HÜCRE KÜLTÜRÜ ORTAMINDA KARVAKROLÜN KOBALT KLORÜR İLE İNDÜKLENEN KİMYASAL HİPOKSİDEKİ KORUYUCU ROLÜ

Year 2023, , 354 - 361, 23.09.2023

Ahmi Öz

https://doi.org/10.17343/sdutfd.1261969

Abstract

Amaç
Hipoksi nöronal hasar açısından en önemli faktörlerden
biridir. Nöronlarda eksprese edilen TRPM7 katyon
kanallarının hipoksi ve hücresel pH değişimleri
dahil birçok faktörle aktive olduğu bilinmektedir. Bu
nedenle bu araştırmada deneysel çalışmalarda hipoksi
modeli oluşturmak için sıklıkla kullanılan kobalt
klorür (CoCl2) ile indüklenen in vitro hipoksi modelinde
TRPM7 katyon kanallarının güçlü blokörü karvakrolün
hücresel sağkalım ve ölüm parametreleri üzerine etkisinin
araştırılması amaçlanmıştır.
Gereç ve Yöntem
SH-SY5Y hücreleri kültür flasklarında çoğaltıldı. Hücrelere
hipoksi uygulaması için 200 μM CoCl2 içeren
medyum ile 24 saat inkübasyon yapıldı. Karvakrolün
etkisinin sınandığı grupta ise hücreler TRPM7 kanal
inhibisyonunu sağlamak üzere 1 saat karvakrol (250
μM) içeren medyum ile inkübe edildikten sonra hipoksi
uygulanarak inkübasyon tamamlandı. Ardından kültür
kaplarından kaldırılan hücreler, apoptoz testi, MTT
hücre canlılığı analizi, reaktif oksijen türleri (ROT)
üretimi tayini, mitokondriyal membran depolarizasyonu
(MMD) tayini ve kaspaz 3, 8 ve 9 enzim aktiviteleri
tayini yapıldı.
Bulgular
Kontrole kıyasla hipoksi uygulaması yapılan grupta
hücre canlılığı azalırken canlılığın azaldığını gösteren
diğer parametrelerde (apoptoz, ROT üretimi, MMD
ve kaspaz aktivitelerinde) artış olduğu belirlendi. Karvakrol
ön inkübasyonu sonrasında CoCl2 ile hipoksi
uygulanan grupta ise hücresel canlılığın düştüğünü
gösteren parametrelerin yalnız CoCl2 uygulanan gruba
kıyasla anlamlı ölçüde azaldığı gözlemlendi.
Sonuç
Karvakrol uygulaması ile hipoksik koşulların ortaya çıkardığı
anormal hücresel hasar durumu ve hücresel
ölüm mekanizmaları yavaşlatılabilmektedir. TRPM7
katyon kanal ekspresyonu iyi bilinen SH-SY5Y hücrelerinde
karvakrolün olumlu etkilerinin daha çok
TRPM7 kanalları aracılı gerçekleştiği düşünülmektedir.

Keywords

Karvakrol, Hipoksi, TRPM7 Katyon Kanalları, Nöroproteksiyon

PROTECTIVE ROLE OF CARVACROL IN COBALT CHLORIDE-INDUCED CHEMICAL HYPOXIA IN CELL CULTURE MEDIA

Abstract

Objective
Hypoxia is among the most important factors
regarding to neuronal injury. TRPM7 cation channels,
which are expressed in neurons, and it is well
known that TRPM7 channels are activated by many
factors including hypoxia and cellular pH changes.
Therefore, in this study, it was aimed to investigate
the effect of carvacrol, a potent inhibitor of TRPM7
cation channels, on cellular survival and apoptotic
parameters in an in vitro hypoxia model induced by
cobalt chloride (CoCl2), which is frequently used to do
hypoxia in experimental studies.
Material and Method
The SH-SY5Y cells were grown in cell culture flasks.
Cells were incubated for 24 hours with medium
containing 200 μM CoCl2 to induction of hypoxia. In
the group, in which the effect of carvacrol was tested,
the cells were incubated with a medium containing
carvacrol (250 μM) for 1 h to TRPM7 channel
inhibition, and the incubation was completed by
applying hypoxia for 24 h. Then, the cells detached
from the culture flasks and apoptosis test, MTT cell
viability analysis, reactive oxygen species (ROT)
production determination, mitochondrial membrane
depolarization (MMD) determination and caspase 3,
8 and 9 enzyme activities were performed.
Results
It was demonstrated that while cell viability decreased
in the hypoxia-induced group compared to the
control, there was an increase in other parameters
(apoptosis, ROS production, MMD and caspase
activities) that reduced to cell viability. It was observed
that the parameters reducing cellular viability were
significantly decreased in the hypoxia-treated group
upon carvacrol pre-incubation compared to the CoCl2
group.
Conclusion
Carvacrol administration can be slowed down to
abnormal cellular damage and cell death mechanisms
caused by hypoxic conditions. It is thought that the
positive effects of carvacrol in SH-SY5Y cells, whose
TRPM7 cation channel expression is well known, are
mediated by mostly TRPM7 channels.

Keywords

Carvacrol, Hypoxia, TRPM7 Cation Channels, Neuroprotection

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