Ceylan Verda Bitirim, Kardelen Genç, Zeynep Özer, Belma Turan

N-asetilsisteinle Ön-muamele Edilmiş Kardiyomyositlerden Salgılanan Eksozomların Yaşlanmaya Bağlı ROS Üretimi Üzerindeki Etkisi

Amaç: Bu çalışmada bir antioksidan olan N-asetilsisteinin (NAC), kardiyomyositlerden salınan eksozomlar aracılığı ile kardiyomyositlerde yaşlanmaya bağlı artan reaktif oksijen türleri (ROS) üretimi ve mitokondri membran potansiyeli (MMP) üzerindeki düzenleyici etkisinin moleküler düzeyde incelenmesi hedeflenmiştir. Gereç ve Yöntem: Yaşlanma modeli, sıçan sol ventrikül hücre hattı H9c2’leri D-galaktoz (D-Gal; 50 mg/mL) ile 48-saat inkübasyon yapılarak gerçekleştirilmiştir. NAC ile ön-muameleye tabi tutulan sağlıklı H9c2’lerden salınan eksozomlar yaşlı kardiyomyositler ile inkübe edilerek, yaşlanma ile artan ROS üretimindeki artışa ve MMP depolarizasyonuna etkileri hem normal kontrol hücreleri ve hem de eksozom verilmemiş yaşlı hücrelerle karşılaştırılarak incelenmiştir. ROS ve MMP ölçümleri spesifik floresan boyalar (DCFDA ve FCCP) kullanılarak konfokal mikroskobunda floresan şiddetinde değişim olarak görüntülenerek bağıl olarak hesaplanmıştır. ROS belirteci olarak da kabul edilen miR-21 ifadesi, eksozomal miRNA izolasyonunu takiben yapılan cDNA sentezi qRT-PCR ölçümleri gerçekleştirilmiştir. Bulgular: Yaşlanma modeli oluşturulmuş kardiyomyositlerin, NAC ile inkübe edilmiş H9c2’lerden izole edilen eksozomlar ile 72-saat inkübasyonu yaşlanmaya bağlı artan ROS üretiminin belirgin şekilde baskılanmasına, depolarize olan MMP’nin normalize olmasına neden olmaktadır. Diğer yandan, NAC muamelesi normal sağlıklı kardiyomyositlerde eksozomal miR-21 ifadesini anlamlı olarak baskılamaktadır. Sonuç: Bu bulgular ışığında, kalp hasar tedavisinde kullanılan ROS hedefli ajanların eksozom-temelli parakrin bir mekanizma aracılığı ile kontrolsüz olarak artan mitokondriyon kaynaklı ROS üretiminin spesifik bir şekilde baskılıyor olabileceğini işaret etmektedir. Çalışmamız, ayrıca, literatürde ilk kez olmak üzere, NAC ile ön-muamele edilmiş kardiyomyositlerde eksozomal miRNA modülasyonuna neden olduğunu göstermektedir. Bu bulgular, antioksidan sistemi kuvvetlendirilmiş kardiyomiyositlerden elde edilen eksozom uygulamasının yaşlanmaya bağlı yetersiz kalp fonksiyonu için yeni bir tedavi yaklaşımı olduğunu göstermektedir.

Effect of Exosomes Secreted from N-acetylcysteine Pretreated Cardiomyocytes on Aging- induced ROS Production

Objectives: In this study, it was aimed to investigate the regulatory effect of N-acetylcysteine (NAC), an antioxidant, on the aging-related reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) in cardiomyocytes via exosomes released from cardiomyocytes at the molecular level. Materials and Methods: The aging model was performed by incubating rat left ventricular cell line H9c2 with D-galactose (D-Gal; 50 mg/mL) for 48 hours. Exosomes released from H9c2 pretreated with NAC were incubated with aged cardiomyocytes and their effects on the enhanced ROS production and MMP depolarization with aging were compared with both normal control cells and aged cells without exosomes. ROS and MMP measurements were calculated relative to the confocal microscope using specific fluorescent dyes (DCFDA and FCCP) by visualizing the change in fluorescence intensity. MiR-21 expression, which is also accepted as a ROS marker, and cDNA synthesis qRT-PCR measurements were performed following exosomal miRNA isolation. Results: Seventy-two-hour incubation of aging-modeled cardiomyocytes with exosomes isolated from H9c2 incubated with NAC significantly suppresses the increased ROS production associated with aging and normalizes depolarized MMP. On the other hand, NAC treatment significantly suppressed exosomal miR-21 expression in normal healthy cardiomyocytes. Conclusion: These findings indicate that ROS-targeted agents used in the treatment of heart damage may specifically suppress mitochondrion- derived ROS production, which increases uncontrollably through an exosome-based paracrine mechanism. Our study also demonstrates, for the first time in the literature, that NAC induces exosomal miRNA modulation in pretreated cardiomyocytes. These findings indicate that the administration of exosomes obtained from cardiomyocytes with enhanced antioxidant system is a new treatment approach for aging-related inadequate heart function.

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