Modulação da doença periodontal por curcumin modificado quimicamente. Estudo de dose-resposta em roedor
Arquivos
Data
2016-03-18
Autores
Brandão, Dayane de Almeida [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Curcumin é um polifenol amarelo extraído do rizoma de uma planta tropical, do tipo herbácea. Possui ações anti-inflamatória, antioxidante, antiangiogênica, imunomodulatória, citotóxica, antimicrobiana e antiapoptótica. A aplicação terapêutica do curcumin vem sendo avaliada em modelos pré-clínicos e estudos de várias doenças. As limitações da eficácia do curcumin in vivo são atribuídas à sua má solubilidade em veículos aquosos e baixa taxa de absorção no trato gastrointestinal. O objetivo desse trabalho foi avaliar o efeito dose-resposta do composto sintético análogo ao curcumin (CMC2.24) em diferentes doses (1 mg, 3 mg, 10 mg, 30 mg) sobre o processo inflamatório e osteoclastogênese em modelo de doença periodontal in vivo e in vitro, para analisar qual é a dose mínima necessária para que o composto tenha o efeito biológico. A doença periodontal foi induzida em ratos por meio de injeção de 30 µg de LPS de Escherichia coli, realizadas 3x/semana durante 4 semanas. Os controles receberam injeções do mesmo volume do veículo de diluição do LPS (PBS). A administração de CMC2.24 (1, 3, 10 e 30mg/kg) foi feita por via intragástrica (gavagem oral) diariamente, durante 29 dias (um dia antes da primeira injeção de LPS/PBS). A expressão de fosfatase ácido tartarato resistente (TRAP), indicativa de diferenciação osteoclástica, foi avaliada por meio de imunohistoquímica, a proporção de células inflamatórias, em cortes corados com H/E, por estereometria, a presença de citocinas através do PCR em tecido gengival e a reabsorção óssea por análise de coloração por azul de metileno e microtomografia computadorizada. Nos experimentos in vitro, macrófagos foram estimulados com microrganismos e tratados com CMC2.24 em concentração de 1, 3, 10 e 30 µM. A expressão gênica foi avaliada através de PCR e ELISA, enquanto fagocitose e quantificação de espécies reativas de oxigênio foram avaliadas por atividade fagocitária e produção de ROS, respectivamente. De acordo com os resultados, CMC2.24 reduziu significativamente o infiltrado inflamatório. Além disso, CMC diminuiu significativamente a quantidade de células TRAP positiva a partir de 3 mg/kg e a perda óssea alveolar, a partir de 1 mg/kg. In vitro, experimentos utilizando macrófagos demonstraram que o CMC inibe a produção de TNF e IL-10 após estímulos microbianos. Além disso, CMC2.24 também inibiu atividade fagocitária e estimulou a produção de ROS. CMC2.24 em diferentes doses demonstrou potencial terapêutico para aplicação em doença periodontal, devido à inibição da resposta inflamatória e também foi efetivo na redução da reabsorção óssea inflamatória.
Curcumin is a yellowish polyphenol extracted from the rizome of a herbaceous tropical plant. It has multiple biological activities described, including anti-inflammatory, antioxidant, antiangiogenic, immunomodulatory, cytotoxic, antimicrobial and pro- and antiapoptotic activities. Its therapeutic application is actively evaluated in preclinical models and clinical studies of various diseases. In vivo use of curcumin is limited by its pharmacodynamic propereties, such as poor solubility in aqueous vehicles, low absorption rate in the gastrointestinal tract and short half-life in the peripheral circulation. The aim of this study was to evaluate the dose-response effect of a synthetic compound that is structurally to natural curcumin (CMC2.24) in a model of experimental periodontal disease that mimics the host-microbial interaction and the resultant inflammation and bone resorption that are hallmarks of this conditions in humans. Periodontal disease was induced in mice by injection of 30 ug LPS of Escherichia coli, carried out 3x / week for 4 weeks. Controls received injections of the same volume of vehicle (PBS). Administration of CMC2.24 (1, 3, 10 and 30mg / kg) was performed daily (starting the day before the first LPS/PBS injection) by oral gavage (1, 3, 10 and 30 mg/Kg) during the whole experimental period. Immunohistochemical detection of tartrate-resistant acid phosphatase (TRAP) was used to identify osteoclasts in the area of interest; the inflammatory infiltrate was assessed by stereometric/morphological analysis in H/E-stained sections; expression of candidate inflammatory cytokines in the gingival tissue determined by RT-PCR and the extent of inflammatory bone resorption quantitated macroscopic analysis of the area of exposed root surface and also by computed microtomography. In in vitro experiments, murine macrophages were stimulated with periodontal diseaseassociated microorganisms in the presence and absence of CMC2.24 (1, 3, 10 and 30 uM). Gene expression was assessed by RT-PCR and ELISA; phagocytic activity was studied by immunocytofluorescence and production of reactive oxygen species was determined by a biochemical assay using a ROS-substrate that emit fluorescence upon cleavage. In vivo, administration of CMC2.24 significantly reduced the inflammatory infiltrate, and the number of osteoclasts starting at the 3 mg/Kg dosage; whereas inflammatory bone resorption was significantly inhibited already at the 1 mg/Kg dosage. In vitro, pre-treatment of macrophages with CMC2.24 markedly inhibited the production of TNF and IL-10 after microbial stimuli. CMC2.24 also inhibited phagocytic activity and stimulated production of ROS. We conclude that CMC2.24 at low doses (3 mg/Kg) has therapeutic potential for application in periodontal disease, due to inhibition of microbial-related inflammation.
Curcumin is a yellowish polyphenol extracted from the rizome of a herbaceous tropical plant. It has multiple biological activities described, including anti-inflammatory, antioxidant, antiangiogenic, immunomodulatory, cytotoxic, antimicrobial and pro- and antiapoptotic activities. Its therapeutic application is actively evaluated in preclinical models and clinical studies of various diseases. In vivo use of curcumin is limited by its pharmacodynamic propereties, such as poor solubility in aqueous vehicles, low absorption rate in the gastrointestinal tract and short half-life in the peripheral circulation. The aim of this study was to evaluate the dose-response effect of a synthetic compound that is structurally to natural curcumin (CMC2.24) in a model of experimental periodontal disease that mimics the host-microbial interaction and the resultant inflammation and bone resorption that are hallmarks of this conditions in humans. Periodontal disease was induced in mice by injection of 30 ug LPS of Escherichia coli, carried out 3x / week for 4 weeks. Controls received injections of the same volume of vehicle (PBS). Administration of CMC2.24 (1, 3, 10 and 30mg / kg) was performed daily (starting the day before the first LPS/PBS injection) by oral gavage (1, 3, 10 and 30 mg/Kg) during the whole experimental period. Immunohistochemical detection of tartrate-resistant acid phosphatase (TRAP) was used to identify osteoclasts in the area of interest; the inflammatory infiltrate was assessed by stereometric/morphological analysis in H/E-stained sections; expression of candidate inflammatory cytokines in the gingival tissue determined by RT-PCR and the extent of inflammatory bone resorption quantitated macroscopic analysis of the area of exposed root surface and also by computed microtomography. In in vitro experiments, murine macrophages were stimulated with periodontal diseaseassociated microorganisms in the presence and absence of CMC2.24 (1, 3, 10 and 30 uM). Gene expression was assessed by RT-PCR and ELISA; phagocytic activity was studied by immunocytofluorescence and production of reactive oxygen species was determined by a biochemical assay using a ROS-substrate that emit fluorescence upon cleavage. In vivo, administration of CMC2.24 significantly reduced the inflammatory infiltrate, and the number of osteoclasts starting at the 3 mg/Kg dosage; whereas inflammatory bone resorption was significantly inhibited already at the 1 mg/Kg dosage. In vitro, pre-treatment of macrophages with CMC2.24 markedly inhibited the production of TNF and IL-10 after microbial stimuli. CMC2.24 also inhibited phagocytic activity and stimulated production of ROS. We conclude that CMC2.24 at low doses (3 mg/Kg) has therapeutic potential for application in periodontal disease, due to inhibition of microbial-related inflammation.
Descrição
Palavras-chave
Periodontia, Curcumina, Reabsorção óssea, Periodontics, Bone resorption