RESUMO
Adherence of Actinobacillus actinomycetemcomitans to human gingival fibroblast cells induces cytoskeletal reorganization. A. actinomycetemcomitans is considered a pathogenic bacteria involved in localized aggressive periodontitis. Studies with epithelial cells have shown an adherent capacity of bacteria that is increased under anaerobic conditions. For adherence to take place, there is a need for interaction between extracellular vesicles and bacterial fimbriae. However, molecular events associated with the adherence process are still unknown. The aim of this study was to investigate whether A. actinomycetemcomitans adherence to human gingival fibroblasts promotes cytoskeletal reorganization. Adherence was determined with light microscopy and scanning electron microscopy. For F-actin visualization, cells were treated with fluorescein-isothiocyanate-phalloidin and samples were examined with epifluorescence optics. Fluorescent was recorded on Kodak T-Max 400 film. We showed that A. actinomycetemcomitans adheres to human gingival fibroblast primary cultures, this property stimulating an increase in the intracellular calcium levels. In human gingival fibroblast primary cultures, we observed that maximal A. actinomycetemcomitans adherence took place 1.5h after culture infection occurred and remained for 6h. The adherence was associated with morphologic alterations and an increased in the intracellular calcium levels. These experiments suggest that A. actinomycetemcomitans adherence cause morphological alterations, induce actin stress fibers and recruitment of intracellular calcium levels.
Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Aderência Bacteriana/fisiologia , Citoesqueleto/metabolismo , Citoesqueleto/microbiologia , Fibroblastos/microbiologia , Fibroblastos/patologia , Gengiva/microbiologia , Aggregatibacter actinomycetemcomitans/ultraestrutura , Aderência Bacteriana/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Ácido Egtázico/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Gengiva/efeitos dos fármacos , Gengiva/patologia , Gengiva/ultraestrutura , Humanos , Microscopia de FluorescênciaRESUMO
Periodontal disease comprises a group of infections that lead to inflammation of the gingiva, periodontal tissue destruction, and in severe cases is accompanied by alveolar bone loss with tooth exfoliation. Actinobacillus actinomycetemcomitans is a Gram-negative microorganism, which possesses and produces lipopolysaccharide (LPS) molecules that play a key role in disease development. Human gingival fibroblasts are the major constituents of gingival connective tissue and may interact directly with bacteria and bacterial products including LPS. Flavonoids possess antioxidant and anti-inflammatory properties that reduce inflammatory molecule expression in macrophages and monocytes. In this study, we evaluated the ability of diverse flavonoids to regulate nitric oxide production of LPS-stimulated human gingival fibroblasts, and studied the effect of luteolin on diminish phosphorylation in mitogen-activated protein kinase (MAPK) family members as well as in protein kinase B (Akt), nuclear factor kappa B (NF-kappaB) activation, inducible nitric oxide synthase (NOS) expression, and nitric oxide (NO) synthesis. We also found that pretreatment with three flavonoids, including quercetin, genistein, and luteolin, blocked nitric oxide synthesis in a dose-dependent fashion. Luteolin exerted the strongest blocking action on expression of this inflammatory mediator. Luteolin pretreatment attenuated LPS-induced extracellular signal-regulated kinase, p38, and Akt phosphorylation. LPS treatment of human gingival fibroblasts resulted in NF-kappaB translocation. Cell pretreatment with luteolin abolished LPS effects on NF-kappaB translocation. In addition, luteolin treatment blocked LPS-induced cellular proliferation inhibition without affecting genetic material integrity. We concluded that luteolin interferes with LPS signaling pathways, reducing activation of several mitogen-activated protein kinase family members, and inhibits inflammatory mediator expression.
Assuntos
Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Luteolina/farmacologia , Polissacarídeos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Flavonoides/farmacologia , Gengiva/citologia , Gengiva/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Morfolinas/farmacologia , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Tyrosine phosphorylation is an early step in lipopolysaccharide (LPS) stimulated monocytes and macrophages that appears to play a key role in signal transduction. We have demonstrated that LPS purified from Actinobacillus actinomycetemcomitans also increases protein tyrosine phosphorylation in human gingival fibroblasts (HGF). This effect was elicited rapidly after LPS stimulation at concentrations that stimulate anti-bacterial responses in human gingival fibroblasts. Two main proteins, with an apparent molecular weight of 44 and 42 kDa, were phosphorylated after LPS stimulation of the human gingival fibroblasts. The phosphorylation was detected after 5 to 15 min and reached the maximum at 30 min of treatment. The increase in tyrosine phosphorylation was apparent following stimulation with LPS at 10 ng/ml and the response was dose dependent up to 10 microg/ml. Pretreatment with the tyrosine kinase inhibitors, herbimycin A and genistein inhibited the LPS-stimulated phosphorylation of p44 and p42 MAP kinases in a dose dependent manner. Pretreatment of human gingival fibroblasts with antibodies anti-CD14 or anti-TLR-4 but not anti-TLR-2 inhibited the LPS-induced tyrosine phosphorylation of p44 and p42. Additionally, LPS-induced p44 and p42 phosphorylation was inhibited by polymyxin treatment. These findings demonstrate that LPS from A. actinomycetemcomintans increases rapidly p44 and p42 phosphorylation (ERK 1 and ERK 2, respectively) in human gingival fibroblasts. Our data also suggest that CD14 and TLR-4 receptors are involved in the LPS effects in human gingival fibroblasts.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Gengiva/efeitos dos fármacos , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor 4 Toll-Like/metabolismo , Antibacterianos/farmacologia , Anticorpos Bloqueadores/farmacologia , Benzoquinonas , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Genisteína/farmacologia , Gengiva/citologia , Gengiva/metabolismo , Humanos , Lactamas Macrocíclicas , Receptores de Lipopolissacarídeos/imunologia , Fosforilação , Polimixinas/farmacologia , Quinonas/farmacologia , Rifabutina/análogos & derivados , Receptor 4 Toll-Like/imunologiaRESUMO
Periodontal disease, a gingival inflammatory disease caused by gram-negative bacteria, is the main cause of tooth loss. Lipopolysaccharides (LPS) present in bacterial cell walls induce human gingival fibroblasts' production of pro-inflammatory cytotoxins such as IL-1beta and TNFalpha. The goal of this study was to determine p38 role in the expression of inducible nitric oxide synthase enzyme (i-NOS) and cyclooxygenase (COX-2), as well as in PGE(2) and nitric oxide synthesis in human gingival fibroblasts challenged with LPS. We found that lipopolysaccharides induced a rapid and significant increase in p38 phosphorylation. After interruption of p38 transduction pathway by pre-treatment with inhibitor SB203580, no response to stimulation with LPS was observed; i-NOS expression and nitric oxide synthesis was completely blocked. However, p38 inhibition only partially blocked COX-2 expression and PGE2 synthesis. We conclude that p38 is critically involved in i-NOS induction, and that it participates in COX-2 expression and in PGE2 synthesis.