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Effects of Estrogen and Phytoestrogen Treatment on an In Vitro Model of Recurrent Stroke on HT22 Neuronal Cell Line

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Abstract

An increase of stroke incidence occurs in women with the decline of estrogen levels following menopause. This ischemic damage may recur, especially soon after the first insult has occurred. We evaluated the effects of estrogen and phytoestrogen treatment on an in vitro recurrent stroke model using the HT22 neuronal cell line. HT22 cells were treated with 17β-estradiol or genistein 1 h after the beginning of the first of two oxygen and glucose deprivation/reoxygenation (OGD/R) cycles. During the second OGD, there was a deterioration of some components of the electron transport chain, such as cytochrome c oxidase subunit 1 with a subsequent increase of reactive oxygen species (ROS) production. Accordingly, there was also an increase of apoptotic phenomena demonstrated by poly(ADP-ribose) polymerase 1 cleavage, Caspase-3 activity, and Annexin V levels. The recurrent ischemic injury also raised the hypoxia-inducible factor 1α and glucose transporter 1 levels, as well as the ratio between the lipidated and cytosolic forms of microtubule-associated protein 1A/1B-light chain 3 (LC3-II/LC3-I). We found a positive effect of estradiol and genistein treatment by partially preserving the impaired cell viability after the recurrent ischemic injury; however, this positive effect does not seem to be mediated neither by blocking apoptosis processes nor by decreasing ROS production. This work contribute to the better understanding of the molecular mechanisms triggered by recurrent ischemic damage in neuronal cells and, therefore, could help with the development of an effective treatment to minimize the consequences of this pathology.

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Abbreviations

OGD/R:

Oxygen and glucose deprivation/reoxygenation

OGD:

Oxygen and glucose deprivation

CO-1:

Cytochrome c oxidase subunit 1

ROS:

Reactive oxygen species

PARP-1:

Poly(ADP-ribose) polymerase 1

HIF-1α:

Hypoxia-inducible factor 1α

GLUT:

Glucose transporter

LC3:

Microtubule-associated protein 1A/1B-light chain 3

I/R:

Ischemia/reperfusion

ETC:

Electron transport chain

PH:

Prolyl hydroxylase

HRE:

Hypoxia response element

ER:

Estrogen receptor

SERM:

Selective estrogen receptor modulator

DMEM:

Dulbecco’s Modified Eagle’s Medium

FBS:

Fetal bovine serum

N:

Cells cultured in complete medium under normoxia

V:

Cells subjected to OGD or OGD/R and treated with vehicle

E:

Cells subjected to OGD or OGD/R and treated with estradiol

G:

Cells subjected to OGD or OGD/R and treated with genistein

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

TBS-T:

Tris-HCL 20 mM pH 7.5, NaCl 150 mM, Tween-20 0,1 % v/v

HRP:

Horseradish peroxidase

ECL:

Enhanced chemiluminescence

DCFDA:

2′,7′-Dichlorofluorescein diacetate

PI:

Propidium iodide

AV:

Annexin V

CNS:

Central nervous system

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Acknowledgments

This study was funded by European Union FEDER funds, Plan de Ciencia, Tecnología e Innovación del Principado de Asturias, FICYT (GRUPIN 14-069).

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Authors and Affiliations

  1. Department of Functional Biology. Physiology Area, University of Oviedo, Av. Julián Clavería, No. 6, 33006, Oviedo, Spain

    Javier Morán, Pablo Garrido, Elena Díaz, Ana Alonso & Celestino González

  2. Unit of Transplants, Cell Therapy and Regenerative Medicine, Central University Hospital of Asturias, Av. Roma s/n, 33011, Oviedo, Spain

    Marcos Perez-Basterrechea & Jesús Otero

  3. Service of Hematology, Central University Hospital of Asturias, Av/Roma s/n, 33011, Oviedo, Spain

    Enrique Colado

  4. Department of Laboratory Medicine, Central University Hospital of Asturias, Av/Roma s/n, 33011, Oviedo, Spain

    Enrique Colado

  5. Department of Clinical Neuroscience and Rehabilitation, Sahlgrenska Academy, University of Gothenburg, Medicinaregatan 9A, Box 440, 413 90, Gothenburg, Sweden

    Javier Morán

  6. Prostate Cancer Institute, National University of Ireland, Galway. Co, Galway, Ireland

    Pablo Garrido

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  1. Javier Morán
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Morán, J., Perez-Basterrechea, M., Garrido, P. et al. Effects of Estrogen and Phytoestrogen Treatment on an In Vitro Model of Recurrent Stroke on HT22 Neuronal Cell Line. Cell Mol Neurobiol 37, 405–416 (2017). https://doi.org/10.1007/s10571-016-0372-1

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