Cell Biology and Toxicology

, Volume 24, Issue 1, pp 75–86 | Cite as

The flavonoid rutin induces astrocyte and microglia activation and regulates TNF-alpha and NO release in primary glial cell cultures

  • A. R. Silva
  • A. M. Pinheiro
  • C. S. Souza
  • S. R. V.-B. Freitas
  • V. Vasconcellos
  • S. M. Freire
  • E. S. Velozo
  • M. Tardy
  • R. S. El-Bachá
  • M. F. D. Costa
  • S. L. CostaEmail author
Original Article


Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 μmol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 μmol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 μmol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld’s staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 μmol/L rutin. Enhanced TNFα release was observed in astrocyte primary cultures treated with 100 μmol/L rutin and in mixed primary cultures treated with 50 and 100 μmol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFα production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.


Astrocyte Flavonoid Microglia NO Rutin TNF-α 



astrocyte primary culture


blood-brain barrier


central nervous system




enzyme-linked immunosorbent assay


glial fibrillary acidic protein


lactate dehydrogenase


mixed primary culture


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


polyacrylamide gel electrophoresis


sodium dodecyl sulfate




tumor necrosis factor



This work was supported by grants from Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), and Banco do Nordeste do Brasil (BNB). We gratefully acknowledge the research support provided by Programa de Pós-Graduação em Imunologia - UFBA, and Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).


  1. Aherne AS, O’Brien NM. Protection by the flavonoids myricetin, quercetin, and rutin against hydrogen peroxide-induced DNA damage in Caco-2 and Hep G2 cells. Nutr Cancer 1999;34:160–6.PubMedCrossRefGoogle Scholar
  2. Allan SM, Rothwell NJ. Cytokines and acute neurodegeneration. Nat Rev Neurosci 2001;2:734–44.PubMedCrossRefGoogle Scholar
  3. Aloisi F. Immune function of microglia. Glia 2001;36:165–79.PubMedCrossRefGoogle Scholar
  4. Aschner M. Astrocytes as mediators of immune and inflammatory responses in the CNS. Neurotoxicology 1998;19(2):269–82.PubMedGoogle Scholar
  5. Benveniste EN. Cytokine actions in the central nervous system. Cytokine Growth Factor Rev 1998;9:259–75.PubMedCrossRefGoogle Scholar
  6. Brahmachari S, Fung YK, Pahan K. Induction of glial fibrillary acidic protein expression in astrocytes by nitric oxide. J Neurosci 2006;26(18):4930–9.PubMedCrossRefGoogle Scholar
  7. Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H. Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta Med 2002;68:445–8.PubMedCrossRefGoogle Scholar
  8. Chen YC, Shen SC, Chen LG, Lee TJF, Yang LL. Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expression induced by nitric oxide synthase inhibitors and lipopolysaccharide. Biochem Pharmacol 2001;61:1417–27.PubMedCrossRefGoogle Scholar
  9. Chen CJ, Raung SL, Liao SL, Chen SY. Inhibition of inducible nitric oxide synthase expression by baicalein in endotoxin/cytokine-stimulated microglia. Biochem Pharmacol 2004;67(5):957–65.PubMedCrossRefGoogle Scholar
  10. Chen TJ, Jeng JY, Lin CW, Wu CY, Chen YC. Quercetin inhibition of ROS-dependent and -independent apoptosis in rat glioma C6 cells. Toxicology 2006;223:113–26.PubMedCrossRefGoogle Scholar
  11. Cookson MR, Pentreath VW. Alterations in the glial fibrillary acidic protein content of primary astrocyte cultures for evaluation of glial cell toxicity. Toxicol In Vitro. 1994;8(3):351–9.CrossRefPubMedGoogle Scholar
  12. Costa SL, Planchenault T, Charrière-Bertrand C, et al. Astroglial permissivity for neurotic outgrowth in neuron-astrocyte cocultures depends on regulation of lamini bioavailability. Glia 2002;37:105–13.PubMedCrossRefGoogle Scholar
  13. Coyle JT, Schwarcz R. Mindglue: implications of glial cell biology for psychiatry. Arch Gen Psychiatry 2000;57(1):90–3.PubMedCrossRefGoogle Scholar
  14. Dawson VL, Brahmbhatt JA, Mong JA, Dawson TM. Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glia cortical cultures. Neuropharmacology 1994;33(11):1425–30.PubMedCrossRefGoogle Scholar
  15. Deschner EE, Ruperto J, Wong G, Newmark HL. Quercetin and rutin as inhibitors of azoxymethanol-induced colonic neoplasia. Carcinogenesis 1991;12:1193.PubMedCrossRefGoogle Scholar
  16. Dickson DW, Lee SC, Mattiace LA, Yen SH, Brosnan C. Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer’s disease. Glia 1993;7:75–83.PubMedCrossRefGoogle Scholar
  17. Dihal AA, de Boer VC, van der Woude H, et al. Quercetin, but not its glycosidated conjugate rutin, inhibits azoxymethane-induced colorectal carcinogenesis in F344 rats. J Nutr 2006;136:2862–7.PubMedGoogle Scholar
  18. Dimayuga FO, Wang C, Clark JM, Dimayuga ER, Dimayuga VM, Bruce-Keller AJ. SOD1 overexpression alters ROS production and reduces neurotoxic inflammatory signaling in microglial cells. J Neuroimmunol 2007;182:89–99.PubMedCrossRefGoogle Scholar
  19. Gebicke-Haerter PJ. Microglia in neurodegeneration: molecular aspects. Microsc Res Tech 2001;54:47–58.PubMedCrossRefGoogle Scholar
  20. Giulian D, Leara J, Li J, Keenen C. Phagocytic microglia release cytokines and cyttoxins that regulate the survival of astrocytes and neurons in culture. Neurochem Int 1994;25:227–32.PubMedCrossRefGoogle Scholar
  21. Gonzalez-Scarano F, Baltuch G. Microglia as mediators of inflammatory and degenerative diseases. Annu Rev Neurosci 1999;22:219–40.PubMedCrossRefGoogle Scholar
  22. Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 1989;119:203–10.PubMedCrossRefGoogle Scholar
  23. Havsteen BH. Biochemical and medical significance of flavonoids. Pharmacol Ther 2002;96:67–202.PubMedCrossRefGoogle Scholar
  24. Higuchi T, Shibata H, Nakada T, Sato Y. Pharmaceutical composition for treatment of infection with drug resistant bacterium and disinfectant. U.S. Pat Appl 2006 0229262.Google Scholar
  25. Janbaz KH, Saeed AS, Gilani AH. Protective effect of rutin on paracetamol- and CCl4-induced hepatotoxicity in rodents. Fitoterapia 2002;73:557–63.PubMedCrossRefGoogle Scholar
  26. Lambev I, Belcheva A, Zhelyazkov D. Flavonoids with antioxidant action (naringin and rutin) and the release of mastocytic and nonmastocytic histamine. Acta Physiol Pharmacol Bulg 1980a;6:70–5.PubMedGoogle Scholar
  27. Lambev I, Krushkov I, Zheliazkov D, Nikolov N. Antiexudative effect of naringin in experimental pulmonary edema and peritonitis. Eksp Med Morfol 1980b;19:207–12.PubMedGoogle Scholar
  28. Lin CC, Shieh DE. The anti-inflammatory activity of Scutellaria rivularis extracts and its active components, baicalin, baicalein, and wogonin. Am J Chin Med 1996;24:31–6.PubMedCrossRefGoogle Scholar
  29. Lipkin M, Reddy B, Newmark H, Lamprecht SA. Dietary factors in human colorectal cancer. Annu Rev Nutr 1999;19:545–86.PubMedCrossRefGoogle Scholar
  30. Little AR, O’Callaghan JP. Astrogliosis in the adult and developing CNS: is there a role for proinflammatory cytokines? Neurotoxicology 2001;22:607–18.PubMedCrossRefGoogle Scholar
  31. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75.PubMedGoogle Scholar
  32. Manning P, Cookson MR, McNeil CJ, Figlewicz D, Shaw PJ. Superoxide-induced nitric oxide release from cultured glial cells. Brain Res 2001;911:203–10.PubMedCrossRefGoogle Scholar
  33. Markhan KR. Techniques of flavonoid identification. London: Academic Press; 1982.Google Scholar
  34. Moises HW, Zoega T, Gottesman IL. The glial growth factors deficiency and synaptic destabilization hypothesis of schizophrenia. BMC Psychiatry 2002;2(1):8.PubMedCrossRefGoogle Scholar
  35. Nakayama T, Yamada M, Osawa T, Kawakishi S. Suppression of active oxygen-induced cytotoxicity by flavonoids. Biochem Pharmacol 1993;45:265–7.PubMedCrossRefGoogle Scholar
  36. Nicholson TE, Dibb S, Renton KW. Nitric oxide mediates an LPS-induced depression of cytochrome P450 (CYP1A) activity in astrocytes. Brain Res 2004;1029:148–54.PubMedCrossRefGoogle Scholar
  37. O’Callaghan JP. Assessment of neurotoxicity: use of glial fibrillary acidic protein as a biomarker. Biomed Environ Sci 1991;4:197–206.PubMedGoogle Scholar
  38. Peng L, Wang B, Ren P. Reduction of MTT by flavonoids in the absence of cells. Colloids Surf B Biointerfaces 2005;45:108–11.PubMedCrossRefGoogle Scholar
  39. Raivich G, Jones LL, Werner A, Blathmann H, Doetschmann T, Kreutzberg GW. Molecular signals for glial activation: pro- and anti-inflammatory cytokines in the injured brain. Acta Neurochir 1999;73:21–30.Google Scholar
  40. Rajkowska G, Miguel-Hidalgo JJ, Makkos Z, Metzer H, Overholser J, Stockmeier C. Layer-specific reductions in GFAP-reactive astroglia in the dorsolateral prefrontal cortex in schizophrenia. Schizophr Res 2002;57(2–3):127–38.PubMedCrossRefGoogle Scholar
  41. Raven PH, Evert RF, Eichorn SE. Biologia Vegetal. 6th ed. Rio de Janeiro: Guanabara Koogan; 2001. p. 34–53.Google Scholar
  42. Ridet JL, Malhotra SK, Privat A, Gage FH. Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci 1997;20:570–7.PubMedCrossRefGoogle Scholar
  43. Rosenfeld G. Corante pancrômico para hematologia e citologia clínica: nova combinação dos componentes de May Grunwald e do Giemsa num só corante de emprego prático. Mem Inst Butantã 1947;20:329–35.Google Scholar
  44. Salimi K, Humpel C. Down regulation of complement receptor 3 and major histocompatibility complex I and II antigen-like immunoreactivity accompanies ramification in isolated rat microglia. Brain Res 2002;946:283–9.PubMedCrossRefGoogle Scholar
  45. Sanfeliu C, Crisòfol R, Torán N, Rodrigues-Farré E, Kim SU. Use of human central nervous system cell cultures in neurotoxicity testing. Toxicol In Vitro. 1999;13(4–5):753–9.CrossRefPubMedGoogle Scholar
  46. Schroeter H, Rose S, Jenner P, Rice-Evans CA. Uptake and metabolism of epicatechin and its access to the brain after oral ingestion. Free Radic Biol Med 2002;33:1693–702.PubMedCrossRefGoogle Scholar
  47. Silva AR, Hughes JB, Barreto RA, et al. The flavonoid rutin, extracted from Dimorphandra mollis, inhibits proliferation and stimulates differentiation of GL-15 human glioblastoma cells. In: XVII Simpósio de Plantas Medicinais do Brasil, Cuiabá-MT. Anais do XVII Simpósio de Plantas Medicinais do Brasil. 2002.Google Scholar
  48. Silva AMM, Silva AR, Pinheiro AM, et al. Alkaloids from Prosopis juliflora leaves induce glial activation, cytotoxicity and stimulate NO production. Toxicon. [In Press. Accepted Manuscript. Available online 17 August 2006.Google Scholar
  49. Streit WJ, Graeber MB, Kreutzberg GW. Functional plasticity of microglia: a review. Glia 1988;5:301–7.CrossRefGoogle Scholar
  50. Streit WJ, Walter AS, Pennel NA. Reactive microgliosis. Prog Neurobiol 1999;57:563–81.PubMedCrossRefGoogle Scholar
  51. Takasato Y, Rapoport SI, Smith QR. An in situ brain perfusion technique to study cerebrovascular transort in the rat. Am J Physiol 1984;247:H484–93.PubMedGoogle Scholar
  52. Tardy M. Astrocyte et homeostasie. Med Sci (Paris) 1991;8(7):799–804.Google Scholar
  53. Vairano M, Graziani G, Tentori L, Tringali G, Navarra P, Russo CD. Primary cultures of microglial cells for testing toxicity of anticancer drugs. Toxicol Lett 2004;148:91–4.PubMedCrossRefGoogle Scholar
  54. Vilhardt F. Microglia: phagocyte and glia cell. Int J Biochem Cell Biol 2005;37:17–21.PubMedCrossRefGoogle Scholar
  55. Wang Z, Li D, Liang Y, Wang D, Cai N. Activation of astrocytes by advanced glycation end products: cytokines induction and nitric oxide release. Acta Pharmacol Sin 2002;23(11):974–80.PubMedGoogle Scholar
  56. Yang K, Lamprecht AS, Liu Y, et al. Chemoprevention studies of the flavonoids quercetin and rutin in normal and azoxymethane-treated mouse colon. Carcinogenesis 2000;21:1655–60.PubMedCrossRefGoogle Scholar
  57. Youdim KA, Dobbie MS, Kuhnle G, Proteggente AR, Abbott NJ, Rice-Evans C. Interaction between flavonoids and the blood-brain barrier: in vitro studies. J Neurochem 2003;85(1):180–92.PubMedCrossRefGoogle Scholar
  58. Youdim KA, Shukitt-Hale B, Joseph JA. Flavonoids and the brain: interactions at the blood-brain barrier and their physiological effects on the central nervous system. Free Radic Biol Med 2004;37(11):1683–93.PubMedCrossRefGoogle Scholar
  59. Zieliňska M, Gülden M, Seibert H. Effects of quercetin and quercetin-3-O-glycosides on oxidative damage in rat C6 glioma cells. Environ Toxicol Pharmacol 2003;13:47–53.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • A. R. Silva
    • 1
  • A. M. Pinheiro
    • 1
  • C. S. Souza
    • 1
  • S. R. V.-B. Freitas
    • 1
  • V. Vasconcellos
    • 1
  • S. M. Freire
    • 2
  • E. S. Velozo
    • 3
  • M. Tardy
    • 4
  • R. S. El-Bachá
    • 1
  • M. F. D. Costa
    • 1
  • S. L. Costa
    • 1
    Email author
  1. 1.Laboratório de Neuroquímica e Biologia Celular, Departamento de Biofunção, Instituto de Ciências da SaúdeUniversidade Federal da Bahia (UFBA)SalvadorBrazil
  2. 2.Laboratório de Imunologia, Departamento de Biointeração, Instituto de Ciências da SaúdeUniversidade Federal da Bahia (UFBA)SalvadorBrazil
  3. 3.Laboratório de Pesquisa em Matéria Médica, Faculdade de FarmáciaUFBA Universidade Federal da BahiaSalvadorBrazil
  4. 4.Faculté de Médicine, Val-de-MarneUniversité Paris XIICréteilFrance

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