Green Tea Polyphenols Attenuating Ultraviolet B–Induced Damage to Human Retinal Pigment Epithelial Cells In Vitro

Abstract

Purpose.: To examine the protective effect of green tea polyphenols against ultraviolet B (UVB)–induced damage to retinal pigment epithelial (RPE) cells.

Methods.: Green tea polyphenols (GTP) was used to treat RPE cells before or after exposure to UVB. Viability of RPE cells was tested by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Survivin gene expression was examined by real-time PCR analysis. Ultrastructure of RPE cells was examined by transmission electron microscopy.

Results.: GTP effectively suppressed the decrease in viability of the UVB stressed RPE cells and the UVB suppression of survivin gene expression level. GTP alleviated mitochondria dysfunction and DNA fragmentation induced by UVB.

Conclusions.: GTP protected RPE cells from UVB damage through its increase in the survivin gene expression and its attenuation of mitochondria dysfunction and DNA fragmentation. GTP is a potential candidate for further development as a chemoprotective factor for the primary prevention of age-related eye diseases such as age-related macular degeneration.

Human retinal pigment epithelial (RPE) cells perform important functions in visual process, and their dysfunction causes many retinal degenerative diseases, such as age-related macular degeneration (AMD). Extrinsic oxidative stress, a disturbance in the pro-oxidant–antioxidant balance, is considered to be involved in the process of RPE dysfunction. ^{1 –3}  

Solar ultraviolet (UV) radiation reaching the earth's surface is increasing because of depletion of the ozone layer, among which UVB (280–320 nm) is the most harmful to humans. ^4,5 Excessive exposure to solar UV radiation may lead to the formation of reactive oxygen species (ROS) in RPE cells, resulting in oxidant stress and retinal damage. ^1,6 Studies show that UV radiation induces RPE cell apoptosis in vitro. ^{1,7 –9} UV radiation is believed to be involved in the pathogenesis of AMD through oxidative stress and DNA breakage in RPE cells. ^3,10,11  

Many efforts have been made to fight UV damage to humans. RPE cell apoptosis induced by oxidative stress is alleviated by antioxidants such as ascorbic acid and related substances. ^{12 –15} Many plant materials contain polyphenols that can be used as antioxidants. ¹⁶ Green tea has a high level of polyphenols (25%–35% of dry base). Experiments in vivo and in vitro show that green tea polyphenols (GTP) enhance intracellular antioxidant defense ^17,18 and promote the downregulation of proapoptotic genes, ^19,20 suggesting that GTP might be used to fight against RPE dysfunction induced by UVB radiation. GTP alleviates the UVB-induced stress and the destructive morphologic changes in HaCat cells by both interacting with UVB-induced ROS and attenuating mitochondrion-mediated apoptosis. ²¹ Information on GTP effects on UVB-induced stress in RPE cells, however, has not been available. 

Survivin gene encodes a protein that regulates cell division and suppresses apoptosis, though the exact mechanism behind the ability of survivin to inhibit apoptosis is unclear. ²² The role of survivin as an inhibitor of apoptosis (IAP) that is expressed in keratinocytes under UVB irradiation conditions has been confirmed using transgenic K14-survivin mice, showing that the cell death rate of the K14-survivin mice is 60% lower than in the control group after UVB exposure. The expression of survivin leads to the inhibition of keratinocyte apoptosis, though cell proliferation remains unaffected. ²³  

This study was conducted to investigate the effects of GTP on the viability, microstructures, DNA fragmentation, and expression of survivin genes in UVB-stressed RPE cells to evaluate the protective effect of GTP against UVB-induced RPE cell injuries. 

Materials and Methods

Materials

The human retinal pigment epithelial (RPE) cell line (no. D407) was purchased from the Center of Experiment Animals of Sun Yat-sen University (Guangzhou, China). Use of the no. D407 RPE cell line in the test was verbally consented by the Center of Animal Experiments of Sun Yat-sen University. The RPE cells were seeded in 50-mL flasks (10⁶/flask) containing 5 mL RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U penicillin-streptomycin and were cultured at 37°C in humidified atmosphere of 5% CO₂. When the cells were >70% confluent (70% of the dish bottom surface area covered by cell monolayer), they were washed with 0.5% (vol/vol) trypsin/ EDTA and collected with a 1-mL pipette tip for further use. 

GTP used in the study was supplied by CinoTea Co., Ltd. (Hangzhou, China); its composition is listed in Table 1. The GTP was dissolved in RPMI-1640 medium without FBS supplement as stock solutions at concentrations 0, 14, 70, 140, 700 and 1400 mg/L before use. 

Table 1.

 

Composition of Green Tea Polyphenols (mg/g)

GC	EGC	C	EC	EGCG	GCG	ECG	CG	Total Catechins
44.38	85.47	14.09	41.13	344.73	42.49	103.37	8.80	684.47

 C, (+)-catechin; CG, (+)-catechin-3-gallate; EC, (−)-epicatechin; ECG, (−)-epicatechin-3-gallate; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin-3-gallate; GC, (+)-gallocatechin; GCG, (+)-gallocatecgin-3-gallate.

FBS was purchased from Life Technologies Inc.(Gaithersburg, MD). Reagent (TRIzol) and penicillin-streptomycin were purchased from Invitrogen Corp. (Carlsbad, CA). MTT [3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide], trypsin, and ethylenediaminetetraacetate (EDTA) were purchased from Amresco Inc. (Solon, OH). The other reagents used were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. (Dalian, China). 

Test of GTP Effect on Viability of UVB-Stressed RPE Cells

GTP pretreatment and GTP posttreatment were carried out in 96-well plates, with three plates for each treatment. The RPE cells were seeded onto the plate well (10⁵ cells each well) with 200 μL above RPMI-1640 medium and were cultured under the conditions described until 70% cell confluence. The cells were used for GTP and UVB treatment. 

For GTP pretreatment, the medium solution in the wells was drained, and the cells were washed by PBS. After the PBS was drained, 200 μL of the GTP stock solution was transferred to each well, with 15 wells at each GTP dose. The RPE cells were incubated in GTP solution for 2 hours and then were washed with PBS. The GTP solution was replaced by FBS-free RPMI-1640 medium and then irradiated by 100 μw/cm² UVB for 2 hours with UVB light tubes (Spectronics Corp., Westbury, NY). 

For the GTP posttreatment, the medium was replaced by serum-free medium and the RPE cells were irradiated by 100 μw/cm² UVB for 2 hours. GTP treatment was carried out as described. 

Three plates for control were seeded with RPE cells and incubated in FBS-free medium without GTP and UVB irradiation. The RPE cells were washed with PBS and cultured in RPMI-1640 medium supplemented with 10% FBS and 100 U penicillin-streptomycin at 37°C in humidified atmosphere of 5% CO₂ for 12 hours. 

Cell viability was tested by MTT assay. ²¹ Twenty microliters of MTT solution (0.5 mg/mL, dissolved in PBS) was added to each culture well, and the plates were incubated at 37°C in a humidified atmosphere of 5% CO₂ for 4 hours. The solution was pipetted out, and 200 μL dimethyl sulfoxide was added and then mixed by pipetting. The formazan dye formation was measured at 540 nm using an ELISA reader (Thermo Fisher Scientific Inc., Waltham, MA). Results were presented as viability percentage with respect to control. 

Test of GTP Effect on Cell Morphology, Survivin Gene Expression, and DNA Fragmentation of UVB-Stressed RPE Cells

GTP and UVB Treatment.

RPE cells were seeded in 10-cm glass petri dishes (10⁶ cells each dish) with 5 mL RPMI-1640 medium supplemented with 10% FBS and incubated as described until they were 70% cell confluent. RPE cells in the dishes were washed by PBS. GTP pretreatment, GTP posttreatment, and control were treated as described. Two doses of GTP solution (70 and 140 mg/L) were tested, and 5 mL GTP solution was used for each dish. The UVB treatment was the same as described. 

Transmission Electron Microscopy.

The cells were washed with PBS and centrifuged at 2000g for 5 minutes. The cell pellet specimen was prefixed with 1 mL 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) at 4°C overnight, washed three times with 0.1 M phosphate buffer, finally fixed in 1% OsO₄ for 2 hours, and again washed three times with 0.1 M phosphate buffer. The fixed cell specimen was dehydrated in an increasing graded series of ethanol solutions (50%, 70%, 80%, 90%, 95%, and 100%) in which each dehydration step lasted 15 minutes. The dehydrated specimen was drenched sequentially in acetone for 20 minutes and embedded in Epon-812. Sections of 70- to 90-nm thickness were made on an ultra-microtome (OM U2; Reichert-Jung Co., Heidelberg, Germany) and were stained using uranyl acetate and alkaline lead citrate for 15 minutes. Sections were examined and photographed with a transmission electron microscope (JEM-1230; JEOL Ltd. Akishima, Tokyo, Japan). ²⁴  

Real-Time PCR Analysis of Survivin Gene Expression.

Samples were washed with PBS. Total RNA was extracted using reagent (TRIzol; Invitrogen) according to the manufacturer's protocol. Extracted RNA samples were checked by 1.5% agarose gel electrophoresis. RT-PCR was carried out using an RT-PCR kit (PrimeScript; TaKaRa). RT reaction was carried out in a 20-μL reaction system, including 4 μL 5× buffer (PrimeScript; TaKaRa), 1 μL enzyme mix (PrimeScript RT Enzyme Mix I; TaKaRa), 1 μL oligo dT primer (50 μM), 1 μL random mers (100 μM), and 13 μL total RNA at 37°C for 5 minutes and then 85°C for 5 seconds. Quantitative PCR was performed (iCycle iQ; Bio-Rad Laboratories, Hercules, CA) using the RT reaction solution and primers for generating the survivin gene (forward, 5′-CTGTTTTGATTCCCGGGCTTACCA-3; reverse, 5′-CACCCCGTTTCCCCAATGACTTAG-3′; expected size, 436 bp). The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH; forward, 5′-GGGGAGCCAAAAGGGTCATCATCT-3′; reverse, 5′-GACGCCTGCTTCACCACCTTCTTG-3′; expected size, 457 bp) was used as an endogenous control. The PCR reaction system consisted of 12.5 μL premix reagent (2×) (SYBR Premix Ex Taq; TaKaRa), 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 9.5 μL dd H₂O, and 2 μL RT reaction solution. The reaction was carried out at 94°C for 3 minutes followed by 40 cycles of 94°C for 30 seconds and 65°C for 60 seconds. Samples were tested in triplicate, and the mean values were presented. 

Electrophoresis of RPE Cell DNA Samples.

RPE cells were homogenized in 1 mL CTAB and transferred into a 5-mL tube and incubated in a 65°C water bath for 5 minutes. Then 1 mL mixture of chloroform/isoamyl alcohol (24/1, vol/vol) was added to the tube and mixed. The aqueous supernatant was transferred to a new tube after centrifugation at 12,000 rpm for 10 minutes at room temperature, mixed with 5 mL 95% (vol/vol) ethanol, left to stand for 5 minutes at room temperature, and centrifuged at 12,000 rpm for 3 minutes. The supernatant was drained, and the precipitated DNA was washed twice with 70% ethanol. The DNA sample was dissolved in 0.5 mL water, electrophoresed on 1.5% agarose gel, and photographed. 

Results

Effect of GTP on Viability of RPE Cells Irradiated by UVB

RPE cell viability was greatly influenced by the UVB irradiation. Under the present study conditions, the viability of UVB-irradiated RPE cells decreased by 49.2% compared with unirradiated control (Fig. 1). Effects of GTP on the viability of UVB-stressed RPE cells varied with GTP concentrations. GTP-protective effects were dose dependent up to 140 mg/L. However, cell viability decreased with further increases in GTP concentrations from 700 mg/L to 1400 mg/L (Fig. 1). The 700-mg/L dose could be toxic concentration to the RPE cell. It was also shown that the protective effects of GTP pretreatment were better than those of GTP posttreatment (Fig. 1), suggesting that GTP protection of RPE cells from UVB injury is dependent on GTP concentration and treatment methods. 

Figure 1.

 

GTP protecting RPE cells from UVB injuries. Data are expressed as mean ± SD (n = 3) of the ratios of absorbance of tested sample to that of control, which was not treated by GTP and UVB.

Effect of GTP on Microstructures of RPE Cells Irradiated by UVB

Significant abnormalities in RPE cell microstructures, such as microvilli shedding, nucleolus degeneration, mitochondria deformity, and formation of vesicular structures in cytoplasm, were observed after UVB irradiation. Before UVB irradiation, the intact cytoplasm of the RPE cell was evenly distributed, and the nucleolus was located at the center of the cell. Outer cell membrane microvilli were observed (Fig. 2, control, whole cell structure). The mitochondria were full of regular disposition cristae (Fig. 2, control, organelle structure). After UVB irradiation, the nucleolus shrank and the outer cell microvilli were shed. Deformed mitochondria and many vesicular structures were observed (Fig. 2; 0 GTP+UVB). RPE cells pretreated with 70 and 140 mg/L GTP before UVB irradiation underwent fewer changes in the cell microstructures (Fig. 2; 70 GTP+UVB and 140 GTP+UVB). Microvilli were partially seen. Vesicular structures were smaller. Most of the mitochondria looked normal at the 140-mg/L GTP dose, though a few mitochondria became dumbbell-shaped at the 70-mg/L GTP dose. GTP posttreatment showed the same tendency as GTP pretreatment, but the protective effects were less than those of GTP pretreatment. The dumbbell-shaped and long mitochondria were seen in the GTP posttreatment of both GTP doses (Fig. 2; UVB+GTP), suggesting that GTP attenuated UVB-induced RPE cell damage by both pretreatment and posttreatment. 

Figure 2.

 

Changes in microstructures of UVB-stressed RPE cells. The numbers before GTP indicate each GTP dose (mg/L). GTP+UVB, GTP pretreatment; UVB+ GTP, GTP posttreatment; Nu, nucleolus; MV, cell membrane microvilli. Arrow: normal mitochondria; black triangle: deformed mitochondria; white triangle: vesicular structure.

Effect of GTP on DNA Fragmentation of RPE Cells Irradiated by UVB

A single band was observed in the RPE cell DNA samples before UVB irradiation (Fig. 3, lanes A1 and B1). There were small DNA fragments on the electrophoresis gels after the RPE cells were irradiated by UVB (Fig. 3, lanes A4 and B4). Pretreatment with both 140 mg/L and 70 mg/L GTP partially inhibited the DNA fragmentation (Fig. 3, lanes A2-A3 and B2-B3). However, posttreatment with GTP had a less protective effect against DNA fragmentation, especially at 70 mg/L. These indicate that GTP suppressed the DNA fragmentation induced by UVB and promoted the repair of the damaged DNA. The protective effect was dependent on dosage. 

Figure 3.

 

Effects of GTP on DNA fragmentation of RPE cells. Lane A1, Control: normal cells not treated by UVB and GTP; lane A2, 140 mg/L GTP pretreatment and then UVB irradiation; lane A3, 70 mg/L GTP pretreatment and then UVB irradiation; lane A4, 0 mg/L GTP pretreatment and then UVB irradiation; lane B1, control (normal cells not treated by UVB and GTP); lane B2, UVB irradiation and then 140 mg/L GTP posttreatment; lane B3, UVB irradiation and then 70 mg/L GTP posttreatment; lane B4, UVB irradiation and then 0 mg/L GTP posttreatment.

Effect of GTP on Expression of Survivin Gene in RPE Cells Irradiated by UVB

The expression level of the survivin gene in RPE cells was suppressed by UVB irradiation (Fig. 4). Both GTP pretreatment and GTP posttreatment, however, attenuated the UVB suppression of survivin gene expression. Survivin is a member of the IAP family. The survivin protein function is to inhibit caspase activation, leading to the negative regulation of apoptosis or programmed cell death. ²⁵ Disruption of the survivin induction pathways led to increases in apoptosis, ²⁶ suggesting that the protective effect of GTP against UVB damage to RPE cells might be related to its regulation of survivin gene expression. 

Figure 4.

 

Expression of survivin gene in RPE cells. Relative expression data were presented after normalization against GAPDH. Data are expressed as the mean ± SD (n = 3).

Discussion

The mitochondrion is the power center of cells. One study shows that the morphology and distribution changes of mitochondria cristae not only relate to the supply of cell energy, they play important roles in the process of apoptosis. ²⁷ During the early stages of mitochondrion-mediated apoptosis, dramatic mitochondrial alterations, including crista remodeling, were observed. ^27,28 Mitochondrion-mediated apoptosis is closely related to the reactive oxygen species (ROS), which activate mitochondrial permeability transition pore opening and induce cytochrome c release from cardiolipin and outer membrane rupture by proapoptotic protein translocation. ²⁹ Curcumin induce apoptosis in A549 cells through an ROS-dependent mitochondrial signaling pathway. ³⁰ UVB irradiation decreased the viability of RPE cells, accompanying the formation of deformed mitochondria such as a dumbbell-shaped one (Fig. 2). This was also observed in the UVB-irradiated HaCat cell. ²¹ The implication is that mitochondrion-mediated apoptosis took place in the UVB-stressed RPE cells. GTP alleviated the decline of cell viability and the mitochondrion deformity of RPE cells (Figs. 1, 2), suggesting that GTP suppressed the UVB-induced mitochondrion-mediated apoptosis of RPE cells. GTP is a powerful antioxidant that has strong ROS-scavenging capacity. ^{31 –35} The mechanism by which GTP attenuates mitochondrion-mediated apoptosis might be related to its ROS-scavenging effect. ²¹  

There is evidence that DNA damage leads to nonapoptotic death in yeast. ³⁶ Plasma treatment induced DNA damage such as DNA double-strand breaks and mitochondria dysfunction accompanied by the release of cytochrome c from mitochondria, leading to apoptosis. ³⁷ DNA fragmentation took place in the UVB-irradiated RPE cells, but GTP pretreatment inhibited the DNA fragmentation (Fig. 3). Studies have indicated that (−)-epigallocatechin-3-gallate (EGCG), a major component of GTP, plays a role in protecting against free-radical DNA damage in irradiated cells with low linear energy transfer (LET) radiation such as x-rays. ^31,38 EGCG was the major component of GTP used in the present study (Table 1), and it may play important role in the protective effects against DNA damage. GTP and its (+)-catechin upregulated antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase. ^39,40 On the other hand, GTP posttreatment alleviated the DNA fragmentation in the UVB-stressed RPE cells (Fig. 4), suggesting that GTP promoted the repair of the damaged DNA. The (+)-catechin in GTP increased the expression of poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme. ⁴⁰ The protective effects of GTP against DNA fragmentation of the RPE cells might be exerted by the increased expression of DNA repair enzyme and antioxidant enzyme. 

Survivin is a bifunctional member of the inhibitor of apoptosis protein family because it is able both to inhibit apoptosis and to regulate the cell cycle during cell mitotic division. ⁴¹ Although much research has been focused on evaluation of the prognostic and therapeutic indicators of the Survivin protein in cancer, one study showed that survivin protein expresses in the basal layer of normal human epidermis. Survivin is important for epidermal self-renewal; silencing survivin reduces keratinocyte survival. ²⁶ The detailed molecular mechanisms by which survivin counteracts apoptosis are not fully clear. Studies have shown that survivin acts either alone or in complex with the x-linked inhibitor of apoptosis protein to inhibit caspase-9, thus protecting cells from apoptosis. ⁴² Survivin forms a conserved complex with the chromosomal passenger complex proteins Aurora-B kinase, Borealin, and INCENP to regulate mitotic events. ⁴³ A mitochondrial survivin pool is released on stress stimuli and binds to caspase-9, thus blocking tumor-cell apoptosis, ⁴² and survivin is downregulated during mitochondrial apoptosis in human keratinocytes. ⁴⁴ Silencing survivin activates caspase-3 and results in an increased number of keratinocytes in the G2/M phase. ²⁶  

Survivin plays a critical role in epidermal homeostasis in normal conditions and during UVB exposure. Low doses of UVB increase survivin expression at earlier time points, whereas high doses of UVB downregulate the survivin level, resulting in apoptosis. Overexpression of survivin protects keratinocytes from UVB-induced apoptosis, and silencing of survivin renders keratinocytes more susceptible to UVB-induced cell death. UVB downregulates survivin expression before caspase-3 activation, and silencing survivin augments the rate of UVB-induced apoptosis. ²⁶ (+)-Catechin partially inhibits the activation of caspase-3, resulting in the abolishment of both the cleavage of PARP and the degradation of inhibitor of caspase-activated DNase. ⁴⁰ This study showed that GTP protected RPE cells from UVB damage by suppressing the decrease in survivin expression level and alleviating mitochondrial dysfunction and DNA fragmentation, in agreement with the observation that survivin, which is likely derived from the mitochondrial pool, ⁴⁵ acts primarily through the ‘intrinsic’ apoptotic pathway. 

AMD is a major eye disease throughout the world. In the United States, more than 1.75 million persons have AMD, and 2.95 million more are at substantial risk for its development by 2020. ^46,47 UV light has been found to be a source of oxidative stress, ¹⁰ causing morphologic changes and alterations in size and movement in RPE cells, ⁴⁸ which is believed to be an important factor inducing the retinal damage in vivo. ⁴⁹ Considering the depletion of the ozone layer and the increasing lifetime of exposure to UV radiation, UV-induced AMD has become more threatening. ⁵⁰ This study indicated that GTP protects RPE cells from UVB damage through increases in survivin gene expression level and attenuation of mitochondria dysfunction and DNA fragmentation. This suggests GTP will be effective in preventing UVB-induced damage in RPE cells and may be a suitable alternative for chemoprotection for the primary prevention of age-related eye diseases such as AMD. 

Footnotes

 Supported by Science Foundation of Chinese University Grant 2009QNA6031.

Footnotes

 Disclosure: J.-Y. Xu, None; L.-Y. Wu, None; X.-Q. Zheng, None; J.-L. Lu, None; M.-Y. Wu, None; Y.-R. Liang, None