İnsan kaynaklı karaciğer (HepG2) hücrelerinde palmitat ile oluşturulan yağlanmanın paraoksonaz-1 ve paraoksonaz-3 enzimlerine etkisi

Öz

Amaç: Palmitat, hem sağlıklı bireylerin hem de non-alkolik karaciğer yağlanması hastalarının karaciğerinde en fazla bulunan yağ asitlerinden biridir. HepG2 hücrelerinde palmitat ile oluşturulan yağlanma, karaciğerdeki yağ birikiminin akut zararlı etkilerinin araştırılmasında kullanılan in vitro non-alkolik yağlı karaciğer hastalığı modelidir. Non-alkolik yağlı karaciğer hastalığı ateroskleroz ile yakından ilişkilidir. Paraoksonaz-1 ve paraoksonaz-3 dolaşımda yüksek dansiteli lipoproteine bağlı anti-aterosklerotik enzimlerdir ve esas olarak karaciğerde sentezlenirler. Palmitat ile oluşturulan yağlanmanın paraoksonaz-1 ve paraoksonaz-3 enzimleri üzerine etkisini araştıran bir çalışma bulunmamaktadır. Bu çalışmanın amacı HepG2 hücrelerinde palmitat ile oluşturulan yağlanmanın paraoksonaz-1 ve paraoksonaz-3 enzimlerine etkisini araştırmaktır.

Yöntemler: Yağlanma oluşturmak için hücreler 0.4, 0.7 ve 1 mM palmitat ile 24 saat inkübe edildi. Hücre canlılığı 3-(4,5-Dimetil-2-tiazolil)-2,5-difenil-2H-tetrazolium bromür testi ile değerlendirildi. Hücreler oil red O ile boyandı ve trigliserit düzeyleri ölçüldü. Paraoksonaz-1 ve paraoksonaz-3 protein düzeyleri western blot ile, mRNA’ları ise kantitatif PCR ile ve arilesteraz aktivitesi spektrofotometrik olarak ölçüldü.

Bulgular: Tüm palmitat konsantrasyonları paraoksonaz-1 mRNA düzeylerinde anlamlı bir artışa yol açtı. Palmitat konsantrasyonları paraoksonaz-1 ve paraoksonaz-3 protein düzeylerinde, paraoksonaz-3 mRNA düzeylerinde ve arilesteraz aktivitesinde anlamlı bir değişime yol açmadı.

Sonuç: Çalışmamız, HepG2 hücrelerinde palmitat ile oluşturulan yağlanmanın paraoksonaz-1 mRNA düzeyini arttırdığını, paraoksonaz-1 ve paraoksonaz-3 protein düzeylerine, paraoksonaz-3 mRNA düzeylerine ve arilesteraz aktivitesine etkisi olmadığını gösterdi.

Anahtar Kelimeler

• Palmitat,paraoksonaz-1,paraoksonaz-3,arilesteraz,HepG2,non-alkolik yağlı karaciğer hastalığı

Effect of palmitate-induced steatosis on paraoxonase-1 and paraoxonase-3 enzymes in human-derived liver (HepG2) cells

Abstract

Aim: Palmitate is one of the most abundant fatty acid in both liver of healthy individuals and in patients with non-alcoholic fatty liver disease. Palmitate-induced steatosis in HepG2 cells is an in vitro non-alcoholic fatty liver disease model to investigate acute harmful effects of fat overaccumulation in the liver. Non-alcoholic fatty liver disease is strongly associated with atherosclerosis. Paraoxonase-1 and paraoxonase-3 are anti-atherosclerotic enzymes which are bound to high density lipoprotein in circulation and they are primarily synthesized by liver. There is no study that investigated the effect of palmitate-induced steatosis on paraoxonase-1 and paraoxonase-3 enzymes. The aim of present study was to investigate the effect of palmitate-induced steatosis on paraoxonase-1 and paraoxonase-3 enzymes in HepG2 cells.

Methods: To induce steatosis, cells were incubated with 0.4, 0.7 and 1 mM palmitate for 24 hours. Cell viability was evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Cells were stained with oil red O and triglyceride levels were measured. Paraoxonase-1 and paraoxonase-3 protein levels were measured by western blotting, their mRNA expression were measured by quantitative PCR and arylesterase activity was measured spectrophotometrically.

Results: All palmitate concentrations caused a significant increase on paraoxonase-1 mRNA levels. Palmitate concentrations did not cause a significant change on paraoxonase-1 and paraoxonase-3 protein levels, paraoxonase-3 mRNA levels and arylesterase activities.

Conclusion: Our study showed that palmitate-induced steatosis up-regulates paraoxonase-1 mRNA, has no effect on paraoxonase-1 and paraoxonase-3 protein levels, paraoxonase-3 mRNA and arylesterase activity in HepG2 cells.

Keywords

• Palmitate,paraoxonase-1,paraoxonase-3,arylesterase,HepG2,non-alcoholic fatty liver disease

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