Fingolimod'un HUVEC Hücre Hattında LPS'den Kaynaklanan Enflamatuvar Yanıt Üzerindeki Etkilerinin Araştırılması

Abstract

Amaç: Bu çalışmanın amacı, fingolimodun lipopolisakkarit (LPS) kaynaklı inflamatuar yanıt bağlamında tümör nekroz faktörü-alfa (TNF-α) üretimi üzerindeki etkisini insan göbek kordonu endotel hücreleri (HUVEC) modelinde incelemekti. Materyal ve yöntem: 96 kuyulu plakanın her bir kuyusuna toplam 5×10³ hücre ekildi. Hücreler, kuyunun dibinde yapışma ve yerleşme sağlamak için 24 saat inkübe edildi. Daha sonra hücreler, 12 saat boyunca 10 µg/ml dozunda LPS (Escherichia coli O55:B5'ten L2880 Sigma-Aldrich Lipopolysaccharides) ile muamele edildi. Deney gruplarımız aşağıdaki gibi belirlendi. Sağlıklı grup: LPS ve Fingolimod tedavisi olmayan grup LPS Grubu: Grup 12 saat boyunca 10 μg/ml LPS ile tedavi edildi, ancak Fingolimod ile tedavi edilmedi LPS + Fingolimod 1 µM (LPS + Fin 1) grubu: 12 saat boyunca 10 μg/ml LPS ile tedavi edilen ve 1 µM fingolimod ile tedavi edilen grup. LPS + Fingolimod 0,5 µM (LPS + Fin 0,5) grubu: 12 saat boyunca 10 μg/ml LPS ile tedavi edilen ve 0,5 µM fingolimod ile tedavi edilen grup. LPS + Fingolimod 0,1 µM (LPS + Fin 0,1) grubu: 12 saat boyunca 10 μg/ml LPS ile tedavi edilen ve 0,1 µM fingolimod ile tedavi edilen grup. 12 ve 24 saatte, plakalar MTT yöntemi kullanılarak bir mikro plaka okuyucu spektrofotometre (Epoch Microplate Spectrophotometer, BioTek, ABD) ile 570 nm absorbans değerinde ölçüldü. 12 ve 24 saatte, plakalardaki tüm deney gruplarının süpernatantları toplandı. TNFα seviyeleri, bir Epoch Spektrofotometre Sisteminde ve bir Take3 Plaka cihazında ELISA kitleri kullanılarak kantifize edildi. Sonuç: Çalışmamızın MTT sonuçları analiz edildiğinde, hücre canlılığı yüzdesinin fingolimod tedavi gruplarında LPS grubuna kıyasla önemli ölçüde arttığı görüldü. Ayrıca, TNF-α sonuçları analiz edildiğinde, TNF-α seviyelerinin fingolimod tedavi edilen gruplarda LPS grubuna kıyasla önemli ölçüde azaldığı görüldü. Sonuç: Bulgular, LPS ile uyarılan HUVEC hücrelerinde TNF-α üretiminin belirgin şekilde arttığını göstermektedir. Ancak, fingolimodun uygulanması bu artış üzerinde önemli bir inhibitör etki gösterdi ve doza bağlı bir yanıt sergiledi. Bu gözlemler, fingolimodun inflamatuar yanıtı düzenleme potansiyeline sahip olabileceğini düşündürmektedir.

Keywords

• lps
• fingolimod
• akciğer

Investigation of the Effects of Fingolimod on the LPS-Induced Inflammatory Response in the HUVEC Cell Line

Abstract

Aim: The objective of this study was to examine the impact of fingolimod on tumour necrosis factor-alpha (TNF-α) production in the context of a lipopolysaccharide (LPS)-induced inflammatory response in a human umbilical vein endothelial cells (HUVEC) model. Material and method: A total of 5×10³ cells were seeded into each well of the 96-well plate. The cells were incubated for 24 hours to allow for adhesion and settlement at the bottom of the well. Subsequently, the cells were treated with LPS (L2880 Sigma-Aldrich Lipopolysaccharides from Escherichia coli O55:B5) at a dose of 10 µg/ml for 12 hours. Our experimental groups were determined as follows. Healthy group: Group without LPS and Fingolimod treatment LPS Group: The Group was treated with 10 μg/ml LPS for 12 hours, but not treated with Fingolimod LPS+ Fingolimod 1 µM (LPS+ Fin 1) group: Group treated with 10 μg/ml LPS for 12 hours and treated with 1 µM fingolimod.LPS+ Fingolimod 0.5 µM (LPS+ Fin 0.5) group: Group treated with 10 μg/ml LPS for 12 hours and treated with 0,5 µM fingolimod. LPS+ Fingolimod 0.1 µM (LPS+ Fin 0.1) group: Group treated with 10 μg/ml LPS for 12 hours and treated with 0,1 µM fingolimod. At 12 and 24 hours, the plates were measured at a 570 nm absorbance value with a microplate reader spectrophotometer (Epoch Microplate Spectrophotometer, BioTek, USA) using the MTT method. At 12 and 24 hours, the supernatants of all experimental groups in the plates were collected. The levels of TNFα were quantified using ELISA kits in an Epoch Spectrophotometer System and a Take3 Plate device. Result: When the MTT results of our study were analysed, it was observed that the percentage of cell viability increased significantly in the fingolimod treatment groups compared to the LPS group. In addition, when the TNF-α results were analysed, it was observed that TNF-α levels were significantly decreased in the fingolimod-treated groups compared to the LPS group. Conclusion: The findings demonstrate that the production of TNF-α was markedly elevated in LPS-stimulated HUVEC cells. However, the administration of fingolimod exhibited a notable inhibitory effect on this increase, exhibiting a dose-dependent response. These observations suggest that fingolimod may possess the potential to regulate the inflammatory response.

Keywords

• lps
• fingolimod
• lung

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