Avaliação do efeito combinado da fotobiomodulação e da naringenina na atividade de metaloproteinase da matriz por fibroblastos gengivais
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2022-08-12
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Universidade Estadual Paulista (Unesp)
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A mucosite oral (MO) é uma das principais complicações agudas da quimioterapia ou da radioterapia de cabeça e pescoço podendo ocasionar interrupções e atrasos no tratamento oncológico impactando a qualidade de vida e sobrevida destes pacientes. Seu tratamento ainda é controverso e baseado principalmente em terapias paliativas que apenas reduzem a sintomatologia dolorosa, baseado nisso, torna-se fundamental a avaliação de novas estratégias terapêuticas que atuem prevenindo ou tratando de forma efetiva as lesões. Levando em conta que estudos preliminares sugerem que a aplicação isolada da FBM e dos compostos de NA sobre fibroblastos gengivais resultaram em redução da expressão e síntese de metaloproteinase da matriz (MMP), é esperado que a combinação de ambas as propostas terapêuticas possa resultar em um efeito sinérgico, acelerando o processo de reparo da MO. Dessa forma, o objetivo deste trabalho foi determinar a dose da FBM com LBI necessária para modular a síntese de MMP-2 (estudo 1) e avaliar o efeito combinado da aplicação da NA e da FBM na modulação da síntese e atividade de MMP-2, IL-6 e TIMP-1 por fibroblastos gengivais humanos (HGF), bem como sobre funções celulares relacionadas ao processo de reparo tecidual (estudo 2). Foram utilizadas culturas primárias de HGF que foram obtidas de uma doadora voluntária de acordo com o Comitê de Ética em Pesquisas em Humanos. O fator de necrose tumoral alfa (TNF-α) foi utilizado como controle positivo, por induzir a síntese de MMPs em HGF. Para a irradiação com LBI utilizou-se o dispositivo LASERTable (InGaAsP; 780 nm; 0,025 W; estudo 1: 0,5, 2 e 4 J/cm2; estudo 2: 4 J/cm2). Para o estudo 1, a análise da viabilidade celular foi realizada por meio do ensaio colorimétrico de metiltetrazolium (MTT) e avaliação da síntese de MMP-2 foi realizada por meio do imunoensaio ELISA. Para o estudo 2, utilizou-se a NA sintética, na concentração de 10μg/mL, baseado em estudos anteriores. O efeito da combinação da NA e da FBM na proliferação de HGF foi realizado por meio do ensaio PrestoBlue, na migração por meio do ensaio de cicatrização in vitro, na síntese de MMP-2, IL-6 e TIMP-1 por HGF por meio de imunoensaio ELISA e na atividade gelatinolítica de HGF por meio de zimografia in situ. Os dados foram submetidos à análise de normalidade e homocedasticidade para seleção dos testes estatísticos adequados (ANOVA, Tukey). Os dados da atividade gelatinolítica foram avaliados qualitativamente e apresentados de forma descritiva. Todas as inferências estatísticas foram feitas considerando o nível de significância de 5%. Os experimentos foram realizados em duplicatas, com N=6. De acordo com os resultados obtidos do estudo 1, a terapia com FBM 4 J/cm2 aumentou significativamente a viabilidade dos HFG na presença ou ausência de TNF-α em relação ao controle negativo (p<0,05) e os grupos tratados com FBM 2 J/cm2 e 4 J/cm2 associada ou não ao TNF-α apresentaram diminuição significativa da síntese de MMP- 2 em relação ao controle negativo (p<0,05). Sendo assim, a densidade de 4 J/cm2 foi selecionada para o estudo 2. E no estudo 2, a combinação de FBM+NA aumentou a proliferação e a migração dos HFG na presença ou ausência de TNF-α (p<0,05) e promoveu síntese de MMP-2 similar ao controle negativo (p>0,05). Para a síntese de IL-6, FBM+NA foram estatisticamente semelhantes ao controle negativo (p>0,05) e para FBM+NA na presença de TNF-α apresentaram redução significativa da síntese de IL-6 em relação ao controle positivo (TNF-α) (p>0,05). O tratamento de FBM+NA apresentou síntese de TIMP-1 estatisticamente semelhante ao controle negativo (p>0,05). A FBM+NA na presença de TNF-α, promoveu menor intensidade de fluorescência, indicando modulação da atividade gelatinolítica induzida por essas terapias. Dessa forma, a FBM com 4 J/cm2 associado a NA apresenta um potencial sinérgico in vitro resultando em aumento da proliferação e migração celular dos fibroblastos gengivais humanos e menores níveis de síntese de MMP-2 e IL-6, sem alterar a síntese de TIMP-1. E a associação dessas terapias na presença de TNF-α indicou a modulação da atividade gelatinolítica, favorecendo estas funções celulares que estão relacionadas a aceleração do reparo tecidual.
Oral mucositis is one of the main acute complications of chemotherapy or radiotherapy of head and neck, causing interruptions and delays in the oncological treatment, impacting the quality of life and survival of these patients. Its treatment is still controversial and based mainly on palliative therapies that only reduce the pain symptomatology, based on this, it becomes fundamental to evaluate new therapeutic strategies to prevent or treat lesions effectively. Considering that preliminary studies suggest that the isolated application of PBM and NA compounds on gingival fibroblasts resulted in reduced expression and synthesis of MMPs, it is expected that the combination of both therapeutic proposals may result in a synergistic effect, accelerating the process of oral tissue repair. Thus, the objective of this work was to evaluate the dose of FBM with LLLT necessary to modulate a synthesis of MMP-2 (study 1) and to evaluate the combined effect of the application of NA and PBM in the modulation of MMP-2, IL-6 and TIMP-1 synthesis and activity by human gingival fibroblasts (HGF), as well as on cellular functions related to the tissue repair process (study 2). Primary cultures of HGF were obtained from a voluntary donor according to the Ethics Committee on Human Research. Tumor necrosis factor-alpha (TNF-α) was used as a positive control, by inducing the synthesis of MMPs in HGF. For LLLT irradiation, the LASERTable device was used (InGaAsP; 780 nm; 25 mW; study 1: 0.5, 2 and 4 J/cm2; study 2: 4 J/cm2). For study 1, cell viability analysis was performed using the methyltetrazolium (MTT) colorimetric assay and the evaluation of MMP-2 synthesis was performed using the ELISA immunoassay. For study 2, synthetic NA was used at a concentration of 10 µg/mL, based on previous studies.The effect of the combination of NA and PBM on the proliferation of HGF was performed by PrestoBlue assay, in the migration through the in vitro healing assay, in the synthesis of MMP-2, IL-6 and TIMP1 by HGF by ELISA immunoassay and in the activity gelatinolytic activity of HGF by in situ zymography. The data were submitted to analysis of normality and homoscedasticity to select the appropriate statistical tests (ANOVA, Tukey). The gelatinolytic activity data was evaluated qualitatively and presented in a descriptive way. All statistical inferences were made considering the significance level of 5%. The experiments were performed in duplicates, with N=6. According to the results obtained from study 1, therapy with PBM 4 J/cm2 significantly increased the viability of HFG in the presence or absence of TNF-α in relation to the negative control (p<0.05) and the groups treated with PBM 2 J/cm2 and 4 J/cm2 associated or not with TNF-α showed a significant decrease in MMP-2 synthesis compared to the negative control (p<0.05). Therefore, a density of 4 J/cm2 was selected for study 2. And in study 2, the combination of PBM+NA increased proliferation and migration of HFG in the presence or absence of TNF-α (p<0.05) and promoted synthesis of MMP-2 similar to the negative control (p>0.05). For IL-6 synthesis, PBM+NA were statistically similar to the negative control (p>0.05) and for PBM+ NA in the presence of TNF-α, there was a significant reduction in IL-6 synthesis in relation to the positive control (TNF-α) (p>0.05). The PBM+NA treatment showed TIMP-1 synthesis compared to the negative control (p>0.05). PBM+NA in the presence of TNF-α, promoted lower fluorescence intensity, indicating modulation of gelatinolytic activity induced by these therapies. Thus, PBM with 4 J/cm2 associated with NA has a synergistic potential in vitro resulting in increased proliferation and cell migration of human gingival fibroblasts and lower levels of synthesis of MMP-2 and IL-6, without altering TIMP-1 synthesis. And the association of these therapies in the presence of TNF-α indicated the modulation of gelatinolytic activity, favoring these cellular functions that are related to the acceleration of tissue repair
Oral mucositis is one of the main acute complications of chemotherapy or radiotherapy of head and neck, causing interruptions and delays in the oncological treatment, impacting the quality of life and survival of these patients. Its treatment is still controversial and based mainly on palliative therapies that only reduce the pain symptomatology, based on this, it becomes fundamental to evaluate new therapeutic strategies to prevent or treat lesions effectively. Considering that preliminary studies suggest that the isolated application of PBM and NA compounds on gingival fibroblasts resulted in reduced expression and synthesis of MMPs, it is expected that the combination of both therapeutic proposals may result in a synergistic effect, accelerating the process of oral tissue repair. Thus, the objective of this work was to evaluate the dose of FBM with LLLT necessary to modulate a synthesis of MMP-2 (study 1) and to evaluate the combined effect of the application of NA and PBM in the modulation of MMP-2, IL-6 and TIMP-1 synthesis and activity by human gingival fibroblasts (HGF), as well as on cellular functions related to the tissue repair process (study 2). Primary cultures of HGF were obtained from a voluntary donor according to the Ethics Committee on Human Research. Tumor necrosis factor-alpha (TNF-α) was used as a positive control, by inducing the synthesis of MMPs in HGF. For LLLT irradiation, the LASERTable device was used (InGaAsP; 780 nm; 25 mW; study 1: 0.5, 2 and 4 J/cm2; study 2: 4 J/cm2). For study 1, cell viability analysis was performed using the methyltetrazolium (MTT) colorimetric assay and the evaluation of MMP-2 synthesis was performed using the ELISA immunoassay. For study 2, synthetic NA was used at a concentration of 10 µg/mL, based on previous studies.The effect of the combination of NA and PBM on the proliferation of HGF was performed by PrestoBlue assay, in the migration through the in vitro healing assay, in the synthesis of MMP-2, IL-6 and TIMP1 by HGF by ELISA immunoassay and in the activity gelatinolytic activity of HGF by in situ zymography. The data were submitted to analysis of normality and homoscedasticity to select the appropriate statistical tests (ANOVA, Tukey). The gelatinolytic activity data was evaluated qualitatively and presented in a descriptive way. All statistical inferences were made considering the significance level of 5%. The experiments were performed in duplicates, with N=6. According to the results obtained from study 1, therapy with PBM 4 J/cm2 significantly increased the viability of HFG in the presence or absence of TNF-α in relation to the negative control (p<0.05) and the groups treated with PBM 2 J/cm2 and 4 J/cm2 associated or not with TNF-α showed a significant decrease in MMP-2 synthesis compared to the negative control (p<0.05). Therefore, a density of 4 J/cm2 was selected for study 2. And in study 2, the combination of PBM+NA increased proliferation and migration of HFG in the presence or absence of TNF-α (p<0.05) and promoted synthesis of MMP-2 similar to the negative control (p>0.05). For IL-6 synthesis, PBM+NA were statistically similar to the negative control (p>0.05) and for PBM+ NA in the presence of TNF-α, there was a significant reduction in IL-6 synthesis in relation to the positive control (TNF-α) (p>0.05). The PBM+NA treatment showed TIMP-1 synthesis compared to the negative control (p>0.05). PBM+NA in the presence of TNF-α, promoted lower fluorescence intensity, indicating modulation of gelatinolytic activity induced by these therapies. Thus, PBM with 4 J/cm2 associated with NA has a synergistic potential in vitro resulting in increased proliferation and cell migration of human gingival fibroblasts and lower levels of synthesis of MMP-2 and IL-6, without altering TIMP-1 synthesis. And the association of these therapies in the presence of TNF-α indicated the modulation of gelatinolytic activity, favoring these cellular functions that are related to the acceleration of tissue repair
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Palavras-chave
Técnicas de cultura de células., Metaloproteinase da matriz., Inibidores teciduais de metaloproteinases, Terapia com luz de baixa intensidade, Flavanonas, Cell culture techniques, Matrix metalloproteinase, Tissue inhibitor of metalloproteinases, Low-level light therapy, Flavonones