Hidrojen peroksit ile indüklenmiş beas-2b hücrelerinde yeni sentezlenen benzimidazol türevi bileşiğin endoplazmik retikulum stresi ve UPR sinyal yolağı üzerine etkisi
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Tarih
2019
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Hatay Mustafa Kemal Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Solunum epiteli solunumla alınan zararlı partiküller ve enfeksiyon ajanlarına karşı ilk savunma bariyerini oluşturur. Solunum epitel hücrelerinde reaktif oksijen türlerinin neden olduğu oksidatif stres, kronik obstrüktif akciğer hastalığı (KOAH) gibi kronik solunum yolu hastalıklarının patofizyolojisinde önemli rol oynamaktadır. Bu tez çalışmasında, insan bronş epitel hücre hattı olan BEAS-2B hücrelerinde oksidatif stres koşulları altında, endoplazmik retikulum (ER) stresinin katlanmamış protein cevabı (UPR)'nin hangi yolağı üzerinden gerçekleştiğinin ve yeni sentezlenen benzimidazol türevi bileşiğin (RHE-231) ER stresi ve UPR sinyal yolağında koruyucu bir etkisinin olup olmadığının gen ve protein düzeyinde aydınlatılması amaçlandı. Bu doğrultuda, BEAS-2B hücreleri farklı konsantrasyonlar (0-200 μM) ve sürelerde (24 ve 48 saat) hidrojen peroksit (H2O2)'ye maruz bırakılarak uygun konsantrasyonlar (10 ve 20 μM) ve süre (24 saat) belirlendikten sonra oksidatif hasarlı hücre modeli oluşturuldu. Daha sonra BEAS-2B hücreleri farklı konsantrasyonlarda (0-100 µM) RHE-231'e 24 saat süresince maruz bırakılarak uygun konsantrasyonlar (5 ve 10 μM) belirlendi. Çalışmada, oksidatif hasarlı hücre modeli, malondialdehit (MDA) düzeyinin spektrofotometrik ölçümüyle teyit edildi. 24 saat süresince 10 ve 20 μM konsantrasyonlarda H2O2'ye maruz bırakılan BEAS-2B hücrelerinde MDA düzeyi kontrol grubuna kıyasla anlamlı olarak yüksek bulundu (10 μM konsantrasyonda p=0.000; 20 μM konsantrasyonda p=0.000). UPR yolağı parametrelerinden, IRE1α (20 µM H2O2 + 5 µM RHE-231 konsantrasyonda 11.95 kat p=0.026; 20 µM H2O2 + 10 µM RHE-231 konsantrasyonda 14.31 kat p=0.000037), PERK (20 µM H2O2 + 5 µM RHE-231 konsantrasyonda 3.65 kat p=0.032; 20 µM H2O2 + 10 µM RHE-231 konsantrasyonda 39.27 kat p=0.012) gen ekspresyon seviyelerinin kontrol grubuna kıyasla anlamlı derecede arttığı gözlenirken, ATF6 ve GRP78 gen ekspresyon seviyelerinde gruplar arasında anlamlı bir fark gözlenmedi (p>0.05). Western blot analizi sonuçlarına göre, IRE1α ve ATF4 miktarlarında 20 µM H2O2, 20 µM H2O2 + 5 µM RHE-231 ve 20 µM H2O2 + 10 µM RHE-231 konsantrasyonlarında kontrol grubuna kıyasla anlamlı bir artış gözlendi (p<0.01 ve p<0.001). Sonuç olarak, bu çalışmada, BEAS-2B hücrelerinde, H2O2 aracılı oksidatif stresin ER stresini uyardığını ve UPR yolağını aktif hale getirdiği, ancak yeni sentezlenen benzimidazol türevi bileşik olan RHE-231'in ER stresini hafifletmede etkili olmadığı ortaya konmuştur.
The airway epithelium forms the first defense barrier against inhaled harmful particles and infectious agents. Oxidative stress caused by reactive oxygen species in airway epithelial cells plays an important role in the pathophysiology of chronic airway diseases such as COPD. In this thesis, the human bronchial epithelial cell line BEAS-2B cells, under the conditions of oxidative stress, it aimed to clarify at gene and protein levels endoplasmic reticulum (ER) stress occurs through which UPR pathway and whether the newly synthesized benzimidazole derivative compound (RHE-231) has a protective effect on ER stress and unfolded protein response (UPR) signaling pathway. In this respect, BEAS-2B cells were exposed to hydrogen peroxide (H2O2) at different concentrations (0-200 μM) and durations (24 and 48 hours), and after appropriate concentrations (10 and 20 µM) and duration (24 hours) have been determined, oxidative damaged cell model created. Then, BEAS-2B cells were exposed to RHE-231 for 24 hours at different concentrations (0-100 μM) and appropriate concentrations (5 and 10 μM) were determined. In this study, the oxidative damaged cell model was confirmed by spectrophotometric measurement of malondialdehyde (MDA) level. MDA levels were significantly higher in the BEAS-2B cells exposed to H2O2 at concentrations of 10 and 20 μM for 24 hours compared to the control group (p=0.000 at a concentration of 10 μM; p=0.000 at a concentration of 20 μM). Among the UPR pathway parameters, IRE1α (11.95 fold p=0.026 at 20 µM H2O2 + 5 µM RHE-231 concentration; 14.31 fold p=0.000037 at 20 µM H2O2 + 10 µM RHE-231 concentration), PERK (20 µM H2O2 + 5 µM RHE-231 3.65 times p=0.032; 20 µM H2O2 + 10 µM RHE-231 39.27 times p=0.012), but there was no significant difference between ATF6 and GRP78 gene expression levels (p>0.05). According to the results of the Western blot analysis, a significant increase was observed in the amounts of 20 μM H2O2, 20 μM H2O2 + 5 μM RHE-231 and 20 μM H2O2 + 10 μM RHE-231 in the amounts of IRE1α and ATF4 compared to the control group (p<0.01 and p<0.001). In conclusion, in this study, it was demonstrated that H2O2 mediated oxidative stress in BEAS-2B cells stimulated ER stress and activated the UPR pathway, but the newly synthesized benzimidazole derivative compound RHE 231 was not effective in relieving ER stress.
The airway epithelium forms the first defense barrier against inhaled harmful particles and infectious agents. Oxidative stress caused by reactive oxygen species in airway epithelial cells plays an important role in the pathophysiology of chronic airway diseases such as COPD. In this thesis, the human bronchial epithelial cell line BEAS-2B cells, under the conditions of oxidative stress, it aimed to clarify at gene and protein levels endoplasmic reticulum (ER) stress occurs through which UPR pathway and whether the newly synthesized benzimidazole derivative compound (RHE-231) has a protective effect on ER stress and unfolded protein response (UPR) signaling pathway. In this respect, BEAS-2B cells were exposed to hydrogen peroxide (H2O2) at different concentrations (0-200 μM) and durations (24 and 48 hours), and after appropriate concentrations (10 and 20 µM) and duration (24 hours) have been determined, oxidative damaged cell model created. Then, BEAS-2B cells were exposed to RHE-231 for 24 hours at different concentrations (0-100 μM) and appropriate concentrations (5 and 10 μM) were determined. In this study, the oxidative damaged cell model was confirmed by spectrophotometric measurement of malondialdehyde (MDA) level. MDA levels were significantly higher in the BEAS-2B cells exposed to H2O2 at concentrations of 10 and 20 μM for 24 hours compared to the control group (p=0.000 at a concentration of 10 μM; p=0.000 at a concentration of 20 μM). Among the UPR pathway parameters, IRE1α (11.95 fold p=0.026 at 20 µM H2O2 + 5 µM RHE-231 concentration; 14.31 fold p=0.000037 at 20 µM H2O2 + 10 µM RHE-231 concentration), PERK (20 µM H2O2 + 5 µM RHE-231 3.65 times p=0.032; 20 µM H2O2 + 10 µM RHE-231 39.27 times p=0.012), but there was no significant difference between ATF6 and GRP78 gene expression levels (p>0.05). According to the results of the Western blot analysis, a significant increase was observed in the amounts of 20 μM H2O2, 20 μM H2O2 + 5 μM RHE-231 and 20 μM H2O2 + 10 μM RHE-231 in the amounts of IRE1α and ATF4 compared to the control group (p<0.01 and p<0.001). In conclusion, in this study, it was demonstrated that H2O2 mediated oxidative stress in BEAS-2B cells stimulated ER stress and activated the UPR pathway, but the newly synthesized benzimidazole derivative compound RHE 231 was not effective in relieving ER stress.
Açıklama
Anahtar Kelimeler
Biyokimya, Biochemistry ; Genetik
