| Purpose:Docosahexaenoic acid (22:6, n-3, DHA) is highly concentrated in RPE and can be converted into the active oxygenate derivative protectin DX (PDX). It remains controversial whether DHA can reduce the risk of progression to advanced AMD. The current study investigated the anti-inflammatory and antioxidant effects of PDX on oxidative stress-induced RPE cells and extrapolated these results and mechanisms.Methods:1) Cell cultureThe human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The ARPE-19 cells were grown and maintained in DMEM-F12 medium supplemented with 10% FBS,100 U/ml penicillin, and 100μg/ml streptomycin (hereafter referred to as "complete DMEM/F12") and incubated at 37℃ with a constant supply of 5% CO2. Cells were used between passages 16 and 18.2) Experimental treatmentsARPE-19 cells were seeded and treated in 96-well plates for the ELISA analysis or in 6-well plates for the ROS detection, T-AOC and SOD measurements, and western blot and real-time quantitative PCR analyses. Specifically, cells were seeded in 96-well plates (2×104/well in a volume of 0.1ml) or 6-wellplates (4×105/well ina volume of 2.0 ml) in complete DMEM/F12. After reaching～80% confluence, the cells were starved in serum- free DMEM/F12 for 24 h. Before the ARPE-19 cells were stimulated with 200 μM t-BH in serum-free DMEM/F12 for 2 h, they were pretreated with the vehicle or indicated concentrations of PDX for 20 min. After stimulation, the cells were rinsed with serum-free DMEM/F12 to remove excess t-BH and inactivated PDX and then 1) incubated in DMEM/F12 supplemented with 1% FBS for another 24 h, after which time the supernatants were collected for ELISA (0.2 ml/well in 96-well plates); and 2) the cells were collected for the detection of T-AOC and SOD or for western blot analysis (2 ml/well in 6-well plates); 3) or incubated in DMEM/F12 supplemented with 1% FBS (2 ml/well in 6-well plates) for another 2 h and collected for real-time quantitative PCR analysis; 4) incubated with 1 μM DCFDA (2 ml/well in 6-well plates) for 30 min at 37℃ in the dark for the detection of intracellular ROS.3) Detection of intracellular ROSROS were estimated using the fluorescent dye 2’-7’-dichlorofluorescin diacetate (DCFDA), which is converted into a highly fluorescent compound within the cell in the presence of ROS. After treatment with 1 μM DCFDA for 30 min at 37℃ in the dark, as described above, the cells were examined under a fluorescence microscope and were collected, washed with PBS and analyzed using a flow cytometer with excitation at 488 nm and emission at 530 nm. The density of stained cells was determined for each sample after the fluorescence of unstained cells was subtracted.4) Measurement of T-AOC and SODIntracellular total antioxidant capability (T-AOC) and superoxide dismutase (SOD) activity were measured with the respective commercial kits. The T-AOC assay used a spectrometric method. Ferric ions were reduced with antioxidant-reducing agents, and the optical density was measured at 520 nm with a microplate reader. Intracellular SOD activity was measured using a xanthine and xanthine oxidase system to produce superoxide. The superoxide oxidizes hydroxylamine to nitrite to forma carmine-colored reagent, and the optical density at 550 nm was measured with a microplate reader.5) ELISAThe levels of VEGF-A and TNF-a secreted by RPE cells were measured using ELIS As according to the manufacturer’s instructions. The absorbance at a 450-nm wavelength was read with a microplate reader with a reference wavelength of 570 nm for VEGF-A and a reference wavelength of 620 nm for TNF-a. The VEGF-A ELISA kit was able to detect VEGF-A concentrations up to 1000 pg/ml. The TNF-a ELISA kit was able to detect TNF-a concentrations varying from 0 to 500 pg/ml6) Real-time quantitative PCR(qPCR)Real-time qPCR was used to quantify mRNA expression levels of VEGF-A, TNF-a and PGC-la. Total RNA was extracted from the cells using TRIzol reagent. First-strand cDNA was synthesized with 1 μg of DNA-free total RNA for each sample and an oligo-(dT) primer using a TaKaRa RNA PCR Kit. The primers were designed by Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). Real-time qPCR was performed using 2 μl of cDNA mixed with SYBR Primix Ex TaqTM and the StepOnePlus Real-Time PCR system. The relative fold change in gene expression was calculated using the 2-AACt method. GAPDH was used as an internal control for sample normalization.7) Western blot analysisWestern blot analysis was used to detect the expression of PGC-1α protein. RPE cells were processed to determine the protein content using standard protocols. The proteins were transferred onto activated polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 10% nonfat dried milk in PBS containing 0.2% Tween-20 (PBST). The blocked membranes were incubated overnight with primary antibodies diluted in an albumin solution at 4℃. The membranes were then rinsed three times in PBST and incubated with the corresponding peroxidase-conjugated secondary antibody for 1 h at room temperature. Densitometry was performed with background subtraction, and protein bands were normalized against the GAPDH band of the same sample.8) Statistical analysisAll assays were performed using at least three separate experiments and in triplicate, and data are expressed as the mean±standard deviation. The statistical analysis was performed using SPSS software (version 16.0 for Windows; SPSS Inc., Chicago, IL, USA). Analysis of variance was performed using one-way ANOVA procedures. P< 0.05 was considered statistically significant.Results:1) PDX inhibits t-BH-induced ROS productionCompared with the baseline, increased DCFDA fluorescence was observed in t-BH-stimulated ARPE-19 cells, indicating a marked increase in intracellular ROS production that can be significantly attenuated by pretreatment with 10μM PDX. The inhibitory effect of PDX on t-BH-induced ROS production can be better demonstrated using flow cytometry:on the histogram of relative fluorescence intensity of DCFDA, pretreatment with 5 μM or 10 μM PDX significantly attenuated ROS production in t-BH-induced RPE cells, although this inhibitory effect was not observed with PDX at the concentration of 1 μM. This result suggests that 5 μM and 10μM PDX can effectively reduce oxidative stress in RPE cells.2) PDX enhances intracellular SOD activity and T-AOC in t-BH-induced RPE cellsThe detoxification of ROS is counterbalanced by the ability of all of the antioxidants in a cell (including SOD, which is an important antioxidant) to counteract oxidation (measured by T-AOC). It is possible that PDX’s role in inhibiting ROS production is mediated by enhancing the activities of known antioxidant enzymes, such as SOD. Indeed, our results showed that treatment with t-BH did not lead to a significant alteration in SOD activity or T-AOC; however, pretreatment with PDX significantly increased intracellular SOD activity and T-AOC in a concentration-dependent manner (P < 0.05), suggesting that PDX enhances the antioxidant capability of ARPE-19 cells.3) PDX downregulates t-BH-induced TNF-α and VEGF-A secretion by RPE cellsTNF-α is well known as a major mediator of early inflammatory response, and VEGF-A, an important mediator of angiogenesis, is closely involved in inflammation. TNF-α secretion was completely abrogated by pretreatment with PDX at concentrations from 0.05μM to 10 μM, while VEGF-A secretion showed a less remarkable inhibitory profile with PDX, with significant inhibition detected only at higher concentrations (1 μM to 10 μM). This result suggests that PDX may attenuate AMD-related inflammation during oxidative stress in RPE cells.4) PDX downregulates TNF-α expression in t-BH-induced RPE cellsThe mRNA levels of TNF-α and VEGF-A in t-BH-induced ARPE-19 cells were analyzed using real-time qPCR analysis. Treatment of cells with t-BH increased the levels of TNF-α and VEGF-A mRNA by 5.5-and 9-fold, respectively, compared to the control. While PDX inhibited the excessive expression of TNF-α mRNA in t-BH-induced ARPE-19 cells in a dose-response manner, it had no significant effect on VEGF-A mRNA expression.5) PDX upregulates PGC-1α mRNA and protein levels in t-BH-induced RPE cellsPGC-1α has been shown to have a regulatory effect on the expression of endogenous antioxidant proteins such as SOD, and it might be involved in the antioxidant mechanism of PDX. We found that pretreatment with PDX significantly upregulated PGC-1α mRNA expression in a dose-dependent manner. The western blot analysis showed that t-BH stimulation led to a marked downregulation of PGC-1α protein, which was abrogated by pretreatment with 10 μM PDX. This result suggests that PDX may exhibit its antioxidative effect by upregulating PGC-1α protein expression.Conclusions:Protectin DX counteracts oxidative stress and associated pro-inflammatory responses in RPE cells. These results suggest the potential application of active oxygenated DHA derivatives in the prevention and treatment of AMD and other oxidative stress-related retina diseases, such as diabetic retinopathy. |
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