International Journal of Radiation Biology, October 2014; 90(10): 928–935 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.911990
Phloroglucinol inhibits ultraviolet B radiation-induced oxidative stress in the mouse skin Mei Jing Piao1*, Mee Jung Ahn1*, Kyoung Ah Kang1, Ki Cheon Kim1, Jian Zheng1, Cheng Wen Yao1, Ji Won Cha1, Chang Lim Hyun1, Hee Kyoung Kang1, Nam Ho Lee2 & Jin Won Hyun1 1School of Medicine and Institute for Nuclear Science and Technology, and 2Department of Chemistry, College of Natural
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Sciences, Jeju National University, Jeju, 690-756, Korea
Conclusion: These results suggest that phloroglucinol safeguards the mouse skin against UVB-induced oxidative stress and apoptosis.
Abstract Purpose: Previously we demonstrated that phloroglucinol (1,3,5trihydroxybenzene) protected human HaCaT keratinocytes against ultraviolet B (UVB, 280–320 nm)-induced oxidative stress in vitro by scavenging intracellular reactive oxygen species (ROS). The current study investigated whether phlo roglucinol could similarly protect the mouse skin against UVB-induced oxidative tissue damage in vivo. Materials and methods: Male 7-week-old Balb/c mice were divided into the following untreated normal control, phloroglucinol only-treated, vehicle plus UVB (30 or 60 mJ/cm2)exposed, and phloroglucinol (10 or 50 mg/ml) plus UVB (30 or 60 mJ/cm2)-treated groups. Following UVB exposure, phloroglucinol or phosphate buffered saline vehicle was applied to the dorsal skin of each mouse daily for 3 days. Studies were conducted at 24 h after the last of the UVB exposures. Histopathological analyses of dorsal skin lesions were performed on all mice. In addition, the levels of UVB-provoked injury to cellular components, including DNA, proteins, and lipids were detected by levels of 8-oxoguanine (8-oxoG), protein carbonyls, and 8-isoprostane. Apoptosis were assessed by using western blot for B-cell lymphoma-2-associated X protein (Bax) and activated caspase-3 expression, by using immunohistochemistry. Results: UVB radiation increased the thickness of the epidermis and the dermis, and also stimulated the accumulation of mast cells in the irradiated skin. However, treatment with phloroglucinol significantly decreased all of these parameters. Furthermore, phloroglucinol decreased UVB-provoked injury to cellular components, including DNA, proteins, and lipids; down-regulated the expression of phospho-histone H2A.X in the injured skin; and reduced the UVB-generated levels of 8-oxoG, protein carbonyls, and 8-isoprostane, which are all markers of oxidative stress. In addition, phloroglucinol attenuated the UVB-induced expression of the pro-apoptotic proteins, Bax protein, and activated caspase-3.
Keywords: Phloroglucinol, ultraviolet B, oxidative stress, apoptosis, skin tissue
Introduction Excessive exposure of the skin to ultraviolet (UV) radiation results in the generation of debilitating levels of intracellular reactive oxygen species (ROS). These ROS quickly overwhelm the tissue antioxidant systems and other oxidant-degrading pathways (Bickers and Athar 2006, Lyu and Park 2012). UV radiation also induces thickening of the epidermis and the dermis (Kim et al. 2005), as well as accumulation of mast cells in the damaged skin. Mast cells participate in the initiation of UV-provoked inflammation and oxidative stress, and contribute to the continued production of elevated levels of ROS (Malaviya et al. 1994). UV radiation consists of ultraviolet A (UVA, 320–400 nm), ultraviolet B (UVB, 280–320 nm), and ultraviolet C (UVC, 100–280 nm) light. Experimental studies indicate that UVB light is particularly likely to stimulate ROS generation and oxidative stress in the skin (Katiyar et al. 2001, Fonseca et al. 2010). UVB-induced ROS can cause irreversible damages to cellular macromolecules, including DNA, proteins, and lipids. The net result is the formation of oxidized products, such as 8-oxoguanine (8-oxoG) DNA lesions, protein carbonyls, and lipid hydroperoxides. These oxidized products are implicated in the onset of skin cancers (Vayalil et al. 2003, Tomaino et al. 2006) and apoptosis. The detrimental effects of UVB exposure have led to the search for exogenous compounds that can activate endogenous enzymatic and non-enzymatic antioxidant defense systems, thereby protecting the skin from UVB-induced oxidative stress (Sharma et al. 2007).
*These authors contributed equally to this paper. Correspondence: Prof. Jin Won Hyun, School of Medicine, Jeju National University, Jeju 690-756, Korea. E-mail: [email protected] (Received 17 November 2013; revised 20 March 2014; accepted 27 March 2014)
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Phloroglucinol reduces UVB-evoked skin damage 929 Brown algae, such as Ecklonia cava, E. stolonifera, and Eisenia bicyclis, are rich in phloroglucinol, phloroglucinol derivatives (Okada et al. 2004), and other antioxidant compounds. Phloroglucinol (1,3,5-trihydroxybenzene) directly inhibits the generation of ROS (Kim and Kim 2010). Moreover, phloroglucinol ameliorates hydrogen peroxide (H2O2)mediated cell damage and reduces gamma ray-induced oxidative stress via the quenching of ROS and the activation of antioxidant systems (Kang et al. 2006, 2010). We recently demonstrated that phloroglucinol attenuates the production of matrix metalloproteinase-1 in UVBirradiated human HaCaT keratinocytes (Piao et al. 2012). The compound also protects human keratinocytes against UVB-induced oxidative stress by scavenging ROS in vitro (Kim et al. 2012). Therefore, the present study evaluated whether phloroglucinol can similarly prevent oxidative damage in vivo in the UVB-exposed mouse skin.
Materials and methods Reagents Phloroglucinol and the primary antibody against actin were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The primary antibodies against phospho-histone H2A.X (Ser 139) and caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA), and the primary antibody against B-cell lymphoma-2-associated X protein (Bax) (B-9) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All other chemicals were of analytical grade.
Animals and UVB source Male 7-week-old Balb/c mice (Orient Bio Inc., Gyeonggi-Do, Korea) were used for all experiments. The animals were maintained at a controlled temperature of 25–28°C with a 12 h/12 h light/dark cycle and were provided with a standard diet and drinking water ad libitum. All experimental procedures were conducted in accordance with the guidelines for the care and use of laboratory animals at Jeju National University (Jeju, Korea) (permission number: 2012-0006). The source of UVB light was a UVP UV lamp (UVP. LLC., Upland, CA, USA) and the UVB dose was quantified using a FotoRadiometro HD2102.2 instrument (Delta Ohm Srl, Caselle di Selvazzano, Italy). The intensity of the light source used was 364 3 mW/cm2. To expose to the dorsal skin of each mouse at a dose of 30 or 60 mJ/cm2 daily for 3 days, the exposure time for UVB irradiation was 86 or 172 sec, respectively.
Treatment of animals Balb/c mice were divided into the following eight groups (n 5 mice/group): (1) Untreated normal control group; (2) phloroglucinol (50 mg/ml) only-treated group; (3) and (4) vehicle plus UVB (30 or 60 mJ/cm2)-exposed groups; and (5–8) phloroglucinol (10 or 50 mg/ml) plus UVB (30 or 60 mJ/cm2)-treated groups. Following UVB exposure, phloroglucinol or phosphate buffered saline (PBS) vehicle (200 ml) was applied to the dorsal skin of each mouse daily for 3 days. Studies were conducted at 24 h after the last of the UVB exposures.
Histopathology For the histopathological analysis of the skin, skin samples were obtained from control and UVB- and/or phloroglucinol-treated mice, fixed in 10% formalin, and embedded in paraffin. The paraffin sections were cut to a thickness of 5 mm, and then stained with hematoxylin and eosin to determine epidermal and dermal thickness, or stained with toluidine blue to quantify mast cells. The thicknesses of the epidermis (from the stratum basale to the stratum corneum) and the dermis (including the papillary layer and the reticular layer) were determined in approximately ten randomly chosen fields taken from five representative sections per group, followed by microscopic evaluation at 100 optical magnification with the aid of a digital camera ProgRes CFscan (Jenoptik, Jena, Germany). Measurements were made using Prog Res CapturePro 2.5 software (Jenoptik). The numbers of mast cells were counted using a BX-50 microscope ( 20 magnification; Olympus, Center Valley, PA, USA) in five different sections taken from five animals per group. Mast cell counts were conducted by three investigators who were blind to the experimental conditions.
Western blot analysis Mouse skin tissue was lysed by incubating the tissue on ice for 10 min in PRO-PREP protein extraction solution (100 ml; iNtRON Biotechnology, Gyeonggi-Do, Korea). The tissue lysates were centrifuged at 16,000 g for 5 min. The supernatants were collected from the lysates, and protein concentrations were determined. Aliquots of the lysates (30 mg of protein) were boiled for 5 min and electrophoresed in a 10% sodium dodecyl sulfate-polyacrylamide gel. The electrophoresed proteins were then transferred onto nitrocellulose membranes, and the membranes were subsequently incubated with primary antibodies against phospho-histone H2A.X, Bax, caspase-3 or actin. Following the reaction with primary antibodies, the membranes were further incubated with secondary anti-immunoglobulinG-horseradish peroxidase conjugates (Pierce, Rockford, IL, USA). Protein bands were detected using an Amersham ECL plus Western blotting detection system (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) according to the manufacturer’s instructions.
Detection of 8-oxoG
DNA was isolated from mouse skin tissue using a Wizard genomic DNA purification kit (Promega Corporation, Madison, WI, USA) and quantified using a spectrophotometer. The amount of 8-oxoG in the isolated DNA was determined using a Bioxytech 8-OHdG-ELISA Kit (OXIS Health Products, Portland, OR, USA) according to the manufacturer’s instructions.
Detection of protein carbonyls Protein carbonyls are products of protein oxidation. Protein carbonyl formation was determined using an Oxiselect protein carbonyl ELISA kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions.
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Detection of 8-isoprostane The isoprostanes are eicosanoid-like compounds that are non-enzymatically produced through the oxygen radicalinduced peroxidation of tissue phospholipids and lipoproteins. Levels of 8-isoprostane were determined using an OxiSelect 8-iso-prostaglandin F2a ELISA kit (Cell Biolabs, San Diego, CA, USA), according to the manufacturer’s instructions.
Immunohistochemistry
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Mouse skin tissue sections were deparaffinized and rehydrated in xylene, followed by a series of ethanol solutions containing decreasing concentrations of ethanol. Tissue sections were incubated with a methanol (0.3%)-H2O2 solution
for 30 min to block endogenous peroxidase activity. The sections were then incubated overnight at 4°C with a primary antibody against caspase-3 (1:50 dilution). The sections were washed with PBS (three times, 5 min each time), and the slides were incubated for 30 min at room temperature with a biotin-conjugated F(ab’)2 fragment derived from a goat antirabbit immunoglobulin G antibody (Molecular Probes Inc., Eugene, OR, USA) plus horseradish peroxidase-conjugated streptavidin (Invitrogen, Camarillo, CA, USA). Tissue sections were incubated with diaminobenzidine for 5 min for color development. Counterstaining was performed with hematoxylin for 5 min. The slides were then dehydrated in a series of increasingly concentrated ethanol solutions, followed by xylene.
Figure 1. Phloroglucinol inhibits UVB-induced histological alterations in the dorsal mouse skin tissue. (A) Mast cells (arrows) were counted in dorsal skin samples. Cellular morphology was viewed at 20 magnification. Scale bar 50 mm. (B) The thickness of the epidermis and the dermis were measured. Tissue morphology was viewed at 20 magnification. Scale bar 100 mm. *,**Significantly different from the normal control group (p 0.05) and the UVB-irradiated group at 30 mJ/cm2 (p 0.05), respectively. And #,##significantly different from the normal control group (p 0.05), and the UVB-irradiated group at 60 mJ/cm2 (p 0.05), respectively. Error bars indicate the standard error of the mean (SEM) for n 5.
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Phloroglucinol reduces UVB-evoked skin damage 931
Figure 1. (Continued)
Statistical analysis All measurements were made in three independent experiments, and all values are given as the mean the standard error (SE). The results were subjected to an analysis of variance and Tukey’s post-hoc test to analyze differences between conditions. In all cases, p 0.05 was considered statistically significant.
Results Phloroglucinol inhibits UVB-induced histopathological alterations in the mouse dorsal skin UVB irradiation induces an increase in intracellular ROS levels in cultured human keratinocytes, as well as in the
human skin (Hanson and Clegg 2002, Beak et al. 2004). ROS play important roles in mast cell activation and degranulation (Santos et al. 2000, Suzuki et al. 2005) while ROSstimulated mast cells release higher quantities of ROS (Swindle et al. 2004). Accordingly, mast cells are important effectors of the deleterious actions of UV light (Park et al. 2007), and the number of mast cells in the dermis near blood and lymphatic vessels and nerves is increased in UV-exposed skin (El-Abaseri et al. 2013). In agreement with these observations, histological analyses revealed large numbers of toluidine blue-stained mast cells in the dorsal skin of mice exposed to UVB radiation (30 or 60 mJ/cm2). On the other hand, topical administration of phloroglucinol (10 or 50 mg/ml) immediately after UVB exposure significantly
932 M. J. Piao et al. decreased the number of mast cells in the irradiated mouse skin, but the compound had no appreciable effect on its own (Figure 1A). Epidermal thickness is used as a quantitative parameter for the assessment of inflammation and photo-aging in the skin (Gilchrest 1996); therefore, we next investigated the thickness of the epidermis and the dermis after UVB and/or phloroglucinol treatment. UVB radiation (30 or 60 mJ/cm2) significantly increased the thickness of both skin tissue layers in the mouse; however, phloroglucinol (10 or 50 mg/ml) significantly moderated the actions of UVB light (Figure 1B).
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Phloroglucinol protects DNA, proteins, and lipids from UVB-provoked damage in the mouse dorsal skin The ability of phloroglucinol to protect against cellular DNA damage, protein carbonyl formation, and membrane lipid peroxidation in the UVB-irradiated dorsal mouse skin was next investigated. DNA damage, including that stemming from UVB light, results in the rapid phosphorylation of histone H2A.X at Ser 139 by phosphoinositide 3-kinase-like kinases (Burma et al. 2001). UVB radiation (60 mJ/cm2) dramatically increased the expression levels of phospho-histone H2A.X relative to those in untreated normal control skin; however, phloroglucinol (10 or 50 mg/ml) markedly reduced phospho-histone H2A.X content in the UVB-exposed skin (Figure 2A). Furthermore, 8-oxoG content, a hallmark of oxidative stress-induced DNA base damage, was significantly increased from 97 pg/ml in the untreated normal control group to 158 pg/ml in the UVB-irradiated group (Figure 2B). However, phloroglucinol completely overturned this increase, resulting in 8-oxoG levels of 96 pg/ml and 81 pg/ml at phloroglucinol concentrations of 10 mg/ml and 50 mg/ml, respectively (Figure 2B). Protein carbonylation is an indication of oxidative damage to proteins. The protein carbonyl content was significantly increased in the mouse skin after UVB exposure (60 mJ/cm2), reaching a concentration of 12 nmol/mg in the irradiated skin versus 6 nmol/mg in the normal control skin. By contrast, treatment with 10 mg/ml and 50 mg/ ml of phloroglucinol prevented protein carbonylation in the UVB-irradiated group, yielding protein carbonyl levels of 7 nmol/mg and 6 nmol/mg, respectively (Figure 2C). Finally, 8-isoprostane, a by-product of ROS-induced lipid peroxidation and a specific index of cellular lipoperoxidation (Belli et al. 2005), was elevated in the mouse dorsal skin after exposure to UVB radiation. The 8-isoprostane concentration was 10,863 pg/ml in the irradiated skin tissue and 1,654 pg/ml in the normal control skin. Nevertheless, treatment with phloroglucinol at concentrations of 10 mg/ml or 50 mg/ml attenuated the UVB-induced 8-isoprostane levels to 6,953 pg/ml and 4,900 pg/ml, respectively.
Phloroglucinol down-regulates the expression of apoptotic mediators in the UVB-irradiated mouse dorsal skin Previous studies demonstrated the occurrence of UVB-stimulated apoptosis in the mouse skin (Chun et al. 2007, Hwang et al. 2007). Therefore, we investigated the expression pattern
Figure 2. Phloroglucinol protects against UVB-induced damage to cellular components in the dorsal mouse skin. The levels of (A) phospho-histone H2A.X and (B) 8-oxoG (markers of oxidative DNA damage), (C) protein carbonyls (markers of protein oxidation), and (D) 8-isoprostane (a marker of lipid peroxidation) were detected in normal control, UVB-exposed, and UVB/phloroglucinol-treated mouse skin tissue. Actin (42 kDa) was used as a loading control for phospho-histone H2A.X (15 kDa) levels in (A). *Significantly different from normal control group (p 0.05), and **significantly different from UVB-irradiated group at 60 mJ/cm2 (p 0.05). Error bars indicate the standard error of the mean (SEM) for n 3 independent experiments.
of Bax, a pro-apoptotic regulator, and activated (cleaved) caspase-3, a key apoptotic executor, in the UVB-exposed mouse skin. Western blotting of dorsal skin tissue samples (Figure 3A) and immunohistochemistry analysis of intact dorsal skin (Figure 3B) revealed significantly increased levels of Bax and cleaved caspase-3, following UVB exposure; however, phloroglucinol reversed the UVB-induced increase in both pro-apoptotic mediators (Figure 3A, 3B).
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Phloroglucinol reduces UVB-evoked skin damage 933
Figure 3. Phloroglucinol reduces the expression levels of UVB-induced pro-apoptotic mediators in the mouse skin. (A) Expression of Bax (23 kDa) and active caspase-3 (17 and 19 kDa) was evaluated by Western blotting with each specific primary antibody. Actin (42 kDa) was used as a loading control. (B) Expression of active caspase-3 (arrows) was assessed by immunohistochemistry in normal control, UVB-exposed, and UVB/ phloroglucinol-treated mouse skin tissue ( 10 magnification). Scale bar 500 mm.
Discussion We recently demonstrated that the polyphenolic compound, phloroglucinol, attenuated cellular damage induced by oxidative stress through the quenching of ROS and the activation of antioxidant systems in vitro (Kang et al. 2006, 2010, Kim et al. 2012). Therefore, the present study focused on the role of phloroglucinol in UVB-induced oxidative stress and apoptosis in mice skin tissue in vivo. The skin, as the outermost barrier of the body, is exposed to various exogenous sources of oxidative stress, in particular UV radiation (Podda and Grundmann-Kollmann 2001) and UV-generated ROS. In the current study, exposure of the mouse skin to UVB light (30 or 60 mJ/cm2) increased the number of accumulated mast cells by about 3-fold relative to the normal control, but phloroglucinol (10 and 50 mg/ml) significantly reduced their numbers (Figure 1A). Given that mast cells generate and secrete copious amounts of ROS (Malaviya et al. 1994, Swindle et al. 2004), this observation led us to speculate that mast cells might participate in the maintenance of the UVBinduced ROS pool. UV exposure promotes an increase in epidermal and dermal thickness, which is a defensive response that helps to protect the skin from further UV damage (Kim et al. 2005). UVB radiation similarly increased the thickness of the mouse epidermis and dermis in the current study, but this action was partially overturned by phloroglucinol (Figure 1B). As noted above, exposure of the skin to UV radiation stimulates the production of excessive amounts of intracellular ROS (e.g., superoxide and H2O2). This imbalance in the production of ROS and the antioxidant defense systems culminates in oxidative stress (Birch-Machin and Swalwell 2010). Oxidative stress can damage cellular macromolecules, leading to DNA strand breaks, DNA base
and protein modifications, and lipid peroxidation. Such DNA base modifications include the oxidation of guanine residues to 8-oxoG, which is the most frequently detected and studied DNA lesion (Neeley and Essigmann 2006). On the other hand, histone H2A.X is critical for DNA repair following double-strand DNA breaks and is phosphorylated on Ser 139 within minutes following DNA damage. In the present study, the levels of phospho-histone H2A.X (Figure 2A) and 8-oxoG (Figure 2B) were both augmented in the mouse skin after exposure to UVB light. However, phloroglucinol substantially reduced their content (Figure 2A, 2B). Levels of protein oxidation products are also increased in the UV-irradiated skin of animals and humans (Sharma et al. 2007, Pandel et al. 2013). For example, oxidation of certain amino acid residues (i.e., lysine, arginine, and proline) results in the formation of protein carbonyl derivatives, which affect the nature and function of the parent proteins (Stadtman 2001). Here, protein carbonyl levels were elevated in UVBexposed mouse skin tissue and subsequently decreased by phloroglucinol treatment (Figure 2C). In addition, 8-isoprostane is a stable prostaglandin-like product of lipid peroxidation that is formed from arachidonic acid by the non-enzymatic actions of ROS. Therefore, 8-isoprostane, along with 8-oxoG, is considered to be a reliable biomarker of oxidative stress (Zieba et al. 2001). Like 8-oxoG, UVB radiation stimulated the production of 8-isoprostane in the mouse skin. Nonetheless, phloroglucinol attenuated UVB-induced lipid peroxidation, as evidenced by reduced levels of 8-isoprostane (Figure 2D). Finally, UVB-provoked oxidative stress induces apoptosis in cultured epithelial cells, keratinocytes, and intact mouse skin (Afaq et al. 2007, Cao et al. 2012). B-cell lymphoma 2 (Bcl-2) family proteins play critical roles in
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934 M. J. Piao et al. the regulation of apoptosis by functioning as promoters (e.g., Bax) or inhibitors (e.g., Bcl-2 or B-cell lymphomaextra-large) of the cell death process (Adams and Cory 2001). Furthermore, activated (cleaved) caspase-3 acts as the key executer of apoptosis, cleaving and inactivating vital cellular proteins, such as poly (ADP-ribose) polymerase (Yan and Shi 2005). According to the results of our Western blotting and immunohistochemistry analyses, the expression levels of Bax and activated caspase-3 were both increased in UVB-exposed mouse skin (Figure 3). Conversely, phloroglucinol significantly reduced the levels of these pro-apoptotic mediators following UVB exposure (Figure 3). In summary, the current study demonstrated that phloroglucinol safeguarded the mouse skin against UVB radiation-induced oxidative damage. The compound exerted its actions by decreasing mast cell recruitment and epidermal/ dermal thickness; by down-regulating the expression of phospho-histone H2A.X; by reducing the UVB-generated levels of 8-oxoG, protein carbonyls, and 8-isoprostane; and by attenuating the UVB-induced expression of Bax and activated caspase-3. These observations suggest that phloroglucinol could be developed as an efficacious therapeutic agent to mitigate the effects of excessive sun exposure in the skin.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-C1ABA001-2011-0021037).
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Phloroglucinol inhibits ultraviolet B radiation-induced oxidative stress in the mouse skin Mei Jing Piao1*, Mee Jung Ahn1*, Kyoung Ah Kang1, Ki Cheon Kim1, Jian Zheng1, Cheng Wen Yao1, Ji Won Cha1, Chang Lim Hyun1, Hee Kyoung Kang1, Nam Ho Lee2 & Jin Won Hyun1 1School of Medicine and Institute for Nuclear Science and Technology, and 2Department of Chemistry, College of Natural
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Sciences, Jeju National University, Jeju, 690-756, Korea
Conclusion: These results suggest that phloroglucinol safeguards the mouse skin against UVB-induced oxidative stress and apoptosis.
Abstract Purpose: Previously we demonstrated that phloroglucinol (1,3,5trihydroxybenzene) protected human HaCaT keratinocytes against ultraviolet B (UVB, 280–320 nm)-induced oxidative stress in vitro by scavenging intracellular reactive oxygen species (ROS). The current study investigated whether phlo roglucinol could similarly protect the mouse skin against UVB-induced oxidative tissue damage in vivo. Materials and methods: Male 7-week-old Balb/c mice were divided into the following untreated normal control, phloroglucinol only-treated, vehicle plus UVB (30 or 60 mJ/cm2)exposed, and phloroglucinol (10 or 50 mg/ml) plus UVB (30 or 60 mJ/cm2)-treated groups. Following UVB exposure, phloroglucinol or phosphate buffered saline vehicle was applied to the dorsal skin of each mouse daily for 3 days. Studies were conducted at 24 h after the last of the UVB exposures. Histopathological analyses of dorsal skin lesions were performed on all mice. In addition, the levels of UVB-provoked injury to cellular components, including DNA, proteins, and lipids were detected by levels of 8-oxoguanine (8-oxoG), protein carbonyls, and 8-isoprostane. Apoptosis were assessed by using western blot for B-cell lymphoma-2-associated X protein (Bax) and activated caspase-3 expression, by using immunohistochemistry. Results: UVB radiation increased the thickness of the epidermis and the dermis, and also stimulated the accumulation of mast cells in the irradiated skin. However, treatment with phloroglucinol significantly decreased all of these parameters. Furthermore, phloroglucinol decreased UVB-provoked injury to cellular components, including DNA, proteins, and lipids; down-regulated the expression of phospho-histone H2A.X in the injured skin; and reduced the UVB-generated levels of 8-oxoG, protein carbonyls, and 8-isoprostane, which are all markers of oxidative stress. In addition, phloroglucinol attenuated the UVB-induced expression of the pro-apoptotic proteins, Bax protein, and activated caspase-3.
Keywords: Phloroglucinol, ultraviolet B, oxidative stress, apoptosis, skin tissue
Introduction Excessive exposure of the skin to ultraviolet (UV) radiation results in the generation of debilitating levels of intracellular reactive oxygen species (ROS). These ROS quickly overwhelm the tissue antioxidant systems and other oxidant-degrading pathways (Bickers and Athar 2006, Lyu and Park 2012). UV radiation also induces thickening of the epidermis and the dermis (Kim et al. 2005), as well as accumulation of mast cells in the damaged skin. Mast cells participate in the initiation of UV-provoked inflammation and oxidative stress, and contribute to the continued production of elevated levels of ROS (Malaviya et al. 1994). UV radiation consists of ultraviolet A (UVA, 320–400 nm), ultraviolet B (UVB, 280–320 nm), and ultraviolet C (UVC, 100–280 nm) light. Experimental studies indicate that UVB light is particularly likely to stimulate ROS generation and oxidative stress in the skin (Katiyar et al. 2001, Fonseca et al. 2010). UVB-induced ROS can cause irreversible damages to cellular macromolecules, including DNA, proteins, and lipids. The net result is the formation of oxidized products, such as 8-oxoguanine (8-oxoG) DNA lesions, protein carbonyls, and lipid hydroperoxides. These oxidized products are implicated in the onset of skin cancers (Vayalil et al. 2003, Tomaino et al. 2006) and apoptosis. The detrimental effects of UVB exposure have led to the search for exogenous compounds that can activate endogenous enzymatic and non-enzymatic antioxidant defense systems, thereby protecting the skin from UVB-induced oxidative stress (Sharma et al. 2007).
*These authors contributed equally to this paper. Correspondence: Prof. Jin Won Hyun, School of Medicine, Jeju National University, Jeju 690-756, Korea. E-mail: [email protected] (Received 17 November 2013; revised 20 March 2014; accepted 27 March 2014)
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Phloroglucinol reduces UVB-evoked skin damage 929 Brown algae, such as Ecklonia cava, E. stolonifera, and Eisenia bicyclis, are rich in phloroglucinol, phloroglucinol derivatives (Okada et al. 2004), and other antioxidant compounds. Phloroglucinol (1,3,5-trihydroxybenzene) directly inhibits the generation of ROS (Kim and Kim 2010). Moreover, phloroglucinol ameliorates hydrogen peroxide (H2O2)mediated cell damage and reduces gamma ray-induced oxidative stress via the quenching of ROS and the activation of antioxidant systems (Kang et al. 2006, 2010). We recently demonstrated that phloroglucinol attenuates the production of matrix metalloproteinase-1 in UVBirradiated human HaCaT keratinocytes (Piao et al. 2012). The compound also protects human keratinocytes against UVB-induced oxidative stress by scavenging ROS in vitro (Kim et al. 2012). Therefore, the present study evaluated whether phloroglucinol can similarly prevent oxidative damage in vivo in the UVB-exposed mouse skin.
Materials and methods Reagents Phloroglucinol and the primary antibody against actin were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The primary antibodies against phospho-histone H2A.X (Ser 139) and caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA), and the primary antibody against B-cell lymphoma-2-associated X protein (Bax) (B-9) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All other chemicals were of analytical grade.
Animals and UVB source Male 7-week-old Balb/c mice (Orient Bio Inc., Gyeonggi-Do, Korea) were used for all experiments. The animals were maintained at a controlled temperature of 25–28°C with a 12 h/12 h light/dark cycle and were provided with a standard diet and drinking water ad libitum. All experimental procedures were conducted in accordance with the guidelines for the care and use of laboratory animals at Jeju National University (Jeju, Korea) (permission number: 2012-0006). The source of UVB light was a UVP UV lamp (UVP. LLC., Upland, CA, USA) and the UVB dose was quantified using a FotoRadiometro HD2102.2 instrument (Delta Ohm Srl, Caselle di Selvazzano, Italy). The intensity of the light source used was 364 3 mW/cm2. To expose to the dorsal skin of each mouse at a dose of 30 or 60 mJ/cm2 daily for 3 days, the exposure time for UVB irradiation was 86 or 172 sec, respectively.
Treatment of animals Balb/c mice were divided into the following eight groups (n 5 mice/group): (1) Untreated normal control group; (2) phloroglucinol (50 mg/ml) only-treated group; (3) and (4) vehicle plus UVB (30 or 60 mJ/cm2)-exposed groups; and (5–8) phloroglucinol (10 or 50 mg/ml) plus UVB (30 or 60 mJ/cm2)-treated groups. Following UVB exposure, phloroglucinol or phosphate buffered saline (PBS) vehicle (200 ml) was applied to the dorsal skin of each mouse daily for 3 days. Studies were conducted at 24 h after the last of the UVB exposures.
Histopathology For the histopathological analysis of the skin, skin samples were obtained from control and UVB- and/or phloroglucinol-treated mice, fixed in 10% formalin, and embedded in paraffin. The paraffin sections were cut to a thickness of 5 mm, and then stained with hematoxylin and eosin to determine epidermal and dermal thickness, or stained with toluidine blue to quantify mast cells. The thicknesses of the epidermis (from the stratum basale to the stratum corneum) and the dermis (including the papillary layer and the reticular layer) were determined in approximately ten randomly chosen fields taken from five representative sections per group, followed by microscopic evaluation at 100 optical magnification with the aid of a digital camera ProgRes CFscan (Jenoptik, Jena, Germany). Measurements were made using Prog Res CapturePro 2.5 software (Jenoptik). The numbers of mast cells were counted using a BX-50 microscope ( 20 magnification; Olympus, Center Valley, PA, USA) in five different sections taken from five animals per group. Mast cell counts were conducted by three investigators who were blind to the experimental conditions.
Western blot analysis Mouse skin tissue was lysed by incubating the tissue on ice for 10 min in PRO-PREP protein extraction solution (100 ml; iNtRON Biotechnology, Gyeonggi-Do, Korea). The tissue lysates were centrifuged at 16,000 g for 5 min. The supernatants were collected from the lysates, and protein concentrations were determined. Aliquots of the lysates (30 mg of protein) were boiled for 5 min and electrophoresed in a 10% sodium dodecyl sulfate-polyacrylamide gel. The electrophoresed proteins were then transferred onto nitrocellulose membranes, and the membranes were subsequently incubated with primary antibodies against phospho-histone H2A.X, Bax, caspase-3 or actin. Following the reaction with primary antibodies, the membranes were further incubated with secondary anti-immunoglobulinG-horseradish peroxidase conjugates (Pierce, Rockford, IL, USA). Protein bands were detected using an Amersham ECL plus Western blotting detection system (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) according to the manufacturer’s instructions.
Detection of 8-oxoG
DNA was isolated from mouse skin tissue using a Wizard genomic DNA purification kit (Promega Corporation, Madison, WI, USA) and quantified using a spectrophotometer. The amount of 8-oxoG in the isolated DNA was determined using a Bioxytech 8-OHdG-ELISA Kit (OXIS Health Products, Portland, OR, USA) according to the manufacturer’s instructions.
Detection of protein carbonyls Protein carbonyls are products of protein oxidation. Protein carbonyl formation was determined using an Oxiselect protein carbonyl ELISA kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions.
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Detection of 8-isoprostane The isoprostanes are eicosanoid-like compounds that are non-enzymatically produced through the oxygen radicalinduced peroxidation of tissue phospholipids and lipoproteins. Levels of 8-isoprostane were determined using an OxiSelect 8-iso-prostaglandin F2a ELISA kit (Cell Biolabs, San Diego, CA, USA), according to the manufacturer’s instructions.
Immunohistochemistry
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Mouse skin tissue sections were deparaffinized and rehydrated in xylene, followed by a series of ethanol solutions containing decreasing concentrations of ethanol. Tissue sections were incubated with a methanol (0.3%)-H2O2 solution
for 30 min to block endogenous peroxidase activity. The sections were then incubated overnight at 4°C with a primary antibody against caspase-3 (1:50 dilution). The sections were washed with PBS (three times, 5 min each time), and the slides were incubated for 30 min at room temperature with a biotin-conjugated F(ab’)2 fragment derived from a goat antirabbit immunoglobulin G antibody (Molecular Probes Inc., Eugene, OR, USA) plus horseradish peroxidase-conjugated streptavidin (Invitrogen, Camarillo, CA, USA). Tissue sections were incubated with diaminobenzidine for 5 min for color development. Counterstaining was performed with hematoxylin for 5 min. The slides were then dehydrated in a series of increasingly concentrated ethanol solutions, followed by xylene.
Figure 1. Phloroglucinol inhibits UVB-induced histological alterations in the dorsal mouse skin tissue. (A) Mast cells (arrows) were counted in dorsal skin samples. Cellular morphology was viewed at 20 magnification. Scale bar 50 mm. (B) The thickness of the epidermis and the dermis were measured. Tissue morphology was viewed at 20 magnification. Scale bar 100 mm. *,**Significantly different from the normal control group (p 0.05) and the UVB-irradiated group at 30 mJ/cm2 (p 0.05), respectively. And #,##significantly different from the normal control group (p 0.05), and the UVB-irradiated group at 60 mJ/cm2 (p 0.05), respectively. Error bars indicate the standard error of the mean (SEM) for n 5.
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Phloroglucinol reduces UVB-evoked skin damage 931
Figure 1. (Continued)
Statistical analysis All measurements were made in three independent experiments, and all values are given as the mean the standard error (SE). The results were subjected to an analysis of variance and Tukey’s post-hoc test to analyze differences between conditions. In all cases, p 0.05 was considered statistically significant.
Results Phloroglucinol inhibits UVB-induced histopathological alterations in the mouse dorsal skin UVB irradiation induces an increase in intracellular ROS levels in cultured human keratinocytes, as well as in the
human skin (Hanson and Clegg 2002, Beak et al. 2004). ROS play important roles in mast cell activation and degranulation (Santos et al. 2000, Suzuki et al. 2005) while ROSstimulated mast cells release higher quantities of ROS (Swindle et al. 2004). Accordingly, mast cells are important effectors of the deleterious actions of UV light (Park et al. 2007), and the number of mast cells in the dermis near blood and lymphatic vessels and nerves is increased in UV-exposed skin (El-Abaseri et al. 2013). In agreement with these observations, histological analyses revealed large numbers of toluidine blue-stained mast cells in the dorsal skin of mice exposed to UVB radiation (30 or 60 mJ/cm2). On the other hand, topical administration of phloroglucinol (10 or 50 mg/ml) immediately after UVB exposure significantly
932 M. J. Piao et al. decreased the number of mast cells in the irradiated mouse skin, but the compound had no appreciable effect on its own (Figure 1A). Epidermal thickness is used as a quantitative parameter for the assessment of inflammation and photo-aging in the skin (Gilchrest 1996); therefore, we next investigated the thickness of the epidermis and the dermis after UVB and/or phloroglucinol treatment. UVB radiation (30 or 60 mJ/cm2) significantly increased the thickness of both skin tissue layers in the mouse; however, phloroglucinol (10 or 50 mg/ml) significantly moderated the actions of UVB light (Figure 1B).
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Phloroglucinol protects DNA, proteins, and lipids from UVB-provoked damage in the mouse dorsal skin The ability of phloroglucinol to protect against cellular DNA damage, protein carbonyl formation, and membrane lipid peroxidation in the UVB-irradiated dorsal mouse skin was next investigated. DNA damage, including that stemming from UVB light, results in the rapid phosphorylation of histone H2A.X at Ser 139 by phosphoinositide 3-kinase-like kinases (Burma et al. 2001). UVB radiation (60 mJ/cm2) dramatically increased the expression levels of phospho-histone H2A.X relative to those in untreated normal control skin; however, phloroglucinol (10 or 50 mg/ml) markedly reduced phospho-histone H2A.X content in the UVB-exposed skin (Figure 2A). Furthermore, 8-oxoG content, a hallmark of oxidative stress-induced DNA base damage, was significantly increased from 97 pg/ml in the untreated normal control group to 158 pg/ml in the UVB-irradiated group (Figure 2B). However, phloroglucinol completely overturned this increase, resulting in 8-oxoG levels of 96 pg/ml and 81 pg/ml at phloroglucinol concentrations of 10 mg/ml and 50 mg/ml, respectively (Figure 2B). Protein carbonylation is an indication of oxidative damage to proteins. The protein carbonyl content was significantly increased in the mouse skin after UVB exposure (60 mJ/cm2), reaching a concentration of 12 nmol/mg in the irradiated skin versus 6 nmol/mg in the normal control skin. By contrast, treatment with 10 mg/ml and 50 mg/ ml of phloroglucinol prevented protein carbonylation in the UVB-irradiated group, yielding protein carbonyl levels of 7 nmol/mg and 6 nmol/mg, respectively (Figure 2C). Finally, 8-isoprostane, a by-product of ROS-induced lipid peroxidation and a specific index of cellular lipoperoxidation (Belli et al. 2005), was elevated in the mouse dorsal skin after exposure to UVB radiation. The 8-isoprostane concentration was 10,863 pg/ml in the irradiated skin tissue and 1,654 pg/ml in the normal control skin. Nevertheless, treatment with phloroglucinol at concentrations of 10 mg/ml or 50 mg/ml attenuated the UVB-induced 8-isoprostane levels to 6,953 pg/ml and 4,900 pg/ml, respectively.
Phloroglucinol down-regulates the expression of apoptotic mediators in the UVB-irradiated mouse dorsal skin Previous studies demonstrated the occurrence of UVB-stimulated apoptosis in the mouse skin (Chun et al. 2007, Hwang et al. 2007). Therefore, we investigated the expression pattern
Figure 2. Phloroglucinol protects against UVB-induced damage to cellular components in the dorsal mouse skin. The levels of (A) phospho-histone H2A.X and (B) 8-oxoG (markers of oxidative DNA damage), (C) protein carbonyls (markers of protein oxidation), and (D) 8-isoprostane (a marker of lipid peroxidation) were detected in normal control, UVB-exposed, and UVB/phloroglucinol-treated mouse skin tissue. Actin (42 kDa) was used as a loading control for phospho-histone H2A.X (15 kDa) levels in (A). *Significantly different from normal control group (p 0.05), and **significantly different from UVB-irradiated group at 60 mJ/cm2 (p 0.05). Error bars indicate the standard error of the mean (SEM) for n 3 independent experiments.
of Bax, a pro-apoptotic regulator, and activated (cleaved) caspase-3, a key apoptotic executor, in the UVB-exposed mouse skin. Western blotting of dorsal skin tissue samples (Figure 3A) and immunohistochemistry analysis of intact dorsal skin (Figure 3B) revealed significantly increased levels of Bax and cleaved caspase-3, following UVB exposure; however, phloroglucinol reversed the UVB-induced increase in both pro-apoptotic mediators (Figure 3A, 3B).
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Phloroglucinol reduces UVB-evoked skin damage 933
Figure 3. Phloroglucinol reduces the expression levels of UVB-induced pro-apoptotic mediators in the mouse skin. (A) Expression of Bax (23 kDa) and active caspase-3 (17 and 19 kDa) was evaluated by Western blotting with each specific primary antibody. Actin (42 kDa) was used as a loading control. (B) Expression of active caspase-3 (arrows) was assessed by immunohistochemistry in normal control, UVB-exposed, and UVB/ phloroglucinol-treated mouse skin tissue ( 10 magnification). Scale bar 500 mm.
Discussion We recently demonstrated that the polyphenolic compound, phloroglucinol, attenuated cellular damage induced by oxidative stress through the quenching of ROS and the activation of antioxidant systems in vitro (Kang et al. 2006, 2010, Kim et al. 2012). Therefore, the present study focused on the role of phloroglucinol in UVB-induced oxidative stress and apoptosis in mice skin tissue in vivo. The skin, as the outermost barrier of the body, is exposed to various exogenous sources of oxidative stress, in particular UV radiation (Podda and Grundmann-Kollmann 2001) and UV-generated ROS. In the current study, exposure of the mouse skin to UVB light (30 or 60 mJ/cm2) increased the number of accumulated mast cells by about 3-fold relative to the normal control, but phloroglucinol (10 and 50 mg/ml) significantly reduced their numbers (Figure 1A). Given that mast cells generate and secrete copious amounts of ROS (Malaviya et al. 1994, Swindle et al. 2004), this observation led us to speculate that mast cells might participate in the maintenance of the UVBinduced ROS pool. UV exposure promotes an increase in epidermal and dermal thickness, which is a defensive response that helps to protect the skin from further UV damage (Kim et al. 2005). UVB radiation similarly increased the thickness of the mouse epidermis and dermis in the current study, but this action was partially overturned by phloroglucinol (Figure 1B). As noted above, exposure of the skin to UV radiation stimulates the production of excessive amounts of intracellular ROS (e.g., superoxide and H2O2). This imbalance in the production of ROS and the antioxidant defense systems culminates in oxidative stress (Birch-Machin and Swalwell 2010). Oxidative stress can damage cellular macromolecules, leading to DNA strand breaks, DNA base
and protein modifications, and lipid peroxidation. Such DNA base modifications include the oxidation of guanine residues to 8-oxoG, which is the most frequently detected and studied DNA lesion (Neeley and Essigmann 2006). On the other hand, histone H2A.X is critical for DNA repair following double-strand DNA breaks and is phosphorylated on Ser 139 within minutes following DNA damage. In the present study, the levels of phospho-histone H2A.X (Figure 2A) and 8-oxoG (Figure 2B) were both augmented in the mouse skin after exposure to UVB light. However, phloroglucinol substantially reduced their content (Figure 2A, 2B). Levels of protein oxidation products are also increased in the UV-irradiated skin of animals and humans (Sharma et al. 2007, Pandel et al. 2013). For example, oxidation of certain amino acid residues (i.e., lysine, arginine, and proline) results in the formation of protein carbonyl derivatives, which affect the nature and function of the parent proteins (Stadtman 2001). Here, protein carbonyl levels were elevated in UVBexposed mouse skin tissue and subsequently decreased by phloroglucinol treatment (Figure 2C). In addition, 8-isoprostane is a stable prostaglandin-like product of lipid peroxidation that is formed from arachidonic acid by the non-enzymatic actions of ROS. Therefore, 8-isoprostane, along with 8-oxoG, is considered to be a reliable biomarker of oxidative stress (Zieba et al. 2001). Like 8-oxoG, UVB radiation stimulated the production of 8-isoprostane in the mouse skin. Nonetheless, phloroglucinol attenuated UVB-induced lipid peroxidation, as evidenced by reduced levels of 8-isoprostane (Figure 2D). Finally, UVB-provoked oxidative stress induces apoptosis in cultured epithelial cells, keratinocytes, and intact mouse skin (Afaq et al. 2007, Cao et al. 2012). B-cell lymphoma 2 (Bcl-2) family proteins play critical roles in
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934 M. J. Piao et al. the regulation of apoptosis by functioning as promoters (e.g., Bax) or inhibitors (e.g., Bcl-2 or B-cell lymphomaextra-large) of the cell death process (Adams and Cory 2001). Furthermore, activated (cleaved) caspase-3 acts as the key executer of apoptosis, cleaving and inactivating vital cellular proteins, such as poly (ADP-ribose) polymerase (Yan and Shi 2005). According to the results of our Western blotting and immunohistochemistry analyses, the expression levels of Bax and activated caspase-3 were both increased in UVB-exposed mouse skin (Figure 3). Conversely, phloroglucinol significantly reduced the levels of these pro-apoptotic mediators following UVB exposure (Figure 3). In summary, the current study demonstrated that phloroglucinol safeguarded the mouse skin against UVB radiation-induced oxidative damage. The compound exerted its actions by decreasing mast cell recruitment and epidermal/ dermal thickness; by down-regulating the expression of phospho-histone H2A.X; by reducing the UVB-generated levels of 8-oxoG, protein carbonyls, and 8-isoprostane; and by attenuating the UVB-induced expression of Bax and activated caspase-3. These observations suggest that phloroglucinol could be developed as an efficacious therapeutic agent to mitigate the effects of excessive sun exposure in the skin.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-C1ABA001-2011-0021037).
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