrowth group, intensive staining for PAI-1 expression was observed mainly in the cytoplasm of fibroblasts, endothelial cells and inflammatory cells. CONCLUSION: These findings suggest that the up-regulation of PAI-1 may play an important part in the molecular pathogenesis of cyclosporin A-induced gingival overgrowth.

Mechanisms of cytotoxicity of eugenol in human osteoblastic cells in vitro.

AIM: To evaluate the mechanisms of cytotoxicity of eugenol in human osteoblastic cells in vitro. METHODOLOGY: Cytotoxicity and cell proliferation assays were performed to elucidate the toxic effects of eugenol on the human osteoblastic cell line U2OS. Furthermore, the effects of antioxidants catalase (scavenger of H2O2), superoxide dismutase (SOD, an extracellular superoxide free radical scavenger) and N-acetyl-L-cysteine (NAC, a cell-permeable glutathione precursor) were added to discover the possible mechanisms of eugenol-induced cytotoxicity. Paired Student's t-test was applied for the statistical analysis of the results. RESULTS: Eugenol demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P < 0.05). The 50% inhibition concentration of eugenol was approximately 0.75 mmol L(-1). Eugenol also inhibited cell proliferation during a 4-day culture period (P < 0.05). Addition of NAC extracellularly protected the cells from eugenol-induced cytotoxicity (P < 0.05). Neither, SOD nor catalase provided any protective effects on eugenol-induced cytotoxicity (P > 0.05). CONCLUSIONS: The levels of eugenol tested inhibited growth and proliferation of U2OS cells. Eugenol has significant potential for periapical toxicity. These inhibitory effects were associated with glutathione levels.

Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cells.

AIM: To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. METHODOLOGY: The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. RESULTS: The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P > 0.05). CONCLUSIONS: Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.

Upregulation of tissue-type plasminogen activator in inflamed human dental pulps.

AIM: To compare tissue-type plasminogen activator (t-PA) expression in normal human pulp and inflamed human pulp tissue specimens. METHODOLOGY: Thirty pulpal tissue specimens (13 normal and 17 inflamed pulps) were obtained from extracted third molars. The levels of t-PA between normal pulp and inflamed pulp tissues were compared using the quantitative reverse-transcriptase polymerase chain reaction analysis. In addition, immunohistochemistry was used to identify the in situ localization of t-PA expression in pulp specimens. Wilcoxon-Mann-Whitney rank sum test was applied for the statistical analysis of the results. RESULTS: t-PA mRNA gene was found more in inflamed pulps when compared with normal pulp tissue (P<0.05). The results from immunohistochemistry demonstrated that t-PA expression was significantly higher in the inflamed pulp (P=0.025). t-PA stain was detected in the fibroblasts, inflammatory infiltrates and endothelial cells. CONCLUSIONS: t-PA expression was significantly higher in inflamed pulp tissue. t-PA may play an important role in the pathogenesis of pulpal inflammation.

Induction of vascular endothelial growth factor expression in human pulp fibroblasts stimulated with black-pigmented Bacteroides.

AIM: To investigate the effect of black-pigmented Bacteroides on the expression of vascular endothelial growth factor (VEGF) gene in human pulp fibroblasts. METHODOLOGY: The supernatants of Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia were used to evaluate VEGF gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis. RESULTS: Black-pigmented Bacteroides induced significantly high levels of VEGF mRNA gene expression in human pulp fibroblasts (P < 0.05). In addition, the expression of VEGF depended on the bacteria tested. CONCLUSIONS: Black-pigmented Bacteroides may be involved in developing pulpal disease through the stimulation of VEGF production that would lead to the expansion of the vascular network coincident to progression of the inflammation.

The effect of thermocycling and dentine pre-treatment on the durability of the bond between composite resin and dentine.

The high bond strength between restorative resin and dentine plays an important role in long-term performance of restorations in the oral environment. A variety of treatment techniques have been described to enhance the bond strength of composite resin to dentine. Unfortunately, few studies have reported available bond durability of adhesive resins to dentine. The purpose of this research was to study the shear bond strength of composite resin to dentine pre-treated with phosphoric acid, self-etching agent or Nd:YAP laser irradiation. The durability of bond strength between resin and dentine stored in the artificial saliva thermocycling between 5 and 55 degrees C was also evaluated. The scanning electron microscope was used to assess the treated-dentine surfaces. The mean value of the shear bond strength in the acid-etching group (18.2 +/- 4.9 MPa) was the highest among the three dentine treatments (self-etching system: 12.6 +/-3.0 MPa, Nd:YAP laser: 13.4 +/- 3.3 MPa) prior to thermocycling. After thermocycling, shear strength values of all treated dentines decreased with increasing number of the cycles. When subjected to 3000 thermocycles, the mean bond strengths of these pre-treated samples to composite resin became 9.1 +/- 1.4, 7.8 +/- 1.8, and 8.1 +/- 1.7 MPa for acid-etching, self-etching and laser-irradiation, respectively, with a significant reduction of 38-50%.

Cytotoxic effects of gingival retraction cords on human gingival fibroblasts in vitro.

The objective of this study was to determine the cytocompatibility of three different extracts of gingival retraction cords and to compare the cytotoxic effect of these materials on human gingival fibroblasts. Gingival retraction cords impregnated with aluminium sulphate (Gingi-Aid), dl-adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-Plain) were eluted with culture medium for 10 min and 24 h. Cytotoxicity was judged using a tetrazolium bromide reduction assay. Our data demonstrated that gingival retraction cords applied alone almost completely inhibited cell viability (P < 0.05). In addition, the results also showed that the eluates from aluminium sulphate-impregnated cord, dl-adrenaline HCl-impregnated cord and non-drug-impregnated cord were cytotoxic to primary human gingival fibroblast cultures (P < 0.05). The cell viability of incubation of gingival fibroblasts containing 10-min eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 61, 21 and 70%, respectively. The cell viability of incubation of gingival fibroblasts containing 24 h eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 68, 58 and 72%, respectively. It was found that dl-adrenaline HCl-impregnated gingival retraction cord was the most toxic gingival retraction cord among the materials tested in all cultures (P < 0.05). The cytotoxicity decreased in an order of dl-adrenaline HCl-impregnated cord > aluminium sulphate-impregnated cord > non-drug-impregnated cord. The extent or degree of the cytotoxicity depended on the materials tested. Gingival retraction cords have significant potential for gingival toxicity. Careful management of gingiva retraction cords would lower the risk of potential gingival tissue damage during clinical application procedure and thus increase the success of prosthodontic procedures.

Induction of interleukin-8 gene expression by black-pigmented Bacteroides in human pulp fibroblasts and osteoblasts.

AIM: To investigate the effect of black-pigmented Bacteroides on the expression of interleukin (IL)-8 gene in human pulp fibroblasts and osteoblasts. METHODOLOGY: The supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-8 gene expression in human pulp fibroblasts and osteoblasts. The levels of mRNAs were measured by the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. RESULTS: Investigations of the time-dependence of IL-8 mRNA expression in black-pigmented Bacteroides-treated pulp fibroblasts and osteoblasts revealed a rapid accumulation of the transcript after 2 h of exposure, and remained elevated throughout the 24-h incubation period. In addition, IL-8 mRNA gene expression was also found in human osteoblasts stimulated with black-pigmented Bacteroides. However, black-pigmented Bacteroides was found to be more effective in the induction of IL-8 mRNA gene expression in osteoblasts than in pulp fibroblasts (P < 0.05). CONCLUSIONS: Black-pigmented Bacteroides are capable of amplifying the local immune response and promoting pulpal/periapical tissue inflammation by stimulating pulp fibroblasts and osteoblasts to express IL-8.

Induction of interleukin-6 gene expression by pro-inflammatory cytokines and black-pigmented Bacteroides in human pulp cell cultures.

AIM: To investigate the effect of pro-inflammatory cytokines and black-pigmented Bacteroides on the expression of IL-6 gene in human pulp fibroblasts. METHODOLOGY: IL-1alpha, tumour necrosis factor-alpha (TNF-alpha) and the supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-6 gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis. RESULTS: Investigations of the time dependence of IL-6 mRNA expression in pro-inflammatory cytokines-treated cells revealed a rapid accumulation of the transcript after 2 h of exposure and remained elevated throughout the 24-h incubation period. In addition, black-pigmented Bacteroides also induced IL-6 gene expression in human pulp fibroblasts. CONCLUSIONS: Pro-inflammatory cytokines and black-pigmented Bacteroides may be involved in developing pulpal inflammation through the stimulation of IL-6 production.