Gallic and vanillic acid suppress inflammation and promote myelination in an in vitro mouse model of neurodegeneration

  • Sonia Siddiqui
  • Aisha Kamal
  • Faisal Khan
  • Khawar Saeed Jamali
  • Zafar Saeed Saify
Original Article


Neuroinflammation affects millions of people around the world as a result of injury or stress. Neuroinflammation represents almost all types of neurological diseases such as multiple sclerosis and Alzheimer’s disease. Neurodegenerative diseases comprise demyelination and synaptic loss. The inflammatory response is further propagated by the activation of glial cells and modulation of constitutively expressed extracellular matrix proteins. The aim of the present study was to identify the anti-inflammatory effects of purified compounds gallic acid (GA, 1.0 µM) and vanillic acid (VA, 0.2 µM) on the lysolecithin (LPC, 0.003%)-induced model of inflammation. Hippocampal neurons were co-cultured with glial cells, and LPC was added to induce inflammation. Neurite outgrowth was measured by morphometry software. The level of myelination and demyelination was identified by immunostaining and sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting techniques using different antibodies. Whole-cell patch clamp recordings were used to observe the sustained repetitive firing pattern. The data showed that GA and VA significantly increased the neurite outgrowth after 48 h in culture. Both compounds significantly reduced the expression of cyclooxygenase-2, NFκB, tenascin-C, chondroitin sulfate proteoglycans and glial fibrillary acidic protein in astrocytes in the LPC-induced model of inflammation. The level of myelin protein in neurites and oligodendrocyte cell bodies was significantly upregulated by GA and VA treatment. The reduction in sustained repetitive firing in the LPC-induced model of inflammation was reversed by both GA and VA treatment. This study supports the hypothesis that VA and GA have anti-inflammatory activities and could be regarded as potential treatments for neurodegenerative disease.


Lysolecithin CNS injury Remyelination GA VA ECM proteins 



The authors are gratified for the Research Grant # 2680 provided by Higher Education Commission (HEC) of Pakistan.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interests.

Ethical approval

The experiments were performed to the Protocol 2015-0007, assigned by Advisory Committee on Animal Standards, ICCBS, University of Karachi.

Supplementary material

11033_2018_4557_MOESM1_ESM.tif (4.7 mb)
Supplementary material 1 (TIF 4782 KB) Supplementary Fig. 1 Dose–response effects of gallic and vanillic acid on hippocampal neurite outgrowth. ad Neurons were cultured in the presence of different concentrations of GA and VA acid (µM: 0.1, 0.2, 0.5, 1, 1.5) for 48 h. a, c Mean average lengths of the 10 longest neurites from each group. b, d Sum of all neurite lengths was calculated as the sum of all neurites in each group (n = 180). Neurite lengths were measured by tracing the entire length of the neurite from the cell body. The results are shown as the means ± SD. Statistical differences were revealed by Mann–Whitney U-test. **p < 0.001
11033_2018_4557_MOESM2_ESM.tif (5.8 mb)
Supplementary material 2 (TIF 5956 KB). Supplementary Fig. 2 Expression of proteins. a and b Primary oligodendrocytes were stained with antibodies against TN-C and CSPGs. Asterisks shows the oligodendrocytes. Scale bar = 50 µm. c Neurons were stained with antibody against neurofilament. d Cortical astrocytes were stained with antibody against GFAP. Arrows represents neurons or astrocytes. Scale bar = 25 µm. e and f Neuroglial co-culture immunostained with antibodies against LN and CSPGs. Arrows represents neurons and arrowheads represents neurites. Scale bar = 25 µm. g and h Neurons were stained with antibodies against CSPG and TN-C. Asterisks shows neurons. Scale bar = 50 µm. i and j Astrocytes were stained with antibodies with LN and TN-C. Asterisks shows the astrocytes. Scale bar = 50 µm


  1. 1.
    Jha MK, Jeon S, Suk K (2012) Glia as a link between neuroinflammation and neuropathic pain. Immune Netw 12:41–47CrossRefGoogle Scholar
  2. 2.
    Skaper SD, Facci L, Giusti P (2014) Mast cells, glia and neuroinflammation: partners in crime? Immunology 141:314–327CrossRefGoogle Scholar
  3. 3.
    Harlow DE, Macklin WB (2014) Inhibitors of myelination: ECM changes, CSPGs and PTPs. Exp Neurol 251:39–46CrossRefGoogle Scholar
  4. 4.
    Calixto JB, Campos MM, Otuki MF, Santos AR (2004) Anti-inflammatory compounds of plant origin. Part II. Modulation of pro-inflammatory cytokines, chemokines and adhesion molecules. Planta Med 70:93–103CrossRefGoogle Scholar
  5. 5.
    Calixto JB, Otuki MF, Santos AR (2003) Anti-inflammatory compounds of plant origin. Part I. Action on arachidonic acid pathway, nitric oxide and nuclear factor κB (NF-κB). Planta Med 69:973–983CrossRefGoogle Scholar
  6. 6.
    Bellik Y, Boukraa L, Alzahrani HA, Bakhotmah BA, Abdellah F, Hammoudi SM, Iguer-Ouada M (2012) Molecular mechanism underlying anti-inflammatory and anti-allergic activities of phytochemicals: an update. Molecules 18:322–353CrossRefGoogle Scholar
  7. 7.
    Kim YS, Young MR, Bobe G, Colburn NH, Milner JA (2009) Bioactive food components, inflammatory targets, and cancer prevention. Cancer Prev Res 2:200–208CrossRefGoogle Scholar
  8. 8.
    Hertog MGL, Feskens EJM, Kromhout D, Hertog MGL, Hollman PCH, Hertog MGL, Katan MB (1993) Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen elderly study. Lancet 342:1007–1011CrossRefGoogle Scholar
  9. 9.
    Prince PS, Dhanasekar K, Rajakumar S (2011) Preventive effects of vanillic acid on lipids, bax, bcl-2 and myocardial infarct size on isoproterenol-induced myocardial infarcted rats: a biochemical and in vitro study. Cardiovasc Toxicol 11:58–66CrossRefGoogle Scholar
  10. 10.
    Liu KY, Hu S, Chan BC, Wat EC, Lau CB, Hon KL, Fung KP, Leung PC, Hui PC, Lam CW, Wong CK (2013) Anti-inflammatory and anti-allergic activities of Pentaherb formula, Moutan Cortex (Danpi) and gallic acid. Molecules 18:2483–2500CrossRefGoogle Scholar
  11. 11.
    Choi KC, Lee YH, Jung MG, Kwon SH, Kim MJ, Jun WJ, Lee J, Lee JM, Yoon HG (2009) Gallic acid suppresses lipopolysaccharide-induced nuclear factor-kappaB signaling by preventing RelA acetylation in A549 lung cancer cells. Mol Cancer Res 7:2011–2021CrossRefGoogle Scholar
  12. 12.
    Venkatesan R, Ji E, Kim SY (2015) Phytochemicals that regulate neurodegenerative disease by targeting neurotrophins: a comprehensive review. Biomed Res Int 2015:814068CrossRefGoogle Scholar
  13. 13.
    Pavelko KD, van Engelen BG, Rodriguez M (1998) Acceleration in the rate of CNS remyelination in lysolecithin-induced demyelination. J Neurosci 18:2498–2505CrossRefGoogle Scholar
  14. 14.
    Hamill CE, Goldshmidt A, Nicole O, McKeon RJ, Brat DJ, Traynelis SF (2005) Special lecture: glial reactivity after damage: implications for scar formation and neuronal recovery. Clin Neurosurg 52:29–44PubMedGoogle Scholar
  15. 15.
    Fitch MT, Silver J (2008) CNS injury, glial scars, and inflammation: inhibitory extracellular matrices and regeneration failure. Exp Neurol 209:294–301CrossRefGoogle Scholar
  16. 16.
    Siddiqui S, Horvat-Broecker A, Faissner A (2009) Comparative screening of glial cell types reveals extracellular matrix that inhibits retinal axon growth in a chondroitinase ABC-resistant fashion. Glia 57:1420–1438CrossRefGoogle Scholar
  17. 17.
    Siddiqui S, Horvat-Brocker A, Faissner A (2008) The glia-derived extracellular matrix glycoprotein tenascin-C promotes embryonic and postnatal retina axon outgrowth via the alternatively spliced fibronectin type III domain TNfnD. Neuron Glia Biol 4:271–283CrossRefGoogle Scholar
  18. 18.
    Stettner M, Wolffram K, Mausberg AK, Albrecht P, Derksen A, Methner A, Dehmel T, Hartung HP, Dietrich H, Kieseier BC (2013) Promoting myelination in an in vitro mouse model of the peripheral nervous system: the effect of wine ingredients. PLoS ONE 8:e66079CrossRefGoogle Scholar
  19. 19.
    Fulmer CG, VonDran MW, Stillman AA, Huang Y, Hempstead BL, Dreyfus CF (2014) Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination. J Neurosci 34:8186–8196CrossRefGoogle Scholar
  20. 20.
    Makar TK, Bever CT, Singh IS, Royal W, Sahu SN, Sura TP, Sultana S, Sura KT, Patel N, Dhib-Jalbut S, Trisler D (2009) Brain-derived neurotrophic factor gene delivery in an animal model of multiple sclerosis using bone marrow stem cells as a vehicle. J Neuroimmunol 210:40–51CrossRefGoogle Scholar
  21. 21.
    Locatelli C, Filippin-Monteiro FB, Creczynski-Pasa TB (2013) Alkyl esters of gallic acid as anticancer agents: a review. Eur J Med Chem 60:233–239CrossRefGoogle Scholar
  22. 22.
    Lirdprapamongkol K, Sakurai H, Kawasaki N, Choo MK, Saitoh Y, Aozuka Y, Singhirunnusorn P, Ruchirawat S, Svasti J, Saiki I (2005) Vanillin suppresses in vitro invasion and in vivo metastasis of mouse breast cancer cells. Eur J Pharm Sci 25:57–65CrossRefGoogle Scholar
  23. 23.
    Hsu JY, Bourguignon LY, Adams CM, Peyrollier K, Zhang H, Fandel T, Cun CL, Werb Z, Noble-Haeusslein LJ (2008) Matrix metalloproteinase-9 facilitates glial scar formation in the injured spinal cord. J Neurosci 28:13467–13477CrossRefGoogle Scholar
  24. 24.
    Fujita Y, Yamashita T (2014) Axon growth inhibition by RhoA/ROCK in the central nervous system. Front Neurosci 8:338CrossRefGoogle Scholar
  25. 25.
    Suzumura A (2013) Neuron–microglia interaction in neuroinflammation. Curr Protein Pept Sci 1:16–20CrossRefGoogle Scholar
  26. 26.
    Kassubek R, Gorges M, Schocke M, Hagenston VAM, Huss A, Ludolph AC, Kassubek J, Tumani H (2017) GFAP in early multiple sclerosis: a biomarker for inflammation. Neurosci Lett 657:166–170CrossRefGoogle Scholar
  27. 27.
    Ji K, Tsirka SE (2012) Inflammation modulates expression of laminin in the central nervous system following ischemic injury. J Neuroinflamm 9:159CrossRefGoogle Scholar
  28. 28.
    Claycomb KI, Winokur PN, Johnson KM, Nicaise AM, Giampetruzzi AW, Sacino AV, Snyder EY, Barbarese E, Bongarzone ER, Crocker SJ (2014) Aberrant production of tenascin-C in globoid cell leukodystrophy alters psychosine-induced microglial functions. J Neuropathol Exp Neurol 73:964–974CrossRefGoogle Scholar
  29. 29.
    Kataria H, Alizadeh A, Shahriary GM, Saboktakin Rizi S, Henrie R, Santhosh KT, Thliveris JA, Karimi-Abdolrezaee S (2018) Neuregulin-1 promotes remyelination and fosters a pro-regenerative inflammatory response in focal demyelinating lesions of the spinal cord. Glia 66:538–561CrossRefGoogle Scholar
  30. 30.
    Yucel-Lindberg T, Nilsson S, Modéer T (1999) Signal transduction pathways involved in the synergistic stimulation of prostaglandin production by interleukin-1beta and tumor necrosis factor alpha in human gingival fibroblasts. J Dent Res 1:61–68CrossRefGoogle Scholar
  31. 31.
    Font-Nieves M, Sans-Fons MG, Gorina R, Bonfill-Teixidor E, Salas-Pérdomo A, Márquez-Kisinousky L, Santalucia T, Planas AM (2012) Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes. J Biol Chem 287:6454–6468CrossRefGoogle Scholar
  32. 32.
    Medeiros R, Figueiredo CP, Pandolfo P, Duarte FS, Prediger RD, Passos GF, Calixto JB (2010) The role of TNF-alpha signaling pathway on COX-2 upregulation and cognitive decline induced by beta-amyloid peptide. Behav Brain Res 209:165–173CrossRefGoogle Scholar
  33. 33.
    Satoh H, Amagase K, Ebara S, Akiba Y, Takeuchi K (2013) Cyclooxygenase (COX)-1 and COX-2 both play an important role in the protection of the duodenal mucosa in cats. J Pharmacol Exp Ther 344:189–195CrossRefGoogle Scholar
  34. 34.
    Lawrence T (2009) The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Ghasemlou N, Jeong SY, Lacroix S, David S (2007) T cells contribute to lysophosphatidylcholine-induced macrophage activation and demyelination in the CNS. Glia 55(3):294–302CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Institute of Biomedical SciencesDow University of Health SciencesKarachiPakistan
  2. 2.Department of Neuroscience, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological SciencesUniversity of KarachiKarachiPakistan
  3. 3.Department of SurgeryDow University of Health SciencesKarachiPakistan
  4. 4.HEJ Research Institute of Chemistry, International Center for Chemical and Biological SciencesUniversity of KarachiKarachiPakistan

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