Resveratrol Protects Against Ultraviolet A-Mediated Inhibition of ... - Core

RESVERATROL PROTECTS AGAINST ULTRAVIOLET A-MEDIATED INHIBITION OF THE PHAGOCYTIC FUNCTION OF HUMAN RETINAL PIGMENT EPITHELIAL CELLS VIA LARGE-CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM CHANNELS 1

Shwu-Jiuan Sheu1,2,3 and Tsung-Tien Wu1,2 Department of Ophthalmology, Kaohsiung Veterans General Hospital, Kaohsiung, 2 School of Medicine, National Yang-Ming University, Taipei; and 3 Yuhing Junior College of Health Care and Management, Kaohsiung, Taiwan.

This study was undertaken to examine the protective effect of resveratrol on human retinal pigment epithelial (RPE) cell phagocytosis against ultraviolet irradiation damage. Cultured RPE cells were exposed to ultraviolet A (UVA, 20 minutes) irradiation, and treated with meclofenamic acid (30 μM, 20 minutes), paxilline (100 nM, 20 minutes) or resveratrol (10 μM, 20 minutes). Meclofenamic acid and resveratrol were given after exposure to UVA. Pretreatment with meclofenamic acid, resveratrol or paxilline before UVA irradiation was also performed. Fluorescent latex beads were then fed for 4 hours and the phagocytotic function was assessed by flow cytometry. UVA irradiation inhibited the phagocytic function of human RPE cells. The large-conductance calcium-activated potassium channel activator meclofenamic acid ameliorated the damage caused by UVA irradiation. Pretreatment with resveratrol acid also provided protection against damage caused by UVA. Posttreatment with meclofenamic acid offered mild protection, whereas resveratrol did not. In conclusion, the red wine flavonoid resveratrol ameliorated UVA-mediated inhibition of human RPE phagocytosis. The underlying mechanism might involve the large-conductance calciumactivated potassium channels.

Key Words: large-conductance calcium-activated potassium-channels, phagocytosis, resveratrol, retinal pigment epithelium, ultraviolet irradiation (Kaohsiung J Med Sci 2009;25:381–8) The results have been presented in part at the annual meeting of The Association of Research in Vision and Ophthalmology (ARVO), 2008, Fort Lauderdale, FL, USA.

Age-related macular degeneration (AMD) is a major cause of adult visual loss in developed countries. The

Received: Feb 25, 2009 Accepted: Apr 29, 2009 Address correspondence and reprint requests to: Dr Shwu-Jiuan Sheu, Department of Ophthalmology, Kaohsiung Veterans General Hospital, 386 Ta-Chung 1st Road, Kaohsiung 813, Taiwan. E-mail: [email protected]

Kaohsiung J Med Sci July 2009 • Vol 25 • No 7 © 2009 Elsevier. All rights reserved.

impaired vision in AMD patients results from photoreceptor cell death in the central retina. In the early stage, there is degeneration of retinal pigment epithelial (RPE) cells [1,2]. RPE cells are responsible for phagocytosis and degradation of shed photoreceptor outer segments. It has been demonstrated that in AMD, RPE cells die through apoptosis [3]. Although the exact cause of RPE cell death is not clear, current evidence supports an important role of oxidative stress in RPE 381

S.J. Sheu and T.T. Wu

apoptosis [4,5]. Normally, the material phagocytosed by RPE cells undergoes lysosomal degradation. With age, the lysosomal system becomes incapable of degrading efficiently oxidized lipid constituents, which are thought to contribute to the formation of lipofuscin. These eventually form drusen deposits, which compromise the exchange between RPE and choriocapillaries, and underlie the observed morphologic changes of RPE with age [6–8]. Ultraviolet (UV) irradiation can induce DNA breakdown and cause the production of reactive oxygen species. UV-induced RPE damage, including apoptosis, DNA damage and cellular changes have been reported [9–11]. UV exposure has also been implicated as a risk factor for AMD, although the results of studies are inconsistent [12–14]. Large-conductance calcium-activated potassium channels (BKCa channels) have been found in many tissues, and participate in a variety of cellular processes including K+ transport. BKCa channels may provide an important pathway for transcellular K+ transport, which has been demonstrated in RPE [15]. BKCa channels can be distinguished from most K+ channels by their dual-control activation, i.e. they are activated by either an increase in intracellular Ca2+ or membrane depolarization. These channels can also control Ca2+ influx and a wide array of Ca2+-dependent physiologic processes. A better understanding of the role of BKCa channels in a variety of excitable and non-excitable cells has led to a plethora of publications on the subject, and an ever-increasing number of newly developed compounds [16–18]. Our previous study showed that oxidizing agents suppress the activity of BKCa channels in RPE cells, suggesting that BKCa channels are involved in oxidative damage of human RPE cells [19]. Resveratrol (3,5,4’-trihydroxystilbene), a flavonoid found in red wine, exhibits antioxidant and antiproliferative activities in vitro [20–22]. A number of potential health benefits, including reduced risk of cancer and heart disease, are thought to be associated with the consumption of resveratrol [23]. Moreover, resveratrol was reported to reduce oxidation and proliferation of human RPE cells by inhibiting extracellular signalregulated kinase [24]. Our previous study also showed that resveratrol significantly reduced oxidative damage of the phagocytic function of human RPE R-50 cells. One of the underlying mechanisms might be linked to the activity of BKCa channels in the RPE cells [25]. 382

This study was undertaken to examine the protective effect of resveratrol on human RPE cell phagocytosis against UVA irradiation damage.

MATERIALS AND METHODS Human RPE R-50 cells were cultured, as previously described [26], in Ham’s F-12 medium (Life Technologies Inc., Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 2 mM L-glutamate (Life Technologies Inc.). The cell cultures were maintained in T-75 flasks in a humidified incubator at 37°C with 5% CO2. The medium was changed every 2 days to provide sufficient nutrition. For the phagocytosis assay, RPE cells were seeded in a 24-well culture plate at a concentration of 5 × 104 cells/well. All experiments were performed after the cells had reached near confluence. Each well of the confluent RPE explants was inoculated with 700 μL of Ham’s F-12 medium containing 107 amine-modified fluorescent orange latex beads (Sigma-Aldrich Inc., St Louis, MO, USA), and then incubated for 4 hours at 37°C in an atmosphere of 5% CO2 and 95% air. To assess the role of BKCa channels, the channel activator meclofenamic acid (Sigma-Aldrich Inc.) and the channel blocker paxilline (Sigma-Aldrich Inc.), were used in the experiments. The UVA lamp emits ultraviolet rays at between 355 nm and 375 nm, with a peak luminosity at 365 nm. The cultured RPE cells were exposed to UVA (20 mW/cm2, 20 minutes) irradiation and treated with meclofenamic acid (30 μM, 20 minutes), paxilline (100 nM, 20 minutes) or resveratrol (10 μM, 20 minutes; Sigma-Aldrich Inc.). Meclofenamic acid and resveratrol were administered after exposure to UVA. Pretreatment with meclofenamic acid, resveratrol or paxilline before UVA irradiation was also performed. The combined effect of UVA and paxilline was tested by treating RPE cells with meclofenamic acid, resveratrol or paxilline and UVA irradiation in different sequences, UVA meclofenamic acid paxilline, UVA paxilline meclofenamic acid, UVA resveratrol paxilline, UVA paxilline resveratrol, meclofenamic acid paxilline UVA, paxilline meclofenamic acid UVA, resveratrol paxilline UVA, or paxilline resveratrol UVA. The dose and time of exposure remained the same in all experiments. The RPE cells were washed Kaohsiung J Med Sci July 2009 • Vol 25 • No 7

Resveratrol protects UVA damage on RPE cells

Kaohsiung J Med Sci July 2009 • Vol 25 • No 7

RESULTS UVA irradiation and paxilline strongly inhibited the phagocytic function of human RPE cells. However, the BKCa channel activator meclofenamic acid (30 μM) offered protection against the inhibitory effects of paxilline on RPE phagocytosis (Figure 1). Pretreatment with meclofenamic acid protected RPE cells against damage caused by UVA, increasing the phagocytosis index from 20.23 ± 1.02% to 44.89 ± 0.44% (p = 0.004). Pretreatment with resveratrol also protected against damage caused by UVA, increasing the phagocytosis index from 20.23 ± 1.02% to 35.11 ± 0.10% (p = 0.004). A protective effect was not observed in the paxilline group. Posttreatment with meclofenamic acid offered mild protection whereas resveratrol did not (Figure 2). Combined treatment of paxilline and UVA did not produce further damage. Pretreatment with meclofenamic acid or resveratrol protected RPE cells against 100

80 Phagocytosis index (%)

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twice with phosphate-buffered saline between stimulation. The cells were then challenged with fluorescent latex beads for 4 hours and the phagocytic function was assessed by flow cytometry. The concentrations of all the agents chosen in this study were found to have minimal toxicity in cultured RPE cells (data not shown). The samples were prepared for flow cytometry using a previously described protocol [27]. The cells were detached using 0.05% trypsin with ethylenediamine tetraacetic acid for 2–4 minutes. The externally adhering latex beads were largely removed by this procedure, as confirmed by fluorescence microscopy. The cells were recovered by centrifugation at 1,500 revolutions/minute for 5 minutes. The resulting cell pellet was resuspended in phosphate-buffered saline and assessed immediately by flow cytometry. Fluoresceinated latex bead uptake was measured using a fluorescence-activated cell sorter (FACStar Plus; Becton Dickson, Mountain View, CA, USA). A 15-W argon laser tuned to 488 nm at 200 mW was used to excite the fluorescent beads. Fluorescence emission was collected using a selective band-pass filter of 585–620 nm. Negative and positive controls were included in each experiment. The negative control consisted of untreated RPE R-50 cells only, while the positive control comprised untreated RPE R-50 cells challenged with fluorescent beads. The data were printed out as a histogram. Using the curve for the negative control, we set the gate for each experiment to obtain the percentage of phagocytosis-positive cells. Each flow cytometry run comprised 10,000 scattering events. The data were presented as the phagocytic index, as modified from previous reports [28], which was calculated as follows: mean fluorescence (total fluorescence/ number of cells sorted) × percentage of phagocytosispositive cells. The results are shown as the percentage of the untreated positive control. Experiments were performed in triplicate. All assays were repeated three times to ensure reproducibility. Analysis of variance was performed to assess the differences in mean values across the treatment groups involving cell culture experiments with multiple treatments. The Wilcoxon signed-rank test was used to identify significant differences between the paired treatments. Data processing and analysis were performed using SPSS version 12 (SPSS Inc., Chicago, IL, USA). Differences between the values were considered statistically significant at p < 0.05.

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Figure 1. Effect of oxidative stress and radiation damage on the phagocytic function of human retinal pigment epithelial (RPE) cells. The phagocytic function of human RPE cells was inhibited by exposure to ultraviolet A (UVA; 20 mW/cm2, 20 minutes) or paxilline (100 nM, 20 minutes). Meclofenamic acid (30 mM) ameliorated the inhibitory effects of paxilline on RPE phagocytosis. *Statistically significant compared with UVA stimulation. UVA = ultraviolet A; meclofenamic acid paxilline = administration of meclofenamic acid (30 mM, 20 minutes) followed by paxilline (100 nM, 20 minutes).

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Figure 2. Protective effect of meclofenamic acid and resveratrol on ultraviolet A (UVA)-induced damage of human retinal pigment epithelial (RPE) cells. Phagocytosis of human RPE cells was inhibited by UVA. Pretreatment with meclofenamic acid (30 mM, 20 minutes) or resveratrol (10 mM, 20 minutes), but not paxilline, protected cell phagocytic function against damage caused by UVA irradiation. *Statistically significant compared with UVA stimulation. UVA meclofenamic acid = UVA (20 mW/cm2, 20 minutes) exposure followed by administration of meclofenamic acid (30 mM, 20 minutes); UVA resveratrol = UVA exposure followed by administration of resveratrol (10 mM, 20 minutes); UVA paxilline = UVA exposure followed by administration of paxilline (100 nM, 20 minutes); meclofenamic acid UVA = administration of meclofenamic acid followed by UVA exposure; resveratrol UVA = administration of resveratrol followed by UVA exposure; paxilline UVA = administration of paxilline followed by UVA exposure.

damage caused by paxilline and UVA and increased the phagocytosis index from 19.64 ± 0.23% to 39.97 ± 0.37% in the meclofenamic acid group and to 29.43 ± 0.62% in the resveratrol group (p = 0.004). Pretreatment with paxilline before UVA irradiation markedly decreased the protective effects of meclofenamic acid and resveratrol. Posttreatment with meclofenamic acid still decreased the effect of palliative, but much less than that with meclofenamic acid pretreatment; this occurred irrespective of whether meclofenamic acid was administered before or after paxilline. Even in the presence of the combined effects of UVA and

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Figure 3. Protective effects of meclofenamic acid against the combined effects of ultraviolet A (UVA) and paxilline on phagocytic function of human retinal pigment epithelial (RPE) cells. Pretreating RPE cells with meclofenamic acid was protective, even in the presence of combined exposure to paxilline and UVA. However, the protective effect of meclofenamic acid was reduced if the effect of meclofenamic acid was blocked by paxilline before UVA irradiation. Posttreatment with meclofenamic acid still showed a palliative effect, but the efficacy was much less than that with meclofenamic acid pretreatment. *Statistically significant compared with UVA paxilline. UVA paxilline = UVA exposure (20 mW/cm2, 20 minutes) followed by paxilline (100 nM, 20 minutes); UVA meclofenamic acid paxilline = UVA exposure followed by administration of meclofenamic acid (30 mM, 20 minutes) and then of paxilline; UVA paxilline meclofenamic acid = UVA exposure followed by administration of paxilline and then of meclofenamic acid; meclofenamic acid paxilline UVA = administration of meclofenamic acid followed by administration of paxilline and then of UVA exposure; paxilline meclofenamic acid UVA = administration of paxilline followed by administration of meclofenamic acid and then of UVA exposure.

paxilline, meclofenamic acid and resveratrol still protected RPE phagocytosis, particularly with pretreatment (Figures 3 and 4). These effects suggest that the BKCa channels are involved in human RPE R-50 phagocytosis. Therefore, the underlying mechanism for the inhibitory action of UV irradiation might be associated with the activity of BKCa channels present in these cells. Kaohsiung J Med Sci July 2009 • Vol 25 • No 7

Resveratrol protects UVA damage on RPE cells 100


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Figure 4. Protective effects of resveratrol against the combined effects of ultraviolet A (UVA) and paxilline on phagocytic function of human retinal pigment epithelial (RPE) cells. Pretreating RPE cells with resveratrol was protective on RPE phagocytic function, even in the presence of combined exposure to paxilline and UVA. However, the protective effect of resveratrol was remarkably reduced if the resveratrol effect was blocked by paxilline before UVA irradiation. Posttreatment with resveratrol showed only a mild amelioration effect. *Statistically significant compared with UVA paxilline. UVA paxilline = UVA exposure (20 mW/cm2, 20 minutes) followed by administration of paxilline (100 nM, 20 minutes); UVA resveratrol paxilline = UVA exposure followed by administration of resveratrol (10 mM, 20 minutes) and then of paxilline; UVA paxilline resveratrol = UVA exposure followed by administration of paxilline and then of resveratrol; resveratrol paxilline UVA = administration of resveratrol, followed by administration of paxilline and then of UVA exposure; paxilline resveratrol UVA = administration of paxilline followed by administration of resveratrol and then of UVA exposure.

DISCUSSION Our results showed that the underlying mechanism for the inhibitory action of UVA irradiation might be associated with the activity of BKCa channels present in these cells. Pretreatment with resveratrol offered protection against irradiation damage, which might involve the activity of BKCa channels. Although the exact role of sun exposure in causing AMD remains inconclusive, UV protection is generally accepted to protect against these changes in Kaohsiung J Med Sci July 2009 • Vol 25 • No 7

eyes that occur due to aging [12,14]. Normally, the lens filters UV light to the retina. With the aging of the general population and the advanced techniques used for cataract surgery, people are becoming more susceptible to UV exposure, with increased risk of associated damage after cataract extraction. The increased progression of AMD after cataract extraction has been documented by several studies [29,30]. A blue-light filtering intraocular lens has recently gained much attention [31]. The results of our study confirmed that UVA irradiation had deleterious effects on an important function of human RPEs, namely phagocytosis. The results also revealed the possibility that BKCa channels are involved, which is similar to the oxidative damage of RPE cells in our previous study [25]. UVA irradiation can induce DNA breakdown and stimulate the production of reactive oxygen species, which cause oxidative damage to the RPE cells [9–11]. Several in vitro studies have shown that oxidative stress induced by chemical oxidants, such as hydrogen peroxide, leads to RPE damage and death [4,32,33]. Recent clinical studies have suggested that adequate intake of dietary antioxidants may delay or prevent the development of retinal diseases [34,35]. Resveratrol, a flavonoid in red wine, is an effective scavenger of free radicals, including singlet oxygen and superoxide anions. Moreover, resveratrol was reported to reduce oxidation and proliferation of human RPE cells via extracellular signal-regulated kinase inhibition, and elicit dilation of isolated porcine retinal arterioles [24,36]. Moderate red wine consumption has been reported to have a protective effect on the development of AMD [37,38]. Our previous study showed that resveratrol protects human RPE phagocytosis against H2O2 damage, possibly via the BKCa channels in RPE cells [25]. In this study, the BKCa channel activator meclofenamic acid protected the phagocytotic function of human RPE cells against UVA irradiation. In other words, the underlying mechanism of UV irradiation damage on RPE phagocytosis might involve the BKCa channels. Moreover, resveratrol showed a protective effect in the pretreatment model, but not in the posttreatment model. This finding led to the speculation that the red wine flavonoid resveratrol can ameliorate the UVA irradiation damage on human RPE phagocytosis and help prevent degeneration of RPE cells at a very early stage in the pathogenesis of AMD. AMD is one of the major causes of adult visual loss in developed countries. 385


Its incidence and treatment have become more and more important with the increase in the aging population worldwide. Treatments such as anti-VEGF, photodynamic therapy and surgical intervention are currently only available for exudative AMD [2]. Even with advanced treatment, complete visual recovery is still impossible. Prevention offers a better approach to prevent visual loss due to AMD, and will reduce the socioeconomic burden from the disease treatment and the disability-related cost. Our results support the hypothesis that moderate wine consumption can protect RPE cells from oxidative damage and, thereby, decrease the risk of developing retinal diseases. This offers an alternative approach for the prevention and management of AMD. In conclusion, the red wine flavonoid resveratrol significantly reduced UVA irradiation damage of human RPE phagocytosis. The underlying mechanism might involve BKCa channels.

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ACKNOWLEDGMENTS

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The study was supported by grants from the National Science Council (NSC97-2314-B-075B-011) and Kaohsiung Veterans General Hospital (VGHKS 98-063), Taiwan.

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葡萄紅素可經由大傳導鈣活化性鉀離子通道保護人類視網膜色素上皮細胞對抗紫外線之傷害許淑娟 1

1,2,3

吳宗典

1,2

2

高雄榮民總醫院眼科陽明大學醫學院 3

育英醫護管理專科學校

本研究目的在探討葡萄紅素對人類視網膜色素上皮細胞對抗紫外線傷害之保護作用。實驗室中培養的人類視網膜色素上皮細胞株分別以 ultraviolet A (UVA)、 meclofenamic acid (30 μM)、paxilline (100 nM) 或 resveratrol (10 μM) 處理 20 分鐘。此外對 UVA 曝露 20 分鐘後的細胞分別再以 meclofenamic acid 或 resveratrol 處理。有些則於 UVA 照射前以 meclofenamic acid、resveratrol 或 paxilline 先處理。完成上述步驟後再餵予螢光珠 4 小時後以流式細胞儀檢測細胞之吞噬作用的表現與定量。結果發現 UVA 照射會抑制人類視網膜色素上皮細胞的吞噬作用。大傳導鈣活化性鉀離子通道活化劑 meclofenamic acid 可減緩 UVA 照射所造成的傷害 , 事後給藥也有保護作用但較差。事前給予葡萄紅素亦表現出顯著的保護作用但事後給藥效果不顯著。根據實驗結果 , 我們推測紅酒類黃酮葡萄紅素應可提供人類視網膜色素上皮細胞對抗紫外線傷害些許保護、其作用機轉可能涉及大傳導鈣活化性鉀離子通道。關鍵詞：大傳導鈣活化性鉀離子通道，吞噬作用，葡萄紅素，視網膜色素上皮，紫外線 ( 高雄醫誌 2009;25:381–8)

收文日期：98 年 2 月 25 日接受刊載：98 年 4 月 29 日通訊作者：許淑娟醫師高雄榮民總醫院眼科高雄市大中一路 386 號 388
