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

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

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

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