Photodermatology, Photoimmunology & Photomedicine
ORIGINAL ARTICLE
The protective effect of Kaempferia parviflora extract on UVB-induced skin photoaging in hairless mice Ji-Eun Park1, Hee-Bong Pyun2, Seon Wook Woo1, Jae-Hong Jeong1 & Jae-Kwan Hwang1,2
1 Department of Biotechnology, Yonsei University, Seoul, Korea. 2 Department of Biomaterials Science and Engineering, Yonsei University, Seoul, Korea.
Key words: hairless mice; Kaempferia parviflora; matrix metalloproteinases (MMPs); NF-κB; photoaging
Correspondence: Prof Jae-Kwan Hwang, PhD, Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea. Tel: +822 2123 5881 Fax: +822 362 7265 e-mail: [email protected]
Accepted for publication: 3 December 2013
Conflicts of interest: None declared.
SUMMARY Background Chronic skin exposure to ultraviolet (UV) light increases reactive oxygen species (ROS) and stimulates the expression of matrix metalloproteinases (MMPs) through c-Jun and c-Fos activation. These signaling cascades induce the degradation of extracellular matrix (ECM) components, resulting in photoaging. Methods This study evaluated the preventive effect of the ethanol extract of Kaempferia parviflora Wall. ex. Baker (black ginger) on UVB-induced photoaging in vivo. To investigate the antiphotoaging effect of K. parviflora extract (KPE), UVBirradiated hairless mice administered oral doses of KPE (100 or 200 mg/kg/ day) for 13 weeks. Results In comparison to the UVB control group, KPE significantly prevented wrinkle formation and the loss of collagen fibers with increased type I, III, and VII collagen genes (COL1A1, COL3A1, and COL7A1). The decrease in wrinkle formation was associated with a significant reduction in the UVB-induced expression of MMP-2, MMP-3, MMP-9, and MMP-13 via the suppression of c-Jun and c-Fos activity. KPE also increased the expression of catalase, which acts as an antioxidant enzyme in skin. In addition, expression of inflammatory mediators, such as nuclear factor kappa B (NF-κB), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2), was significantly reduced by KPE treatment. Conclusion The results show that oral administration of KPE significantly prevents UVBinduced photoaging in hairless mice, suggesting its potential as a natural antiphotoaging material. Photodermatol Photoimmunol Photomed 2014; 30: 237–245
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/phpp.12097
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Two distinct biological processes are thought to underlie skin aging: intrinsic aging by the passage of time and extrinsic aging induced by environmental factors, especially ultraviolet (UV) light. Extrinsic aging through UV irradiation is also referred to as photoaging, and during photoaging, UV irradiation induces the breakdown of skin components like collagen, elastin, and ground substances (1). Specifically, UV irradiation causes collagen breakdown in dermal connective tissue and significantly reduces the expression of type I collagen, the most abundant structural protein of skin (2). UV irradiation also causes the production of reactive oxygen species (ROS), direct or indirect DNA damage, and damage to extracellular matrix (ECM) integrity (3). When human skin is exposed to acute UV irradiation, it causes altered pigmentation, inflammation, immune suppression, sunburn, and dermal connective tissue damage. In contrast, chronic exposure of human skin to UV irradiation breaks down the normal architecture of skin connective tissue, impairs skin function, and ultimately causes photoaging (4). During UV-induced ROS production, activation of the mitogen-activated protein kinases/associated protein-1 (MAPK/AP-1) pathway is triggered. UV irradiation induces the phosphorylation of three MAPK proteins: extracellular signal regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Also activated by UV irradiation are c-Jun and c-Fos, which constitute the AP-1 protein complex and leads to the overexpression of matrix metalloproteinases (MMPs) (2). MMPs are in a large family of zinc and calciumdependent endopeptidases, and they play pivotal roles in tumor invasion, inflammation, and skin aging. UV-induced MMPs are especially responsible for tissue remodeling via the degradation of ECM components including collagen, elastin, and fibrillin-1 (4). Recent studies have demonstrated that the inhibition of MMP production suppresses UV-induced epidermal thickening and wrinkle formation (5, 6). Therefore, the regulation of MMPs may be an effective strategy for the prevention and treatment of photoaging. In addition to MMPs, UV irradiation activates nuclear factor kappa B (NF-κB), a transcription factor which stimulates the transcription of pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and cyclooxygenase-2 (COX-2) in human skin. These cytokines activate their cell surface receptors, resulting in epidermal proliferation, various UV-induced cellular responses, and inflammation. This UV-induced inflammation is characterized by erythema, immunosuppression, and edema (7). 238
Kaempferia parviflora Wall. ex Baker, commonly known as black ginger, belongs to the Zingiberaceae family. Black to purple rhizomes of K. parviflora have been used as dietary supplements and traditional medicines for the treatment of various illnesses in tropical countries (8), and previous studies confirm a variety of K. parviflora’s bioactivities. K. parviflora confers known antioxidative, anti-inflammatory, anticancer, antiviral, and gastroprotective effects (9–11), but the antiphotoaging effects of K. parviflora have not been reported to date. Therefore, we examined the protective effect of orally administrated K. parviflora extract on UVB-induced photoaging in vivo by evaluating various parameters such as wrinkle formation, skinfold thickness, collagen reduction, MMP regulation through c-Jun and c-Fos inhibition, catalase expression, and pro-inflammatory cytokine levels.
MATERIALS AND METHODS Preparation of KPE Rhizomes of K. parviflora were collected in Bangkok, Thailand. A voucher specimen was deposited at the Department of Biotechnology, Yonsei University, Seoul, Korea. The dried rhizomes were ground and then soaked in 95% ethanol for 24 h at room temperature. K. parviflora filtrate was concentrated under reduced pressure using a rotary evaporator (Heidolph Instruments GmbH & Co. KG., Schwabach, Germany) to obtain K. parviflora extract (KPE) with a yield of 9.56%.
Animal experiments Five-week-old female hairless mice (SKH-1) were purchased from Daehan Biolink Ltd. (Eumsung, Korea). The mice were housed in temperature (23 ± 2°C), humidity (55 ± 10%), and light (12 h day/night) controlled rooms at Yonsei Laboratory Animal Research Center (YLARC, Seoul, Korea). Twenty mice were randomly allocated to each group (five mice per group, four total groups): (i) control group (Normal), (ii) UVB-irradiated group (UVB), (iii) UVB-irradiated and 100 mg/kg/day KPEtreated group (KPE-100), and (iv) UVB-irradiated and 200 mg/kg/day KPE-treated group (KPE-200). The mice in the KPE-treated groups (100 or 200 mg/kg/day) were administered oral doses of KPE daily for 13 weeks with concurrent exposure to UVB irradiation three times per week on the back of the mice. UVB was supplied by the UV crosslinker CL-1000 M (UVP, Cambridge, UK), using 302 nm UV wavelength. UVB irradiation began with a starting dose of 75 mJ/cm2 during the first week, and then Photodermatol Photoimmunol Photomed 2014; 30: 237–245 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Antiphotoaging effect of Kaempferia parviflora
the dose was increased weekly by one minimal erythema dose (MED) until reaching three MEDs, which was then maintained until 13 weeks of total treatment time. After 13 weeks of treatment, the mice were anesthetized and sacrificed by intraperitoneal injection of a mixture of zoletil and rompun (One Bio, Daejeon, Korea). Skin biopsy samples were obtained from the dorsal skin of the hairless mice, rapidly frozen in liquid nitrogen, and stored at −70°C. Samples for histological analysis were fixed in 10% buffered formalin for optical microscopy.
The membranes were then detected with primary antibodies against catalase, c-Jun, c-Fos, MMP-3, MMP-13, NF-κB, IL-1β, COX-2, and α-tubulin (Cell Signaling, Beverly, MA, USA). Bound antibodies were detected with a horseradish peroxidase-linked secondary antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA). Blotted antibody signals were detected with ECL detection solution and were visualized by the G:BOX Image Analysis System (Syngene, Cambridge, UK).
Gelatin zymography Reverse transcription-polymerase chain reaction (RT-PCR) PCR amplification of cDNA products (5 μl) was performed with PCR premix and the following primer pairs (Bioneer, Daejeon, Korea): mouse β-actin (forward, 5'-CCA GCC AGC CAC CAT CGC TC-3'; reverse, 5'-TGA CCT TGG CCA GGG GTG CA-3'), mouse COL1A1 (forward, 5'-GTC CCC AAT GGT GAG ACG TG-3'; reverse, 5'-GCA CGG AAA CTC CAG CTG AT-3'), mouse COL3A1 (forward, 5'-AGC GGC TGA GTT TTA TGA CG-3'; reverse, 5'-AGC ACA GGA GCA GGT GTA GA-3'), mouse COL7A1 (forward, 5'-AAG CCG AGA TTA AGG GCT GG-3'; reverse, 5'-CAC CAA ATG GAG CAC AGC AG-3'), mouse MMP-3 (forward, 5'-TAG CAG GTT ATC CTA AAA GCA-3'; reverse, 5'-CCA GCT ATT GCT CTT CAA T-3'), and mouse MMP-13 (forward, 5'-CAT CCA TCC CGT GAC CTT AT-3'; reverse, 5'-GCA TGA CTC TCA CAA TGC GA-3'). Before PCR amplification, the primers were denatured at 94°C for five minutes. Amplification consisted of 25 cycles: denaturation at 94°C for 30 s, annealing at 56°C for one minute, extension at 72°C for one minute, then a final five-minute extension at 72°C. PCR was performed in the Gene Amp PCR System 2700 (Applied Biosystems, Foster City, CA, USA). The PCR products were separated by 1.5% agarose gel electrophoresis and visualized by 6X Loading STAR solution and UV illumination using the G:BOX Image Analysis System (Syngene, Cambridge, UK).
Western blot analysis Homogenized skin sections were lysed in RIPA lysis buffer (ELPIS-Biotech, Daejeon, Korea) with a protease inhibitor cocktail (Sigma-Aldrich Co., St. Louis, MO, USA). Protein concentrations were determined by the Bradford assay. For Western blotting, equal amounts of protein (30 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST). Photodermatol Photoimmunol Photomed 2014; 30: 237–245 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
MMP-2 and MMP-9 activity in the skin sections was assessed by gelatin zymography. Samples were mixed with standard gel loading buffer containing 2% SDS without β-mercaptoethanol and loaded on the gel without prior heating. The samples were separated by electrophoresis on a 10% polyacrylamide gel containing SDS and 1% gelatin at 85 V for two hours in a Bio-Rad Mini PROTEAN 3 Cell electrophoretic apparatus (Bio-Rad Laboratories, Seoul, Korea). After electrophoresis, the gel was washed with 2.5% Triton X-100 for one hour to remove SDS and then incubated in reaction buffer (50 mM Tris-HCl [pH 7.5] containing 10 mM CaCl2 and 0.15 M NaCl) at 37°C. After 20 h, the gel was stained with Coomassie® Brilliant Blue R-250 and then destained with 30% methanol and 10% acetic acid. MMP-2 and MMP-9 were detected at 72 kDa and 92 kDa, respectively, as a clear zone against the dark background.
Evaluation of skin wrinkle formation After 13 weeks of KPE treatment, the dorsal skin surface impressions of anesthetized hairless mice were measured using the Visioline VL650 Replica full kit (Epigem, Seoul, Korea) and analyzed with Visioline VL650 (CK Electronics GmbH, Cologne, Germany). The analyzed data represented the number, depth, length, and total area of the wrinkles.
Histological analysis After sacrifice, the skin samples were fixed in 10% formalin for 24 h and stained with hematoxylin and eosin (H&E) for skin layers and Masson’s trichrome for collagen fibers. The stained sections were analyzed using the Eclipse TE2000U Inverted Microscope with twin CCD cameras (Nikon, Tokyo, Japan).
Total collagen assay Skin tissues (400 mg) were homogenized with glass beads in 1 ml of 6 N HCl and hydrolyzed at 105°C for 18 h. The 239
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Fig. 1. The effect of KPE on wrinkle formation. (a) The dorsal skin surface of hairless mice was exposed to UVB irradiation three times a week for 13 weeks. Before sacrifice, the skin replica samples of the dorsal areas were taken. (b) Wrinkle values were obtained from the skin replica analysis. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, **P < 0.01.
collagen content was measured using a total collagen assay kit (Quickzyme Bioscience, Leiden, Netherlands).
Statistical analysis Results are expressed as the mean ± standard deviation (SD) of five mice in each group. All groups were compared by one-way analysis of variance (ANOVA), followed by the Duncan test (SPSS 12.0, SPSS Inc., Chicago, IL, USA). ## P < 0.01, #P < 0.05, **P < 0.01, and *P < 0.05 were considered to be statistically significant.
The effect of KPE on UVB-induced skin thickness Epidermal thickness is known as a quantitative parameter of the inflammatory response in skin photoaging. Chronic UV irradiation induces skin inflammation, which causes excessive epidermal proliferation or skin thickening (3). H&E staining showed that the epidermal thickness was significantly reduced by KPE treatment as compared to the UVB control group (Fig. 2), indicating KPE’s inhibitory effects on UVB-induced skin thickening.
RESULTS
The effect of KPE on UVB-induced collagen degradation
The effect of KPE on UVB-induced wrinkle formation
During skin aging, UV irradiation reduces the spatial density of collagen bundles. Collagen content is reduced either by a decrease in collagen synthesis or an increase in collagen degradation (2). UVB irradiation stimulated the degradation of collagen fibers in the UVB control group as represented by the smaller blue-stained area, while KPE significantly inhibited collagen degradation (Fig. 3A). A quantitative assay measuring total collagen content also supported the preventive effects of KPE on collagen degradation (Fig. 3B). mRNA levels of COL1A1, COL3A1, and COL7A1, which synthesize types I, III, and VII collagen, were significantly increased by KPE compared to the
Repetitive UV irradiation increases the production of MMPs, which can result in wrinkle formation by degrading various ECM components (2). In the dorsal skin of hairless mice, the oral administration of KPE significantly reduced UVB-induced wrinkle formation as compared to the UVB control group (Fig. 1A). Quantitative analysis of wrinkle values, which represent the number, depth, length, and total area of the wrinkles, was consistent with the photographs (Fig. 1B). These results indicate that KPE effectively attenuates UVB-induced wrinkle formation. 240
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Fig. 2. The effect of KPE on skin thickness. (a) Skin tissue sections were stained with H&E, representing epidermal thickness (original magnification ×400). (b) Skinfold thickness was measured with a caliper mid-way between neck and hips. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, **P < 0.01.
Fig. 3. The effect of KPE on collagen fibers. (a) Skin tissue sections were stained with Masson’s trichrome, identifying collagen fibers (original magnification ×400). (b) The amount of collagen was estimated after UVB irradiation for 13 weeks. (c) mRNA levels of COL1A1, COL3A1, and COL7A1 was evaluated by RT-PCR. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, *P < 0.05, **P < 0.01.
UVB control group (Fig. 3C). These results suggest that KPE prevents UVB-induced collagen degradation and promotes collagen synthesis.
The effect of KPE on UVB-induced MMP expression UV irradiation induces MMP expression, resulting in the degradation of various ECM components and characteristic changes associated with skin photoaging (3). Levels of MMP-3 and MMP-13 mRNA and protein were significantly reduced in the KPE-treated group as compared to the UVB control group (Fig. 4A,B). Gelatin zymography analysis showed that KPE also reduced MMP-2 and MMP-9 activity levels (Fig. 4C). Taken together, these data demonstrate that KPE significantly reduces UVB-induced MMP expression.
The effect of KPE on AP-1 complex and catalase expression MMP secretion is caused by the activation of the AP-1 complex. Activated c-Jun and c-Fos, which form the AP-1 Photodermatol Photoimmunol Photomed 2014; 30: 237–245 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
complex, are associated with the transcription of MMPs (2). In this study, c-Jun and c-Fos protein levels were highly activated in the UVB control group, while KPE significantly attenuated the expression of c-Jun and c-Fos in a dose-dependent manner (Fig. 5A). These results suggest that KPE prevents AP-1 activation. Catalase protects the skin against the deleterious effects of UV-induced oxidative stress (12). Chronic UVB exposure reduced catalase expression in hairless mice, while KPE significantly increased the level of catalase protein compared to the UVB control group (Fig. 5B). These results suggest that KPE may effectively scavenge ROS by increasing catalase expression.
The effect of KPE on UVB-induced inflammatory responses NF-κB is activated by UV irradiation and further stimulates the transcription of pro-inflammatory cytokines, leading to cellular responses, epidermal proliferation, and inflammation (7). The UVB control group showed higher levels of inflammatory factors such as NF-κB, IL-1β and 241
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Fig. 4. The effect of KPE on MMP expression. (a) The level of mRNA expression and (b) protein levels of MMP-3 and MMP-13 were determined by RT-PCR and Western blotting, respectively. (c) MMP-2 and MMP-9 activity was analyzed by gelatin zymography. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, *P < 0.05, **P < 0.01.
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Fig. 5. The effects of KPE on AP-1 and catalase expression. (a) c-Jun and c-Fos expression and (b) Catalase expression was evaluated by Western blotting. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, **P < 0.01.
Fig. 6. The effect of KPE on NF-κB, IL-1β, and COX-2 expression. NF-κB, IL-1β, and COX-2 expression levels were determined by Western blotting. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, *P < 0.05, **P < 0.01.
COX-2. However, KPE significantly inhibited their expression in a dose-dependent manner (Fig. 6). These results indicate that KPE protects skin from UVB-induced inflammation by suppressing NF-κB, IL-1β, and COX-2 expression.
DISCUSSION In the present study, we investigated the preventive effect of KPE against UVB-induced photoaging by analyzing clinical and histological changes in hairless mice (Figs 1 and 2). In mice and humans, hyperpigmentation, roughness, dryness, coarse elastosis, laxity, and wrinkling are the Photodermatol Photoimmunol Photomed 2014; 30: 237–245 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
clinical symptoms of skin photoaging (6). Subclinically, skin aging is further characterized skin thickening. These clinical and subclinical changes are the result of skin cell hyperproliferation and collagen degradation, specifically, these phenomena are associated with reduced levels of collagen, a basic component of the skin’s dermal layer. Our study showed that oral administration of KPE increased total collagen levels of the dorsal skin of hairless mice by increasing COL1A1, COL3A1, and COL7A1 mRNA expression (Fig. 3). Collagen, a fibrous protein, is a major constituent of connective skin tissues, and the functional properties of the skin are highly dependent on collagen integrity in the dermis (13). Several different types of collagen are involved in ECM formation. Most collagen fibers in the dermis are types I and III collagens. On the other hand, type VII collagen plays an important role in supporting the skin barrier, acting like an anchoring protein at the dermal epidermal junction (DEJ) (1). One mechanism by which photoaging may occur is through abnormal increases in MMP production. Therefore, natural materials which reduce MMP production may successfully protect against photoaging (14). As shown in Fig. 4, UVB-induced expression of MMP-2, MMP-3, MMP-9, and MMP-13 (also known as gelatinase-A, stromelysin-1, gelatinase-B, and collagenase-3, respectively) were significantly inhibited by oral administration of KPE. UV irradiation induces the synthesis of MMP-3, which degrades types III and VII collagen, and MMP-13, which initiates the breakdown of interstitial type I collagen in rodents. MMP-2 and MMP-9, which degrade types IV and VII collagen, are also induced by UV irradiation. Overall, these MMPs are associated with the degradation of various types of collagen which are the major components of the skin dermis (15). Because MMPs play a role in photoaging, understanding their mechanism of action, however complex, is important. UV irradiation induces ROS activation, which leads 243
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to the degradation of ECM components and to detrimental chemical modifications, resulting in the upregulation of c-Jun and c-fos (AP-1). Increased expression of c-Jun and c-fos results in increased MMP expression (16). In our study, KPE significantly reduced the expression of c-Jun and c-Fos, which is likely the pathway by which the levels of several MMPs were reduced. Since wrinkle formation is closely associated with MMPs, their regulation has been targeted as a strategy to prevent photoaging (2), and KPE may be a viable candidate by which to reduce this MMPinduced photoaging. Of notable mention, some natural materials have also been reported to possess effective antiphotoaging activities in vitro by attenuating MMP expression through c-Jun and c-fos (AP-1) signaling inhibition (16, 17), so our in vivo results are consistent with this work. Previous studies have also demonstrated that major antioxidant enzymes such as catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) protect UV-induced oxidative effects. Among these antioxidant enzymes, catalase is a representative epidermal differentiation marker in redox damage, and it acts to reduce photooxidative stress (18). In the present study, KPE significantly increased catalase protein levels (Fig. 5), which may be one mechanism by which photoaging was reduced. One way in which oxidative stress might contribute to photoaging is by inducing NF-κB signaling, which is associated with MMP expression in the skin. After UV irradiation, NF-κB, a transcription factor, is activated and increases MMP production. In the present study, KPE effectively inhibited UVB-induced inflammatory signaling by reducing the expression of NF-κB, IL-1β, and COX-2
(Fig. 6). Because UV irradiation induces inflammatory mediators which enhance photoaging (7), KPE’s inhibition of this process is valuable information. It is possible that KPE may be an appropriate supplement to such NF-κB inhibitors, or may be an appropriate alternative treatment. Although we have illustrated some ways by which KPE might confer its beneficial effects, further work exploring KPE’s other mechanisms of action are necessary. It has been reported that the rhizomes of K. parviflora contain high amounts of polymethoxyflavones, such as 5,7-dimethoxyflavone, 3,5,7trimethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone (19, 20), and also some kaempferiaosides and phenolic glycosides (21, 22). Previously, we identified the inhibitory effects of 5,7-dimethoxyflavone on UVB-induced skin photoaging in vitro (17). However, one future approach would be to clarify whether 5,7dimethoxyflavone also effectively functions as an antiphotoaging material in vivo.
CONCLUSION Oral administration of KPE significantly attenuated UVBinduced wrinkle formation, skin thickening, and collagen degradation in hairless mice, and also increased COL1A1, COL3A1, and COL7A1 expression. In addition, KPE effectively reduced MMP expression by inhibiting the activation of c-Jun and c-Fos. KPE also increased catalase expression and markedly suppressed UVB-induced inflammatory responses. Overall, K. parviflora has the potential to be an effective natural anti-photoaging material.
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ORIGINAL ARTICLE
The protective effect of Kaempferia parviflora extract on UVB-induced skin photoaging in hairless mice Ji-Eun Park1, Hee-Bong Pyun2, Seon Wook Woo1, Jae-Hong Jeong1 & Jae-Kwan Hwang1,2
1 Department of Biotechnology, Yonsei University, Seoul, Korea. 2 Department of Biomaterials Science and Engineering, Yonsei University, Seoul, Korea.
Key words: hairless mice; Kaempferia parviflora; matrix metalloproteinases (MMPs); NF-κB; photoaging
Correspondence: Prof Jae-Kwan Hwang, PhD, Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea. Tel: +822 2123 5881 Fax: +822 362 7265 e-mail: [email protected]
Accepted for publication: 3 December 2013
Conflicts of interest: None declared.
SUMMARY Background Chronic skin exposure to ultraviolet (UV) light increases reactive oxygen species (ROS) and stimulates the expression of matrix metalloproteinases (MMPs) through c-Jun and c-Fos activation. These signaling cascades induce the degradation of extracellular matrix (ECM) components, resulting in photoaging. Methods This study evaluated the preventive effect of the ethanol extract of Kaempferia parviflora Wall. ex. Baker (black ginger) on UVB-induced photoaging in vivo. To investigate the antiphotoaging effect of K. parviflora extract (KPE), UVBirradiated hairless mice administered oral doses of KPE (100 or 200 mg/kg/ day) for 13 weeks. Results In comparison to the UVB control group, KPE significantly prevented wrinkle formation and the loss of collagen fibers with increased type I, III, and VII collagen genes (COL1A1, COL3A1, and COL7A1). The decrease in wrinkle formation was associated with a significant reduction in the UVB-induced expression of MMP-2, MMP-3, MMP-9, and MMP-13 via the suppression of c-Jun and c-Fos activity. KPE also increased the expression of catalase, which acts as an antioxidant enzyme in skin. In addition, expression of inflammatory mediators, such as nuclear factor kappa B (NF-κB), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2), was significantly reduced by KPE treatment. Conclusion The results show that oral administration of KPE significantly prevents UVBinduced photoaging in hairless mice, suggesting its potential as a natural antiphotoaging material. Photodermatol Photoimmunol Photomed 2014; 30: 237–245
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Two distinct biological processes are thought to underlie skin aging: intrinsic aging by the passage of time and extrinsic aging induced by environmental factors, especially ultraviolet (UV) light. Extrinsic aging through UV irradiation is also referred to as photoaging, and during photoaging, UV irradiation induces the breakdown of skin components like collagen, elastin, and ground substances (1). Specifically, UV irradiation causes collagen breakdown in dermal connective tissue and significantly reduces the expression of type I collagen, the most abundant structural protein of skin (2). UV irradiation also causes the production of reactive oxygen species (ROS), direct or indirect DNA damage, and damage to extracellular matrix (ECM) integrity (3). When human skin is exposed to acute UV irradiation, it causes altered pigmentation, inflammation, immune suppression, sunburn, and dermal connective tissue damage. In contrast, chronic exposure of human skin to UV irradiation breaks down the normal architecture of skin connective tissue, impairs skin function, and ultimately causes photoaging (4). During UV-induced ROS production, activation of the mitogen-activated protein kinases/associated protein-1 (MAPK/AP-1) pathway is triggered. UV irradiation induces the phosphorylation of three MAPK proteins: extracellular signal regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Also activated by UV irradiation are c-Jun and c-Fos, which constitute the AP-1 protein complex and leads to the overexpression of matrix metalloproteinases (MMPs) (2). MMPs are in a large family of zinc and calciumdependent endopeptidases, and they play pivotal roles in tumor invasion, inflammation, and skin aging. UV-induced MMPs are especially responsible for tissue remodeling via the degradation of ECM components including collagen, elastin, and fibrillin-1 (4). Recent studies have demonstrated that the inhibition of MMP production suppresses UV-induced epidermal thickening and wrinkle formation (5, 6). Therefore, the regulation of MMPs may be an effective strategy for the prevention and treatment of photoaging. In addition to MMPs, UV irradiation activates nuclear factor kappa B (NF-κB), a transcription factor which stimulates the transcription of pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and cyclooxygenase-2 (COX-2) in human skin. These cytokines activate their cell surface receptors, resulting in epidermal proliferation, various UV-induced cellular responses, and inflammation. This UV-induced inflammation is characterized by erythema, immunosuppression, and edema (7). 238
Kaempferia parviflora Wall. ex Baker, commonly known as black ginger, belongs to the Zingiberaceae family. Black to purple rhizomes of K. parviflora have been used as dietary supplements and traditional medicines for the treatment of various illnesses in tropical countries (8), and previous studies confirm a variety of K. parviflora’s bioactivities. K. parviflora confers known antioxidative, anti-inflammatory, anticancer, antiviral, and gastroprotective effects (9–11), but the antiphotoaging effects of K. parviflora have not been reported to date. Therefore, we examined the protective effect of orally administrated K. parviflora extract on UVB-induced photoaging in vivo by evaluating various parameters such as wrinkle formation, skinfold thickness, collagen reduction, MMP regulation through c-Jun and c-Fos inhibition, catalase expression, and pro-inflammatory cytokine levels.
MATERIALS AND METHODS Preparation of KPE Rhizomes of K. parviflora were collected in Bangkok, Thailand. A voucher specimen was deposited at the Department of Biotechnology, Yonsei University, Seoul, Korea. The dried rhizomes were ground and then soaked in 95% ethanol for 24 h at room temperature. K. parviflora filtrate was concentrated under reduced pressure using a rotary evaporator (Heidolph Instruments GmbH & Co. KG., Schwabach, Germany) to obtain K. parviflora extract (KPE) with a yield of 9.56%.
Animal experiments Five-week-old female hairless mice (SKH-1) were purchased from Daehan Biolink Ltd. (Eumsung, Korea). The mice were housed in temperature (23 ± 2°C), humidity (55 ± 10%), and light (12 h day/night) controlled rooms at Yonsei Laboratory Animal Research Center (YLARC, Seoul, Korea). Twenty mice were randomly allocated to each group (five mice per group, four total groups): (i) control group (Normal), (ii) UVB-irradiated group (UVB), (iii) UVB-irradiated and 100 mg/kg/day KPEtreated group (KPE-100), and (iv) UVB-irradiated and 200 mg/kg/day KPE-treated group (KPE-200). The mice in the KPE-treated groups (100 or 200 mg/kg/day) were administered oral doses of KPE daily for 13 weeks with concurrent exposure to UVB irradiation three times per week on the back of the mice. UVB was supplied by the UV crosslinker CL-1000 M (UVP, Cambridge, UK), using 302 nm UV wavelength. UVB irradiation began with a starting dose of 75 mJ/cm2 during the first week, and then Photodermatol Photoimmunol Photomed 2014; 30: 237–245 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Antiphotoaging effect of Kaempferia parviflora
the dose was increased weekly by one minimal erythema dose (MED) until reaching three MEDs, which was then maintained until 13 weeks of total treatment time. After 13 weeks of treatment, the mice were anesthetized and sacrificed by intraperitoneal injection of a mixture of zoletil and rompun (One Bio, Daejeon, Korea). Skin biopsy samples were obtained from the dorsal skin of the hairless mice, rapidly frozen in liquid nitrogen, and stored at −70°C. Samples for histological analysis were fixed in 10% buffered formalin for optical microscopy.
The membranes were then detected with primary antibodies against catalase, c-Jun, c-Fos, MMP-3, MMP-13, NF-κB, IL-1β, COX-2, and α-tubulin (Cell Signaling, Beverly, MA, USA). Bound antibodies were detected with a horseradish peroxidase-linked secondary antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA). Blotted antibody signals were detected with ECL detection solution and were visualized by the G:BOX Image Analysis System (Syngene, Cambridge, UK).
Gelatin zymography Reverse transcription-polymerase chain reaction (RT-PCR) PCR amplification of cDNA products (5 μl) was performed with PCR premix and the following primer pairs (Bioneer, Daejeon, Korea): mouse β-actin (forward, 5'-CCA GCC AGC CAC CAT CGC TC-3'; reverse, 5'-TGA CCT TGG CCA GGG GTG CA-3'), mouse COL1A1 (forward, 5'-GTC CCC AAT GGT GAG ACG TG-3'; reverse, 5'-GCA CGG AAA CTC CAG CTG AT-3'), mouse COL3A1 (forward, 5'-AGC GGC TGA GTT TTA TGA CG-3'; reverse, 5'-AGC ACA GGA GCA GGT GTA GA-3'), mouse COL7A1 (forward, 5'-AAG CCG AGA TTA AGG GCT GG-3'; reverse, 5'-CAC CAA ATG GAG CAC AGC AG-3'), mouse MMP-3 (forward, 5'-TAG CAG GTT ATC CTA AAA GCA-3'; reverse, 5'-CCA GCT ATT GCT CTT CAA T-3'), and mouse MMP-13 (forward, 5'-CAT CCA TCC CGT GAC CTT AT-3'; reverse, 5'-GCA TGA CTC TCA CAA TGC GA-3'). Before PCR amplification, the primers were denatured at 94°C for five minutes. Amplification consisted of 25 cycles: denaturation at 94°C for 30 s, annealing at 56°C for one minute, extension at 72°C for one minute, then a final five-minute extension at 72°C. PCR was performed in the Gene Amp PCR System 2700 (Applied Biosystems, Foster City, CA, USA). The PCR products were separated by 1.5% agarose gel electrophoresis and visualized by 6X Loading STAR solution and UV illumination using the G:BOX Image Analysis System (Syngene, Cambridge, UK).
Western blot analysis Homogenized skin sections were lysed in RIPA lysis buffer (ELPIS-Biotech, Daejeon, Korea) with a protease inhibitor cocktail (Sigma-Aldrich Co., St. Louis, MO, USA). Protein concentrations were determined by the Bradford assay. For Western blotting, equal amounts of protein (30 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST). Photodermatol Photoimmunol Photomed 2014; 30: 237–245 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
MMP-2 and MMP-9 activity in the skin sections was assessed by gelatin zymography. Samples were mixed with standard gel loading buffer containing 2% SDS without β-mercaptoethanol and loaded on the gel without prior heating. The samples were separated by electrophoresis on a 10% polyacrylamide gel containing SDS and 1% gelatin at 85 V for two hours in a Bio-Rad Mini PROTEAN 3 Cell electrophoretic apparatus (Bio-Rad Laboratories, Seoul, Korea). After electrophoresis, the gel was washed with 2.5% Triton X-100 for one hour to remove SDS and then incubated in reaction buffer (50 mM Tris-HCl [pH 7.5] containing 10 mM CaCl2 and 0.15 M NaCl) at 37°C. After 20 h, the gel was stained with Coomassie® Brilliant Blue R-250 and then destained with 30% methanol and 10% acetic acid. MMP-2 and MMP-9 were detected at 72 kDa and 92 kDa, respectively, as a clear zone against the dark background.
Evaluation of skin wrinkle formation After 13 weeks of KPE treatment, the dorsal skin surface impressions of anesthetized hairless mice were measured using the Visioline VL650 Replica full kit (Epigem, Seoul, Korea) and analyzed with Visioline VL650 (CK Electronics GmbH, Cologne, Germany). The analyzed data represented the number, depth, length, and total area of the wrinkles.
Histological analysis After sacrifice, the skin samples were fixed in 10% formalin for 24 h and stained with hematoxylin and eosin (H&E) for skin layers and Masson’s trichrome for collagen fibers. The stained sections were analyzed using the Eclipse TE2000U Inverted Microscope with twin CCD cameras (Nikon, Tokyo, Japan).
Total collagen assay Skin tissues (400 mg) were homogenized with glass beads in 1 ml of 6 N HCl and hydrolyzed at 105°C for 18 h. The 239
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Fig. 1. The effect of KPE on wrinkle formation. (a) The dorsal skin surface of hairless mice was exposed to UVB irradiation three times a week for 13 weeks. Before sacrifice, the skin replica samples of the dorsal areas were taken. (b) Wrinkle values were obtained from the skin replica analysis. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, **P < 0.01.
collagen content was measured using a total collagen assay kit (Quickzyme Bioscience, Leiden, Netherlands).
Statistical analysis Results are expressed as the mean ± standard deviation (SD) of five mice in each group. All groups were compared by one-way analysis of variance (ANOVA), followed by the Duncan test (SPSS 12.0, SPSS Inc., Chicago, IL, USA). ## P < 0.01, #P < 0.05, **P < 0.01, and *P < 0.05 were considered to be statistically significant.
The effect of KPE on UVB-induced skin thickness Epidermal thickness is known as a quantitative parameter of the inflammatory response in skin photoaging. Chronic UV irradiation induces skin inflammation, which causes excessive epidermal proliferation or skin thickening (3). H&E staining showed that the epidermal thickness was significantly reduced by KPE treatment as compared to the UVB control group (Fig. 2), indicating KPE’s inhibitory effects on UVB-induced skin thickening.
RESULTS
The effect of KPE on UVB-induced collagen degradation
The effect of KPE on UVB-induced wrinkle formation
During skin aging, UV irradiation reduces the spatial density of collagen bundles. Collagen content is reduced either by a decrease in collagen synthesis or an increase in collagen degradation (2). UVB irradiation stimulated the degradation of collagen fibers in the UVB control group as represented by the smaller blue-stained area, while KPE significantly inhibited collagen degradation (Fig. 3A). A quantitative assay measuring total collagen content also supported the preventive effects of KPE on collagen degradation (Fig. 3B). mRNA levels of COL1A1, COL3A1, and COL7A1, which synthesize types I, III, and VII collagen, were significantly increased by KPE compared to the
Repetitive UV irradiation increases the production of MMPs, which can result in wrinkle formation by degrading various ECM components (2). In the dorsal skin of hairless mice, the oral administration of KPE significantly reduced UVB-induced wrinkle formation as compared to the UVB control group (Fig. 1A). Quantitative analysis of wrinkle values, which represent the number, depth, length, and total area of the wrinkles, was consistent with the photographs (Fig. 1B). These results indicate that KPE effectively attenuates UVB-induced wrinkle formation. 240
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Fig. 2. The effect of KPE on skin thickness. (a) Skin tissue sections were stained with H&E, representing epidermal thickness (original magnification ×400). (b) Skinfold thickness was measured with a caliper mid-way between neck and hips. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, **P < 0.01.
Fig. 3. The effect of KPE on collagen fibers. (a) Skin tissue sections were stained with Masson’s trichrome, identifying collagen fibers (original magnification ×400). (b) The amount of collagen was estimated after UVB irradiation for 13 weeks. (c) mRNA levels of COL1A1, COL3A1, and COL7A1 was evaluated by RT-PCR. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, *P < 0.05, **P < 0.01.
UVB control group (Fig. 3C). These results suggest that KPE prevents UVB-induced collagen degradation and promotes collagen synthesis.
The effect of KPE on UVB-induced MMP expression UV irradiation induces MMP expression, resulting in the degradation of various ECM components and characteristic changes associated with skin photoaging (3). Levels of MMP-3 and MMP-13 mRNA and protein were significantly reduced in the KPE-treated group as compared to the UVB control group (Fig. 4A,B). Gelatin zymography analysis showed that KPE also reduced MMP-2 and MMP-9 activity levels (Fig. 4C). Taken together, these data demonstrate that KPE significantly reduces UVB-induced MMP expression.
The effect of KPE on AP-1 complex and catalase expression MMP secretion is caused by the activation of the AP-1 complex. Activated c-Jun and c-Fos, which form the AP-1 Photodermatol Photoimmunol Photomed 2014; 30: 237–245 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
complex, are associated with the transcription of MMPs (2). In this study, c-Jun and c-Fos protein levels were highly activated in the UVB control group, while KPE significantly attenuated the expression of c-Jun and c-Fos in a dose-dependent manner (Fig. 5A). These results suggest that KPE prevents AP-1 activation. Catalase protects the skin against the deleterious effects of UV-induced oxidative stress (12). Chronic UVB exposure reduced catalase expression in hairless mice, while KPE significantly increased the level of catalase protein compared to the UVB control group (Fig. 5B). These results suggest that KPE may effectively scavenge ROS by increasing catalase expression.
The effect of KPE on UVB-induced inflammatory responses NF-κB is activated by UV irradiation and further stimulates the transcription of pro-inflammatory cytokines, leading to cellular responses, epidermal proliferation, and inflammation (7). The UVB control group showed higher levels of inflammatory factors such as NF-κB, IL-1β and 241
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Fig. 4. The effect of KPE on MMP expression. (a) The level of mRNA expression and (b) protein levels of MMP-3 and MMP-13 were determined by RT-PCR and Western blotting, respectively. (c) MMP-2 and MMP-9 activity was analyzed by gelatin zymography. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, *P < 0.05, **P < 0.01.
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Fig. 5. The effects of KPE on AP-1 and catalase expression. (a) c-Jun and c-Fos expression and (b) Catalase expression was evaluated by Western blotting. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, **P < 0.01.
Fig. 6. The effect of KPE on NF-κB, IL-1β, and COX-2 expression. NF-κB, IL-1β, and COX-2 expression levels were determined by Western blotting. Data are expressed as mean ± SD of five mice in each group; ##P < 0.01, *P < 0.05, **P < 0.01.
COX-2. However, KPE significantly inhibited their expression in a dose-dependent manner (Fig. 6). These results indicate that KPE protects skin from UVB-induced inflammation by suppressing NF-κB, IL-1β, and COX-2 expression.
DISCUSSION In the present study, we investigated the preventive effect of KPE against UVB-induced photoaging by analyzing clinical and histological changes in hairless mice (Figs 1 and 2). In mice and humans, hyperpigmentation, roughness, dryness, coarse elastosis, laxity, and wrinkling are the Photodermatol Photoimmunol Photomed 2014; 30: 237–245 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
clinical symptoms of skin photoaging (6). Subclinically, skin aging is further characterized skin thickening. These clinical and subclinical changes are the result of skin cell hyperproliferation and collagen degradation, specifically, these phenomena are associated with reduced levels of collagen, a basic component of the skin’s dermal layer. Our study showed that oral administration of KPE increased total collagen levels of the dorsal skin of hairless mice by increasing COL1A1, COL3A1, and COL7A1 mRNA expression (Fig. 3). Collagen, a fibrous protein, is a major constituent of connective skin tissues, and the functional properties of the skin are highly dependent on collagen integrity in the dermis (13). Several different types of collagen are involved in ECM formation. Most collagen fibers in the dermis are types I and III collagens. On the other hand, type VII collagen plays an important role in supporting the skin barrier, acting like an anchoring protein at the dermal epidermal junction (DEJ) (1). One mechanism by which photoaging may occur is through abnormal increases in MMP production. Therefore, natural materials which reduce MMP production may successfully protect against photoaging (14). As shown in Fig. 4, UVB-induced expression of MMP-2, MMP-3, MMP-9, and MMP-13 (also known as gelatinase-A, stromelysin-1, gelatinase-B, and collagenase-3, respectively) were significantly inhibited by oral administration of KPE. UV irradiation induces the synthesis of MMP-3, which degrades types III and VII collagen, and MMP-13, which initiates the breakdown of interstitial type I collagen in rodents. MMP-2 and MMP-9, which degrade types IV and VII collagen, are also induced by UV irradiation. Overall, these MMPs are associated with the degradation of various types of collagen which are the major components of the skin dermis (15). Because MMPs play a role in photoaging, understanding their mechanism of action, however complex, is important. UV irradiation induces ROS activation, which leads 243
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to the degradation of ECM components and to detrimental chemical modifications, resulting in the upregulation of c-Jun and c-fos (AP-1). Increased expression of c-Jun and c-fos results in increased MMP expression (16). In our study, KPE significantly reduced the expression of c-Jun and c-Fos, which is likely the pathway by which the levels of several MMPs were reduced. Since wrinkle formation is closely associated with MMPs, their regulation has been targeted as a strategy to prevent photoaging (2), and KPE may be a viable candidate by which to reduce this MMPinduced photoaging. Of notable mention, some natural materials have also been reported to possess effective antiphotoaging activities in vitro by attenuating MMP expression through c-Jun and c-fos (AP-1) signaling inhibition (16, 17), so our in vivo results are consistent with this work. Previous studies have also demonstrated that major antioxidant enzymes such as catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) protect UV-induced oxidative effects. Among these antioxidant enzymes, catalase is a representative epidermal differentiation marker in redox damage, and it acts to reduce photooxidative stress (18). In the present study, KPE significantly increased catalase protein levels (Fig. 5), which may be one mechanism by which photoaging was reduced. One way in which oxidative stress might contribute to photoaging is by inducing NF-κB signaling, which is associated with MMP expression in the skin. After UV irradiation, NF-κB, a transcription factor, is activated and increases MMP production. In the present study, KPE effectively inhibited UVB-induced inflammatory signaling by reducing the expression of NF-κB, IL-1β, and COX-2
(Fig. 6). Because UV irradiation induces inflammatory mediators which enhance photoaging (7), KPE’s inhibition of this process is valuable information. It is possible that KPE may be an appropriate supplement to such NF-κB inhibitors, or may be an appropriate alternative treatment. Although we have illustrated some ways by which KPE might confer its beneficial effects, further work exploring KPE’s other mechanisms of action are necessary. It has been reported that the rhizomes of K. parviflora contain high amounts of polymethoxyflavones, such as 5,7-dimethoxyflavone, 3,5,7trimethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone (19, 20), and also some kaempferiaosides and phenolic glycosides (21, 22). Previously, we identified the inhibitory effects of 5,7-dimethoxyflavone on UVB-induced skin photoaging in vitro (17). However, one future approach would be to clarify whether 5,7dimethoxyflavone also effectively functions as an antiphotoaging material in vivo.
CONCLUSION Oral administration of KPE significantly attenuated UVBinduced wrinkle formation, skin thickening, and collagen degradation in hairless mice, and also increased COL1A1, COL3A1, and COL7A1 expression. In addition, KPE effectively reduced MMP expression by inhibiting the activation of c-Jun and c-Fos. KPE also increased catalase expression and markedly suppressed UVB-induced inflammatory responses. Overall, K. parviflora has the potential to be an effective natural anti-photoaging material.
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