RESUMO
Streptococcus mutans is the main early colonizing cariogenic bacteria because it recognizes salivary pellicle receptors. The Antigen I/II (Ag I/II) of S. mutans is among the most important adhesins in this process, and is involved in the adhesion to the tooth surface and the bacterial co-aggregation in the early stage of biofilm formation. However, this protein has not been used as a target in a virtual strategy search for inhibitors. Based on the predicted binding affinities, drug-like properties and toxicity, molecules were selected and evaluated for their ability to reduce S. mutans adhesion. A virtual screening of 883,551 molecules was conducted; cytotoxicity analysis on fibroblast cells, S. mutans adhesion studies, scanning electron microscopy analysis for bacterial integrity and molecular dynamics simulation were also performed. We found three molecules ZINC19835187 (ZI-187), ZINC19924939 (ZI-939) and ZINC19924906 (ZI-906) without cytotoxic activity, which inhibited about 90% the adhesion of S. mutans to polystyrene microplates. Molecular dynamic simulation by 300 nanoseconds showed stability of the interaction between ZI-187 and Ag I/II (PDB: 3IPK). This work provides new molecules that targets Ag I/II and have the capacity to inhibit in vitro the S. mutans adhesion on polystyrene microplates.
Assuntos
Antígenos de Bactérias/imunologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Fibroblastos/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Streptococcus mutans/efeitos dos fármacos , Proteínas de Bactérias/imunologia , Biofilmes/efeitos dos fármacos , Células Cultivadas , Simulação por Computador , Fibroblastos/imunologia , Fibroblastos/microbiologia , Humanos , Técnicas In Vitro , Ligamento Periodontal/imunologia , Ligamento Periodontal/microbiologia , Streptococcus mutans/crescimento & desenvolvimento , Streptococcus mutans/imunologiaRESUMO
BACKGROUND: Porphyromonas gingivalis (Pg) is a major periodontal pathogen that contains immunostimulatory components. Periodontal ligament mesenchymal stem cells (PDLMSCs) are responsible for regeneration of the periodontium that is lost due to periodontitis. Pathologic factors within the microenvironment that impair resident PDLMSCs are not well understood. The present study investigates in vitro the effects of Pg protein extract (PgPE) on biologic properties of CD105-enriched PDL progenitor cell populations (PDL-CD105+). METHODS: Five populations of PDL-CD105+ cells were exposed to PgPE and assessed for cell viability, apoptosis, and proinflammatory gene expression (interleukin-1ß [IL-1ß], tumor necrosis factor-alpha [TNF-α], and IL-6) by quantitative reverse transcription polymerase chain reaction, IL-6 immunostaining, activation of IL-6/signal transducer and activator of transcription (STAT) 3 signaling pathway, and osteogenic differentiation potential. RESULTS: PgPE treatment (2 µg/mL) did not affect cell viability or survival but induced a significant increase in IL-1ß, TNF-α, and IL-6 messenger RNA (mRNA) expression and positive staining for IL-6. A total of 29 genes from the IL-6/STAT3 pathway were upregulated on PgPE stimulation. These genes are related to biologic processes involved in the control of cell survival (B-cell lymphoma 2 [BCL2]), cell proliferation (hepatocytehepatocyte growth factor), cytokine-mediated signaling pathway (suppressor of cytokine signaling 3, C-X-C ligand 8 [CXCL8]), and response to stress (CXCL8, mitogen-activated protein kinase 3, BCL2-associated X protein, and BCL2). Additionally, PgPE treatment caused an increase in alkaline phosphatase mRNA expression in PDL-CD105+ cells after 7 days of osteogenic induction, although mineral nodule formation was comparable to the control group. CONCLUSIONS: These results suggest that the inflammatory profile induced by PgPE treatment in PDL-CD105+ cells did not affect cell viability, apoptosis, or osteogenic differentiation, perhaps due to increased expression of genes involved in the control of cell proliferation and protection against cell death.
Assuntos
Proteínas de Bactérias/farmacologia , Diferenciação Celular , Células-Tronco Mesenquimais/fisiologia , Osteogênese , Ligamento Periodontal/crescimento & desenvolvimento , Porphyromonas gingivalis/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Microambiente Celular , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/microbiologia , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/microbiologia , Adulto JovemRESUMO
OBJECTIVES: This in vitro study was established to examine whether visfatin thought to be a link between periodontitis and obesity is produced by periodontal ligament (PDL) cells and, if so, whether its synthesis is modulated by microbial and/or biomechanical signals. MATERIALS AND METHODS: PDL cells seeded on BioFlex® plates were exposed to the oral pathogen Fusobacterium nucleatum ATCC 25586 and/or subjected to biomechanical strain for up to 3 days. Gene expression of visfatin and toll-like receptors (TLR) 2 and 4 was analyzed by RT-PCR, visfatin protein synthesis by ELISA and immunocytochemistry, and NFκB nuclear translocation by immunofluorescence. RESULTS: F. nucleatum upregulated the visfatin expression in a dose- and time-dependent fashion. Preincubation with neutralizing antibodies against TLR2 and TLR4 caused a significant inhibition of the F. nucleatum-upregulated visfatin expression at 1 day. F. nucleatum stimulated the NFκB nuclear translocation. Biomechanical loading reduced the stimulatory effects of F. nucleatum on visfatin expression at 1 and 3 days and also abrogated the F. nucleatum-induced NFκB nuclear translocation at 60 min. Biomechanical loading inhibited significantly the expression of TLR2 and TLR4 at 3 days. The regulatory effects of F. nucleatum and/or biomechanical loading on visfatin expression were also observed at protein level. CONCLUSIONS: PDL cells produce visfatin, and this production is enhanced by F. nucleatum. Biomechanical loading seems to be protective against the effects of F. nucleatum on visfatin expression. CLINICAL RELEVANCE: Visfatin produced by periodontal tissues could play a major role in the pathogenesis of periodontitis and the interactions with obesity and other systemic diseases.