Experimental Gerontology 61 (2015) 147–155

Contents lists available at ScienceDirect

Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero

The protective effect of 18β-Glycyrrhetinic acid against UV irradiation induced photoaging in mice Song-Zhi Kong a,b,1, Hai-Ming Chen b,1, Xiu-Ting Yu b, Xie Zhang b, Xue-Xuan Feng b, Xin-Huang Kang a, Wen-Jie Li c, Na Huang a, Hui Luo c,⁎, Zi-Ren Su b,⁎ a b c

College of Science, Guangdong Ocean University, Zhanjiang, Guangdong, People's Republic of China School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People's Republic of China School of Pharmacy, Guangdong Medical College, Zhanjiang, Guangdong, People's Republic of China

a r t i c l e

i n f o

Article history: Received 6 November 2014 Received in revised form 1 December 2014 Accepted 9 December 2014 Available online 11 December 2014 Section Editor: B. Grubeck-Loebenstein Keywords: 18β-Glycyrrhetinic acid Skin photoaging Ultraviolet Anti-inflammation Anti-oxidant Matrix metalloproteinases

a b s t r a c t It has been confirmed that repeated exposure of skin to ultraviolet (UV) radiation results in cutaneous oxidative stress and inflammation, which act in concert to cause premature skin aging, well known as photoaging. 18βGlycyrrhetinic acid (GA), widely used to treat various tissue inflammations, is the main active component of licorice root, and has also been shown to possess favorable anti-oxidative property and modulating immunity function. In the present study, we investigated the potential protective effect of GA on UV-induced skin photoaging in a mouse model. During the experimental period of ten consecutive weeks, the dorsal depilated skin of mice was treated with topical GA for 2 hours prior to UV irradiation. The results showed that GA pretreatment significantly alleviated the macroscopic and histopathological damages in mice skin caused by UV. Meanwhile, the data also indicated that GA markedly up-regulated the activities of the antioxidant enzymes (SOD, GSH-Px), and increased the content of skin collagen, while obviously decreased malonaldehyde level and inhibited high expressions of matrix metalloproteinase-1 (MMP-1) and -3 (MMP-3), as well as down-regulated the expression of inflammatory cytokines such as IL-6, TNF-α and IL-10. Taken together, these findings amply demonstrate that GA observably attenuates UV-induced skin photoaging mainly by virtue of its antioxidative and antiinflammatory properties, as well as regulating the abnormal expression of MMP-1 and MMP-3. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Photoaging is a well-known term that denotes the cutaneous clinical, histological and functional changes induced by chronic and repeated ultraviolet (UV) radiation exposure, especially directly exposed to UVB (290–320 nm) and UVA (320–400 nm) (Battie et al., 2014; Chung, 2003; Sanches Silveira and Myaki Pedroso, 2014). The clinical characteristics of photoaged skin have been known for a long time, which mainly includes coarse wrinkles, laxity, irregular pigmentation and a leathery appearance (Berneburg et al., 2000), while the histological changes have been observed including stratum corneum hyperkeratosis, Abbreviations: UV, ultraviolet; GA, 18β-Glycyrrhetinic acid; MMPs, matrix metalloproteinases; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; NC, naive control group; SC, shaved control group; MC, model control group; VC, vehicle control group; GA-L, GA group at the dose of 2.25 mg/mouse; GA-M, GA group at the dose of 4.5 mg/ mouse; GA-H, GA group at the dose of 9 mg/mouse; ECM, extracellular matrix; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-10, interleukin-10; Hyp, hydroxyproline; MED, minimal erythemal dose; DEJ, dermal-epidermal junction; NF-κB, nuclear factor-κB; AP-1, activator protein 1. ⁎ Corresponding authors. E-mail addresses: [email protected] (H. Luo), [email protected] (Z.-R. Su). 1 Song-Zhi Kong and Hai-Ming Chen contributed equally to this work.

http://dx.doi.org/10.1016/j.exger.2014.12.008 0531-5565/© 2014 Elsevier Inc. All rights reserved.

abnormal deposited elastic fibers and the disorganization of collagen fibrils (Feng et al., 2014; Fisher et al., 1997; Lavker, 1995). Extensive studies for the last several decades have proved that UV-induced oxidative stress, resulting from excessive production of reactive oxygen species (ROS), as well as its induced various inflammatory and immunosuppressive factors play crucial roles in the pathogenesis of photoaging (Herrling et al., 2006; Scharffetter-Kochanek et al., 2000). Specifically, UV-induced oxidative stress depletes and damages non-enzymatic and enzymatic antioxidant defense systems of the skin, and oxidatively modifies DNA, lipids and proteins finally leading to skin cell apoptosis (Fisher et al., 2002; Kasapoglu and Ozben, 2001). Meanwhile, the oxidative stress activates mitogen-activated protein kinase (MAPK) signaling transduction pathways, and then causes the release of inflammatory and immunosuppressive cytokines such as IL-6 and IL-10, as well as induces the expression of matrix metalloproteinases (MMPs) in skin cells (Nichols and Katiyar, 2010; Pillai et al., 2005). MMPs are the key enzymes in the degradation of extracellular matrix (ECM), an essential structural framework for skin cell, which mainly includes collagen and elastic fibers and is responsible for the formation of coarse wrinkles and sagging skin (Rabe et al., 2006). Based on these mechanism analyses of skin photoaging, the agents with anti-oxidant, anti-inflammatory and/or modulating immunity functions might be of preventive or

148

S.-Z. Kong et al. / Experimental Gerontology 61 (2015) 147–155

potential therapeutic value in this disease. In addition, the recent history of skin anti-aging treatment has witnessed an upsurging interest in the use of natural herbal compounds not only because of their characteristic of amicable safety profile, but also because of their high biological activities, especially antioxidative and anti-inflammatory functions (Hwang et al., 2011; Pillai et al., 2005). Licorice species are perennial herbs native to the Mediterranean region, central to southern Russia, and Asia Minor to Iran, now widely cultivated throughout Europe, the Middle East and Asia (Blumenthal et al., 2000; Gong et al., 2008). Licorice has been widely used in traditional Chinese medicinal preparations since ancient times, and described as ‘the grandfather of herbs’ (Ody, 2000). Its major bioactive compound 18β-Glycyrrhetinic acid (GA, the chemical structure is shown in Fig. 1), an aglycone and active metabolite of glycyrrhizic acid, has been proved to possess a variety of pharmacological effects such as anti-inflammatory, antiallergic, anti-carcinogenic and antiimmunemediated cytotoxicity by numerous prior researches (Carmeli et al., 2008; Maitraie et al., 2009). Moreover, in recent years, this compound has also been reported to inhibit the inception and growth of skin tumors (Gong et al., 2008; Wang et al., 1991), and has a good antioxidant property as evidenced by increased antioxidant status and decreased lipid peroxidation (Kalaiarasi and Pugalendi, 2011; Maitraie et al., 2009). However, no report has been issued on the protective effects of this compound against UV-induced skin aging in vivo. In view of its above bioactivities, whether GA also possesses protective effect against this high-profile disease is clearly worth exploring. Hence, the present study aimed to investigate if GA has a photo-protective activity by topical application in UV-induced photoaged mice skin mainly via measuring the parameters of oxidative stress and inflammation, as well as macroscopic and histopathological evaluation.

well as tumor necrosis factor (TNF)-α, interleukins (IL)-6 and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA), and the ELISA kits were obtained from eBioscience, Inc. (San Diego, CA, USA). All other chemicals and reagents used in the study were of analytical grade. 2.2. Animals Eight-week-old female SPF KM mice, non-inbred closed colony (Shang et al., 2009; Zhang et al., 2007), weighted approximately 20 to 22 g, were purchased from the animal center of Guangzhou University of Chinese Medicine (Guangzhou, China). After their arrival, animals were housed at the laboratory animal research center at the Guangzhou University of Chinese Medicine following the Guide for the Care and Use of Laboratory Animals. The mice were maintained under controlled temperature (23 ± 2 °C), humidity (55 ± 10%), automatic lighting (alternating 12-h periods of light and dark, without any ultraviolet emission) and given food and water ad libitum throughout the study period. All experimental protocols were approved by the Committee for Animal Care and Use at Guangzhou University of Chinese Medicine. 2.3. Grouping of experimental animals Mice were acclimated for 1 week before starting the experiment and then were randomly divided into seven groups of nine mice each according to Table 1. At the beginning of the experiment, dorsal hair of mice was shaved with lady shavers (Philips®) for a 2.5 × 3 cm2 skin area after anesthetized by ether inhalation. Thereafter, the shaving operation was performed as required (usually once a day).

2. Materials and methods

2.4. Preparation of photoaged mouse model and GA treatment

2.1. Materials and chemicals

In order to establish photoaged mouse model, the mice were irradiated as described in our previous research with slight modification (Kong et al., 2013). Briefly, simulated solar irradiation was provided by an array of seven UVB lamps, with an emission spectrum between 285 and 350 nm (peak at 310–315 nm), surrounding three UVA lamps (Waldmann UV800, Germany) emitting exclusively UVA in the range of 320–400 nm (peak at 365 nm). The integrated UV irradiance was measured with a Waldmann UV meter (Waldmann Lichttechnik GmbH, Germany), and the minimal erythemal dose (MED) was determined to be approximately 75 mJ/cm2 for the mice skin. During the five times weekly (except Thursday and Sunday) UV exposure, the mice were group-housed in a flat stainless steel irradiation chamber under the UV lamp keeping the distance at 30 cm. The initial exposure dose was 1 MED (75 mJ/cm2) during the first week, then was increased weekly by 1 MED up to 4 MED (300 mJ/cm2), which was maintained for the rest weeks, yielding a total dose of 12.75 J/cm2. The non-irradiated control group was treated identically except the UV lamps were switched off. Two hours before exposure to UV, the shaved dorsal skins of mice were pretreated with the sample solutions (three different doses of

GA, white crystal powder, was purchased from Sigma-Aldrich (St Louis, MO, USA), and its purity (N98%) was determined by high performance liquid chromatography (HPLC). GA was dissolved in propylene glycol–ethanol (7:3, v/v) to yield three different concentrations: 20 mg/ml, 40 mg/ml and 80 mg/ml. Commercial kits used to detect the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, as well as malondialdehyde (MDA) and mouse hydroxyproline (Hyp) contents were purchased from Jiancheng institution of Biotechnology (Nanjing, China). In addition, the contents of matrix metalloproteinase (MMP)-1 and -3, as

Table 1 Treatment schedule of the study.

Fig. 1. Chemical structure of 18β-Glycyrrhetinic acid (GA).

Groups

Shave

UV radiation

Vehicle 120 μl/mouse

GA (mg/mouse) 2.25

4.5

9

Naive control (NC) Shaved control (SC) Model control (MC) Vehicle control (VC) GA low dose (GA-L) GA middle dose (GA-M) GA high dose (GA-H)

− + + + + + +

− − + + + + +

− − − + + + +

− − − − + − −

− − − − − + −

− − − − − − +

+: with treatment, −: without treatment.

S.-Z. Kong et al. / Experimental Gerontology 61 (2015) 147–155

GA) or the vehicle (120 μL per mouse), which were administered daily as the treatment schedule displayed in Table 1. 2.5. Macroscopic evaluation of dorsal skin Visible diversification of dorsal skin in mice was examined and photographed while the mouse was under anesthesia once a week for 10 weeks. The grade of dorsal skins was determined by an observer who was blind to the grouping according to evaluation criteria exhibited in Table 2, modified from Bissett et al. (1990). 2.6. Skin elasticity test (pinch test) For testing skin elasticity, pinch test was carried out weekly according to the modified protocol of Tsukahara et al. (2005). In detail, the dorsal skin at the midline of anesthetized mouse was picked up with fingers as much as possible until the lower limbs of mouse almost off the ground, and then released the fingers. Subsequently, the time(s) of dorsal skin recovered to its original state was measured. 2.7. Histological examination As previously described (Kong et al., 2013), dorsal skin samples (approximately 1 × 0.4 cm) were obtained by quickly stripped at the end of 10th week, fixed in 10% buffered formalin, embedded in paraffin and finally sectioned at 5 μm-thickness. After cleared in Histo-clear (ASONE, Tokyo, Japan), these samples were stained by Hematoxylin–eosin (H&E) for routine examination of the tissue and quantification of epidermal hyperplasia, as well as stained by Gomori's aldehyde fuchsin method for evaluating the skin elasticity. To quantify epidermal hyperplasia, the thickness of the epidermis was measured at 10 randomly selected locations per slide using an optical microscope (Leica DMLB) with 200 × magnification, which was photographed under a Leica DC 300 camera. Histological alterations were evaluated and quantified through the image analysis program Image J 1.36 (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) (Gaspar and Maia Campos, 2003; Ouhtit et al., 2000). 2.8. Determination of SOD, GSH-Px and MDA in the skin At the end of 10th week, skin samples were harvested from the sacrificed mice and were averaged into two portions (about 0.4 g for each). One portion was homogenized (10,000 rpm, 20 s) with Ultra Turrax (T18 Basic, IKA) in 9 volumes of normal saline at 4 °C to get the 10% skin tissue homogenate. Before centrifugation, 0.15 ml of the skin tissue homogenate was taken out for the MDA assay, and the rest was centrifuged at 3000 × g for 20 min at 4 °C. The total supernatant was saved for subsequent SOD and GSH-Px assays. All of the biochemical assays mentioned above were performed as the manufacturer's protocols of corresponding diagnostic kits (Nanjing Jiancheng Bioengineering Inst., Nanjing, China). Furthermore, protein concentration was also estimated with a BCA kit (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, China) as per the protocols. Table 2 Grading scale for evaluation of photoaging. Grade

Evaluation criteria

0 1 2 3 4 5 6

No wrinkles or laxity; fine striations running the length of the body Fine striations Disappearance of all fine striations Shallow wrinkles A few deep wrinkles and laxity Increased deep wrinkles Severe wrinkles; development of tumors/lesions

0: normal skin, 6: severely photo-damaged skin.

149

2.9. Determination of IL-6, IL-10, TNF-α and MMP-1, MMP-3 in the skin The other portion of skin tissue (0.4 g), as described above, was homogenized (10,000 rpm, 20 s) in 9 volumes of PBS (4 °C) to afford the 10% homogenate, then centrifuged at 3000 ×g for 20 min at 4 °C. The total supernatant was employed to estimate the secreted IL-10, IL-6, TNF-α as well as MMP-1 and MMP-3 by using ELISA kits (eBioscience, San Diego, CA, USA) according to the instructions. Meanwhile, protein concentration was also measured using the above-mentioned method. 2.10. Determination of collagen content Hydroxyproline (Hyp) is a characteristic amino acid of mammalian collagen, from which the collagen content could be converted by multiplying a conversion factor 7.46 (Neuman and Logan, 1950). Hence, in the present study, Hyp in the skin of mice was measured by Hyp ELISA kit according to the manufacturer's instructions (Jiancheng Inst. of Biotechnology, Nanjing, China) for the determination of collagen content. 2.11. Statistical analysis All quantitative data were presented as mean ± standard deviation (SD). Experimental values were analyzed by one-way ANOVA. A value of p b 0.05 was considered to be statistically significant. All analyses were performed using Statistical Analysis Software (SPSS 17.0). 3. Results 3.1. GA ameliorated the macroscopic appearance of photoaged mice skin Macroscopic effects of UV on mice skin were proved in Fig. 2. As the mice in the naive control (NC) group were never shaved and irradiated with UV, they were not photoed for this macroscopical examination every week. Thus, the NC group is omitted in Fig. 2. As displayed in Fig. 2, during the whole experiment period, no significant macroscopic changes but age-related slight wrinkles were observed in the skin of nonirradiated mice (SC group), demonstrating that the shaving did not cause macroscopic skin damages. Starting from week 4, skin damages such as edema, erythema, thickening and coarse wrinkles were observed in eight out of nine (88.9%) MC group mice. Similar to MC, seven out of nine (77.78%) mice in VC group showed apparent erythema, deep-wrinkled, even leathery appearance. However, these UV-induced skin changes were partially restored by treatment with topical GA starting from week 6 (as exhibited in Fig. 2). At the 10th week, healthy skin with smoothness and some shallow wrinkles were exhibited in the mice of GA-H group; meanwhile, a few shallow wrinkles and slightly erythema were observed in the dorsal skin of mice in GA-M group. Additionally, in the GA-L group, 5 mice (55.56%) showed slight flesh-colored lesions, and the other 4 exhibited several deep coarse wrinkles and slightly erythema. Statistically, at week 10, for visual scores, there was no significant difference between MC and VC groups, but both were markedly higher than that in SC group (as shown in Fig. 2B), revealing that vehicle treatment did not affect this macroscopic indicator. However, remarkable decrease in score was observed in GA groups when comparing with that of VC group, indicating that topical treatment of GA prevented these UV-induced macroscopic skin damages. 3.2. GA promoted skin elasticity in pinch test After ten-week treatment, the dorsal skin elasticity of the mice were evaluate by the pinch test weekly, and the photographs of mice dorsal skins after being stretched were demonstrated in Fig. 3A. The recovery times of mice in between MC and VC groups were no significant difference; when compared with SC group, they were observably prolonged

150

S.-Z. Kong et al. / Experimental Gerontology 61 (2015) 147–155

Fig. 2. GA ameliorated visual appearance of photoaged skin. (A) Macroscopic appearance of mice skin with different treatments at the end of experiment period. (B) Results of visual score of different groups during the 10-week study period. Data represent means ± SD (n = 9). #p b 0.05 compared with the SC group; *p b 0.05 compared with the VC group.

(all p b 0.05), which manifested starting from week 4 (as shown in Fig. 3B). Compared with VC group, treatment with GA at doses of 4.5– 9 mg/mouse showed remarkable reduction in the recovery time, while the time of GA-L group showed no statistical difference from VC group. These data indicated that GA promoted the mice to regain its initial shape after deformation, namely improving skin elasticity. 3.3. GA prevented UV-induced skin structure damage Photoaged skin is a complex biologic process involving various layers of the skin, mainly characterized by epidermal hyperplasia, flattening of the dermal–epidermal junction (DEJ), the presence of inflammatory infiltrates, as well as dermal elastosis and collagen degradation (Fisher et al., 1997; Sanches Silveira and Myaki Pedroso, 2014; Scharffetter-Kochanek et al., 2000). As exhibited in H&E staining images (Fig. 4) and elastic fiber staining images (Fig. 5), mice skin in groups NC and SC showed almost similar features, and displayed regular skin layers including normal epidermis covered by thin layer of stratum corneum, a wavy DEJ line, regularly distributed hair follicles, wellordered collagen bundles that interweaved closely, as well as welldistributed elastic fibers, the filaments which were stained deep purple (Fig. 5 NC and SC). Besides, inflammatory infiltration was not observed in these two groups. After ten weeks of UV-irradiation, prominent histopathological features of photodamaged skin were displayed in the MC and VC groups, which were quite similar. Epidermis showed abnormal hyperplasia with thickened stratum corneum. Furthermore, the flat DEJ could be observed beneath epidermis, which dramatically decreased the contact area between epidermis and dermis. Besides, in dermis, sparse and

disorganized collagen fibers were observed, some of which even showed conspicuous fracture (Fig. 4 MC (a), (b) and VC); elastic fibers were twisted, broken, even abnormally accumulated, the density of which was obviously decreased (Fig. 5 MC and VC). In addition, inflammatory infiltrations were clearly displayed in the entire dermis (Fig. 4 MC (c)). Nevertheless, topical pretreatment with GA (especially at doses of 4.5–9 mg/mouse) markedly alleviated these UV-induced skin structure damages. The skin of mice in the GA-H group showed relatively complete epidermis and dermis (as exhibited in Figs. 4 GA-H and 5 GA-H). In particular, thin layer of stratum corneum, well-organized collagen and elastic fibers, of which the thickness and distribution were uniformed, could be clearly observed. The DEJ returned to nearnormal status and revealed obvious waves, and diffused inflammation was absent in and underneath the dermis. When applied with 4.5 mg/ mouse GA, as demonstrated in Figs. 4 GA-M and 5 GA-M, mice skin exhibited thinner stratum corneum and wavy DEJ with well-marked dermal papillae as compared to VC group. In the dermal layer, there was an orderly arrangement of collagen and elastic fibers, and no inflammatory infiltrates. Additionally, GA at the dose of 2.25 mg/mouse (GA-L group) could also protect mice skin from photo-damages, although the thickness of epidermis was thicker and the compressed DEJ, incomplete collagen, fractured elastic fibers, as well as slight inflammation could still be observed (as revealed in Figs. 4 GA-L and 5 GA-L). 3.4. GA alleviated UV-induced epidermal hyperplasia Epidermal hyperplasia, characterized by irregular thickening of the epidermal layer, is the direct reason for pachulosis and wrinkle

S.-Z. Kong et al. / Experimental Gerontology 61 (2015) 147–155

151

Fig. 3. GA improved skin elasticity as evaluated by pinch testing. (A) Photographs of pinch test, performed at the end of the week 10. (B) Results of recovery time of different experiment groups. Data represent means ± SD (n = 9). #p b 0.05 compared with the SC group; *p b 0.05 compared with the VC group.

formation and usually evaluated by directly measuring the thickness of epidermis in the present study (Fisher et al., 1997; ScharffetterKochanek et al., 2000). As shown in Fig. 6, no statistically significant difference was found either between NC and SC groups, or between MC and VC groups, amply indicating that the hair removal operation and vehicle treatment had no obvious influence on epidermal hyperplasia. However, UV irradiation could significantly increase the epidermal thickness of MC and VC mice (p b 0.05 vs. SC group), while the thickness was observably decreased by topical GA application in a dosedependent manner (all p b 0.05 vs. VC group). These data revealed that GA might possess an inhibitory effect on this kind of epidermal hyperplasia caused by repeated UV irradiation.

3.5. GA restored the activities of skin antioxidant enzymes As illustrated in Table 3, there were no significant differences in the activities of SOD and GSH-Px either between NC and SC groups, or between MC and VC groups (all p N 0.05); meanwhile, the activities of them in MC group showed a remarkable decrease by 25.9% and 32.0% respectively when comparing with SC group. However, compared to VC group, GA treatment (especially at the doses of 9 mg/mouse) could significantly enhance the activities of SOD and GSH-Px (all p b 0.05), although SOD activity of GA-M group showed no distinct difference. These data revealed that GA could protect skin from UV-induced oxidative damage by up-regulating the activities of these key antioxidant enzymes.

3.6. GA decreased MDA level MDA level of the skin tissue is generally considered as a vital parameter to evaluate its degree of lipid oxidation, which is photoagingrelated. As exhibited in Table 3, for MDA value, no remarkable difference was found either between NC and SC groups or between MC and VC groups, while an obvious increase was found in MC group (p b 0.05) when comparing with SC group. Nevertheless, the levels of MDA in three GA groups had decreased in a certain degree as compared to VC group (especially at dose of 9 mg/mouse, p b 0.05). 3.7. GA enhanced skin collagen content As displayed in Table 3, there was no significant difference for collagen contents between NC and SC groups, as well as between MC and VC groups, as its content in MC group was remarkably decreased by 26.4% comparing with SC group. However, the content was prominently elevated by treatment with GA at doses of 4.5–9 mg/mouse when compared to VC group (all p b 0.05). Although 2.25 mg/mouse GA was able to reduce the decrease of collagen level induced by UV, no significantly statistical difference was observed (p N 0.05 vs. VC group). 3.8. GA reversed UV-induced increase of MMPs' content As shown in Table 4, for the contents of MMP-1 and MMP-3, no significant difference was found either between NC and SC groups, or

152

S.-Z. Kong et al. / Experimental Gerontology 61 (2015) 147–155

Fig. 4. Hematoxylin & eosin (H&E) staining of the mouse skin in: NC group; SC group, showing the similar features to that in NC group; MC group, MC (a) indicating routine examination, MC (b) displaying fractured and sparse collagen (white box), MC (c) exhibiting inflammation infiltrates in and underneath the dermis (white circle); VC group, having similar characteristics to that in MC group; GA-L group; GA-M group and GA-H group (Scale bars: 100 μm). a, dermal–epidermal junction; b, epidermis; c, dermis; d, subcutaneous tissue; and e, hair follicle.

between MC and VC groups. However, the expression of them was much higher in MC group than that in SC group (all p b 0.05). When treated with topical GA, the contents of MMP-1 and MMP-3 were

markedly decreased with a distinctly dose-dependent manner (especially at dose of 9 mg/mouse, all p b 0.05 vs. VC group). These data revealed that treatment with GA could protect the skin from UV-

Fig. 5. Gomori's aldehyde fuchsin staining of the mouse skin for elastic fiber examination, which manifested as deep purple fine line (black arrows), in: NC group, NC (a) indicating routine examination, NC (b) displaying the morphology and distribution of skin elastic fibers (a magnified view of the white box); SC group; MC group; VC group; GA-L group; GA-M group and GA-H group (Scale bars: 100 μm). The white circles indicate the fragment of elastic fibers.

S.-Z. Kong et al. / Experimental Gerontology 61 (2015) 147–155

153

Fig. 6. GA suppressed the UV-induced epidermal thickening. (A) Photographs of epidermal thickness observed by H&E staining (Scale bars: 100 μm). The double-headed white arrows indicate the width of epidermis. (B) The average epidermal thickness of the mice skin upon various treatments. Data represent means ± SD (n = 6). #p b 0.01 compared with the SC group; *p b 0.05 compared with the VC group.

induced collagen damage by down-regulating the overexpressions of MMP-1 and MMP-3, which play major roles in the degradation of skin collagen.

attenuate the abnormal expression of IL-6, IL-10 and TNF-α in a certain degree, especially at dose of 9 mg/mouse (all p b 0.05 vs. VC group). 4. Discussion

3.9. GA suppressed the production of inflammatory cytokines Since the aberrant production of inflammatory cytokines such as IL6, IL-10 and TNF-α, has an important role in the process of skin photoaging (Nichols and Katiyar, 2010; Thornfeldt, 2008), these key inflammatory cytokines were examined in the present research. As depicted in Table 4, for the levels of IL-6, IL-10 and TNF-α, no significant differences were observed between NC and SC groups, as well as between MC and VC groups; the levels of them were obviously increased in MC group (all p b 0.05 vs. SC group). However, pretreatment of GA could Table 3 Effects of GA on SOD and GSH-Px activities, as well as MDA and collagen contents of photoaged mice skin. Group

NC SC MC VC GA-L GA-M GA-H

SOD

GSH-PX

MDA

Collagen

UA/mgprot

UB/mgprot

nmol/mgprotC

μg/mgD

41.97 ± 4.47 41.08 ± 10.16 30.44 ± 5.96# 29.9 ± 5.06# 49.8 ± 6.41* 38.34 ± 4.02 49.69 ± 6.47*

47.46 ± 7.3 45.37 ± 8.37 30.87 ± 8.25# 32.32 ± 4.95# 36.41 ± 7.8 40.06 ± 6* 42.23 ± 8.97*

1.05 ± 0.38 1.07 ± 0.25 3.16 ± 0.55# 3.09 ± 0.93# 2.33 ± 0.65 2.29 ± 0.81 1.94 ± 0.6*

18.12 ± 1.82 17.53 ± 1.99 12.9 ± 1.25# 11.97 ± 2.25# 13.52 ± 1.94 14.19 ± 1.21* 14.2 ± 2.34*

Each value represents the mean ± SD of 9 mice per group. #p b 0.05 compared with the SC group; *p b 0.05 compared with the VC group. A One unit of SOD activity was defined as the amount of the enzyme inhibiting the oxidation by 50%. B One unit of glutathione peroxidase was defined as the amount of the enzyme leading 1 μmol GSH oxidized per min. C MDA content was expressed as nmol per mg protein. D Collagen content was expressed as μg per mg skin.

It has been widely accepted that the primary mechanisms of UVinduced skin photoaging involve oxidative stress and inflammatory reaction, which eventually result in the structural and functional alterations of extracellular matrix (ECM), e.g. collagen and elastic fibers (Khavkin and Ellis, 2011; Quan et al., 2009; Sanches Silveira and Myaki Pedroso, 2014). In recent decades, attention has been focused on finding the natural herbal compounds with high biological activities, especially antioxidative and anti-inflammatory functions, for preventing and controlling this kind of disease (Chen et al., 2012; Yaar and Gilchrest, 2007). 18β-Glycyrrhetinic acid (GA), a major bioactive ingredient in Licorice root, has been commonly used as a classic anti-inflammatory medicament, and has also been reported to possess a good antioxidant property (Jayasooriya et al., 2014; Kalaiarasi and Pugalendi, 2011; Wang et al., 2011), indicating a potential for the prevention of photoaging. Thus, in the present study, the protective efficacy of GA in photoaged mice skin was evaluated. The data demonstrated that topical treatment with GA significantly alleviated UV-induced skin macroscopic and histopathological damages mainly by up-regulating the activities of key antioxidant enzymes, suppressing inflammatory response, as well as inhibiting the high expressions of MMP-1 and MMP-3. As mentioned above, UV-induced oxidative stress decreases the antioxidant ability of skin, and causes direct deleterious chemical modifications to the cellular components, including DNA, proteins and lipids, which then promote fibroblasts apoptosis and ultimately denature the collagen and elastin (Herrling et al., 2006; Kasapoglu and Ozben, 2001; Nichols and Katiyar, 2010). Meanwhile, the denatured skin collagen and elastin is manifested as the formation of wrinkles and reduction

154

S.-Z. Kong et al. / Experimental Gerontology 61 (2015) 147–155

Table 4 Effects of GA on MMP-1, MMP-3 and key inflammatory cytokine contents of photoaged mice skin. Group NC SC MC VC GA-L GA-M GA-H

MMP-1

MMP-3

IL-6

IL-10

TNF-α

ng/mgprota

ng/mgprotb

pg/mgprotc

pg/mgprotd

pg/mgprote

9.04 ± 3.49 9.12 ± 1.66 13.52 ± 2.03# 14.18 ± 2.55# 11.88 ± 1.28 11.07 ± 1.82 10.90 ± 2.07⁎

13.03 ± 3.88 15.30 ± 2.97 19.43 ± 5.06# 21.59 ± 3.26# 19.91 ± 5.53 17.26 ± 4.55 16.48 ± 4.34⁎

20.77 ± 4.37 22.38 ± 8.76 42.37 ± 5.70# 39.94 ± 8.14# 32.22 ± 13 27.93 ± 12.8⁎ 24.62 ± 7.5⁎⁎

222.51 ± 67.24 288.51 ± 130.18 486.26 ± 134.56# 490.10 ± 273.27# 447.43 ± 229.85 379.24 ± 141.04 308.46 ± 152.90⁎

21.73 ± 8.08 23.25 ± 9.34 38.76 ± 8.75# 34.49 ± 4.60# 23.45 ± 9.8⁎ 25.6 ± 6.06 23.03 ± 9.42⁎

Each value represents the mean ± SD of 9 mice per group. # p b 0.05 compared with the SC group. ⁎ p b 0.05. ⁎⁎ p b 0.01 compared with the VC group. a MMP-1 content was expressed as ng per mg protein. b MMP-3 content was expressed as ng per mg protein. c IL-6 was expressed as pg per mg protein. d IL-10 was expressed as pg per mg protein. e TNF-a was expressed as pg per mg protein.

in skin elasticity macroscopically, as well as the twisted, disorganized and broken collagen and elastic fibers histopathologically (Kim et al., 2005; Sanches Silveira and Myaki Pedroso, 2014; Yaar and Gilchrest, 2007). Consistent with previous studies, we found that repeated UVirradiation induced an observable decrease in the activities of the key antioxidant enzymes such as SOD and GSH-Px, and markedly aggravated skin lipid peroxidation, manifested as the accumulation of MDA (Kong et al., 2013). Together with that, at the macroscopic level, UVirradiated mice skin displayed obviously wrinkled and sagged appearance, which exhibited higher visual scores and needed more time to recover its original state after being stretched; at the histopathological level, the skin showed tangled and degraded collagen and elastic fibers. However, after pretreatment with topical GA, the activities of these antioxidant enzymes were significantly increased, while the content of MDA was remarkably decreased. Self-evidently, the wrinkled and sagged mice skin caused by UV was significantly alleviated, while the morphology and density of collagen and elastic fibers were maintained to some extent, as particularly shown in Section 3 of our present study. As revealed by previous studies, repeated UV exposure induces activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activation through cell surface receptors of the skin, then leading to the over-expression of proinflammatory cytokines such as TNF-α and IL-6 (Abeyama et al., 2000; Chung et al., 2009; Kim et al., 2011). These over-expressed cytokines further negatively alter the function of skin cells, which increase oxidative stress through their production of free radicals, and undesirably impact the metabolism of skin connective tissue by hindering the degradation of extracellular matrix (ECM) (Kwon et al., 2013; Rittié and Fisher, 2002). Moreover, since the growth of keratinocytes in epidermis is mediated by the cytokines IL-6 and TNF-α, the epidermal hyperplasia can be observed histopathologically when there is an abnormal increase of these cytokines (Cimino et al., 2007; Kondo, 2000; Scharffetter-Kochanek et al., 2000). In addition, UV irradiation has also been implicated in local and systemic immunosuppression, which is partially mediated by altered cytokine expression (Aubin, 2003; Clydesdale et al., 2001). Studies also demonstrated that increased production of the immunosuppressive cytokine IL-10 has been noted in the cutaneous inflammatory after exposure to UV (Aubin, 2003; Weiss et al., 2004). Consist with these researches, our data proved that the levels of IL-6, TNF-α and IL-10 in mice skin were significantly upregulated after exposure to UV, while the skin macroscopically showed leathery appearance and histopathologically displayed epidermal hyperplasia and diffuse inflammation. However, pretreatment of skin with GA (especially at the dose of 9 mg/mouse) significantly inhibited the over-production of IL-6, IL-10 and TNF-α caused by UV, as well as obviously suppressed the UV-induced epidermal hyperproliferation and dermal inflammatory infiltrates. Hence, we have reasons to believe

that GA possesses the ability to inhibit UV-induced epidermal hyperplasia and diffuse inflammation in dermis via its pharmacological effects of anti-inflammatory, and then ease skin photoaging. Besides that, it is well established that the production of matrix metalloproteinases (MMPs), a group of zinc dependent extracellular proteinases, is intimately regulated by both ROS and inflammatory cytokines, the excessive production of which stimulates the transcription of MMP genes (Hwang et al., 2011; Philips et al., 2011; Pillai et al., 2005). MMPs, specifically MMP-1 and MMP-3, break down the ECM, which is an essential structural framework for skin cells and mainly includes collagen and elastic fibers. In particular, MMP-1 (interstitial collagenase-1, the most effective collagenase) initiates the degradations of collagen types I and III, while MMP-3 (stromelysin 1) not only degrades basement membrane type IV collagen but also activates proMMP-1, as well as degrades the non-collagenous components and elastic fibers (Kim et al., 2005; Philips et al., 2003, 2011; Pillai et al., 2005). Consequently, the over-expression of MMP-1 and MMP-3 is widely regarded as the essential reason for the characteristic wrinkling and sagging of photodamaged skin. In the present study, we also found that the levels of MMP-1 and MMP-3 were excessively increased in UVirradiated mice skin, while the skin displayed obviously wrinkled and sagged appearance macroscopically and exhibited tangled and degraded collagen and elastic fibers histopathologically. Furthermore, these findings were further confirmed by Hyp content determination and pinch test. However, GA treatment (especially at the dose of 9 mg/ mouse) markedly suppressed the UV-induced increase in MMP-1 and MMP-3 production. Our data also proved that mice skin in GA treatment groups showed increased collagen content and exhibited welldistributed collagen and elastic fibers, as well as displayed a visually compact and smooth appearance, which matched to the results observed in pinch test and macroscopic evaluation. Taken together, there is every reason to believe that GA is a potent MMP inhibitor which markedly inhibits the expression of MMP-1 and MMP-3, thereby mitigates the degradation of collagen and elastic fibers, and then protects skin from actinic damage. In summary, our experimental findings amply demonstrate that 18β-Glycyrrhetinic acid (GA) observably attenuates UV-induced skin photoaging mainly by virtue of its antioxidative and anti-inflammatory properties, as well as regulating the abnormal expression of MMP-1 and MMP-3. These results provide an available pre-clinical evidence for the clinical application of GA as a therapeutic and cosmetic product against skin photoaging. Conflict of interest The authors declare that there are no conflicts of interest.

S.-Z. Kong et al. / Experimental Gerontology 61 (2015) 147–155

Acknowledgments This work was supported by the program for scientific research start-up funds of Guangdong Ocean University, the grant from the College students' innovation and entrepreneurship training plan of Guangdong Province (No. 201410572091), and the special funds (No. 276(2014)) from the central finance of China in support of the development of local Colleges and University. References Abeyama, K., Eng, W., Jester, J.V., Vink, A.A., Edelbaum, D., Cockerell, C.J., Bergstresser, P.R., Takashima, A., 2000. A role for NF-κB-dependent gene transactivation in sunburn. J. Clin. Invest. 105, 1751–1759. Aubin, F., 2003. Mechanisms involved in ultraviolet light-induced immunosuppression. Eur. J. Dermatol. 13, 515–523. Battie, C., Jitsukawa, S., Bernerd, F., Del Bino, S., Marionnet, C., Verschoore, M., 2014. New insights in photoaging, UVA induced damage and skin types. Exp. Dermatol. 23 (Suppl. 1), 7–12. Berneburg, M., Plettenberg, H., Krutmann, J., 2000. Photoaging of human skin. Photodermatol. Photoimmunol. Photomed. 16 (6), 239–244. Bissett, D.L., Chatterjee, R., Hannon, D.P., 1990. Photoprotective effect of topical anti-inflammatory agents against ultraviolet radiation-induced chronic skin damage in the hairless mouse. Photodermatol. Photo. 7, 153–158. Blumenthal, M., Goldberg, A., Brinckmann, J., 2000. Herbal Medicine: Expanded Commission E Monographs. American Botanical Council, Newton, pp. 233–236. Carmeli, E., Harpaz, Y., Kogan, N.N., Fogelman, Y., 2008. The effect of an endogenous antioxidant glabridin on oxidized LDL. J. Basic Clin. Physiol. Pharmacol. 19, 49–63. Chen, L., Hu, J.Y., Wang, S.Q., 2012. The role of antioxidants in photoprotection: a critical review. J. Am. Acad. Dermatol. 67 (5), 1013–1024. Chung, J.H., 2003. Photoaging in Asians. Photodermatol. Photoimmunol. Photomed. 19, 109–121. Chung, H.Y., Cesari, M., Anton, S., Marzetti, E., Giovannini, S., Seo, A.Y., Carter, C., Yu, B.P., Leeuwenburgh, C., 2009. Molecular inflammation: underpinnings of aging and agerelated diseases. Ageing Res. Rev. 8, 18–30. Cimino, F., Cristani, M., Saija, A., Bonina, F.P., Virgili, F., 2007. Protective effects of a red orange extract on UVB-induced damage in human keratinocytes. Biofactors 30 (2), 129–138. Clydesdale, G.J., Dandie, G.W., Muller, H.K., 2001. Ultraviolet light induced injury: immunological and inflammatory effects. Immunol. Cell Biol. 79 (6), 547–568. Feng, X.X., Yu, X.T., Li, W.J., Kong, S.Z., Liu, Y.H., Zhang, X., Xian, Y.F., Zhang, X.J., Su, Z.R., Lin, Z.X., 2014. Effects of topical application of patchouli alcohol on the UV-induced skin photoaging in mice. Eur. J. Pharm. Sci. 63, 113–123. Fisher, G.J., Wang, Z.Q., Fatta, S.C., Varani, J., Kang, S., Voorhees, J.J., 1997. Pathophysiology of premature skin aging induced by ultraviolet light. N. Engl. J. Med. 337, 1419–1428. Fisher, G.J., Kang, S., Varani, J., Bata-Csorgo, Z., Wan, Y., Datta, S., Voorhees, J.J., 2002. Mechanisms of photoaging and chronological skin aging. Arch. Dermatol. 138, 1462–1470. Gaspar, L.R., Maia Campos, P.M., 2003. Rheological behavior and the SPF of sunscreens. Int. J. Pharm. 250, 35–44. Gong, X.L., Luo, Y., Tang, D.C., Sun, X.F., 2008. Research progress in glycyrrhetinic acid and its derivatives. Strait Pharm. J. 20 (9), 4–7. Herrling, T.H., Jung, K., Fuchs, J., 2006. Measurements of UV-generated free radicals/reactive oxygen species (ROS) in skin. Spectrochim. Acta A 63, 840–845. Hwang, K.A., Yi, B.R., Choi, K.C., 2011. Molecular mechanisms and in vivo mouse models of skin aging associated with dermal matrix alterations. Lab. Anim. Res. 27, 1–8. Jayasooriya, R.G., Dilshara, M.G., Park, S.R., Choi, Y.H., Hyun, J.W., Chang, W.Y., Kim, G.Y., 2014. 18β-Glycyrrhetinic acid suppresses TNF-α induced matrix metalloproteinase9 and vascular endothelial growth factor by suppressing the Akt-dependent NF-κB pathway. Toxicol. in Vitro 28 (5), 751–758. Kalaiarasi, P., Pugalendi, K.V., 2011. Protective effect of 18β-glycyrrhetinic acid on lipid peroxidation and antioxidant enzymes in experimental diabetes. J. Pharm. Res. 4 (1), 107–118. Kasapoglu, M., Ozben, T., 2001. Alterations of antioxidant enzymes and oxidative stress markers in aging. Exp. Gerontol. 36, 209–220. Khavkin, J., Ellis, D.A., 2011. Aging skin: histology, physiology, and pathology. Facial Plast. Surg. Clin. N. Am. 19, 229–234. Kim, H.H., Lee, M.J., Lee, S.R., Kim, K.H., Cho, K.H., Eun, H.C., Chung, J.H., 2005. Augmentation of UV-induced skin wrinkling by infrared irradiation in hairless mice. Mech. Ageing Dev. 126, 1170–1177.

155

Kim, H.S., Kim, A.R., Park, H.J., Park, D.K., Kim, do-K, Ko, N.Y., Kim, B., Choi, D.K., Won, H.S., Shin, W.S., Kim, Y.M., Choi, W.S., 2011. Morus bombycis Koidzumi extract suppresses collagen-induced arthritis by inhibiting the activation of nuclear factor-κB and activator protein-1 in mice. J. Ethnopharmacol. 136 (3), 392–398. Kondo, S., 2000. The roles of cytokines in photoaging. J. Dermatol. Sci. 23 (Suppl. 1), S30–S36. Kong, S.Z., Shi, X.G., Feng, X.X., Li, W.J., Liu, W.H., Chen, Z.W., Xie, J.H., Lai, X.P., Zhang, S.X., Zhang, X.J., Su, Z.R., 2013. Inhibitory effect of hydroxysafflor yellow a on mouse skin photoaging induced by ultraviolet irradiation. Rejuvenation Res. 16, 404–413. Kwon, O.S., Jung, S.H., Yang, B.S., 2013. Topical administration of manuka oil prevents UVB irradiation-induced cutaneous photoaging in mice. Evid. Based Complement. Alternat. Med. 2013, 1–10. Lavker, R.M., 1995. Cutaneous aging: chronologic versus photoaging. In: Gilchrest, B.A. (Ed.), Photoaging. Blackwell, Cambridge, MA, pp. 123–135. Maitraie, D., Hung, C.F., Tu, H.Y., Liou, Y.T., Wei, B.L., Yang, S.C., Wang, J.P., Lin, C.N., 2009. Synthesis, anti-inflammatory, and antioxidant activities of 18 beta-glycyrrhetinic acid derivatives as chemical mediators and xanthine oxidase inhibitors. Bioorg. Med. Chem. 17 (7), 2785–2792. Neuman, R., Logan, M., 1950. The determination of collagen and elastin in tissues. J. Biol. Chem. 186, 549–556. Nichols, J.A., Katiyar, S.K., 2010. Skin photoprotection by natural polyphenols: anti-inflammatory, antioxidant and DNA repair mechanisms. Arch. Dermatol. Res. 302, 71–83. Ody, P., 2000. The Complete Guide Medicinal Herbal. Dorling Kindersley, London, p. 75. Ouhtit, A., Muller, H.K., Davis, D.W., Ullrich, S.E., McConkey, D., Ananthaswamy, H.N., 2000. Temporal events in skin injury and the early adaptive responses in ultraviolet-irradiated mouse skin. Am. J. Pathol. 156, 201–207. Philips, N., Smith, J., Keller, T., Gonzalez, S., 2003. Predominant effects of Polypodium leucotomos on membrane integrity, lipid peroxidation, and expression of elastin and matrixmetalloproteinase-1 in ultraviolet radiation exposed fibroblasts, and keratinocytes. J. Dermatol. Sci. 32 (1), 1–9. Philips, N., Auler, S., Hugo, R., Gonzalez, S., 2011. Beneficial regulation of matrix metalloproteinases for skin health. J. Enzym. Res. 2011, 1–4. Pillai, S., Oresajo, C., Hayward, J., 2005. Ultraviolet radiation and skin aging: roles of reactive oxygen species, inflammation and protease activation, and strategies for prevention of inflammation-induced matrix degradation — a review. Int. J. Cosmet. Sci. 27, 17–34. Quan, T., Qin, Z., Xia, W., Shao, Y., Voorhees, J.J., Fisher, G.J., 2009. Matrix-degrading metalloproteinases in photoaging. J. Investig. Dermatol. Symp. Proc. 14 (1), 20–24. Rabe, J.H., Mamelak, A.J., McElgunn, P.J., Morison, W.L., Sauder, D.N., 2006. Photoaging: mechanisms and repair. J. Am. Acad. Dermatol. 55, 1–19. Rittié, L., Fisher, G.J., 2002. UV-light-induced signal cascades and skin aging. Ageing Res. Rev. 1, 705–720. Sanches Silveira, J.E., Myaki Pedroso, D.M., 2014. UV light and skin aging. Rev. Environ. Health 29 (3), 243–254. Scharffetter-Kochanek, K., Brenneisen, P., Wenk, J., Herrmann, G., Ma, W., Kuhr, L., Meewes, C., Wlaschek, M., 2000. Photoaging of the skin from phenotype to mechanisms. Exp. Gerontol. 35 (3), 307–316. Shang, H., Wei, H., Yue, B., Xu, P., Huang, H., 2009. Microsatellite analysis in two populations of Kunming mice. Lab. Anim. 43 (1), 34–40. Thornfeldt, C.R., 2008. Chronic inflammation is etiology of extrinsic aging. J. Cosmet. Dermatol. 7, 78–82. Tsukahara, K., Moriwaki, S., Hotta, M., Fujimura, T., Sugiyama-Nakagiri, Y., Sugawara, S., Kitahara, T., Takema, Y., 2005. The effect of sunscreen on skin elastase activity induced by ultraviolet — a irradiation. Biol. Pharm. Bull. 28, 2302–2307. Wang, Z.Y., Agarwal, R., Zhou, Z.C., Bickers, D.R., Mukhtar, H., 1991. Inhibition if mutagenecity in Salmonella typhimurium and skin tumor initiating and tumor promoting activities in Senecar mice by glycyrrhetinic acid: comparison of 18α- and 18β-stereoisomers. Carcinogenesis 12, 187–192. Wang, C.Y., Kao, T.C., Lo, W.H., Yen, G.C., 2011. Glycyrrhizic acid and 18β-glycyrrhetinic acid modulate lipopolysaccharide-induced inflammatory response by suppression of NF-κB through PI3K p110δ and p110γ inhibitions. J. Agric. Food Chem. 59 (14), 7726–7733. Weiss, E., Mamelak, A.J., La Morgia, S., Wang, B., Feliciani, C., Tulli, A., Sauder, D.N., 2004. The role of interleukin 10 in the pathogenesis and potential treatment of skin diseases. J. Am. Acad. Dermatol. 50, 657–675. Yaar, M., Gilchrest, B.A., 2007. Photoageing: mechanism, prevention and therapy. Br. J. Dermatol. 157, 874–887. Zhang, X., Zhu, Z., Huang, Z., Tan, P., Ma, R.Z., 2007. Microsatellite genotyping for four expected inbred mouse strains from KM mice. J. Genet. Genomics 34 (3), 214–222.

The protective effect of 18β-Glycyrrhetinic acid against UV irradiation induced photoaging in mice.

It has been confirmed that repeated exposure of skin to ultraviolet (UV) radiation results in cutaneous oxidative stress and inflammation, which act i...
2MB Sizes 0 Downloads 4 Views