Análise in vitro das propriedades da eriocitrina e eriodictiol sobre fibroblastos L929
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Data
2022-03-31
Autores
Bione, Flávia Theresa Soares de Castro
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Universidade Estadual Paulista (Unesp)
Resumo
A cicatrização de feridas é regida por fases sequenciais, e dentre a complexa rede de eventos humorais e celulares destaca-se a atividade de fibroblastos, os quais secretam citocinas e fatores de crescimento, participando ativamente do desenvolvimento de novo tecido e da regulação do processo de reparo. Fatores diversos podem comprometer os mecanismos implicados ao processo de reparo, retardando e/ou impedindo seu curso natural, o que faz com que o tratamento de feridas apresente limitado sucesso e compreenda estratégias de alto custo. Estudos acerca das propriedades cicatrizantes de produtos naturais têm sido realizados pois podem facilitar o processo de cicatrização, por demonstrarem desde ações anti inflamatória, antioxidante, antibacteriana à síntese de pró-colágeno. Os flavonoides são apresentados como alternativas estratégicas para várias afecções imunoinflamatórias, por apresentarem potencial anti-inflamatório e antioxidante. O objetivo do trabalho foi avaliar a hipótese de que os flavonoides, eriocitrina (Erioc) e eriodictiol (Eriod), apresentam propriedades moduladoras sobre a atividade in vitro de fibroblastos L929. Os fibroblastos L929 foram cultivados concomitante a diferentes tratamentos, de acordo com cada grupo – 1-Células (Controle), 2-Células+Erioc1µM, 3-Células+Erioc25µM, 4- Células+Erioc50µM, 5-Células+Eriod1µM, 6- Células+Eriod25µM, 7-Células+Eriod50µM. As análises seguintes foram realizadas: proliferação celular e citotoxicidade (Alamar blue e MTT - 1, 3 e 7 dias); atividade migratória (ensaio de arranhão – 24 e 72hrs); síntese de colágeno (Espectrofotometria – 3 dias); expressão de fatores de crescimento FGF e TGF-β1 (ELISA – 3 e 7 dias). A análise estatística dos dados foi realizada através do software GraphPad Prism 8. Os resultados demonstraram que o tratamento com eriocitrina ou eriodictiol não comprometeu a viabilidade in vitro dos fibroblastos L929 e a proliferação celular manteve-se similar entre os grupos tratados ou não com ambos os flavonoides. A atividade migratória dos fibroblastos, em 24 hrs, foi estimulada pelo tratamento com eriocitrina a 1uM e 25μM (p<0.05) e, após em 72 horas, a administração de eriocitrina 1uM, 25uM e o eriodictiol 1uM promoveram significativa redução da área do arranhão, demonstrando maior atividade de migração celular (p<0.05). Os tratamentos com eriocitrina e eriodictiol na concentração de 1μM promoveram significativo aumento na produção de colágeno, quando comparados aos grupos controle e eriocitrina 25 e 50μM (p<0.05). O tratamento com eriocitrina e eriodictiol modulou apenas a expressão de TGF-β1 aos 3 dias quando comparada ao grupo não tratado, enquanto em 7 dias, os compostos a 25 e 50μM reduziram significativamente a expressão de TGF-β1 e FGF, e o tratamento com eriocitrina a 1 μM aumentou de modo significativo a expressão de TGF-β1 (p<0.05). Os resultados permitem concluir que a eriocitrina e eriodictiol em baixa concentração, modulam a atividade e função in vitro dos fibroblastos L929 nas condições avaliadas, estimulando a migração celular e produção de colágeno in vitro, sugerindo-se uma correlação ao aumento expressivo de TGF- β1.
Wound healing occurs in sequential phases, and among the complex network of humoral and cellular events, fibroblasts show important activity secreting cytokines and growth factors, actively participating in the development of new tissue and in the regulation of the wound healing process. Several factors may compromise the mechanisms involved in the repair process, delaying and/or preventing its natural course, which makes the treatment of wounds with limited success and involves high-cost strategies. Studies on the healing properties of natural products have been performed, as they can facilitate the healing process, demonstrating from anti-inflammatory, antioxidant, antibacterial actions to the synthesis of pro-collagen. Flavonoids are presented as strategic alternatives for various immunoinflammatory disorders, as they have anti-inflammatory and antioxidant potential. The aim of the study was to evaluate the hypothesis that the flavonoids, eriocitrin (Erioc) and eriodictiol (Eriod), have modulating properties on the in vitro activity of L929 fibroblasts. L929 fibroblasts were cultured concomitantly with different treatments, according to each group – 1-Cells (Control), 2-Cells+Erioc1µM, 3-Cells+Erioc25µM, 4- Cells+Erioc50µM, 5-Cells+Eriod1µM, 6-Cells +Eriod25µM, 7-Cells+Eriod50µM. The following analyzes were performed: cell proliferation and cytotoxicity (Alamar blue and MTT - 1, 3 and 7 days); migratory activity (scratch test – 24 and 72 hrs); collagen synthesis (Spectrophotometry – 3 days); expression of growth factors FGF and TGF-β1 (ELISA – 3 and 7 days). Statistical analysis of the data was performed using the GraphPad Prism 8 software. The results showed that treatment with eriocitrin or eriodictiol did not compromise the in vitro viability of L929 fibroblasts and cell proliferation remained similar between groups treated or not with both flavonoids. The migratory activity of fibroblasts, in 24 hrs, was stimulated by the treatment with eriocitrin at 1uM and 25μM (p<0.05) and, after 72 hours, the administration of eriocitrin 1uM, 25uM and eriodictyol 1uM promoted a significant reduction in the scratch area, demonstrating higher cell migration activity (p<0.05). The treatments with eriocitrin and eriodictyol at 1μM promoted a significant increase in collagen production, when compared to the control and eriocitrin 25 and 50μM groups (p<0.05). Treatment with eriocitrin and eriodictiol just modulated the expression of TGF-β1 at 3 days when compared to the untreated group, while at 7 days, compounds at 25 and 50μM significantly reduced the expression of TGF-β1 and FGF, and the treatment with 1 μM eriocitrin significantly increased TGF-β1 expression (p<0.05). The results allow to conclude that eriocitrin and eriodictiol in low concentration modulate the activity and function of L929 fibroblasts in vitro under the conditions evaluated, stimulating cell migration and collagen production in vitro, suggesting a correlation with the expressive increase of TGF-β1.
Wound healing occurs in sequential phases, and among the complex network of humoral and cellular events, fibroblasts show important activity secreting cytokines and growth factors, actively participating in the development of new tissue and in the regulation of the wound healing process. Several factors may compromise the mechanisms involved in the repair process, delaying and/or preventing its natural course, which makes the treatment of wounds with limited success and involves high-cost strategies. Studies on the healing properties of natural products have been performed, as they can facilitate the healing process, demonstrating from anti-inflammatory, antioxidant, antibacterial actions to the synthesis of pro-collagen. Flavonoids are presented as strategic alternatives for various immunoinflammatory disorders, as they have anti-inflammatory and antioxidant potential. The aim of the study was to evaluate the hypothesis that the flavonoids, eriocitrin (Erioc) and eriodictiol (Eriod), have modulating properties on the in vitro activity of L929 fibroblasts. L929 fibroblasts were cultured concomitantly with different treatments, according to each group – 1-Cells (Control), 2-Cells+Erioc1µM, 3-Cells+Erioc25µM, 4- Cells+Erioc50µM, 5-Cells+Eriod1µM, 6-Cells +Eriod25µM, 7-Cells+Eriod50µM. The following analyzes were performed: cell proliferation and cytotoxicity (Alamar blue and MTT - 1, 3 and 7 days); migratory activity (scratch test – 24 and 72 hrs); collagen synthesis (Spectrophotometry – 3 days); expression of growth factors FGF and TGF-β1 (ELISA – 3 and 7 days). Statistical analysis of the data was performed using the GraphPad Prism 8 software. The results showed that treatment with eriocitrin or eriodictiol did not compromise the in vitro viability of L929 fibroblasts and cell proliferation remained similar between groups treated or not with both flavonoids. The migratory activity of fibroblasts, in 24 hrs, was stimulated by the treatment with eriocitrin at 1uM and 25μM (p<0.05) and, after 72 hours, the administration of eriocitrin 1uM, 25uM and eriodictyol 1uM promoted a significant reduction in the scratch area, demonstrating higher cell migration activity (p<0.05). The treatments with eriocitrin and eriodictyol at 1μM promoted a significant increase in collagen production, when compared to the control and eriocitrin 25 and 50μM groups (p<0.05). Treatment with eriocitrin and eriodictiol just modulated the expression of TGF-β1 at 3 days when compared to the untreated group, while at 7 days, compounds at 25 and 50μM significantly reduced the expression of TGF-β1 and FGF, and the treatment with 1 μM eriocitrin significantly increased TGF-β1 expression (p<0.05). The results allow to conclude that eriocitrin and eriodictiol in low concentration modulate the activity and function of L929 fibroblasts in vitro under the conditions evaluated, stimulating cell migration and collagen production in vitro, suggesting a correlation with the expressive increase of TGF-β1.
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Palavras-chave
Cicatrização, Fibroblastos, Inflamação, Flavonoides, Wound healing, Fibroblasts, Inflammation, Flavonoids