Retinal pigment epithelial (RPE) cell is a monolayer of multifunctional cells between the retina and the choroid (uveal vasculature). RPE cells form a blood-retinal barrier that limits infiltration of blood cells and serum proteins to the retina, as well as transport nutrients from the vascular choroid to photoreceptors. On one hand, RPE cell death can be induced by both exogenous and endogenous reactive oxygen species (ROS) such as peroxynitrite (ONOO-).1-3 On the other hand, RPE cells have properties similar to macrophages., the most important among which being induced immunity by complement components and complement receptors.4 Our study focuses on expressions of inducible nitric oxide synthase (iNOS) and Fas/FasL with complement 3 (C3) in RPE cells of mouse in response to ONOO- stimulation, and the antagonism of puerarin in this process.
Animals and cell cultures
Forty pathogen-free C57BL/6 mice aged 2-3 weeks were used in this study. All animals were treated in accordance with The Association for Research in Vision and Ophthalmology (ARVO) Resolution Statement for the use of Animals in Ophthalmic and Vision Research in USA and China. Donor C57BL/6 mice were decapitated and their eyes were immediately enucleated and placed in ice-cold Dulbecco's Modified Eagle's Medium (DMEM) with 2 mmol/L L-glutamine. The whole eyeballs were incubated for 20 minutes at 37°C in a solution of 2% disperse. The eyes were rinsed twice in DMEM and placed in a Petri dish containing fresh DMEM. Under a dissecting microscope, the eyeballs were cut open along the edge of the cornea. After the lens and iris being removed, the RPE cells and its attached neural retina were gently separated from the eye cup and transferred to a new petri dish containing fresh DMEM. After two changes of fresh medium and incubation for 15 minutes at room temperature, the entire RPE cell sheets were easily separated from the neural retina. Separated RPE cell sheets were cut into pieces and then transferred into culture flasks precoated with 1 μg/ml fibronectin and grew at 37°C in a 5% CO2 atmosphere in standard DMEM containing 10% fetal bovine serum (FBS). All cell culture material and chemicals were purchased from Invitrogen (Carlsbad, CA, USA).
Immunohistochemistry to confirm the identity of RPE cultures
Passages 2 to 3 RPE cells and their supernatants were used and all these cells were routinely stained with antibodies against a broad range of epidermal keratins (AE1/AE3; RDI, Flanders, NJ07836, Sigma, USA) for determination of their epithelial origins. The RPE cells were seeded on a chamber slide, washed in phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) at room temperature for 15 minutes. Subsequently, nonspecific binding was blocked with normal sheep serum (Serotec, Raleigh, NC, USA) at room temperature for 30 minutes. The primary antibody (mouse anticytokeratin RDI: PRO 61835=1:100) used to characterize the cells was diluted in PBS with 0.1% Tween 20 and incubated for 60 minutes at room temperature. After the cells being washed three times for 5 minutes each in PBS with 0.1% Tween 20, the secondary antibody (Sheep anti-Mouse-Cy3; C-2181; 1:400, Sigma) was applied for 30 minutes. Subsequent to be repeated washing, the number of cells in four random visual fields was counted and the number of positive cells was used to calculate the positive cells. Universal negative control mice (DAKO, NP015) were for negative control. All cell culture materials and chemicals were purchased from Invitrogen (Carlsbad, USA).
After that, RPE cells were divided into control group, ONOO- group and puerarin groups. Heat-pathogen free saline (NS, 200 μl) was added into control group, ONOO- (200 μl) was added into ONOO- group and the terminal concentration was 0.5 mmol/L. At the same time, ONOO- (5 μg/ml) and puerarin (10 μg/ml) were added into puerarin group . The total amount of the agents was 200 μl in each group. Cells were continued to be cultured after treated with above agents and collected at 6, 12, 24 hours for further study.
Synthesis of ONOO-
ONOO- was obtained by reacting ice-cold solutions of sodium nitrite (0.6 mol/L) with H2O2 (0.7 mol/L) in acidic medium (0.6 mol/L HCl) and rapidly quenching the reaction in NaOH (1.5 mol/L), as described previously.5,6 The reaction mixture solution was frozen at -20°C, and the ONOO- concentrated in the upper layer was collected. Its concentration was measured at 302 nm using a molar extinction coefficient of 1670 mol-1·cm-1.
Western blotting for detecting nitrotyrosine (NT) and C3
RPE cells were prepared as described, which were homogenized and solubilized in ice cold phosphate buffer saline (PBS) containing protease inhibitors, phenylmethylsulfonyl fluoride (1 μg/ml), aprotinin (1 μg/ml), leupeptin (1 μg/ml), pepstatin A (1 μg/ml) and EDTA (1 mmol/L). The homogenate was centrifuged at 15 000 r/min at 4°C for 10 minutes. The protein content of the supernatants was determined by the Bradford method.7 After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% linear slab gel was over, under reducing conditions, separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using a semidry electrophoretic transfer cell (Trans-blot; Bio-Rad, Richmond, CA, USA). Blot was stained at room temperature with 1:600 dilution of monoclonal mouse anti-NT and mouse anti-C3 antibody over night at 4°C respectively. After washing and incubation with horseradish peroxidase-conjugated 1:1000 dilution secondary antibody, blot was developed using the enhanced chemiluminescence and analyzed with Western blotting analysis detection system (ECL Plus; Amersham Pharmacia Biotech, USA).
The RPE cells were fixed into 70% ethanol for 24 hours, then examined the apoptotic cell number and percentage in RPE cells. Cells were washed 3 times in PBS and resuspended in PBS at 2×107 cells per milliliter. After staining with primary and secondary antibodies, cells were analyzed with Fluorescence Activated Cell Sorting (FACS) scan. The optical system was 2-W laser producer with an output of 300 mW, the exciting wavelength was 488 nm, and the projected wavelength was 605 nm. The data were input into an HP-300 Cosort 30 model computer (Cold Spring Corporation, USA) and processed with related software. The DNA content distributing square pattern and dual parameter dimension pattern were obtained in this way. The cell number was calculated with cell circle analyzing sequence. Chicken erythrocyte was taken as a standard sample to adjust the instrument coefficient (<5.0%).
Immunohistochemistry and Western blotting for detecting Fas/FasL transduction
First, the RPE cells were taken and fixed using immunohistochemistry. After being washed in PBS, the cells were incubated with hydrogen peroxide (Peroxidase Blocking Reagent; Daco, Carpinteria, CA, USA) to block endogenous peroxidase activity. Then the cells were blocked with 10% goat serum for 30 minutes at room temperature to block non-specific antigen. Block cells were rinsed and washed in PBS and incubated with Fas/FasL (1:400 dilution). Then, the slides were incubated in goat biotinylated anti-rat IgG (LSAB2 System; Dako, USA) as a secondary antibody. After being washed with PBS, the slides were incubated in streptavidin conjugated with horseradish peroxidase. The color was developed with streptavidin and biotin chromogen (Liquid DAB+ Substrate-Chromogen System; Dako). And then, the Western blotting was used.
Gene arrays for iNOS
RPE cells used in arrays were dissected free of any contaminating tissue and homogenized in Trizol reagent. RNA extraction was carried out according to the manufacturer's protocol. Concentration and RNA quality were assessed via spectrophotometry and formaldehyde gel electrophoresis. Amplified mRNA was then labeled with Cy3 or Cy5 (Random Primer DNA Labeling Kit (Bao-Boiscience Engineering Corporation, China). Successfully labeled control and experimental targets with 12 housekeeping genes and 12 artificially synthesized 70 mer oligo DNA which served as positive and negative control were then combined and prepared for hybridization. Array slides were incubated for prehybridization for 1 hour at 42°C. Targets were dried via vacuum centrifugation, then resuspended in 50 μl hybridization solution with added 1 μl Cot 1 DNA and 1 μl poly A oligonucleotide as blocking agents, heated to 95°C for 5 minutes and then added to the face of one slide. The printed face of the second slide of the pair was then placed face to face with the same probe. Slide pairs were then placed on a plastic cover with moistened tissue in a slide box. The slide box was sealed and placed floating in a water bath and hybridized for 24-48 hours at 42°C. Following hybridization, slides were washed in wash solution for 20 minutes and repeated for another 20 minutes, then dipped in nuclease free water and spray dried. Finally, the backs of the slides were cleaned with ddH2O, wiped with 100% ethanol, then wiped dry and scanned by Scan Array Express Scanner (Packard Bioscience Corporation, USA).
RT-PCR array confirmation for iNOS mRNA and Western blotting for iNOS protein
RT-PCR was performed using 2 μg of total RNA for the first-strand synthesis followed by amplification in the presence of specific primers for iNOS (5'-CGCCCTTCCGCAGTTCT-3' and 5'-TCCAGGAGGACATGCAGCAC-3') and β-actin (5'-GAGACCTTCAACACCCAGCC-3') and 5'-GCGGGGCATCGGAACCGCTCA-3'). The amplification consisted of 29 cycles of denaturation for 1 minute at 94°C, annealing for 1 minute at 60°C, and extension for 1 minute at 72°C. Then, the Western blotting of iNOS protein was taken.
Statistical analyses of the data were performed using SPSS 15.0 (SPSS Corp., USA). The results were shown as means ± standard deviation (SD). Statistical significance was determined by one way analysis of variance (ANOVA) followed by the Fisher post hoc test for multiple comparisons. P <0.05 was considered significant. Gene Pix Pro 4.0 photo software (Axon Instruments Corporation, USA) was used for clustering analysis. Two folds higher divergence were regarded as divergence expression of genes. All trials were repeated for at least three times.
Establishing and verifying RPE cell cultures
RPE cell cultures were routinely ≥95% keratin (+). The negative control incubated with isotype matched mouse IgG showed no positive staining. Specific binding was visualized and viewed under fluorescence optics (Olympus, Melville, NY, USA). When examined by phase-contrast microscopy after 2-3 passages, the cells formed a monolayer and displayed an epithelial configuration, being predominately hexagonal in shape (Figure 1).
Comparative studies had shown that the structure and function of RPE cells passaged for 3-5 generations were indistinguishable from those of freshly prepared cells. Thus, in this study, we only used RPE cells that had not been passaged for more than 3 times.
NT and C3 were expressed at different levels between ONOO- and puerarin groups
Western blotting analysis showed low level of NT and C3 expression in control group. In ONOO- group, moderate to strong levels of NT and C3 expression were observed at different stages of the experiment. In puerarin group, expression level of NT and C3 changed gradually from low to high from 6 to 12 hours, then attenuated at 24 hours (Figure 2). Computer-aided photo analysis indicated significant differences in expression levels among three groups (P <0.001, Table 1).
Different level of apoptotic cells between ONOO- and puerarin groups
There were rare apoptotic cells in the control group, but gradually increase apoptotic cells in the ONOO- group.
Moderate to high amounts of apoptotic cells were detected in puerarin group from 6 to 12 hours after treatment, then decreased at 24 hours. Compared to control groups, there were statistically significant differences in the amount and percentage of apoptotic cells in ONOO- and puerarin groups. And there was also statistical difference between ONOO- and puerarin groups (P <0.05, Table 2).
Fas/FasL signals could be observed among control, ONOO- and puerarin groups
Specific signal of Fas/FasL is revealed as yellow, brown-yellow or brown staining in the cell nucleus and cytoplasm with immunohistochemical method. After immunohistochemical staining, in the control group, a very faint yellow color could be observed. In the ONOO- group, staining ranged from yellow to brown-yellow, then to brown in the cell nucleus and cytoplasm at different time points. In puerarin group, gradually decreased staining could be observed from 12 to 24 hours (Figures 3-5).
Differences in up-regulation of iNOS expression between ONOO- and puerarin groups
Cy3 and Cy5 fluorescent intensity values were taken as standard for the X- and Y-axis. Every data spot represented each hybridization signal in the micro-array. If the scatter spot appeared in yellow color, it indicated that the ratio between X and Y was 0.5-2.0, which was no difference in expression. If the scatter spot appeared in red color, it indicated that the ratio between X and Y was beyond 0.5-2.0, which most probably indicated a difference in expression. If the scatter spot appeared in green color, it indicated down-regulated genes.
Compared to control group, there were minor up-regulated iNOS (NOS2) gene at 2.0 folds and nNOS (NOS1) gene at 1.6 folds at 20 days in puerarin treated group. There were continuous up-regulated iNOS gene at 5.0 fold and nNOS gene at 3.5 folds at 12 hours, but there were down-regulated iNOS gene by 2.2 folds and nNOS gene by 2.0 folds at 24 hours in that group; Compared with that of control and puerarin group, there were continuous up-regulated iNOS gene and nNOS gene in ONOO- group. In addition to genes related to NO production, other apoptosis-related geness such as BCL-2 and SOD showed upregulation. TNFR1-FADD-caspase signal transduction pathways showed down-regulated in puerarin group. And opposite results were found in ONOO- group (Figure 6).
We used RT-PCR and Western blotting to detect iNOS mRNA and protein to confirmation our gene array results. Neither iNOS mRNA nor iNOS protein expressed in the control group. After treated with ONOO- and puerarin, iNOS mRNA and iNOS protein distinctly up-regulated in the ONOO- group. iNOS mRNA and iNOS protein in the puerarin group appeared gradually up-regulated during the period of 6 to 12 hours of the experiment, then down-regulated at 24 hours (Figures 7 and 9). Computer-aided photo-analysis showed significant differences among three groups (P <0.001, Figures 8 and 10).
The Fas/FasL system is considered as the major signal transduction pathway to mediate apoptosis.8-11 This study investigated the susceptibility of RPE to Fas/FasL-dependent apoptotic pathways when challenged with different stimuli, including oxidants ONOO-, anti-Fas/FasL antibody and activated iNOS and C3, and the potential antagonistic effect of puerarin to apoptosis. Loss of RPE cells via apoptosis plays a prominent role in several retinal degenerative diseases, such as age-related macular degeneration. That means the occurrence and development of many eye diseases are related to the regulated imbalance of RPE cell apoptosis.12-14 Strategies for preservation of vision that would interrupt the apoptotic signal require knowledge of the molecular events associated with apoptosis.
We found that apoptotic cells continued to increase in the ONOO- group. In the puerarin group after dealing with puerarin, apoptotic cells were increased from 6 to 12 hours, but decreased from 12 to 24 hours, which may indicate a protective role of puerarin on RPE cells. These results are consistent with our previous work.15 Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae ((kudzu root), has been reported to be useful in the treatment of many diseases. Chang et al16 examined the detailed mechanisms underlying the inhibitory effects of puerarin on inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAO) in rats. The authors found that the expressions of iNOS, and active caspase-3 protein as well as the mRNA of tumor necrosis factor-alpha (TNF-α) in ischemic regions were markedly inhibited by the treatment of puerarin, which is a potent neuroprotective agent on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by the inhibition of both HIF-1α and TNF-α activation, followed by the inhibition of inflammatory responses (i.e., iNOS expression), apoptosis formation (active caspase-3), and neutrophil activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Another three authors also found that puerarin significantly decreased the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining cells compared with the control group.17-19 They verified the inhibitory effects of puerarin on angiopoiesis of endometriotic tissue and the regulatory effects of puerarin on tumor-related gene expression of endometriosis as well as reduced the occurrence of apoptosis and improved neurotrophic function of astrocytes, which may be related with its antioxidant effects during oxidative stress.
The classical oxygen-free radical stress model focus more on the role of hydrogen-peroxide (H2O2), nitric-oxide (NO) and superoxide-anion (O•2-), whereas the new model includes ONOO-, a product from rapid reaction of NO and O•2-, an potentially important mediator of cytotoxicity in oxidation.20,21 It is also highly reactive and interacts with cellular constituents inflicting damage on cells.22 Our study supports the new model. We observed localization of the ONOO--mediated protein nitration product nitrotyrosine (NT) in RPE cells and its decrease under the intervention of puerarin. We found that NT was greatly increased in ONOO- group. Expression of a small amount of NT in control group provided physiological evidence for the existence of ONOO-. Purerarin could inhibit the expression of iNOS, therefore decreased the formation of ONOO-.23 It is thus possible that iNOS may contribute to oxidation stress through introducing more powerful oxidative agents such as ONOO-. Under pathological conditions, up-regulated of iNOS mRNA and iNOS protein in RPE cells leads to over-production of NO, accompanied by activation of the oxidant enzyme as well as increasing the O•2-. Extra NO and O2- produce extra ONOO- which acts as a strong oxidant24-25 Recent studies26 also supported our results. With gene array analysis, we found that iNOS divergence genes appeared in six micro-array slides (NOS2). They were up-regulated in ONOO- group and down-regulated in puerarin group. These results verified our previous works. It is wothy of noting that nNOS is regulated in a similar manner as iNOS. nNOS was expressed in normal physiological conditions and mainly located in the brain and its activation depends on Ca2+ and calmodine (CaM). Many studies have shown that iNOS could affects apoptosis in eyes and other organs of human body.27,28 Therefore, the relationship between iNOS and nNOS still needs further investigation.
Cell apoptosis is the result of cascade gene expression. Up to date, more genes contribute to production and regulation of cell apoptosis. It is believed that gene products localized in the inner layer of the cell directly regulate the initiation and progression of apoptosis, while iproducts in the outer layer of the cell affect gene expression through signal transduction pathways.29,30
Our results also suggested that RPE cells may play important roles in regulated complement activation which attributed to the apoptotic events. Increased complement activation in the RPE cells may be important for retinal homeostasis in the context of photoreceptor waste production accumulation. Complement activation is involved in the pathogenesis of age-related macular degeneration.31,32
In our study, apoptosis in RPE cell culture induced by ONOO- could be alleviated by puerarin. Zacks et al11 studied the inhibition of Fas signaling using Fas receptor-neutralizing antibody, siFas, or LPR mice resulted in a significant reduction in the number of TUNEL-positive photoreceptor cells as well as in a significant preservation of outer nuclear layer cell counts and thickness as compared with retina/RPE separation in eyes with intact Fas signaling. They pointed out that Fas-pathway inhibition might serve as a novel mechanism for preserving photoreceptor cells during retinal disease. Maaijwee et al33 showed angiographic evidence of Fas/FasL for revascularization of an rpe-choroid graft in patients with AMD. Zhou et al31 verified that bisretinoid pigments of retinal pigment epithelial lipofuscin, subsequent to photoactivation and cleavage, serve to activate complement. Complement activation by this mechanism is dependent on the alternative pathway and can be modulated by an inhibitor of C3 cleavage. These events in the setting of complement dysregulation could contribute to the chronic inflammation that underlies AMD pathogenesis. Rohrer et al34 observed a targeted inhibitor of the alternative complement pathway reduces angiogenesis in a mouse model of AMD. Therefore, It could be speculated that Fas/Fasl cell signal transduction route and C3 activation and many other apoptotic factors probably affect and strengthen the apoptosis process mediated by ONOO-.
In conclusion, we demonstrate that puerarin is a potent retina protective agent on ONOO--induced RPE disfunction in vitro. Thus, puerarin treatment may represent a novel approach to lowering the risk of or improving recovery in RPE or retina injury-related disorders.
We thanked Dr. Henry J. Kaplan and Prof. Nalini S. Bora from Kentucky Lions Eye Center of USA for their help.
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Keywords:© 2011 Chinese Medical Association
retinal pigment epithelial cells; oxidative; cell signal; complement; puerarin