RESUMO
Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 µM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 µM (p<0.05) and 250 µM (p<0.01). The POH increased ROS production at both 10 µM and 100 µM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 µM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 µM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.
Assuntos
Antibacterianos/farmacologia , Fusobacterium nucleatum/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Monoterpenos/farmacologia , Porphyromonas/efeitos dos fármacos , Espécies Reativas de Oxigênio/análise , Animais , Arginase/análise , Produtos Biológicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Fusobacterium nucleatum/crescimento & desenvolvimento , Expressão Gênica , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Porphyromonas/crescimento & desenvolvimento , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fatores de Tempo , Fator de Necrose Tumoral alfa/análiseRESUMO
Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.
Assuntos
Animais , Camundongos , Fusobacterium nucleatum/efeitos dos fármacos , Espécies Reativas de Oxigênio/análise , Porphyromonas/efeitos dos fármacos , Monoterpenos/farmacologia , Macrófagos/efeitos dos fármacos , Antibacterianos/farmacologia , Arginase/análise , Fatores de Tempo , Produtos Biológicos/farmacologia , Testes de Sensibilidade Microbiana , Expressão Gênica , Lipopolissacarídeos/farmacologia , Reprodutibilidade dos Testes , Fator de Necrose Tumoral alfa/análise , Fusobacterium nucleatum/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Porphyromonas/crescimento & desenvolvimento , Proliferação de Células/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Citometria de Fluxo , Células RAW 264.7 , Macrófagos/metabolismoRESUMO
Candida albicans (Ca), Staphylococcus aureus (Sa), Streptococcus sanguis (Ss), Actinomyces naeslundii (An), Actinomyces odontolyticus (Ao), Porphyromona spp (P spp), Candida glabrata (Cg), Candida krusei (Ck), and Rhodotorula spp (R spp) were tested with equal pieces of biodegradable membranes. Membranes pretreated with saliva or clorhexidine and nontreated control membranes were tested in three different culture media containing 0.1 mL homologous suspension for each strain under study. Incubation was performed at 37 degrees C for 48 hours for aerobiosis and for five days for anaerobiosis. Macroscopy and microscopy were carried out. Membranes were removed, washed, and resuspended. Samples were sonicated, and the supernatant was disseminated on brain heart infusion broth or blood agar. Incubation was repeated, colony-forming unit counts were performed, and statistical analysis was carried out using analysis of variance transforming results to Log10 (x + 1), the highest interaction level was used to calculate standard error. Orthogonal contrast was used to compare the different microorganisms under study. Highest adhesion was found with Ca, Cg, Ck, Sa, and Ss. A sufficient quantity of Actinomyces could not be recovered from the membranes. Results with P spp were poor, confirming lower gram-negative adhesion. Replicate flasks with Ss and Ca were cultivated. Membranes were removed after washing and subjected to scanning electron microscopy, as were untreated control pieces. A cavelike surface was observed. Streptococcus sanguis adhering to the membranes showed extracellular projections. Candida and gram-positive cocci showed great recovery capacity.
Assuntos
Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Implantes Dentários/microbiologia , Membranas Artificiais , Boca/microbiologia , Actinomyces/crescimento & desenvolvimento , Actinomyces/fisiologia , Análise de Variância , Biodegradação Ambiental , Candida/crescimento & desenvolvimento , Candida/fisiologia , Contagem de Colônia Microbiana , Meios de Cultivo Condicionados , Microscopia Eletrônica de Varredura , Porphyromonas/crescimento & desenvolvimento , Porphyromonas/fisiologia , Rhodotorula/crescimento & desenvolvimento , Rhodotorula/fisiologia , Staphylococcus/crescimento & desenvolvimento , Staphylococcus/fisiologia , Streptococcus/crescimento & desenvolvimento , Streptococcus/fisiologiaRESUMO
A small animal model was evaluated to study the interrelationships between microorganisms after their implantation in root canals (inferior central incisors) using germ-free (GF) and conventional (CV) mice. The selected microorganisms were: Porphyromonas endodontalis (ATCC 35406), Eubacterium lentum (ATCC 25559), Peptostreptococcus anaerobius (ATCC 27337), Fusobacterium nucleatum (ATCC 10953), Escherichia coli (ATCC 25922), and Enterococcus faecalis (ATCC 4083). Only P. anaerobius, E. coli, and E. faecalis, respectively, were able to colonize when inoculated alone into the root canal of both CV and GF mice. E. lentum, when inoculated alone colonized only in CV animals. P. endodontalis and F. nucleatum were unable to colonize in CV and GF animals after single inoculation. It is concluded that the experimental animal model presented herein is valuable for ecological studies of root canal infections and that only some strict anaerobic bacteria are able to colonize mice root canals when inoculated by themselves alone in pure culture.