Neurotoxicity Research

, Volume 36, Issue 4, pp 844–858 | Cite as

Protective Effect of Semisynthetic and Natural Flavonoid on Aged Rat Microglia–enriched Cultures

  • Nataša Mrvová
  • Martin Škandík
  • Štefan Bezek
  • Lucia RačkováEmail author
Original Article


The ROS-mediated lysosomal dysfunction and coinciding deterioration of mitochondrial function are thought to be the prominent mechanisms responsible for aging. Microglia, the resident macrophages in the central nervous system, were postulated to belong to the major targets vulnerable to these detrimental processes, acting as principal drivers in brain aging. The present study investigated the potential protective effect of the semisynthetic flavonoid 3′-O-(3-chloropivaloyl) quercetin (CPQ) and quercetin (Q) on microglia-enriched mixed brain cultures (MBCs) established from aged Wistar rats. Both flavonoids tested suppressed the development of lipofuscin-related autofluorescence in aged cells. Further ensuing protective effects included reduction of protein oxidation markers in aged cells. Moreover, unlike Q, CPQ significantly suppressed sensitivity of aged cells to stimulation of superoxide burst. Other activation markers, cellular hypertrophy and isolectin B4 binding, were also downregulated by treatment with both CPQ and Q. In conclusion, results of our study suggest that both flavonoids tested may protect microglia with a quite comparable efficacy against aging-related accumulated alterations. The protective mechanism can include interference with the ROS-mediated vicious cycles involving lysosomal dysfunction. Nevertheless, the lipophilized quercetin, CPQ, a compound with proposed enhanced biological availability compared to parent molecule, can represent an agent potentially useful for new effective pharmaceutical intervention against brain aging, overcoming the limitations of clinical applicability of quercetin.


Microglia Aging Flavonoids Lipofuscin Mitochondria ROS 



The study was supported by VEGA 2/0041/17, VEGA 2/0031/12, VEGA 2/0029/16, APVV-18-0336, APVV-15-0308. The work was supported by The Agency of the Ministry of Education of the Slovak Republic for the Structural Funds of EU, OP R&D of ERDF as a part of the Project: “Evaluation of natural substances and their selection for prevention and treatment of lifestyle diseases” (ITMS 26240220040).

Compliance with Ethical Standards

The study was performed in compliance with the Principles of Laboratory Animal Care and was approved by the institutional ethics committee and by the State Veterinary and Food Administration of the Slovak Republic (Act No. Ro-2590/11-221).

Conflict of Interest

The authors declare that there is no conflict of interest.

Supplementary material

12640_2019_71_MOESM1_ESM.pdf (597 kb)
Online Resource 1 (PDF 596 kb)
12640_2019_71_MOESM2_ESM.pdf (104 kb)
Online Resource 2 (PDF 104 kb)


  1. Acarin L, González B, Castellano B, Castro AJ (1997) Quantitative analysis of microglial reaction to a cortical excitotoxic lesion in the early postnatal brain. Exp Neurol 147(2):410–417. CrossRefPubMedGoogle Scholar
  2. Biasutto L, Marotta E, de Marchi U, Zoratti M, Paradisi C (2007) Ester-based precursors to increase the bioavailability of quercetin. J Med Chem 50:241–253. CrossRefPubMedGoogle Scholar
  3. Brunk UT, Terman A (2002) The mitochondrial-lysosomal axis theory of ageing. Accumulation of damaged mithochondria as a result of imperfect autophagocytosis. Eur J Biochem 269:1996–2002. CrossRefPubMedGoogle Scholar
  4. Caldwell ST, McPhail DB, Duthie GG, Hartley RC (2012) Synthesis of polyhydroxylated flavonoids bearing a lipophilic decyl tail as potential therapeutic antioxidants. Can J Chem 90(1):23–33. CrossRefGoogle Scholar
  5. Chen S-H, Oyarzabal EA, Hong J-S (2013) Preparation of rodent primary cultures for neuron–glia, mixed glia, enriched microglia, and reconstituted cultures with microglia. Methods Mol Biol 1041:231–240. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cherry JD, Olschowka JA, O’Banion MK (2014) Neuroinflammation and M2 microglia: the good, the bad, and the inflamed. J Neuroinflammation 11:98. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Choi DY, Lee YJ, Hong JT, Lee HJ (2012) Antioxidant properties of natural polyphenols and their therapeutic potentials for Alzheimer’s disease. Brain Res Bull 87:144–153. CrossRefPubMedGoogle Scholar
  8. Chondrogianni N, Kapeta S, Chinou I, Vassilatou K, Papassideri I, Gonos ES (2010) Anti-ageing and rejuvenating effects of quercetin. Exp Gerontol 45:763–771. CrossRefPubMedGoogle Scholar
  9. Cruz L, Fernandes VC, Araújo P, Mateus N, de Freitas V (2015) Synthesis, characterisation and antioxidant features of procyanidin B4 and malvidin-3-glucoside stearic acid derivatives. Food Chem 174:480–486. CrossRefPubMedGoogle Scholar
  10. de Vellis J, Cole R (2012) Preparation of mixed glial cultures from postnatal rat brain. Methods Mol Biol 814:49–59. CrossRefPubMedGoogle Scholar
  11. Devore EE, Kang JH, Breteler MM, Grodstein F (2012) Dietary intakes of berries and flavonoids in relation to cognitive decline. Ann Neurol 72:135–143. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Figueroa-Espinoza MC, Villeneuve P (2005) Phenolic acids enzymatic lipophilization. J Agric Food Chem 53:2779–2787. CrossRefPubMedGoogle Scholar
  13. Flanary BE, Streit WJ (2004) Progressive telomere shortening occurs in cultured rat microglia, but not astrocytes. Glia 45:75–88. CrossRefPubMedGoogle Scholar
  14. Flanary BE, Streit WJ (2005) Effects of axotomy on telomere length, telomerase activity, and protein in activated microglia. J Neurosci Res 82:160–171. CrossRefPubMedGoogle Scholar
  15. Flanary BE, Sammons NW, Nguyen C, Walker D, Streit WJ (2007) Evidence that ageing and amyloid promote microglial cell senescence. Rejuvenation Res 10:61–74. CrossRefPubMedGoogle Scholar
  16. Ginhoux F, Lim S, Hoeffel G, Low D, Huber T (2013) Origin and differentiation of microglia. Front Cell Neurosci 7.
  17. Giulian D, Baker TJ (1986) Characterization of ameboid microglia isolated from developing mammalian brain. J Neurosci 6:2163–2178. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Grune T, Reinheckel T, Davies KJ (1997) Degradation of oxidized proteins in mammalian cells. FASEB J 11:526–534. CrossRefPubMedGoogle Scholar
  19. Harman D (1956) Ageing: a theory based on free radical and radiation chemistry. J Gerontol 11(3):298–300. CrossRefPubMedGoogle Scholar
  20. Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621. CrossRefPubMedGoogle Scholar
  21. Jang S, Johnson RW (2010) Can consuming flavonoids restore old microglia to their youthful state? Nutr Rev 68:719–728. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Jansson D, Rustenhoven J, Feng S, Hurley D, Oldfield RL, Bergin PS, Mee EW, Faull RL, Dragunow M (2014) A role for human brain pericytes in neuroinflammation. J Neuroinflammation 11:104. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Jeong H-K, Ji K, Min K, Joe E-H (2013) Brain inflammation and microglia: facts and misconceptions. Exp Neurobiol 22(2):59–67. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Jung T, Bader N, Grune T (2007) Lipofuscin: formation, distribution, and metabolic consequences. Ann N Y Acad Sci 1119:97–111. CrossRefPubMedGoogle Scholar
  25. Jung T, Höhn A, Grune T (2010) Lipofuscin: detection and quantification by microscopic techniques. Methods Mol Biol 594:173–193. CrossRefPubMedGoogle Scholar
  26. Keil VC, Funke F, Zeug A, Schild D, Müller M (2011) Ratiometric high-resolution imageing of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria. Pflugers Arch 462(5):693–708. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Koch K, Havermann S, Ch B, Wätjen W (2014) Caenorhabditis elegans as model system in pharmacology and toxicology: effects of flavonoids on redox-sensitive signalling pathways and ageing. Sci World J 2014:1–15. CrossRefGoogle Scholar
  28. Kuniaková M, Mrvová N, Knezl V, Račková L (2015) Effect of novel quercetin pivaloyl ester on functions of adult rat microglia. Biologia (Bratisl) 70:690–702. CrossRefGoogle Scholar
  29. Kurz T, Terman A, Gustafsson B, Brunk UT (2008) Lysosomes and oxidative stress in ageing and apoptosis. Biochim Biophys Acta 1780:1291–1303. CrossRefPubMedGoogle Scholar
  30. Lorentz C, Dulac A, Pencreac'h G, Ergan F, Richomme P, Soultani-Vigneron S (2010) Lipase-catalyzed synthesis of two new antioxidants: 4-O- and 3-O-palmitoyl chlorogenic acids. Biotechnol Lett 32(12):1955–1960. CrossRefPubMedGoogle Scholar
  31. Ma W, Coon S, Zhao L, Fariss RN, Wong WT (2013) A2E accumulation influences retinal microglial activation and complement regulation. Neurobiol Ageing 34:943–960. CrossRefGoogle Scholar
  32. Mangold CA, Wronowski B, Du M, Masser DR, Hadad N, Bixler GV, Brucklacher RM, Ford MM, Sonntag WE, Freeman WM (2017) Sexually divergent induction of microglial-associated neuroinflammation with hippocampal ageing. J Neuroinflammation 14(1):141. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Marschallinger J, Irving Mosher I, Wyss-Coray T (2017) Microglial dysfunction in brain ageing and neurodegeneration. In: Fulop T, Franceschi C, Hirokawa K, Pawelec G (eds) Handbook of immunosenescence, basic understanding and clinical implications. Springer, Cham, pp 1–15. CrossRefGoogle Scholar
  34. McCarthy KD, de Vellis J (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85(3):890–902. CrossRefPubMedGoogle Scholar
  35. Mellou F, Lazari D, Skaltsa H, Tselepis AD, Kolisis FN, Stamatis H (2005) Biocatalytic preparation of acylated derivatives of flavonoid glycosides enhances their antioxidant and antimicrobial activity. J Biotechnol 116(3):295–304. CrossRefPubMedGoogle Scholar
  36. Milackova I, Rackova L, Majekova M, Mrvova N, Stefek M (2015) Protection or cytotoxicity mediated by a novel quinonoid-polyphenol compound? Gen Physiol Biophys 34(1):51–64. CrossRefPubMedGoogle Scholar
  37. Miller KR, Streit WJ (2017) The effects of ageing, injury and disease on microglial function: a case for cellular senescence. Neuron Glia Biol 3(3):245–253. CrossRefGoogle Scholar
  38. Miquel J (1998) An update on the oxygen stress-mitochondrial mutation theory of ageing: genetic and evolutionary implications. Exp Gerontol 33(1–2):113–126. CrossRefPubMedGoogle Scholar
  39. Miquel J, Economos AC, Fleming J, Johnson JE (1980) Mitochondrial role in cell ageing. Exp Gerontol 15:575–591. CrossRefPubMedGoogle Scholar
  40. Mrvová N, Škandík M, Kuniaková M, Račková L (2015) Modulation of BV-2 microglia functions by novel quercetin pivaloyl ester. Neurochem Int 90:246–254. CrossRefPubMedGoogle Scholar
  41. Nakanishi H, Wu Z (2009) Microglia-ageing: roles of microglial lysosome- and mitochondria-derived reactive oxygen species in brain ageing. Behav Brain Res 201:1–7. CrossRefPubMedGoogle Scholar
  42. Nakanishi H, Hayashi Y, Wu Z (2011) The role of microglial mtDNA damage in age-dependent prolonged LPS-induced sickness behavior. Neuron Glia Biol 7(1):17–23. CrossRefPubMedGoogle Scholar
  43. Ni M, Aschner M (2010) Neonatal rat primary microglia: isolation, culturing, and selected applications. Curr Protoc Toxicol Chapter 12:Unit 12.17. CrossRefPubMedGoogle Scholar
  44. Norden DM, Godbout JP (2013) Microglia of the aged brain: primed to be activated and resistant to regulation. Neuropathol Appl Neurobiol 39:19–34. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Olah M, Biber K, Vinet J, Boddeke HWGM (2011) Microglia phenotype diversity. CNS Neurol Disord - Drug Targets 10:108–118. CrossRefPubMedGoogle Scholar
  46. Olovnikov AM (1996) Telomeres, telomerase, and ageing: origin of the theory. Exp Gerontol 31:443–448. CrossRefPubMedGoogle Scholar
  47. Paolicelli RC, Bisht K, Tremblay M-È (2014) Fractalkine regulation of microglial physiology and consequences on the brain and behaviour. Front Cell Neurosci 8.
  48. Reinheckel T, Sitte N, Ullrich O, Kuckelkorn U, Davies KJ, Grune T (1998) Comparative resistance of the 20S and 26S proteasome to oxidative stress. Biochem J 335(Pt 3):637–642. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Rook GA, Steele J, Umar S, Dockrell HM (1985) A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon. J Immunol Methods 82(1):161–167. CrossRefPubMedGoogle Scholar
  50. Safaiyan S, Kannaiyan N, Snaidero N, Brioschi S, Biber K, Yona S, Edinger AL, Jung S, Rossner MJ, Simons M (2016) Age-related myelin degradation burdens the clearance function of microglia during ageing. Nat Neurosci 19:995–998. CrossRefPubMedGoogle Scholar
  51. Saura J, Tusell JM, Serratosa J (2003) High-yield isolation of murine microglia by mild trypsinization. Glia 44(3):183–189. CrossRefPubMedGoogle Scholar
  52. Shamsi FA, Boulton M (2001) Inhibition of RPE lysosomal and antioxidant activity by the age pigment lipofuscin. IOVS 42:3041–3046Google Scholar
  53. Shiau CE, Kaufman Z, Meireles AM, Talbot WS (2015) Differential requirement for irf8 in formation of embryonic and adult macrophages in zebrafish. PLoS One 10:e0117513. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Sierra A, Gottfried-Blackmore AC, McEwen BS, Bulloch K (2007) Microglia derived from ageing mice exhibit an altered inflammatory profile. Glia 55:412–424. CrossRefPubMedGoogle Scholar
  55. Spagnuolo C, Moccia S, Russo GL (2018) Anti-inflammatory effects of flavonoids in neurodegenerative disorders. Eur J Med Chem 153:105–115. CrossRefPubMedGoogle Scholar
  56. Stein VM, Baumgärtner W, Schröder S, Zurbriggen A, Vandevelde M, Tipold A (2007) Differential expression of CD45 on canine microglial cells. J Vet Med A Physiol Pathol Clin Med 54(6):314–320. CrossRefPubMedGoogle Scholar
  57. Stolzing A, Widmer R, Jung T, Voss P, Grune T (2006) Tocopherol-mediated modulation of age-related changes in microglial cells: turnover of extracellular oxidized protein material. Free Radic Biol Med 40:2126–2135. CrossRefPubMedGoogle Scholar
  58. Sun GY, Chen Z, Jasmer KJ, Chuang DY, Gu Z, Hannink M, Simonyi A (2015) Quercetin attenuates inflammatory responses in BV-2 microglial cells: role of MAPKs on the Nrf2 pathway and induction of heme oxygenase-1. PLoS One 10:e0141509. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Tang Y, Le W (2016) Differential roles of M1 and M2 microglia in neurodegenerative diseases. Mol Neurobiol 53:1181–1194. CrossRefPubMedGoogle Scholar
  60. Veverka M, Gallovič J, Švajdlenka E, Veverková E, Pronayová N, Miláčková I, Štefek M (2013) Novel quercetin derivatives: synthesis and screening for antioxidant activity and aldose reductase inhibition. Chem Papers 67(1):76–83. CrossRefGoogle Scholar
  61. von Bernhardi R, Eugenín-von Bernhardi L, Eugenín J (2015) Microglial cell dysregulation in brain ageing and neurodegeneration. Front Ageing Neurosci 7:1–21. CrossRefGoogle Scholar
  62. von Leden RE, Khayrullina G, Moritz KE, Byrnes KR (2017) Age exacerbates microglial activation, oxidative stress, inflammatory and NOX2 gene expression, and delays functional recovery in a middle-aged rodent model of spinal cord injury. J Neuroinflammation 14:161. CrossRefGoogle Scholar
  63. Wong WT (2013) Microglial ageing in the healthy CNS: phenotypes, drivers, and rejuvenation. Front Cell Neurosci.
  64. Xu H, Chen M, Manivannan A, Lois N, Forrester JV (2008) Age-dependent accumulation of lipofuscin in perivascular and sub-retinal microglia in experimental mice. Ageing Cell 7:58–68. CrossRefGoogle Scholar
  65. Zhang X, Yu L, Xu H (2016) Lysosome calcium in ROS regulation of autophagy. Autophagy 10:1954–1955. CrossRefGoogle Scholar
  66. Zizkova P, Stefek M, Rackova L, Prnova M, Horakova L (2017) Novel quercetin derivatives: from redox properties to promising treatment of oxidative stress related diseases. Chem Biol Interact 265:36–46. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Center of Experimental Medicine, Institute of Experimental Pharmacology and ToxicologySlovak Academy of SciencesBratislavaSlovak Republic

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