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Ethanol extract of Dalbergia odorifera protects skin keratinocytes against ultraviolet B-induced photoaging by suppressing production of reactive oxygen species a
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Sun Ah Ham , Jung Seok Hwang , Eun Sil Kang , Taesik Yoo , Hyun Ho Lim , Won Jin Lee , b
Kyung Shin Paek & Han Geuk Seo a
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Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea
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Department of Nursing, Semyung University, Jecheon, Republic of Korea Published online: 03 Jan 2015.
Click for updates To cite this article: Sun Ah Ham, Jung Seok Hwang, Eun Sil Kang, Taesik Yoo, Hyun Ho Lim, Won Jin Lee, Kyung Shin Paek & Han Geuk Seo (2015): Ethanol extract of Dalbergia odorifera protects skin keratinocytes against ultraviolet B-induced photoaging by suppressing production of reactive oxygen species, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.993916 To link to this article: http://dx.doi.org/10.1080/09168451.2014.993916
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Bioscience, Biotechnology, and Biochemistry, 2014
Ethanol extract of Dalbergia odorifera protects skin keratinocytes against ultraviolet B-induced photoaging by suppressing production of reactive oxygen species Sun Ah Ham1, Jung Seok Hwang1, Eun Sil Kang1, Taesik Yoo1, Hyun Ho Lim1, Won Jin Lee1, Kyung Shin Paek2 and Han Geuk Seo1,* Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea; 2Department of Nursing, Semyung University, Jecheon, Republic of Korea
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Received October 8, 2014; accepted November 15, 2014 http://dx.doi.org/10.1080/09168451.2014.993916
Dalbergia odorifera T. Chen (Leguminosae), an indigenous medicinal herb, has been widely used in northern and eastern Asia to treat diverse diseases. Here, we investigated the anti-senescent effects of ethanolic extracts of Dalbergia odorifera (EEDO) in ultraviolet (UV) B-irradiated skin cells. EEDO significantly inhibited UVB-induced senescence of human keratinocytes in a concentration-dependent manner, concomitant with inhibition of reactive oxygen species (ROS) generation. UVB-induced increases in the levels of p53 and p21, biomarkers of cellular senescence, were almost completely abolished in the presence of EEDO. Sativanone, a major constituent of EEDO, also attenuated UVB-induced senescence and ROS generation in keratinocytes, indicating that sativanone is an indexing (marker) molecule for the anti-senescence properties of EEDO. Finally, treatment of EEDO to mice exposed to UVB significantly reduced ROS levels and the number of senescent cells in the skin. Thus, EEDO confers resistance to UVB-induced cellular senescence by inhibiting ROS generation in skin cells. Key words:
Dalbergia odorifera; ultraviolet; reactive oxygen species; sativanone; senescence
Cellular senescence is a phenotypic change in gene expression, function, and morphology accompanied by permanent and irreversible growth arrest of cultured cells.1,2) Replicative senescence, which occurs in primary cultured cells, is characterized by shortened telomere length, ultimately resulting in incomplete chromosomal replication due to telomeric fusion or loss of telomere-bound factors.3) By contrast, stress-induced premature senescence is triggered by diverse factors that cause cellular stress, such as ultraviolet (UV) radiation,4) chemical agents that induce DNA damage,5,6)
and inappropriate activation of oncogenes.7) Among these factors, UV radiation, in particular UVB (wavelength, 280–315 nm), can cause a range of acute and chronic damage including sunburn, premature aging (photoaging), and skin cancer; the specific manifestation depends on the exposure time and intensity.8) In vitro, UVB radiation of human skin dermal fibroblasts stimulates intracellular production of reactive oxygen species (ROS).9) Several lines of evidence suggest that ROS play important roles in mediating UV-induced cellular senescence by breaking DNA strands and modifying DNA bases.2,10) Excess production and ineffective elimination of ROS production have thus been implicated in skin photoaging induced by UV radiation.11,12) Dalbergia odorifera T. Chem (Leguminosae) is an indigenous medicinal herb that has been widely used in China and Korea for the treatment of ischemia, swelling, necrosis, rheumatic pain, and blood disorders.13) Several components from the heartwood of D. odorifera, including flavonoids, quinines, and phenolic constituents, exhibit diverse biological activities.14,15) These effects include up-regulation of anti-inflammatory heme oxygenase-1 expression in murine macrophages,16,17) antibacterial activity in Ralstonia solanacearum,18) and protection against glutamate-induced oxidative injury in mouse hippocampal HT22 cells.19) Furthermore, sativanone, a polyphenol compound having two benzene rings (MW301.1) from D. odorifera, inhibits LPS-stimulated nitric oxide release and tumor necrosis factor-α secretion in RAW264.7 cells.20,21) However, the molecular mechanisms underlying the cellular actions of D. odorifera extracts, including sativanone, have not been fully elucidated. The diverse pharmacological functions of a variety of components of D. odorifera have been studied previously, and it would therefore be interesting to elucidate the anti-senescence properties of D. odorifera extracts. In this study, we demonstrated that ethanolic extract of
*Corresponding author. Email: [email protected] Abbreviations: DHE, dihydroethidium; EEDO, ethanolic extracts of Dalbergia odorifera; KGM, keratinocyte growth medium; PML, promyelocytic leukemia protein; ROS, reactive oxygen species; SA β-gal, senescence-associated β-galactosidase; UV, ultraviolet. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Dalbergia odorifera (EEDO) prevents premature senescence and reduces ROS production in both cultured human keratinocytes in vitro and mouse skin in vivo.
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Materials and methods Materials. The sativanone standard was generously provided by Dr. Hiroshi Noguchi (School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan). Polyclonal antibodies specific for p53 and p21 were obtained from cell signaling (Beverly, MA, USA). Monoclonal antibodies specific for p16 and promyelocytic leukemia protein (PML) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), polyclonal rabbit anti-β-actin antibody, and dihydroethidium (DHE) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Keratinocyte growth medium (KGM) and keratinocyte growth supplement was purchased from Lonza Biologics (Slough, UK). Preparation of EEDO. Commercially available heartwood of D. odorifera was purchased from a Korean medicinal herb store in Seoul in May 2012. An authenticated voucher specimen (KULBM-1205) was deposited in the Herbarium at the College of Animal Bioscience and Technology, Konkuk University (Seoul, Korea). Dried heartwood of D. odorifera (100 g) was extracted thrice with 60% ethanol under rotation at 120 rpm for 1 h. After evaporation of the solvent under vacuum, the extract (10 g) was reconstituted in dimethyl sulfoxide (DMSO) to a concentration of 10 mg/mL, and then stored at −20 °C until use. HPLC analysis. The main constituents in EEDO were analyzed by high-performance liquid chromatography (HPLC). The samples were injected into an HPLC system (Waters, Washington, USA) fitted with a CAPCEL PAK RP-C18 column (Φ4.6 mm × 250 mm) (Shiseido, Tokyo, Japan). Detection wavelength was set at 275 nm, and the column temperature was maintained at 20 °C. The mobile phase was a gradient consisting of solvent A (acetonitrile) and B (0.3% acetic acid, v/v): 25% A at 0–18 min, 25–46% A at 18–55 min, and 46–80% A at 55–60 min. The flow rate was 0.8 mL min−1; aliquots of 10 μL were injected. Cell culture and detection of intracellular superoxide. Human primary keratinocytes (donor age 11, passage 3) were obtained from Welskin (Seoul, Korea). The cells were cultured in KGM containing keratinocyte growth supplement at 37 °C in an atmosphere of 95% air and 5% CO2, based on the manufacturer’s recommendations. For detection of intracellular superoxide production in keratinocytes, cells seeded on 35 mm cover-glass bottom dishes (SPL Life Sciences) were treated with 5 μg/mL EEDO for 24 h, and then exposed to 40 mJ/cm2 UVB irradiation for 30 min. The red fluorescence corresponding to the levels of intracellular
superoxide was detected through a 580 nm long-pass filter on an Olympus FV-1000 fluorescence microscope. UVB irradiation. UVB irradiation was performed as previously described.22) Briefly, cells were incubated for the indicated times in the presence or absence of the indicated reagents, and then washed twice with phosphate-buffered saline (PBS, pH 7.2) before UVB exposure to avoid the photosensitizing effects of components in the culture medium. UVB irradiation was performed on serum-starved monolayer cultures using an FS20 Lamp (National Biological, Twinsburg, OH, USA). The UVB source was a bank of two FS20 Lamps emitting a continuous spectrum from 270 to 390 nm, with a peak emission at 313 nm; approximately 65% of the radiation was within the UVB wavelength range (280–315 nm). UVB strength was monitored using a Goldilux model 70234 photometer (Lynntech Inc., TX, USA). Senescence-associated (SA) β-galactosidase staining. Senescent cells were detected using an SA-β-galactosidase staining kit, according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). Briefly, human keratinocytes pretreated with 5 μg/mL EEDO or 1 μM sativanone for 24 h were exposed to UVB irradiation, and then incubated for 24 h. The cells were washed with ice-cold PBS and fixed. After two washes with ice-cold PBS, the cells were incubated in staining solution (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2, 40 mM citric acid, 40 mM sodium phosphate, and 1 mg/mL X-gal; pH 6.0) at 37 °C in the absence of CO2 overnight. Senescent cells were visualized using an Olympus JP/1X71 fluorescence microscope.
Determination of cell viability. Human primary keratinocytes were seeded in 24-well plates and treated with the indicated concentrations of EEDO for 24 h. After incubation, MTT (final 0.1 mg/mL) was added to the culture medium, and cells were incubated for an additional 4 h. After removal of the medium, formazan crystals (formed after the reduction of MTT by mitochondrial dehydrogenases in living cells) were solubilized in acidified isopropanol and measured spectrophotometrically at 570 nm. Western blot analysis. Cells treated with the indicated reagents were washed in ice-cold PBS and lysed in PRO-PREP protein extraction solution (iNtRON Biotechnology, Seoul, Korea). Aliquots of cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto a Hybond-P+ polyvinylidene difluoride membrane (GE Healthcare, UK). Membranes were blocked overnight at 4 °C with 5% nonfat milk in TBS (Tris-buffered saline; 50 mM Tris, 150 mM NaCl, pH 7.6) containing 0.1% Tween-20, incubated overnight at 4 °C with the indicated specific antibodies
D. odorifera induced UVB-induced photoaging
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in TBS containing 1% BSA and 0.05% Tween-20, and then incubated for 2 h at room temperature with peroxidase-conjugated goat antibody diluted 1:3000. After extensive washing in TBS containing 0.1% BSA and 0.1% Tween-20, immuno-reactive bands were detected using West-ZOL Plus (iNtRON Biotechnology).
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Measurement of intracellular ROS. To assess the levels of intracellular accumulation of ROS, the fluorescent probe chloromethyl-2′,7′-dichlorofluorescein diaceMolecular Probes/Life tate (CM-H2DCF-DA; Technologies, Grand Island, NY, USA) was used, as described previously.22) Cells were seeded on 35 mm cover glass-bottom dishes (SPL Life Sciences, Seoul, Korea), treated with 5 μg/mL EEDO or 1 μM sativanone for 24 h, and then exposed to 40 mJ/cm2 UVB irradiation for 30 min. The cells were subsequently incubated with 10 μM CM-H2DCF-DA (peroxide-sensitive dye) for a final 30 min at 37 °C, and green fluorescence corresponding to the levels of intracellular ROS was detected through a 520 nm long-pass filter on an Olympus FV-1000 laser fluorescence microscope (Tokyo, Japan).
four weeks, and then with 100 J/m UVB three times a week for the last two weeks. Six weeks later, the mice were sacrificed, and squares of dorsal skin (ca. 1 cm × 1 cm) were excised from each mouse for further examination. For serial sectioning, tissues were fixed in 4% paraformaldehyde, cryoprotected in 20% sucrose, embedded in OCT (Sakura Finetech, Japan), snap-frozen in iso-pentene, and prechilled in liquid nitrogen prior to sectioning. For hematoxylin and eosin (H&E) counterstaining, serial cryosections (7 μm) were incubated sequentially at room temperature in 50% hematoxylin solution for 20 min, differentiated in 1% HCl solution for 1 s, and then transferred directly to 0.5% eosin solution and stained for 20 min. The sections were then washed once with distilled water and incubated in a graded alcohol series, ending with xylene, to dehydrate the tissue. Finally, the tissue sections were mounted under coverslips using Permount (Fisher Scientific, Somerville, NJ, USA). For detection of senescent cells in the dorsal skin of UVB-irradiated hairless mice, serial cryosections mounted on gelatin-coated slides were incubated in staining solution, and the activity of SA-β-galactosidase was visualized using an Olympus JP/1X71 fluorescence microscope.
Animal study. All animal studies were approved by the Institutional Animal Care Committee of Konkuk University. Six-week-old hairless mice (HR-1) were obtained from Japan SLC Inc. and maintained under controlled environmental conditions with a 12 h light/ dark cycle. The mice received food and tap water ad libitum. For mice receiving topical treatment, vehicle (cream base, LAB Research Ltd., Hungary) or EEDO-containing cream (2.5% EEDO in cream base) was applied evenly on the dorsal skin of mice at least 30 min before UVB radiation. Mice were irradiated with 50 J/m2 UVB three times a week for the first
Assay for intracellular superoxide. Intracellular superoxide production was measured using a fluorescent indicator, DHE, as described previously.23) Briefly, serial cryosections (7 μm) of frozen tissue samples obtained from the dorsal skin of UVB-irradiated HR-1 hairless mice were mounted on gelatin-coated slides, washed with ice-cold PBS, and incubated for 30 min at 37 °C with 10 μM DHE in PBS. Following incubation in a humidified chamber protected from light, red fluorescence corresponding to the levels of intracellular superoxide was detected through a 580 nm long-pass filter on an Olympus FV-1000 fluorescence microscope.
Fig. 1. EEDO inhibits UVB-induced senescence in human keratinocytes. Notes: (A) and (B) cells treated with the indicated doses of EEDO for 24 h were exposed to UVB, incubated for an additional 24 h, and subjected to SA β-gal staining. Bars indicate 100 μm. (C) cells were incubated with indicated doses of EEDO for 24 h, and subjected to the MTT assay. (D) cells pretreated with EEDO for 24 h were exposed to UVB irradiation, incubated for 24 h, and subjected to western blot analyses. Results are expressed as means ± SE (n = 4). *, p < 0.01 compared with the untreated group; #, p < 0.01 compared with the UVB-exposed group.
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Fig. 2. EEDO suppresses UVB-induced generation of ROS and intracellular superoxide in human keratinocytes. Notes: (A)–(C) Cells pretreated with EEDO for 24 h were exposed to UVB. After incubation for 30 min, the cells were treated with 10 μM H2DCF-DA (A, left panels) or 10 μM DHE (A, right panels), during the final 30 min of incubation. Intracellular ROS or superoxide levels were detected (A) by confocal laser fluorescence microscopy and quantitated (B and C). Bars indicate 100 μm. Results are expressed as means ± SE (n = 4). *, p < 0.01 compared with the untreated group; #, p < 0.01 compared with the UVB-exposed group.
Statistical analysis. Data are expressed as means ± SE. Statistical significance was determined by Student’s t-test or ANOVA with a post hoc Bonferroni test. A value of p < 0.05 was considered statistically significant.
Results Effects of EEDO on the UVB-induced senescence of human keratinocytes We investigated whether EEDO affects premature senescence of cultured human keratinocytes exposed to UVB. Keratinocytes exposed to UVB radiation exhibited a significant (p < 0.01) increase in senescence-associated β-galactosidase (SA-β-gal) activity, a biomarker of cellular senescence, relative to unexposed
control cells. However, this increase was significantly (p < 0.01) suppressed by EEDO in a concentrationdependent manner (Fig. 1(A) and (B)). At the concentrations of EEDO used in these experiments, cells retained high viability, as determined by MTT assay, within the time frame examined (Fig. 1(C)). To characterize the molecular mechanisms by which EEDO suppresses cellular senescence, we examined the expression levels of key proteins such as p53, p21, p16, and PML, which are biomarkers of cellular senescence.24) As shown in Fig. 1(D), UVB-induced increase in the levels of p53 and p21 was almost completely diminished by pretreatment of EEDO, but the levels of p16 and PML were less affected by EEDO in the keratinocytes exposed to UVB.
Fig. 3. HPLC analysis of EEDO. Notes: EEDO was separated by HPLC on an RP18 column eluted with an acetonitrile gradient (25–80%) at 0.8 mL/min and monitored with a Waters Refraction Index Detector at 275 nm. Sativanone (peak 7) was identified using a standard marker.
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Fig. 4. Sativanone inhibits UVB-induced senescence and ROS generation in human keratinocytes. Notes: (A) and (B) cells pretreated with sativanone for 24 h were exposed to UVB, incubated for 24 h, subjected to SA β-gal staining (A), and quantitated (B). (C) and (D) cells were pretreated with sativanone for 24 h and then exposed to UVB. After incubation for 30 min, the cells were treated with a peroxide-sensitive dye, H2DCF-DA (10 μM), during the final 30 min of incubation. Intracellular ROS levels were detected (C) and quantitated (D) by confocal laser fluorescence microscopy. Bars indicate 100 μm. Results are expressed as means ± SE (n = 4). *, p < 0.01 compared with the untreated group; #, p < 0.01 compared with the UVB-exposed group.
Fig. 5. EEDO prevents UVB-induced cellular senescence in HR-1 hairless mice. Notes: (A) representative cross-sections of the dorsal skin of UVB-exposed HR-1 hairless mice treated with EEDO-containing cream or vehicle for six weeks. Frozen tissue sections were subjected to staining for H&E (A, left panels), SA-β-gal (A, middle panels), and DHE (A, right panels). Bars indicate 100 μm. Representative images from four independent experiments are shown. (B)–(E) the levels of SA β-gal activity (B), DHE fluorescence (C), and epidermal/dermal thickness (D and E) were quantitated. Results are expressed as means ± SE (n = 4). *, p < 0.01 compared with the untreated group; #, p < 0.01 compared with the UVB-exposed group.
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Effects of EEDO on the UVB-induced generation of ROS and superoxide We examined the effects of EEDO on ROS and superoxide production in keratinocytes exposed to UVB using DCF and DHE dye, respectively. UVB irradiation significantly (p < 0.01) increased ROS and superoxide generation, but pretreatment with EEDO for 24 h significantly (p < 0.01) suppressed these effects (Fig. 2(A)–(C)). HPLC profile of EEDO We analyzed the contents of major constituents of EEDO by HPLC. Fig. 3 shows a typical HPLC profile of EEDO, showing ten identifiable peaks. One of the major components was identified as sativanone by comparison with a standard marker. Anti-senescent and antioxidant effects of sativanone Because sativanone was identified as a major component of EEDO, we examined whether sativanone alone could affect premature senescence and ROS generation in cultured human keratinocytes exposed to UVB. As expected, sativanone significantly (p < 0.01) inhibited UVB-induced SA-β-gal activity and generation of ROS in human keratinocytes (Fig. 4). Effects of EEDO on UVB-induced skin aging in HR1 hairless mice To verify in vivo the findings we made in cultured human keratinocytes, we next investigated cellular senescence in skin of HR-1 hairless mice exposed to UVB. In the dorsal skin of hairless mice, UVB irradiation significantly (p < 0.01) induced both cellular senescence and the generation of intracellular superoxide, as determined by SA-β-gal activity, dihydroethidium (DHE) red fluorescence, and epidermal/dermal thickness, respectively (Fig. 5). In line with results from the in vitro studies, treatment of EEDO significantly (p < 0.01) attenuated UVB-induced cellular senescence, superoxide production, and epidermal/dermal thickness.
Discussion In this study, we demonstrated that EEDO attenuates UVB-induced premature senescence in keratinocytes and skin cells. These effects of EEDO were mediated by inhibition of ROS generation, a key factor in premature senescence induced by UVB irradiation.11,12) Treatment of EEDO to HR-1 hairless mice exposed to UVB significantly attenuated the number of senescent cells and reduced ROS levels in the skin. To our knowledge, this is first report demonstrating that EEDO inhibits premature senescence of human keratinocytes and skin cells exposed to UVB. The antisenescence effect of EEDO may be due to its antioxidant properties, as demonstrated in this study. In line with the findings reported here, An et al. reported that flavonoids of D. odorifera protect mouse hippocampal cells from glutamate-induced oxidative injury.19) Butein, an active component of D. odorifera, serves as a powerful antioxidant against lipid and LDL
peroxidation via its versatile free-radical scavenging activity.20) In addition, an anti-inflammatory activity of sativanone, a major component of D. odorifera, is also demonstrated in the LPS-stimulated RAW264.7 cells.20) Other compounds from D. odorifera, such as isoliquiritigenin, 9-hydroxy-6,7-dimethoxydalbergiquinol, and 4,2′,5′-trihydroxy-4′-methoxychalcone, induce expression of heme oxygenase-1, an important antioxidant protein, thereby promoting anti-inflammatory responses in murine macrophages and microglial cells.16,17,25) Although we cannot exclude the possibility that EEDO and sativanone activate intracellular anti-senescence pathways other than antioxidant systems, our findings indicate that the primary role of EEDO and sativanone in preventing UVB-induced cellular senescence is as an antioxidant. In skin cells exposed to UVB, ROS is involved in cellular senescence.4,26) ROS lead to accumulation of DNA damage and induction of premature senescence via activation of p53, PML, and cyclin-dependent kinase inhibitors such as p21 and p16.2,27,28) Consistent with this, in our experiments, expression of these senescence-related proteins was elevated in keratinocytes exposed to UVB radiation, but this elevation was markedly attenuated in keratinocytes treated with EEDO. These results suggest that EEDO regulates the expression of these senescence-related proteins, possibly through suppression of UVB-induced ROS generation. In summary, we have shown that the EEDO changes the cellular responses of keratinocytes to UVB irradiation in vitro and in vivo. The data are consistent with the hypothesis that EEDO facilitates cytoprotection by suppressing ROS generated during UVB-induced oxidative stress. Although it remains unclear how EEDO affects the level of ROS generated by UVB irradiation, our results show that EEDO significantly attenuates cellular senescence in primary cultured keratinocytes and mouse skin exposed to UVB radiation.
Acknowledgments This work was supported in part by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2014R1A2 A2A01004847); and the Korean Health Technology R&D Project, Ministry of Health and Welfare (HI12C0802), Republic of Korea.
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Ethanol extract of Dalbergia odorifera protects skin keratinocytes against ultraviolet B-induced photoaging by suppressing production of reactive oxygen species a
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Sun Ah Ham , Jung Seok Hwang , Eun Sil Kang , Taesik Yoo , Hyun Ho Lim , Won Jin Lee , b
Kyung Shin Paek & Han Geuk Seo a
a
Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea
b
Department of Nursing, Semyung University, Jecheon, Republic of Korea Published online: 03 Jan 2015.
Click for updates To cite this article: Sun Ah Ham, Jung Seok Hwang, Eun Sil Kang, Taesik Yoo, Hyun Ho Lim, Won Jin Lee, Kyung Shin Paek & Han Geuk Seo (2015): Ethanol extract of Dalbergia odorifera protects skin keratinocytes against ultraviolet B-induced photoaging by suppressing production of reactive oxygen species, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.993916 To link to this article: http://dx.doi.org/10.1080/09168451.2014.993916
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Bioscience, Biotechnology, and Biochemistry, 2014
Ethanol extract of Dalbergia odorifera protects skin keratinocytes against ultraviolet B-induced photoaging by suppressing production of reactive oxygen species Sun Ah Ham1, Jung Seok Hwang1, Eun Sil Kang1, Taesik Yoo1, Hyun Ho Lim1, Won Jin Lee1, Kyung Shin Paek2 and Han Geuk Seo1,* Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea; 2Department of Nursing, Semyung University, Jecheon, Republic of Korea
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Received October 8, 2014; accepted November 15, 2014 http://dx.doi.org/10.1080/09168451.2014.993916
Dalbergia odorifera T. Chen (Leguminosae), an indigenous medicinal herb, has been widely used in northern and eastern Asia to treat diverse diseases. Here, we investigated the anti-senescent effects of ethanolic extracts of Dalbergia odorifera (EEDO) in ultraviolet (UV) B-irradiated skin cells. EEDO significantly inhibited UVB-induced senescence of human keratinocytes in a concentration-dependent manner, concomitant with inhibition of reactive oxygen species (ROS) generation. UVB-induced increases in the levels of p53 and p21, biomarkers of cellular senescence, were almost completely abolished in the presence of EEDO. Sativanone, a major constituent of EEDO, also attenuated UVB-induced senescence and ROS generation in keratinocytes, indicating that sativanone is an indexing (marker) molecule for the anti-senescence properties of EEDO. Finally, treatment of EEDO to mice exposed to UVB significantly reduced ROS levels and the number of senescent cells in the skin. Thus, EEDO confers resistance to UVB-induced cellular senescence by inhibiting ROS generation in skin cells. Key words:
Dalbergia odorifera; ultraviolet; reactive oxygen species; sativanone; senescence
Cellular senescence is a phenotypic change in gene expression, function, and morphology accompanied by permanent and irreversible growth arrest of cultured cells.1,2) Replicative senescence, which occurs in primary cultured cells, is characterized by shortened telomere length, ultimately resulting in incomplete chromosomal replication due to telomeric fusion or loss of telomere-bound factors.3) By contrast, stress-induced premature senescence is triggered by diverse factors that cause cellular stress, such as ultraviolet (UV) radiation,4) chemical agents that induce DNA damage,5,6)
and inappropriate activation of oncogenes.7) Among these factors, UV radiation, in particular UVB (wavelength, 280–315 nm), can cause a range of acute and chronic damage including sunburn, premature aging (photoaging), and skin cancer; the specific manifestation depends on the exposure time and intensity.8) In vitro, UVB radiation of human skin dermal fibroblasts stimulates intracellular production of reactive oxygen species (ROS).9) Several lines of evidence suggest that ROS play important roles in mediating UV-induced cellular senescence by breaking DNA strands and modifying DNA bases.2,10) Excess production and ineffective elimination of ROS production have thus been implicated in skin photoaging induced by UV radiation.11,12) Dalbergia odorifera T. Chem (Leguminosae) is an indigenous medicinal herb that has been widely used in China and Korea for the treatment of ischemia, swelling, necrosis, rheumatic pain, and blood disorders.13) Several components from the heartwood of D. odorifera, including flavonoids, quinines, and phenolic constituents, exhibit diverse biological activities.14,15) These effects include up-regulation of anti-inflammatory heme oxygenase-1 expression in murine macrophages,16,17) antibacterial activity in Ralstonia solanacearum,18) and protection against glutamate-induced oxidative injury in mouse hippocampal HT22 cells.19) Furthermore, sativanone, a polyphenol compound having two benzene rings (MW301.1) from D. odorifera, inhibits LPS-stimulated nitric oxide release and tumor necrosis factor-α secretion in RAW264.7 cells.20,21) However, the molecular mechanisms underlying the cellular actions of D. odorifera extracts, including sativanone, have not been fully elucidated. The diverse pharmacological functions of a variety of components of D. odorifera have been studied previously, and it would therefore be interesting to elucidate the anti-senescence properties of D. odorifera extracts. In this study, we demonstrated that ethanolic extract of
*Corresponding author. Email: [email protected] Abbreviations: DHE, dihydroethidium; EEDO, ethanolic extracts of Dalbergia odorifera; KGM, keratinocyte growth medium; PML, promyelocytic leukemia protein; ROS, reactive oxygen species; SA β-gal, senescence-associated β-galactosidase; UV, ultraviolet. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Dalbergia odorifera (EEDO) prevents premature senescence and reduces ROS production in both cultured human keratinocytes in vitro and mouse skin in vivo.
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Materials and methods Materials. The sativanone standard was generously provided by Dr. Hiroshi Noguchi (School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan). Polyclonal antibodies specific for p53 and p21 were obtained from cell signaling (Beverly, MA, USA). Monoclonal antibodies specific for p16 and promyelocytic leukemia protein (PML) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), polyclonal rabbit anti-β-actin antibody, and dihydroethidium (DHE) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Keratinocyte growth medium (KGM) and keratinocyte growth supplement was purchased from Lonza Biologics (Slough, UK). Preparation of EEDO. Commercially available heartwood of D. odorifera was purchased from a Korean medicinal herb store in Seoul in May 2012. An authenticated voucher specimen (KULBM-1205) was deposited in the Herbarium at the College of Animal Bioscience and Technology, Konkuk University (Seoul, Korea). Dried heartwood of D. odorifera (100 g) was extracted thrice with 60% ethanol under rotation at 120 rpm for 1 h. After evaporation of the solvent under vacuum, the extract (10 g) was reconstituted in dimethyl sulfoxide (DMSO) to a concentration of 10 mg/mL, and then stored at −20 °C until use. HPLC analysis. The main constituents in EEDO were analyzed by high-performance liquid chromatography (HPLC). The samples were injected into an HPLC system (Waters, Washington, USA) fitted with a CAPCEL PAK RP-C18 column (Φ4.6 mm × 250 mm) (Shiseido, Tokyo, Japan). Detection wavelength was set at 275 nm, and the column temperature was maintained at 20 °C. The mobile phase was a gradient consisting of solvent A (acetonitrile) and B (0.3% acetic acid, v/v): 25% A at 0–18 min, 25–46% A at 18–55 min, and 46–80% A at 55–60 min. The flow rate was 0.8 mL min−1; aliquots of 10 μL were injected. Cell culture and detection of intracellular superoxide. Human primary keratinocytes (donor age 11, passage 3) were obtained from Welskin (Seoul, Korea). The cells were cultured in KGM containing keratinocyte growth supplement at 37 °C in an atmosphere of 95% air and 5% CO2, based on the manufacturer’s recommendations. For detection of intracellular superoxide production in keratinocytes, cells seeded on 35 mm cover-glass bottom dishes (SPL Life Sciences) were treated with 5 μg/mL EEDO for 24 h, and then exposed to 40 mJ/cm2 UVB irradiation for 30 min. The red fluorescence corresponding to the levels of intracellular
superoxide was detected through a 580 nm long-pass filter on an Olympus FV-1000 fluorescence microscope. UVB irradiation. UVB irradiation was performed as previously described.22) Briefly, cells were incubated for the indicated times in the presence or absence of the indicated reagents, and then washed twice with phosphate-buffered saline (PBS, pH 7.2) before UVB exposure to avoid the photosensitizing effects of components in the culture medium. UVB irradiation was performed on serum-starved monolayer cultures using an FS20 Lamp (National Biological, Twinsburg, OH, USA). The UVB source was a bank of two FS20 Lamps emitting a continuous spectrum from 270 to 390 nm, with a peak emission at 313 nm; approximately 65% of the radiation was within the UVB wavelength range (280–315 nm). UVB strength was monitored using a Goldilux model 70234 photometer (Lynntech Inc., TX, USA). Senescence-associated (SA) β-galactosidase staining. Senescent cells were detected using an SA-β-galactosidase staining kit, according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). Briefly, human keratinocytes pretreated with 5 μg/mL EEDO or 1 μM sativanone for 24 h were exposed to UVB irradiation, and then incubated for 24 h. The cells were washed with ice-cold PBS and fixed. After two washes with ice-cold PBS, the cells were incubated in staining solution (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2, 40 mM citric acid, 40 mM sodium phosphate, and 1 mg/mL X-gal; pH 6.0) at 37 °C in the absence of CO2 overnight. Senescent cells were visualized using an Olympus JP/1X71 fluorescence microscope.
Determination of cell viability. Human primary keratinocytes were seeded in 24-well plates and treated with the indicated concentrations of EEDO for 24 h. After incubation, MTT (final 0.1 mg/mL) was added to the culture medium, and cells were incubated for an additional 4 h. After removal of the medium, formazan crystals (formed after the reduction of MTT by mitochondrial dehydrogenases in living cells) were solubilized in acidified isopropanol and measured spectrophotometrically at 570 nm. Western blot analysis. Cells treated with the indicated reagents were washed in ice-cold PBS and lysed in PRO-PREP protein extraction solution (iNtRON Biotechnology, Seoul, Korea). Aliquots of cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto a Hybond-P+ polyvinylidene difluoride membrane (GE Healthcare, UK). Membranes were blocked overnight at 4 °C with 5% nonfat milk in TBS (Tris-buffered saline; 50 mM Tris, 150 mM NaCl, pH 7.6) containing 0.1% Tween-20, incubated overnight at 4 °C with the indicated specific antibodies
D. odorifera induced UVB-induced photoaging
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in TBS containing 1% BSA and 0.05% Tween-20, and then incubated for 2 h at room temperature with peroxidase-conjugated goat antibody diluted 1:3000. After extensive washing in TBS containing 0.1% BSA and 0.1% Tween-20, immuno-reactive bands were detected using West-ZOL Plus (iNtRON Biotechnology).
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Measurement of intracellular ROS. To assess the levels of intracellular accumulation of ROS, the fluorescent probe chloromethyl-2′,7′-dichlorofluorescein diaceMolecular Probes/Life tate (CM-H2DCF-DA; Technologies, Grand Island, NY, USA) was used, as described previously.22) Cells were seeded on 35 mm cover glass-bottom dishes (SPL Life Sciences, Seoul, Korea), treated with 5 μg/mL EEDO or 1 μM sativanone for 24 h, and then exposed to 40 mJ/cm2 UVB irradiation for 30 min. The cells were subsequently incubated with 10 μM CM-H2DCF-DA (peroxide-sensitive dye) for a final 30 min at 37 °C, and green fluorescence corresponding to the levels of intracellular ROS was detected through a 520 nm long-pass filter on an Olympus FV-1000 laser fluorescence microscope (Tokyo, Japan).
four weeks, and then with 100 J/m UVB three times a week for the last two weeks. Six weeks later, the mice were sacrificed, and squares of dorsal skin (ca. 1 cm × 1 cm) were excised from each mouse for further examination. For serial sectioning, tissues were fixed in 4% paraformaldehyde, cryoprotected in 20% sucrose, embedded in OCT (Sakura Finetech, Japan), snap-frozen in iso-pentene, and prechilled in liquid nitrogen prior to sectioning. For hematoxylin and eosin (H&E) counterstaining, serial cryosections (7 μm) were incubated sequentially at room temperature in 50% hematoxylin solution for 20 min, differentiated in 1% HCl solution for 1 s, and then transferred directly to 0.5% eosin solution and stained for 20 min. The sections were then washed once with distilled water and incubated in a graded alcohol series, ending with xylene, to dehydrate the tissue. Finally, the tissue sections were mounted under coverslips using Permount (Fisher Scientific, Somerville, NJ, USA). For detection of senescent cells in the dorsal skin of UVB-irradiated hairless mice, serial cryosections mounted on gelatin-coated slides were incubated in staining solution, and the activity of SA-β-galactosidase was visualized using an Olympus JP/1X71 fluorescence microscope.
Animal study. All animal studies were approved by the Institutional Animal Care Committee of Konkuk University. Six-week-old hairless mice (HR-1) were obtained from Japan SLC Inc. and maintained under controlled environmental conditions with a 12 h light/ dark cycle. The mice received food and tap water ad libitum. For mice receiving topical treatment, vehicle (cream base, LAB Research Ltd., Hungary) or EEDO-containing cream (2.5% EEDO in cream base) was applied evenly on the dorsal skin of mice at least 30 min before UVB radiation. Mice were irradiated with 50 J/m2 UVB three times a week for the first
Assay for intracellular superoxide. Intracellular superoxide production was measured using a fluorescent indicator, DHE, as described previously.23) Briefly, serial cryosections (7 μm) of frozen tissue samples obtained from the dorsal skin of UVB-irradiated HR-1 hairless mice were mounted on gelatin-coated slides, washed with ice-cold PBS, and incubated for 30 min at 37 °C with 10 μM DHE in PBS. Following incubation in a humidified chamber protected from light, red fluorescence corresponding to the levels of intracellular superoxide was detected through a 580 nm long-pass filter on an Olympus FV-1000 fluorescence microscope.
Fig. 1. EEDO inhibits UVB-induced senescence in human keratinocytes. Notes: (A) and (B) cells treated with the indicated doses of EEDO for 24 h were exposed to UVB, incubated for an additional 24 h, and subjected to SA β-gal staining. Bars indicate 100 μm. (C) cells were incubated with indicated doses of EEDO for 24 h, and subjected to the MTT assay. (D) cells pretreated with EEDO for 24 h were exposed to UVB irradiation, incubated for 24 h, and subjected to western blot analyses. Results are expressed as means ± SE (n = 4). *, p < 0.01 compared with the untreated group; #, p < 0.01 compared with the UVB-exposed group.
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Fig. 2. EEDO suppresses UVB-induced generation of ROS and intracellular superoxide in human keratinocytes. Notes: (A)–(C) Cells pretreated with EEDO for 24 h were exposed to UVB. After incubation for 30 min, the cells were treated with 10 μM H2DCF-DA (A, left panels) or 10 μM DHE (A, right panels), during the final 30 min of incubation. Intracellular ROS or superoxide levels were detected (A) by confocal laser fluorescence microscopy and quantitated (B and C). Bars indicate 100 μm. Results are expressed as means ± SE (n = 4). *, p < 0.01 compared with the untreated group; #, p < 0.01 compared with the UVB-exposed group.
Statistical analysis. Data are expressed as means ± SE. Statistical significance was determined by Student’s t-test or ANOVA with a post hoc Bonferroni test. A value of p < 0.05 was considered statistically significant.
Results Effects of EEDO on the UVB-induced senescence of human keratinocytes We investigated whether EEDO affects premature senescence of cultured human keratinocytes exposed to UVB. Keratinocytes exposed to UVB radiation exhibited a significant (p < 0.01) increase in senescence-associated β-galactosidase (SA-β-gal) activity, a biomarker of cellular senescence, relative to unexposed
control cells. However, this increase was significantly (p < 0.01) suppressed by EEDO in a concentrationdependent manner (Fig. 1(A) and (B)). At the concentrations of EEDO used in these experiments, cells retained high viability, as determined by MTT assay, within the time frame examined (Fig. 1(C)). To characterize the molecular mechanisms by which EEDO suppresses cellular senescence, we examined the expression levels of key proteins such as p53, p21, p16, and PML, which are biomarkers of cellular senescence.24) As shown in Fig. 1(D), UVB-induced increase in the levels of p53 and p21 was almost completely diminished by pretreatment of EEDO, but the levels of p16 and PML were less affected by EEDO in the keratinocytes exposed to UVB.
Fig. 3. HPLC analysis of EEDO. Notes: EEDO was separated by HPLC on an RP18 column eluted with an acetonitrile gradient (25–80%) at 0.8 mL/min and monitored with a Waters Refraction Index Detector at 275 nm. Sativanone (peak 7) was identified using a standard marker.
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Fig. 4. Sativanone inhibits UVB-induced senescence and ROS generation in human keratinocytes. Notes: (A) and (B) cells pretreated with sativanone for 24 h were exposed to UVB, incubated for 24 h, subjected to SA β-gal staining (A), and quantitated (B). (C) and (D) cells were pretreated with sativanone for 24 h and then exposed to UVB. After incubation for 30 min, the cells were treated with a peroxide-sensitive dye, H2DCF-DA (10 μM), during the final 30 min of incubation. Intracellular ROS levels were detected (C) and quantitated (D) by confocal laser fluorescence microscopy. Bars indicate 100 μm. Results are expressed as means ± SE (n = 4). *, p < 0.01 compared with the untreated group; #, p < 0.01 compared with the UVB-exposed group.
Fig. 5. EEDO prevents UVB-induced cellular senescence in HR-1 hairless mice. Notes: (A) representative cross-sections of the dorsal skin of UVB-exposed HR-1 hairless mice treated with EEDO-containing cream or vehicle for six weeks. Frozen tissue sections were subjected to staining for H&E (A, left panels), SA-β-gal (A, middle panels), and DHE (A, right panels). Bars indicate 100 μm. Representative images from four independent experiments are shown. (B)–(E) the levels of SA β-gal activity (B), DHE fluorescence (C), and epidermal/dermal thickness (D and E) were quantitated. Results are expressed as means ± SE (n = 4). *, p < 0.01 compared with the untreated group; #, p < 0.01 compared with the UVB-exposed group.
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Effects of EEDO on the UVB-induced generation of ROS and superoxide We examined the effects of EEDO on ROS and superoxide production in keratinocytes exposed to UVB using DCF and DHE dye, respectively. UVB irradiation significantly (p < 0.01) increased ROS and superoxide generation, but pretreatment with EEDO for 24 h significantly (p < 0.01) suppressed these effects (Fig. 2(A)–(C)). HPLC profile of EEDO We analyzed the contents of major constituents of EEDO by HPLC. Fig. 3 shows a typical HPLC profile of EEDO, showing ten identifiable peaks. One of the major components was identified as sativanone by comparison with a standard marker. Anti-senescent and antioxidant effects of sativanone Because sativanone was identified as a major component of EEDO, we examined whether sativanone alone could affect premature senescence and ROS generation in cultured human keratinocytes exposed to UVB. As expected, sativanone significantly (p < 0.01) inhibited UVB-induced SA-β-gal activity and generation of ROS in human keratinocytes (Fig. 4). Effects of EEDO on UVB-induced skin aging in HR1 hairless mice To verify in vivo the findings we made in cultured human keratinocytes, we next investigated cellular senescence in skin of HR-1 hairless mice exposed to UVB. In the dorsal skin of hairless mice, UVB irradiation significantly (p < 0.01) induced both cellular senescence and the generation of intracellular superoxide, as determined by SA-β-gal activity, dihydroethidium (DHE) red fluorescence, and epidermal/dermal thickness, respectively (Fig. 5). In line with results from the in vitro studies, treatment of EEDO significantly (p < 0.01) attenuated UVB-induced cellular senescence, superoxide production, and epidermal/dermal thickness.
Discussion In this study, we demonstrated that EEDO attenuates UVB-induced premature senescence in keratinocytes and skin cells. These effects of EEDO were mediated by inhibition of ROS generation, a key factor in premature senescence induced by UVB irradiation.11,12) Treatment of EEDO to HR-1 hairless mice exposed to UVB significantly attenuated the number of senescent cells and reduced ROS levels in the skin. To our knowledge, this is first report demonstrating that EEDO inhibits premature senescence of human keratinocytes and skin cells exposed to UVB. The antisenescence effect of EEDO may be due to its antioxidant properties, as demonstrated in this study. In line with the findings reported here, An et al. reported that flavonoids of D. odorifera protect mouse hippocampal cells from glutamate-induced oxidative injury.19) Butein, an active component of D. odorifera, serves as a powerful antioxidant against lipid and LDL
peroxidation via its versatile free-radical scavenging activity.20) In addition, an anti-inflammatory activity of sativanone, a major component of D. odorifera, is also demonstrated in the LPS-stimulated RAW264.7 cells.20) Other compounds from D. odorifera, such as isoliquiritigenin, 9-hydroxy-6,7-dimethoxydalbergiquinol, and 4,2′,5′-trihydroxy-4′-methoxychalcone, induce expression of heme oxygenase-1, an important antioxidant protein, thereby promoting anti-inflammatory responses in murine macrophages and microglial cells.16,17,25) Although we cannot exclude the possibility that EEDO and sativanone activate intracellular anti-senescence pathways other than antioxidant systems, our findings indicate that the primary role of EEDO and sativanone in preventing UVB-induced cellular senescence is as an antioxidant. In skin cells exposed to UVB, ROS is involved in cellular senescence.4,26) ROS lead to accumulation of DNA damage and induction of premature senescence via activation of p53, PML, and cyclin-dependent kinase inhibitors such as p21 and p16.2,27,28) Consistent with this, in our experiments, expression of these senescence-related proteins was elevated in keratinocytes exposed to UVB radiation, but this elevation was markedly attenuated in keratinocytes treated with EEDO. These results suggest that EEDO regulates the expression of these senescence-related proteins, possibly through suppression of UVB-induced ROS generation. In summary, we have shown that the EEDO changes the cellular responses of keratinocytes to UVB irradiation in vitro and in vivo. The data are consistent with the hypothesis that EEDO facilitates cytoprotection by suppressing ROS generated during UVB-induced oxidative stress. Although it remains unclear how EEDO affects the level of ROS generated by UVB irradiation, our results show that EEDO significantly attenuates cellular senescence in primary cultured keratinocytes and mouse skin exposed to UVB radiation.
Acknowledgments This work was supported in part by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2014R1A2 A2A01004847); and the Korean Health Technology R&D Project, Ministry of Health and Welfare (HI12C0802), Republic of Korea.
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