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 H2 DCFDA,and immunofluorescence technology was adopted to detect the nuclear translocation of nuclear facotor-E2-related factor 2 (Nrf2).The expression of Nrt2 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 μ mol/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 μ mol/L can protect RPE cells from oxidative stress by inducting the expression of HO-1 in the cells via ROS/Nrf2 pathway.
ABSTRACT
Objective To observe the effect of triamcinolone acetonide(TA) on activation and barrier function of human retinal pigment epithelium (RPE). Methods ARPE-19 cells were cultured in 96-well tissue culture plate. Four weeks later, TA with different concentration (0.02 and 0.05 mg/ml)was added to the cells and culture for 3 or 7 days. The activation of ARPE-19 cells was assessed by methyl thiazolyl tetrazolium (MTT). ARPE-19 cells were cultured on polyester microporous filters for 4 weeks, and the transepithelial resistance (TER) was recorded. TA (0.02 and 0.05 mg/ml) was added to the culture fluid respectively, and after cultured for 1 week TER was measured again. The RPE permeability was detected by enzyme-linked immunosorbent assay (ELISA) with horse radish peroxidase as the tracer. [WTHZ]Results In the culture fluid with 0.02 mg/ml TA cultured for 3 or 7 days, the average survival rate of ARPE-19 cells was 93.70% and 90.63% respectively, without statistic difference compared with the control (P=0.147, 0.091). While in the 0.05 mg/ml TA group after cultured for the same duration, the activation of ARPE-19 cells decreased significantly compared with the control (with the average survival rate of 87.75% and 88.98%; P=0.025, 0.043). One week after cultured with TA, TER decreased significantly while permeability improved obviously in the 2 TA groups compared to the control (P
ABSTRACT
Objective To investigate the effects of cytokines on the expression of syndecan-1 in cultured human retinal pigment epithelial (RPE) cells and the signal transduction pathway. Methods Reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of syndecan-1 mRNA and protein in normal RPE cells. The expression of syndecan-1 in RPE cells stimulated by different cytokines was detected and quantitatively analyzed by image process of immunofluorescence. The stimulation included 7 and 35 ng/ml tumor necrosis factor (TNF)-? for 24 hours, 1 and 6 ?g/ml lipopolysaccharide (LPS) for 11 hours, 7 ng/ml TNF-? for 0 to 24 hours (once per 2 hours, and 13 times in total), and 30% supernatant of monocyte/macrophage strain (THP-1 cells) for 3, 14 and 43 hours. The effect of 30% supernatant of THP-1 cells was assayed after pretreated by PD098059 [the specific inhibitor of extracellular signal regulated kinase(ERK) 1/2] for 2 hours. After exposed to 30% supernatant of THP-1 cells for 3 hours and treated by 0.25% trypsin for 5 minutes, RPE cells attaching was evaluated by methyl thiazolyl tetrazolium assay. Results In normal human RPE cells, expressions of syndecan-1 mRNA and protein were detected, and strong syndecan-1 positive yellowish green fluorescence was found in the cell membrane and cytoplasm while light green fluorescence was in the nucleus. As the concentration and stimulated time of TNF-? or LPS increased, the fluorescence intensity decreased(P