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Neuroprotective Effects of Ethyl Pyruvate against Aluminum Chloride-Induced Alzheimer’s Disease in Rats via Inhibiting Toll-Like Receptor 4


Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the formation of insoluble deposits of β-amyloid (Aβ) plaques within the parenchyma of the brain. The present study aimed to investigate the neuroprotective role of ethyl pyruvate against in vitro and in vivo model of aluminum chloride (AlCl3)-induced AD. Effect of ethyl pyruvate (5, 10, 20, 40 mM) against AlCl3 (1250 μM)-induced neurotoxicity in primary neuron-glial mixed cell culture was evaluated using cell viability assays (MTT assay as well as calcein-AM/propidium iodide fluorescent dyes). In vivo model, AlCl3 (50 mg/kg) were given through intraperitoneal route (i.p.) once daily for 4 weeks in rats and after 2 weeks, ethyl pyruvate (50, 100, 200 mg/kg/day) was co-administered with AlCl3 once daily via the oral route. The present study, in addition to perform histopathology of the brain, also estimated oxidant and antioxidant parameters as well as memory impairment using pole test, plus maze, and Morris water maze test. The binding mode of ethyl pyruvate in the hMD-2 was also studied. Results of in vitro studies showed that the AlCl3 administration resulted in neuronal cell death. AlCl3 administration in rats resulted in memory loss, oxidative stress (increased lipid peroxide and nitric oxide), impairment of antioxidant mechanisms (superoxide dismutase, catalase, and reduced glutathione), and deposition of amyloid plaques in cerebral cortex region of the brain. AlCl3 also resulted in the overexpression of the TLR4 receptors in the brain tissues. Administration of ethyl pyruvate ameliorated the AlCl3-induced neurotoxicity in neuron-glial mixed cell culture as well as histopathological, neurochemical, and behavioral consequences of chronic administration of AlCl3 in the rat. Ethyl pyruvate showed a docking score of 4.048. Thus, ethyl pyruvate is effective against in vitro and in vivo models of AlCl3-induced AD.

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  1. Baylock RL (2012) Aluminium induced immunoexcitotoxicity in neurodevelopmental and neurodegenerative disorders. Curr Inorg Chem 2(1):46–53

  2. Bennett-Guerrero E, Swaminathan M, Grigore AM, Roach GW, Aberle LG, Johnston JM, Fink MP (2009) A phase II multicenter double-blind placebo-controlled study of ethyl pyruvate in high-risk patients undergoing cardiac surgery with cardiopulmonary bypass. J Cardiothorac Vasc Anesth 23(3):324–329

  3. Bledi Y, Domb AJ, Linial M (2000) Culturing neuronal cells on surfaces coated by a novel polyethyleneimine-based polymer. Brain Res Brain Res Protoc 5(3):282–289

  4. Cai B, Brunner M, Wang H, Wang P, Deitch EA, Ulloa L (2009) Ethyl pyruvate improves survival in awake hemorrhage. J Mol Med (Berl) 87(4):423–433

  5. Ciaramelli C, Calabrese V, Sestito SE, Perez-Regidor L, Klett J, Oblak A, Jerala R, Piazza M, Martin-Santamaria S, Peri F (2016) Glycolipid-based TLR4 modulators and fluorescent probes: rational design, synthesis and biological properties. Chem Biol Drug Des 88(2):217–229

  6. Corl CM, Robinson HR, Contreras GA, Holcombe SJ, Cook VL, Sordillo LM (2010) Ethyl pyruvate diminishes the endotoxin-induced inflammatory response of bovine mammary endothelial cells. J Dairy Sci 93(11):5188–5199

  7. Das UN (2006) Is pyruvate an endogenous anti-inflammatory molecule? Nutrition 22(9):965–972

  8. Dong W, Cai B, Pena G, Pisarenko V, Vida G, Doucet D, Lee M, Sharpe S, Lu Q, Xu DZ, Ramos L, Deitch EA, Ulloa LM (2010) Ethyl pyruvate prevents inflammatory responses and organ damage during resuscitation in porcine hemorrhage. Shock 34(2):205–213

  9. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82(1):70–77

  10. Famili A, Ammar DA, Kahook MY (2013) Ethyl pyruvate treatment mitigates oxidative stress damage in cultured trabecular meshwork cells. Mol Vis 19:1304–1309

  11. Ghosh A, Dhumal VR, Tilak AV, Das N, Singh A, Bondekar AA (2011) Evaluation of nootropic and neuroprotective effects of low dose aspirin in rats. J Pharmacol Pharmacother 2(1):3–6

  12. Hanisch UK, Johnson TV, Kipnis J (2008) Toll-like receptors: roles in neuroprotection? Trends Neurosci 31(4):176–182

  13. Hanke ML, Kielian T (2011) Toll-like receptors in health and disease in brain: mechanisms and therapeutic potential. Clin Sci (Lond) 121(9):367–387

  14. Hevel JM, Marletta MA (1994) Nitric-oxide synthase assays. Methods Enzymol 233:250–258

  15. Huh SH, Chung YC, Piao Y, Jin MY, Son HJ, Yoon NS, Hong JY, Pak YK, Kim YS, Hong JK, Hwang O, Jin BK (2011) Ethyl pyruvate rescues nigrostriatal dopaminergic neurons by regulating glial activation in a mouse model of Parkinson’s disease. J Immunol 187(2):960–969

  16. Johansson AS, Johansson-Haque K, Okret S, Palmblad J (2008) Ethyl pyruvate modulates acute inflammatory reactions in human endothelial cells in relation to the NF-kappaB pathway. Br J Pharmacol 154(6):1318–1326

  17. Kawahara M, Muramoto K, Kobayashi K, Mori H, Kuroda Y (1994) Aluminium promotes the aggregation of Alzheimer’s amyloid beta-protein in vitro. Biochem Biophys Res Commun 198(2):531–535

  18. Kawahara M, Kato M, Kuroda Y (2001) Effects of aluminium on the neurotoxicity of primary cultured neurons and on the aggregation of B-amyloid protein. Brain Res Bull 55(2):211–217

  19. Kielian T (2006) Toll-like receptors in central nervous system glial inflammation and homeostasis. J Neurosci Res 83(5):711–730

  20. Kim JB, Yu YM, Kim SW, Lee JK (2005) Anti-inflammatory mechanism is involved in ethyl pyruvate-mediated efficacious neuroprotection in the postischemic brain. Brain Res 1060(1–2):188–192

  21. Kim SW, Jeong JY, Kim HJ, Seo JS, Han PL, Yoon SH, Lee JK (2010) Combination treatment with ethyl pyruvate and aspirin enhances neuroprotection in the postischemic brain. Neurotox Res 17(1):39–49

  22. Lehnardt S, Massillon L, Follett P, Jensen FE, Ratan R, Rosenberg PA, Volpe JJ, Vartanian T (2003) Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Proc Natl Acad Sci U S A 100(14):8514–8519

  23. Mandrekar S, Landreth GE (2010) Microglia and inflammation in Alzheimer’s disease. CNS Neurol Disord Drug Targets 9(2):156–167

  24. Marklund S, Marklund G (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47(3):469–474

  25. Moro N, Sutton RL (2010) Beneficial effects of sodium or ethyl pyruvate after traumatic brain injury in the rat. Exp Neurol 225(2):391–401

  26. Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11(1):47–60

  27. Mutlu O, Akar F, Celikyurt IK, Tanyeri P, Ulak G, Erden F (2015) 7-NI and ODQ disturbs memory in the elevated plus maze, Morris water maze, and radial arm maze tests in mice. Drug Target Insights 9:1–8

  28. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351–358

  29. Ohto U, Fukase K, Miyake K, Satow Y (2007) Crystal structures of human MD-2 and its complex with antiendotoxic lipid Iva. Science 316:1632–1634

  30. Ozacmak HS, Ozacmak VH, Turan I (2018) Ethyl pyruvate prevents from chronic cerebral hypoperfusion via preserving cognitive function and decreasing oxidative stress, caspase 3 activation and IL-1β level. Bratisl Lek Listy 119(8):469–475

  31. Pratico D, Uryu K, Sung S, Tang S, Trojanowski JQ, Lee VM (2002) Aluminium modulates brain amyloidosis through oxidative stress in APP transgenic mice. FASEB J 16(9):1138–1140

  32. Puchtler H, Sweat F, Levine M (1962) On the binding of Congo red by amyloid. J Histochem Cytochem 10:355–364

  33. Qiu Z, He Y, Ming H, Lei S, Leng Y, Xia ZY (2019) Lipopolysaccharide (LPS) aggravates high glucose and hypoxia/reoxygenation-induced injury through activating ROS-dependent NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes. J Diabetes Res 2019:8151836

  34. Schroeder EL, Holcombe SJ, Cook VL, James MD, Gandy JC, Hauptman JG, Sordillo LM (2011) Preliminary safety and biological efficacy studies of ethyl pyruvate in normal mature horses. Equine Vet J 43(3):341–347

  35. Shen H, Hu X, Liu C, Wang S, Zhang W, Gao H, Stetler RA, Gao Y, Chen J (2010) Ethyl pyruvate protects against hypoxic-ischemic brain injury via anti-cell death and anti-inflammatory mechanisms. Neurobiol Dis 37(3):711–722

  36. Singh SK, Srivastav S, Yadav AK, Srikrishna S, Perry G (2016) Overview of Alzheimer’s disease and some therapeutic approaches targeting Aβ by using several synthetic and herb compounds. Oxidative Med Cell Longev 2016:7361613

  37. Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem 47:389–394

  38. Su X, Wang H, Zhao J, Pan H, Mao L (2011) Beneficial effects of ethyl pyruvate through inhibiting high-mobility group box 1 expression and TLR4/NF-ƙB pathway after traumatic brain injury in the rat. Mediat Inflamm 2011:807142

  39. Takeda K, Akira S (2004) TLR signaling pathways. Semin Immunol 16(1):3–9

  40. van de Loosdrecht AA, Beelen RH, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MM (1994) A tetrazolium–based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods 174(1–2):311–320

  41. Vorhees CV, Williams MT (2006) Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1(2):848–858

  42. Vyas SB, Duffy LK (1995) Stabilization of secondary structure of Alzheimer beta-protein by aluminium (III) ions and D-asp substitutions. Biochem Biophys Res Commun 206(2):718–723

  43. Walter S, Letiembre M, Liu Y, Heine H, Penke B, Hao W, Bode B, Manietta N, Walter J, Schulz-Schuffer W, Fassbender K (2007) Role of the toll-like receptor 4 in neuroinflammation in Alzheimer’s disease. Cell Physiol Biochem 20(6):947–956

  44. Yang ZY, Ling Y, Yin T, Tao J, Xiong JX, Wu HS, Wang CY (2008) Delayed ethyl pyruvate therapy attenuates experimental severe acute pancreatitis via reduced serum high mobility group box 1 levels in rats. World J Gastroenterol 14(28):4546–4550

  45. Yang R, Zhu S, Tonnessen TI (2016) Ethyl pyruvate is a novel anti-inflammatory agent to treat multiple inflammatory organ injuries. J Inflamm (Lond) 13:37

  46. Yu YM, Kim JB, Lee KW, Kim SY, Han PL, Lee JK (2005) Inhibition of the cerebral ischemic injury by ethyl pyruvate with a wide therapeutic window. Stroke 36(10):2238–2243

  47. Yuan Y, Su Z, Pu Y, Liu X, Chen J, Zhu F, Zhu Y, Zhang H, He C (2012) Ethyl pyruvate promotes spinal cord repair by ameliorating the glial microenvironment. Br J Pharmacol 166(2):749–763

  48. Zilberter Y, Gubkina O, Ivanov AI (2015) A unique array of neuroprotective effects of pyruvate in neuropathology. Front Neurosci 9:17

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The manuscript is based on M.S. (Pharm.) Dissertation project work done by Ms. Vijaya Durga Chavali from July 2014 to June 2015 at the Department of Pharmacology and Toxicology, NIPER-Ahmedabad. However, the in silico work is done in the Institute of Pharmacy, Nirma University. The authors acknowledge B. V. Patel PERD Centre, Ahmedabad, for institutional support.


Authors recognize the Department of Pharmaceuticals, Ministry of Chemical and Fertilizers, Government of India, and National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Gandhinagar, India, for financial support.

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Correspondence to Bhagawati Saxena.

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Animal housing and handling were performed as per Good Laboratory Practice (GLP) mentioned in CPCSEA guidelines. Institutional Animal Ethics Committee (IAEC) approved the in vitro and in vivo experimentation protocol (Protocol No. PERD/IAEC/2014/005 and PERD/IAEC/2014/006).

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The authors declare that there is no conflict of interest.

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Chavali, V.D., Agarwal, M., Vyas, V.K. et al. Neuroprotective Effects of Ethyl Pyruvate against Aluminum Chloride-Induced Alzheimer’s Disease in Rats via Inhibiting Toll-Like Receptor 4. J Mol Neurosci (2020). https://doi.org/10.1007/s12031-020-01489-9

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  • Alzheimer’s disease
  • Ethyl pyruvate
  • Neuroinflammation
  • Toll-like receptor 4
  • Memory loss