THE EFFECT OF OUABAIN AND BUFALIN ON THE NEURONS OF PRIMARY CULTURES OF RAT CEREBRAL CORTEX UNDER GLUCOSE-OXYGEN DEPRIVATION

DOI: https://doi.org/10.29296/25877313-2019-10-03
Issue: 
10
Year: 
2019

A.V. Lopachev Junior Research Scientist, Research Center of Neurology (Moscow) E-mail: lopsasha@yandex.ru O.M. Lopacheva Engineer, International Biotechnological Center, Lomonosov Moscow State University; Junior Research Scientist, Research Center of Neurology (Moscow) K.N. Kulichenkova Post-graduate Student, Junior Researchch Scientist, Research Center of Neurology (Moscow) O.I. Kulikova Junior Researchch Scientist, Research Center of Neurology (Moscow) T.N. Fedorova Dr.Sc. (Biol.), Head of Laboratory of Clinical and Experimental Neurochemistry, Research Center of Neurology (Moscow)

Objectives: The objective of this project was the comparison of neuroprotective effects of the cardiotonic steroids (CTS) ouabain and bufalin in a primary culture of rat cortical neurons in conditions of glucose-oxygen deprivation. In addition, investigation of the activation of intracellular singnal-ing cascades associated with the viability of rat cortical neurons in a primary culture was conducted. Material and methods: Modeling of 4-hour glucose-oxygen deprivation, followed by a 20-hour re-oxygenation, was performed on a primary culture of rat cortical neurons (10-12 days in vitro). Ouabain and bufalin in concentrations of 10 nM and 100 nM were administrated 30 min before glu-cose-oxygen deprivation. Oxidative stress was induced through a 24-hour long incubation of the culture with 20 mkM rotenone. Viability of the culture after the experimental procedures was evaluated using the MTT-test. The level of Akt kinase activation was evaluated by comparing the ratio of phos-phorylated Akt protein to total protein of the Akt kinase using Western Blot. Results: 30-minute preincubation with 10 nM and 100 nM ouabain, unlike bufalin, protected neurons from death. At the same time, ouabain did not prevent neuronal death when oxidative stress was induced using 20 mkM rotenone. Unlike bufalin, ouabain caused an increase in Akt kinase activa-tion. Conclusion: Ouabain, but not bufalin, has a neuroprotective effect in conditions of glucose-oxygen deprivation. Simultaneously, in our experi-ments ouabain does not have a neuroprotective effect under direct induction of oxidative stress. The neuroprotective effect of ouabain is likely due to its ability to activate the Akt kinase, which bufalin does not do.

Keywords: 
ouabain
bufalin
ischemia
oxidative stress
Akt

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