The Effect of Rosmarinic Acid on Cell Viability, Steatosis, Paraoxonase-1, and Paraoxonase-3 Protein Levels in Palmitate-induced Non-alcoholic Fatty Liver Disease Model in HepG2 Cells

Aim:We aimed to investigate the effect of rosmarinic acid (RA) on cell viability, steatosis, paraoxonase (PON)1, and PON3 protein levels in palmitate-induced non-alcoholic fatty liver disease (NAFLD) model in HepG2 cells.Materials and Methods:To induce an experimental steatosis model, HepG2 cells were incubated with 1 mM palmitate for 24 hours. For the treatment, non-toxic RA concentrations were added to the cell culture medium simultaneously with the palmitate. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. To evaluate steatosis, intracellular triglyceride levels were measured and the cells were examined microscopically with Oil-Red O staining. PON1 and PON3 protein levels were measured by Western blotting.Results:1 mM palmitate caused a significant decrease in cell viability and a significant increase in triglyceride levels, but it did not significantly change PON1 and PON3 protein levels. RA caused a significant increase in cell viability and a significant decrease in triglyceride levels in the palmitate-treated cells. Similar findings with the triglyceride levels of cells were shown in microscopic examination of cells that were stained with Oil-Red O. RA did not significantly change PON1 and PON3 protein levels in neither non-treated cells nor treated cells with palmitate.Conclusion:Our study showed that RA increases cell viability and decreases steatosis, but it does not change PON1 and PON3 protein levels in palmitate-induced NAFLD model in HepG2 cells

Effect of rosmarinic acid on paraoxonase-1 and paraoxonase-3 protein expessions in palmitate-induced steatosis in human-derived hepatoma cells

Bu çalışmanın amacı, palmitat ile non-alkolik yağlı karaciğer hastalığı modeli oluşturulan insan kaynaklı hepatoma hücrelerinde rosmarinik asidin, hücre canlılığına, yağlanmaya, paraoksonaz-1 ve paraoksonaz-3 protein ekspresyonlarına etkisini araştırmaktır. Deneysel yağlanma modeli oluşturmak için; HepG2 hücreleri 1 mM palmitat ile 24 saat inkübe edildi. Tedavi olarak palmitat ile aynı anda hücre kültürü medyumuna HepG2 hücrelerine toksik olmayan rosmarinik asit konsantrasyonları eklendi. Hücre canlılığı 3-(4,5Dimetil-2-tiazolil)-2,5-difenil-2H-tetrazolium bromür testi ile değerlendirildi. Yağlanmanın değerlendirilmesi için hücre içi trigliserit düzeyleri ölçüldü ve hücreler Oil-Red O ile boyandı. Paraoksonaz-1 ve paraoksonaz-3 protein düzeyleri western blot yöntemiyle ölçüldü. 1 mM palmitat hücre canlılığında anlamlı bir azalmaya ve trigliserit düzeylerinde anlamlı bir artşa yol açtı ancak paraoksonaz-1 ve paraoksonaz-3 protein düzeylerini anlamlı olarak değiştirmedi. Palmitat ile oluşturulan deneysel non-alkolik yağlı karaciğer hastalığı modelinde rosmarinik asit 50 µM, 100 µM ve 200 µM konsantrasyonlarda, hücre canlılığını anlamlı olarak arttırdı ve trigliserit düzeylerini anlamlı olarak azalttı. Oil-Red O ile boyanan hücrelerin mikroskopik incelenmelerinde hücrelerin trigliserit düzeylerindeki değişimleri ile benzer bulgular görüldü. Palmitat ile oluşturulan non-alkolik yağlı karaciğer hastalığı modelinde hücre canlılığını en fazla arttırması ve yağlanmayı en fazla azaltması nedeniyle seçilen 100 µM ve 200 µM rosmarinik asit hem palmitat uygulanmayan hem de palmitat uygulanan hücrelerde paraoksonaz-1 ve paraoksonaz-3 protein düzeylerini anlamlı olarak değiştirmedi. Sonuç olarak çalışmamız, rosmarinik asidin palmitat ile non-alkolik yağlı karaciğer hastalığı modeli oluşturulan insan kaynaklı hepatoma hücrelerinde, hücre canlılığını arttırdığını, yağlanmayı azalttığını, ancak paraoksonaz-1 ve paraoksonaz-3 protein ekspresyonlarını değiştirmediğini gösterdi.The aim of this study is to investigate the effect of rosmarinic acid on cell viability, steatosis, paraoxonase-1 and paraoxonase-3 protein expressions in palmitate-induced nonalcoholic fatty liver disease model in human-derived hepatoma cells. To induce experimental steatosis model; HepG2 cells were incubated with 1 mM palmitate for 24 hours. For the treatment, non-toxic rosmarinic acid concentrations were added to cell culture medium simultaneously with the palmitate. Cell viability was evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. To evaluate steatosis, intracellular triglyceride levels were measured and cells were stained with Oil-Red O. Paraoxonase-1 ve paraoxonase-3 protein levels were measured by western blotting. 1 mM palmitate caused a significant decrease on cell viability and a significant increase on triglyceride levels but it did not significantly change paraoxonase-1 ve paraoxonase-3 protein levels. Rosmarinic acid at 50 µM, 100 µM and 200 µM concentrations significantly increased cell viability and significantly decreased triglyceride levels in palmitate-induced non-alcoholic fatty liver disease model. The similar findings with the triglyceride levels of cells were shown in microscopic examination cells which were stained with Oil-Red O. 100 µM and 200 µM rosmarinic acid concentrations, which are choosen because of they are the most increasing cell viability and the most decreasing steatosis in palmitate-induced non-alcoholic fatty liver disease model, did not significantly change paraoxonase-1 ve paraoxonase-3 protein levels neither non-treated cells or treated cells with palmitate. As a result, our study showed that rosmarinic acid increases cell viability and decreases steatosis but it dose not change paraoxonase-1 ve paraoxonase-3 protein expressions in palmitate-induced non-alcoholic fatty liver disease model in human-derived hepatoma cells