Efeito de uma nova Chalcona sobre a reabsorção óssea inflamatória: estudo in vitro e in vivo
Carregando...
Data
2020-03-26
Autores
Fernandes, Natalie Aparecida Rodrigues
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
As chalconas são um grupo de compostos fenólicos derivados de plantas, com diversas propriedades biológicas. Em função dos seus efeitos farmacológicos, diferentes derivados chalcônicos sintéticos, baseados em seus análogos naturais, têm sido investigados como agentes terapêuticos. Estudos clínicos e pré-clínicos têm demonstrado sua efetividade no tratamento de doenças inflamatórias como câncer e artrite, e em patologias ósseas como osteoporose e tumores ósseos. Considerando suas propriedades biológicas e com objetivo de identificar compostos que possam atuar no tratamento de doenças ósseo inflamatórias, avaliamos a habilidade de um novo composto chalcônico, a Chalcona T4, de suprimir a inflamação e osteoclastogênese em um modelo de doença periodontal. No estudo in vivo, um ensaio de toxicidade em camundongos demonstrou que a administração de diferentes doses da chalcona T4 (5, 50, 100 e 200 mg/kg) por um período de 15 dias, via intragástrica, diariamente, não causou alterações físicas ou comportamentais. Análise histopatológica do rim, fígado e estômago destes animais também indicaram ausência de toxicidade em todas as doses avaliadas. Determinada a ausência de toxicidade, periodontite foi experimentalmente induzida em ratos pela colocação de ligaduras ao redor dos primeiros molares inferiores. Chalcona T4 foi administrada diariamente por gavagem intragástrica em duas doses (5 e 50 mg/kg) durante 15 dias. Ao final do período experimental, as amostras contendo tecido gengival ou hemimandíbula da região de interesse foram removidos e utilizados para análise dos seguintes desfechos: determinação da expressão de TNF-α (ELISA); quantificação dos componentes celulares (infiltrado celular, fibras colágenas e vasos sanguíneos) através da análise estereométrica; avaliação da extensão da perda óssea (coloração com azul de metileno e microtomografia computadorizada (μCT)); expressão e imunolocalização de NFATc1, NF-kB e CD45 (imuno-histoquímica), ativação de p38 MAPK (Western Blot); e análise microbiológica das ligaduras (Pg, Fn, Aa) através de RT-qPCR. No estudo in vitro, células RAW 264.7 foram pré-tratadas com diferentes concentrações da chalcona (1, 2.5, 5 e 10 μM) e estimuladas com RANKL para avaliação da expressão dos genes alvos (Mmp-9, Ctsk, Oscar, Nfatc1 e Trap). Os efeitos da chalcona sobre a diferenciação e atividade dos osteoclastos foram avaliados por microscopia de fluorescência e medição das áreas de reabsorção em placas revestidas com fosfato de cálcio sintético, respectivamente. Seu impacto sobre as vias de sinalização NF-ĸB, p38 MAPK, Erk e AKT foram avaliados por Western Blot. Os resultados in vivo indicam que a menor dose da Chalcona T4 reduziu a reabsorção óssea e o infiltrado inflamatório, bem como favoreceu a angiogênese e produção de matriz colágena nos tecidos gengivais periodontalmente doentes, além de modular a expressão de NF-kB. In vitro, a Chalcona T4 foi capaz de inibir a diferenciação e atividade dos osteoclastos, reduzir de maneira dose-dependente a expressão de marcadores da osteoclastogênese, e modular a ativação de vias de sinalização intracelular (ERK, AKT e NF-ҝB). Estes resultados demonstram a habilidade da Chalcona T4 na modulação da resposta imune inflamatória e osteoclastogênese, e indicam o potencial do composto no tratamento de doenças ósseo inflamatórias.
Chalcones are a group of phenolic compounds derived from plants with diverse biological properties. Due to their pharmacological effects, different synthetic chalconic derivatives, based on their natural analogues, have been investigated as therapeutic agents. Clinical and preclinical studies have demonstrated its effectiveness in the treatment of inflammatory diseases such as cancer and arthritis, and in bone pathologies such as osteoporosis and bone tumors. Considering its biological properties and aiming to identify compounds that can act in the treatment of inflammatory bone diseases, we evaluated the ability of a new chalconic compound, Chalcone T4, to suppress inflammation and osteoclastogenesis in a model of periodontal disease. In the in vivo study, a toxicity test in mouse demonstrated that the daily intragastric gavage administration of different doses of chalcone T4 (5, 50, 100 and 200 mg/kg) for a period of 15 days, did not cause any physical or behavioral changes. Histopathological analysis of the kidney, liver and stomach of these animals also indicated absence of toxicity in all evaluated doses. Once the absence of toxicity was determined, periodontitis was experimentally induced in rats by ligature placement around the lower first molars. Chalcone T4 was administered daily by intragastric gavage in two doses (5 and 50 mg/kg) for 15 days. At the end of the experimental period, samples containing gingival or hemimandibular tissue from the region of interest were removed and used for analysis of the following outcomes: determination of TNF-α expression (ELISA); quantification of cellular components (cell infiltrate, collagen fibers and blood vessels) by stereometric analysis; evaluation of the extent of bone loss (methylene blue dye staining and computed microtomography (μCT)); expression of NFATc1, NF-kB and CD45 (immunohistochemistry), activation of p38 MAPK (Western Blot); and microbiological analysis of the ligatures (Pg, Fn, Aa) by RT-qPCR. In the in vitro study, RAW 264.7 cells were pretreated with different concentrations of chalcone (1, 2.5, 5 and 10 μM) and stimulated with RANKL to evaluate the expression of target genes (Mmp-9, Ctsk, Oscar, Nfatc1 and Trap). The effects of chalcone on osteoclast differentiation and activity were evaluated by fluorescence microscopy and measurement of resorption areas on plates coated with synthetic calcium phosphate, respectively. Their impact on the NF-ĸB, p38 MAPK, Erk and AKT signaling pathways were assessed by Western Blot. The in vivo results indicate that the lowest dose of Chalcone T4 reduced bone resorption and inflammatory infiltrate, as well as favoring angiogenesis and collagen matrix production in the gingival tissues, in addition to modulating NF-kB expression. In vitro, Chalcone T4 was able to inhibit osteoclast differentiation and activity, reduce dose-dependent expression of osteoclastogenesis markers, and modulate the activation of intracellular signaling pathways (ERK, AKT and NF-kB). These results demonstrate the ability of Chalcone T4 in modulating the inflammatory immune response and osteoclastogenesis and indicate the potential of the compound in the treatment of inflammatory bone diseases.
Chalcones are a group of phenolic compounds derived from plants with diverse biological properties. Due to their pharmacological effects, different synthetic chalconic derivatives, based on their natural analogues, have been investigated as therapeutic agents. Clinical and preclinical studies have demonstrated its effectiveness in the treatment of inflammatory diseases such as cancer and arthritis, and in bone pathologies such as osteoporosis and bone tumors. Considering its biological properties and aiming to identify compounds that can act in the treatment of inflammatory bone diseases, we evaluated the ability of a new chalconic compound, Chalcone T4, to suppress inflammation and osteoclastogenesis in a model of periodontal disease. In the in vivo study, a toxicity test in mouse demonstrated that the daily intragastric gavage administration of different doses of chalcone T4 (5, 50, 100 and 200 mg/kg) for a period of 15 days, did not cause any physical or behavioral changes. Histopathological analysis of the kidney, liver and stomach of these animals also indicated absence of toxicity in all evaluated doses. Once the absence of toxicity was determined, periodontitis was experimentally induced in rats by ligature placement around the lower first molars. Chalcone T4 was administered daily by intragastric gavage in two doses (5 and 50 mg/kg) for 15 days. At the end of the experimental period, samples containing gingival or hemimandibular tissue from the region of interest were removed and used for analysis of the following outcomes: determination of TNF-α expression (ELISA); quantification of cellular components (cell infiltrate, collagen fibers and blood vessels) by stereometric analysis; evaluation of the extent of bone loss (methylene blue dye staining and computed microtomography (μCT)); expression of NFATc1, NF-kB and CD45 (immunohistochemistry), activation of p38 MAPK (Western Blot); and microbiological analysis of the ligatures (Pg, Fn, Aa) by RT-qPCR. In the in vitro study, RAW 264.7 cells were pretreated with different concentrations of chalcone (1, 2.5, 5 and 10 μM) and stimulated with RANKL to evaluate the expression of target genes (Mmp-9, Ctsk, Oscar, Nfatc1 and Trap). The effects of chalcone on osteoclast differentiation and activity were evaluated by fluorescence microscopy and measurement of resorption areas on plates coated with synthetic calcium phosphate, respectively. Their impact on the NF-ĸB, p38 MAPK, Erk and AKT signaling pathways were assessed by Western Blot. The in vivo results indicate that the lowest dose of Chalcone T4 reduced bone resorption and inflammatory infiltrate, as well as favoring angiogenesis and collagen matrix production in the gingival tissues, in addition to modulating NF-kB expression. In vitro, Chalcone T4 was able to inhibit osteoclast differentiation and activity, reduce dose-dependent expression of osteoclastogenesis markers, and modulate the activation of intracellular signaling pathways (ERK, AKT and NF-kB). These results demonstrate the ability of Chalcone T4 in modulating the inflammatory immune response and osteoclastogenesis and indicate the potential of the compound in the treatment of inflammatory bone diseases.
Descrição
Palavras-chave
Chalcona, Reabsorção óssea, Doença periodontal, Ratos, Macrófagos, Osteoclastos, Chalcone, Bone resorption, Periodontal disease, Rats, Macrófagos, Osteoclasts.