Avaliação molecular dos mecanismos de bioestimulação fibroblástica utilizando ácido hialurônico e extrato de chá verde (Camellia sinensis)

RESUMO

Objetivo: avaliar a capacidade do extrato de chá verde (Camellia sinensis) e ácido hialurônico em modular a expressão de genes relacionados com a sobrevivência e proliferação de fibroblastos. Material e métodos: após manutenção e expansão, fibroblastos humanos receberam diferentes concentrações (variando de 0% a 100% v/v) de extrato de chá verde e ácido hialurônico para avaliação da adesão celular pela metodologia de cristal violeta. Em seguida, a metodologia RT-qPCR foi aplicada para verificar a expressão dos genes AKT, CDK2, CDK4, VEGF e VEGFR1 frente aos genes controle (ß-ACT, GAPDH e 18S) e calculada pelo método CT. O teste Anova foi aplicado para comparar os resultados (5% significância). Resultados: ambos os compostos impactam na sobrevivência de fibroblastos por meio da ativação do gene AKT. Posteriormente, genes relacionados com a proliferação celular foram avaliados e nossos dados demonstraram que os genes CDK2 e CDK4 foram significantemente expressos em resposta a ambos os compostos analisados. Por fim, o estímulo angiogênico desses compostos foi afirmado pela capacidade de estimular a atividade dos genes VEGF e VEGFR1. Conclusão: em conjunto, os resultados apresentados demonstraram a capacidade do extrato da Camellia sinensis e do ácido hialurônico em promover eventos importantes em fibroblastos, os quais são pré-requisitos em mecanismos de regeneração tecidual. Devido às respostas complementares de ambos os compostos, ensaios de sinergia serão necessários para melhor análise biológica com vista ao desenvolvimento de novos produtos na área odontológica.

Palavras-chave – Regeneração tecidual; Extrato de chá verde (Camellia sinensis); Ácido hialurônico; Fibroblastos; Sinalização celular.

ABSTRACT

Objective: to evaluate the ability of both green tea extract (Camellia sinensis) and hyaluronic acid in modulating the expression of genes related to fibroblast survival and proliferation. Material and methods: after maintenance and expansion, human fibroblasts received di! erent concentrations of these compounds (concentrations ranging from 0% – 100% v/v) of green tea and hyaluronic acid to evaluate cell adhesion by using crystal violet dye. Then, the real-time RTqPCR technique was applied to verify the expression of AKT, CDK2, CDK4, VEGF, VEGFR1 genes against control genes (ß-ACT, GAPDH, 18S) and calculated by the “”CT method. The Anova test was used to compare the results (5% significance). Results: both compounds impact fibroblast survival through the activation of the AKT gene. Subsequently, genes related to cell proliferation were evaluated and our data demonstrated that CDK2 and CDK4 genes were significantly expressed in response to both analyzed compounds. Finally, the angiogenic stimulus of these compounds was confirmed by the ability to stimulate the activity of the VEGF and VEGFR1 genes. Conclusion: together, the results presented demonstrate the ability of Camellia sinensis extract and hyaluronic acid to promote important events in fibroblasts, which are prerequisites in tissue regeneration mechanisms. Due to the complementary responses of both compounds, synergy assays will be necessary for a better biological analysis with a view to the development of new products in the dental field.

Key words – Tissue regeneration; Green tea (Camellia sinensis) extract; Hyaluronic acid; Fibroblasts; Cell signaling.

Referências

Norrby K. Angiogenesis: new aspects relating to its initiation and control. APMIS. 1997;105(6):417-37.

Desmoulière A, Redard M, Darby I, Gabbiani G. Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am J Pathol 1995;146(1):56-66.

Appleton I. Wound healing: future directions. IDrugs 2003;6(11):1067-72.

Hall MP, Band PA, Meislin RJ, Jazrawi LM, Cardone DA. Platelet-rich plasma: current concepts and application in sports medicine. J Am Acad Orthop Surg 2009;17(10):602-8.

Qian X, Lin Q, Wei K, Hu B, Jing P, Wang J. Efficacy and safety of autologous blood products compared with corticosteroid injections in the treatment of lateral epicondylitis: a meta-analysis of randomized controlled trials. PM R 2016;8(8):780-91.

Spaková T, Rosocha J, Lacko M, Harvanová D, Gharaibeh A. Treatment of knee joint osteoarthritis with autologous platelet-rich plasma in comparison with hyaluronic acid. Am J Phys Med Rehabil 2012;91(5):411-7.

Katiyar SK, Afaq F, Perez A, Mukhtar H. Green tea polyphenol (-)-epigallocatechin-3-gallate treatment of human skin inhibits ultraviolet radiation-induced oxidative stress. Carcinogenesis 2001;22(2):287-94.

Hsu S. Green tea and the skin. J Am Acad Dermatol 2005;52(6):1049-59.

Kim HR, Rajaiah R, Wu Q-L, Satpute SR, Tan MT, Simon JE et al. Green tea protects rats against autoimmune arthritis by modulating disease-related immune events. J Nutr 2008;138(11):2111-6.

Trigkilidas D, Anand A. The effectiveness of hyaluronic acid intra-articular injections in managing osteoarthritic knee pain. Ann R Coll Surg Engl 2013;95(8):545-51.

Chou W-Y, Ko J-Y, Wang F-S, Huang C-C, Wong T, Wang C-J et al. Effect of sodium hyaluronate treatment on rotator cuff lesions without complete tears: a randomized, double-blind, placebo-controlled study. J Shoulder Elbow Surg 2010;19(4):557-63.

Kim Y-S, Park J-Y, Lee C-S, Lee S-J. Does hyaluronate injection work in shoulder disease in early stage? A multicenter, randomized, single blind and open comparative clinical study. J Shoulder Elb Surg 2012;21(6):722-7.

Bas A, Forsberg G, Hammarström S, Hammarström M-L. Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol 2004;59(6):566-73.

Deng H, Gong Y, Chen Y, Zhang G, Chen H, Cheng T et al. Porphyromonas gingivalis lipopolysaccharide affects the angiogenic function of endothelial progenitor cells via Akt/FoxO1 signaling. J Periodontal Res 2022;57(4):859-68.

Pratt DJ, Bentley J, Jewsbury P, Boyle FT, Endicott JA, Noble MEM. Dissecting the determinants of cyclin-dependent kinase 2 and cyclin-dependent kinase 4 inhibitor selectivity. J Med Chem 2006;49(18):5470-7.

Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA. Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 2004;56(4):549-80.

Failla CM, Carbo M, Morea V. Positive and negative regulation of angiogenesis by soluble vascular endothelial growth factor receptor-1. Int J Mol Sci 2018;19(5):1306.

Stupack DG, Cheresh DA. Get a ligand, get a life: integrins, signaling and cell survival. J Cell Sci 2002;115(Pt 19):3729-38.

Wendel H-G, De Stanchina E, Fridman JS, Malina A, Ray S, Kogan S et al. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature 2004;428(6980):332-7.

Ding L, Cao J, Lin W, Chen H, Xiong X, Ao H et al. The roles of cyclin-dependent kinases in cell-cycle progression and therapeutic strategies in human breast cancer. Int J Mol Sci 2020;21(6).

Shibuya M. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) signaling in angiogenesis: a crucial target for anti- and pro-angiogenic therapies. Genes Cancer 2011;2(12):1097-105.

Weddell JC, Chen S, Imoukhuede PI. VEGFR1 promotes cell migration and proliferation through PLCγ and PI3K pathways. NPJ Syst Biol Appl 2017;4:1.