Nöronal hücrelerde hidrojen peroksit hasarına karşı epigallokateşin gallat ve resveratrolün koruyucu etkilerinin araştırılması
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Tarih
2021
Yazarlar
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Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Nörodejenerasyon nöronal hücrelerin ilerleyici kaybı ve yapısal bütünlüğünün bozulmasıdır. Nörodejeneratif hastalıkların görülme sıklığı ise her geçen gün artmaktadır. Nöronların hasarlanarak kayba uğramasına bağlı olarak nörodejeneratif hastalıkların ortaya çıkmasında genetik faktörler, çevresel etkenler ve oksidatif stres rol oynamaktadır. Doğada bulunan çeşitli fenollerin nörodejenerasyonu geciktirmek ya da engellemek amacı ile kullanımı gündemdedir. Vücudumuzda elektron taşıma sistemlerinin çeşitli noktalarında süperoksit radikali oluşabilmektedir. Hidrojen peroksit ise temel olarak süperoksit radikalinin dismutasyonu ile oluşur. Süperoksit radikali süperoksit dizmutaz enzimi aracılığı ile iki proton alarak hidrojen peroksiti oluşturur. İskemi, travma, hemoraji ve radyoaktivite gibi durumlarda mitokondrideki aerobik fosforilasyon değişir ve buna bağlı olarak elektron taşıma sisteminden oksijen kaçakları artar. Vücudumuzda normal metabolizma esnasında ya da çeşitli dış faktörler aracılığı ile hidrojen peroksit gibi serbest radikaller artmakta, başta kanser ve kalp-damar hastalıkları gibi çeşitli hastalıklara sebep olmaktadır. Bu zararlı türler vücudun endojen antioksidan sistemleri ile kontrol altında tutulmaktadır. Antioksidan sistemlerin diyetle alınacak antioksidan bileşenlerle desteklenmesi oksidan partiküllere bağlı hastalıkların önlenmesinde büyük önem taşımaktadır. Yeşil çayda bulunan epigallokateşin-3-gallat ve üzümde bulunan resveratrol ise güçlü antioksidan bileşiklerdendir. Epigallokateşin-3-gallat (EGCG); DNA hipermetilasyonunu, telomeraz aktivitesini, anjiyogenezi, metastazı, tümör hücresi apopitozisini, tümör baskılayıcı genleri ve onkogenleri etkileyerek çeşitli yolaklar üzerinden antikarsinojen etki gösterir. EGCG'nin antikarsinojen ve güçlü bir antioksidan olmasının yanı sıra antiinflamatuar, antifibrotik, osteojenik etkileri bulunur. Resveratrol ise temelde antioksidan etkisini; NAPH oksidaz ile üretilen reaktif oksijen miktarını, mitokondrial süperoksit oluşumunu, nitrik oksit sentazdan süperoksit oluşumunu, oksidaz aktivitesini ve ekspresyonunu azaltarak sağlamaktadır. EGCG'nin antikarsinojenik yolaklarına ek olarak resveratrol de hücre büyümesini ve bölünmesini, inflamasyonu, apopitozu, metastazı ve anjiyogenezi kontrol eden çeşitli sinyal-transdüksiyon yollarını etkileyerek başlangıçtan ilerlemeye kadar çeşitli kanser aşamalarını etkiler. Bu çalışma SH-SY5Y nöronal hücrelerinde epigallokateşin-3-gallat ve resveratrol antioksidan ajanlarının hidrojen peroksit hasarına karşı koruyucu etkilerinin araştırılması ve kıyaslanması amacıyla yapılmıştır. Bu amaçla literatür taramaları sonucunda önce hidrojen peroksitin etkin dozu ve inkübasyon süresi saptandı, bu doz 250 µM olarak belirlendi ve toksisite uygulanan gruplarda bu doz kullanıldı. SH-SY5Y hücrelerinde epigallokateşin-3-gallat, resveratrol, epigallokateşin-3-gallat ve resveratrol birlikte olmak üzere üç uygulama grubuna 2 ve 24 saatlik ön tedavinin ardından hidrojen peroksit toksisitesi uygulandı. Bu gruplarda MTS testi ile hücre sağ kalımları değerlendirildi. Hücrelerin koloni oluşturma yeteneklerini gözlemlemek için klonojenik test uygulandı ve sonuçlar post-hoc Turkey HSD testi ile değerlendirildi. Her iki değerlendirme sonucunda da epigallokateşin-3-gallat ve resveratrolün düşük dozlarda daha koruyucu olduğu ve koloni oluşturma yeteneğini arttırdığı gözlendi. Bu çalışmanın bir diğer basamağı ise ELISA testi ile hücrelerdeki kaspaz-3 seviyesi üzerinden apopitozis oluşumunun araştırılmasıydı. ELISA testi sonucunda epigallokateşin-3-gallat ve resveratrolün aktif kaspaz-3 seviyelerini azalttığı gözlendi. Elde edilen sonuçlara göre, in vitro ortamda antioksidan ajanlar olan epigallokateşin-3-gallat ve resveratrolün oksidan bir ajan olan hidrojen peroksite karşı hücre sağ kalımını ve koloni şekillendirme yeteneğini arttırarak koruyucu rol oynadığı ve kaspaz-3 düzeylerini azaltarak apopitozu azalttığı saptandı.
Neurodegeneration is the progressive loss of neuronal cells and deterioration of their structural integrity. The incidence of neurodegenerative diseases is increasing day by day. Genetic factors, environmental factors and oxidative stress play a role in the emergence of neurodegenerative diseases due to damage to neurons. The use of various phenols found in nature to delay or prevent neurodegeneration is on the agenda. Superoxide radicals can form at various points of the electron transport systems in our body. Hydrogen peroxide is formed mainly by dismutation of the superoxide radical. The superoxide radical takes two protons via the superoxide dismutase enzyme to form hydrogen peroxide. In conditions such as ischemia, trauma, hemorrhage and radioactivity, aerobic phosphorylation in mitochondria changes and accordingly, oxygen leakage from the electron transport system increases. During normal metabolism in our body or through various external factors, free radicals such as hydrogen peroxide increase and cause various diseases such as cancer and cardiovascular diseases. These harmful species are kept under control by the body's endogenous antioxidant systems. Supporting antioxidant systems with dietary antioxidant components is of great importance in the prevention of diseases related to oxidant particles. Epigallocatechin-3-gallate in green tea and resveratrol in grapes are powerful antioxidant compounds. Epigallocatechin-3-gallate (EGCG); It acts as an anticarcinogen through various pathways by affecting DNA hypermethylation, telomerase activity, angiogenesis, metastasis, tumor cell apoptosis, tumor suppressor genes and oncogenes. In addition to being an anticarcinogen and a powerful antioxidant, EGCG has anti-inflammatory, antifibrotic and osteogenic effects. Resveratrol basically has antioxidant effect; It provides the amount of reactive oxygen produced by NAPH oxidase, mitochondrial superoxide formation, superoxide formation from nitric oxide synthase, reducing oxidase activity and expression. In addition to the anticarcinogenic pathways of EGCG, resveratrol also affects various cancer stages from onset to progression by affecting various signal-transduction pathways that control cell growth and division, inflammation, apoptosis, metastasis, and angiogenesis. This study was conducted to investigate and compare the protective effects of epigallocatechin-3-gallate and resveratrol antioxidant agents against hydrogen peroxide damage in SH-SY5Y neuronal cells. For this purpose, as a result of literature review, first the effective dose and incubation period of hydrogen peroxide were determined, this dose was determined as 250 μM and this dose was used in toxic groups. In SH-SY5Y cells, hydrogen peroxide toxicity was administered to three treatment groups, including epigallocatechin-3-gallate, resveratrol, epigallocatechin-3-gallate and resveratrol, after 2 and 24 hours of pretreatment. Cell survival was evaluated with MTS test in these groups. The clonogenic test was applied to observe the colony forming abilities of the cells and the results were evaluated with the post-hoc Turkey HSD test. As a result of both evaluations, it was observed that epigallocatechin-3-gallate and resveratrol were more protective at low doses and increased the colony forming ability. Another step of this study was to investigate the formation of apoptosis over the level of caspase-3 in cells with the ELISA test. As a result of ELISA test, it was observed that epigallocatechin-3-gallate and resveratrol decreased active caspase-3 levels. According to the results obtained, it was determined that the antioxidant agents epigallocatechin-3-gallate and resveratrol played a protective role against hydrogen peroxide, an oxidant agent, by increasing cell survival and colony forming ability, and decreased apoptosis by decreasing caspase-3 levels in vitro.
Neurodegeneration is the progressive loss of neuronal cells and deterioration of their structural integrity. The incidence of neurodegenerative diseases is increasing day by day. Genetic factors, environmental factors and oxidative stress play a role in the emergence of neurodegenerative diseases due to damage to neurons. The use of various phenols found in nature to delay or prevent neurodegeneration is on the agenda. Superoxide radicals can form at various points of the electron transport systems in our body. Hydrogen peroxide is formed mainly by dismutation of the superoxide radical. The superoxide radical takes two protons via the superoxide dismutase enzyme to form hydrogen peroxide. In conditions such as ischemia, trauma, hemorrhage and radioactivity, aerobic phosphorylation in mitochondria changes and accordingly, oxygen leakage from the electron transport system increases. During normal metabolism in our body or through various external factors, free radicals such as hydrogen peroxide increase and cause various diseases such as cancer and cardiovascular diseases. These harmful species are kept under control by the body's endogenous antioxidant systems. Supporting antioxidant systems with dietary antioxidant components is of great importance in the prevention of diseases related to oxidant particles. Epigallocatechin-3-gallate in green tea and resveratrol in grapes are powerful antioxidant compounds. Epigallocatechin-3-gallate (EGCG); It acts as an anticarcinogen through various pathways by affecting DNA hypermethylation, telomerase activity, angiogenesis, metastasis, tumor cell apoptosis, tumor suppressor genes and oncogenes. In addition to being an anticarcinogen and a powerful antioxidant, EGCG has anti-inflammatory, antifibrotic and osteogenic effects. Resveratrol basically has antioxidant effect; It provides the amount of reactive oxygen produced by NAPH oxidase, mitochondrial superoxide formation, superoxide formation from nitric oxide synthase, reducing oxidase activity and expression. In addition to the anticarcinogenic pathways of EGCG, resveratrol also affects various cancer stages from onset to progression by affecting various signal-transduction pathways that control cell growth and division, inflammation, apoptosis, metastasis, and angiogenesis. This study was conducted to investigate and compare the protective effects of epigallocatechin-3-gallate and resveratrol antioxidant agents against hydrogen peroxide damage in SH-SY5Y neuronal cells. For this purpose, as a result of literature review, first the effective dose and incubation period of hydrogen peroxide were determined, this dose was determined as 250 μM and this dose was used in toxic groups. In SH-SY5Y cells, hydrogen peroxide toxicity was administered to three treatment groups, including epigallocatechin-3-gallate, resveratrol, epigallocatechin-3-gallate and resveratrol, after 2 and 24 hours of pretreatment. Cell survival was evaluated with MTS test in these groups. The clonogenic test was applied to observe the colony forming abilities of the cells and the results were evaluated with the post-hoc Turkey HSD test. As a result of both evaluations, it was observed that epigallocatechin-3-gallate and resveratrol were more protective at low doses and increased the colony forming ability. Another step of this study was to investigate the formation of apoptosis over the level of caspase-3 in cells with the ELISA test. As a result of ELISA test, it was observed that epigallocatechin-3-gallate and resveratrol decreased active caspase-3 levels. According to the results obtained, it was determined that the antioxidant agents epigallocatechin-3-gallate and resveratrol played a protective role against hydrogen peroxide, an oxidant agent, by increasing cell survival and colony forming ability, and decreased apoptosis by decreasing caspase-3 levels in vitro.
Açıklama
Anahtar Kelimeler
Epigallokateşin-3-Gallat, Resveratrol, Nöronal Toksisite, Antioksidan, Hidrojen Peroksit, Epigallocatechin Gallate, Resveratrol, Neuronal Toxicity, Antioxidant, Hydrogen Peroxide
