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Biochemistry (Moscow)

, Volume 81, Issue 5, pp 511–520 | Cite as

Neuroprotective effect of carnosine on primary culture of rat cerebellar cells under oxidative stress

  • A. V. LopachevEmail author
  • O. M. Lopacheva
  • D. A. Abaimov
  • O. V. Koroleva
  • E. A. Vladychenskaya
  • A. A. Erukhimovich
  • T. N. Fedorova
Article

Abstract

Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatographymass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

Keywords

carnosine AAPH rotenone oxidative stress neuron antioxidant neuroprotective effect 

Abbreviations

AAPH

2,2'-azobis(2-amidinopropane)dihy-drochloride carnosine, ß-alanyl-L-histidine

CNS

central nervous system

DCF

dichlorofluorescein

MAPK

mitogen-activated protein kinases

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MTT

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

PEPT1 and PEPT2

peptide transporters 1 and 2

ROS

reactive oxygen species

rotenone

(2R,6aS,12aS)-1,2,6,6a,12,12a-hexahydro-2-iso-propenyl-8,9-dimethoxychromeno[3,4-b]furo(2,3-h)chromen-6-one

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Copyright information

© Pleiades Publishing, Ltd. 2016

Authors and Affiliations

  • A. V. Lopachev
    • 1
    • 2
    Email author
  • O. M. Lopacheva
    • 1
    • 3
  • D. A. Abaimov
    • 1
  • O. V. Koroleva
    • 2
  • E. A. Vladychenskaya
    • 1
    • 4
  • A. A. Erukhimovich
    • 2
  • T. N. Fedorova
    • 1
  1. 1.Research Center of NeurologyMoscowRussia
  2. 2.Bach Institute of Biochemistry, Research Center of BiotechnologyRussian Academy of SciencesMoscowRussia
  3. 3.Lomonosov Moscow State UniversityInternational Biotechnological CenterMoscowRussia
  4. 4.Lomonosov Moscow State UniversityFaculty of BiologyMoscowRussia

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