Induction of docosahexaenoic acid to expression of heme oxygenase-1 in retinal pigment epithelial cells

Abstract

Background Oxydative stress is an important pathogenesis of age-related macular degeneration.Resent evidences indicate that docosahexaenoic acid (DHA) plays an important role during the development of retinal photoreceptor cells and protect the cells against oxydative stress by inducing the expression of heme oxygenase-1 (HO-1). However, whether DHA can induce the expression of HO-1 in human retinal pigment epithelium (RPE) cells is unelucidated. Objective This study was to investigate the effect of DHA on the expression of HO-1 in RPE cells and its molecular mechanism. Methods Human RPE cell line ARPE-19 was cultured in vitro and treated with 30, 50, 100 and 120 μmol/L DHA for 4 to 24 hours, respectively, and the cells were cultured without DHA as the control group.The cytotoxicity of DHA was detected by lactate dehydrogenase(LDH), and the expression of HO-1 mRNA and protein were detected by real-time PCR and Western blot assay, respectively.The enzymatic activity of HO-1 was detected by colorimetry.The reactive oxygen species (ROS) proportion in the cells was detected using fluorescence probe H2DCFDA, and immunofluorescence technology was adopted to detect the nuclear translocation of nuclear facotor-E2-related factor 2 (Nrf2). The expression of Nrf2 protein in the cells was detected by Western blot after intervention of ROS inhibitor N-acetylcysteine (NAC) and transfection of Nrf2 small interfering RNA (siRNA). Results The LDH leakage rate was significantly different after 0, 3, 50, 100 and 120 μmol/L DHA treated the cells for 24 hours (F=8.14, P<0.05), and the LDH leakage rate in the 120 μmol/L DHA group was significantly higher than that of 0, 30, 50 and 100 μmol/L DHA group (all at P<0.05). The relative expression levels of HO-1 mRNA and HO-1 protein or HO-1 enzymatic activity in the cells were significantly different among different concentrations of DHA group in 8 hours after treatment (F=16.24, P<0.05; F=11.34, P<0.05; F=11.81, P<0.05), and the expressions of these factors were considerably higher in the 30, 50 and 100 μmol/L DHA group than those in the 0 μmol/L DHA group (all at P<0.05). The ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were statistically significant among different concentrations of DHA groups (F=11.08, P<0.05; F=16.42, P<0.05), and the ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were evidently higher in the 30, 50 and 100 μmol/L DHA group than those in the 0 μmol/L DHA group (all at P<0.05). The relative expression levels of HO-1 protein and the proportion of nuclear Nrf2 positive cells were significantly lower in the NAC pretreated 100 μmol/L DHA group than those in the 100 μmol/L DHA group.In addition, the HO-1 relative expression level and the positive cells proportion of nuclear Nrf2 were significantly lower in the of Nrf2 siRNA transfection group than those in the blank siRNA transfection group (both at P<0.05). Conclusions DHA with concentration below 100 μmol/L can protect RPE cells from oxidative stress by inducting the expression of HO-1 in the cells via ROS/Nrf2 pathway. Key words: Antioxidants/pharmacology; Cytoprotection/drug effects; Oxidative stress; Pigment epithelium of eye/drug effects; Heme oxygenase-1; Nuclear factor-E2-related factor 2/metabolism; Reactive oxygen species/metabolism; Docosahexaenoic acid