molecules Article
Antiinflammatory and Antiphotodamaging Effects of Ergostatrien-3β-ol, Isolated from Antrodia camphorata, on Hairless Mouse Skin Yueh-Hsiung Kuo 1,2 , Tzu-Yu Lin 3 , Ya-Jhen You 3 , Kuo-Ching Wen 3 , Ping-Jyun Sung 4 and Hsiu-Mei Chiang 3, * 1 2 3 4
*
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan;
[email protected] Department of Biotechnology, Asia University, Taichung 413, Taiwan Department of Cosmeceutics, China Medical University, Taichung 404, Taiwan;
[email protected] (T.-Y.L.);
[email protected] (Y.-J.Y.);
[email protected] (K.-C.W.) National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan;
[email protected] Correspondence:
[email protected]; Tel.: +886-4-2205-3366 (ext. 5302); Fax: +886-4-2207-8083
Academic Editor: Fernando Albericio Received: 24 June 2016; Accepted: 6 September 2016; Published: 10 September 2016
Abstract: Ergostatrien-3β-ol (EK100), isolated from the submerged whole broth of Antrodia camphorata, has antidiabetic, hyperlipidemic, and hepatoprotective activities. However, the antiphotodamage activity of EK100 has still not been revealed. Inflammation and collagen degradation contribute to skin photodamage and premature aging. In the present study, in vivo experiments were designed to investigate the antiinflammatory and antiphotodamaging activities of EK100 in hairless mice by physiological and histological analysis of the skin. Results indicated that topical application of EK100 (25 and 100 µM) for 10 weeks efficiently inhibited ultraviolet B (UVB)-induced wrinkle formation, erythema, and epidermal thickness in the mice skin. EK100 also restored UVB-induced collagen content reduction in hairless mice skin. In addition, the immunohistochemistry results indicated that EK100 significantly inhibited the UVB-induced expression of matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and nuclear factor kappaB (NF-κB) in the mouse skin. The expression of these proteins was similar to the Normal group after 100 µM EK100 treatment. EK100 inhibited collagen degradation in the skin through MMP-1 inhibition and antiinflammation. EK100 significantly reduced the transepidermal water loss (TEWL), indicating that EK100 protected skin from UVB-induced damage. Our findings strongly suggest that EK100 has significant beneficial antiinflammatory and antiphotoaging activities and that EK100 can be developed as an antiphotodamaging agent. Keywords: ergostatrien-3β-ol; photodamage; hyperplasia; erythema; nuclear factor kappaB; transepidermal water loss
1. Introduction The skin is the outermost tissue of the human body and is easily affected by environmental factors, such as solar radiation, smoking, and pollution. Skin aging is a continual and complex process, and many factors are involved in this process. Photoaging is attributed to continuous exposure to ultraviolet (UV). The characteristics of skin aging include coarse wrinkles, hyperplasia, dryness, laxity and hyperpigmentation [1]. Chronic UV exposure increases the number of stratum corneum layers and keratinocytes expressing filaggrin, which is a marker of terminal differentiation [2,3]. In addition, repeated exposure to sunlight causes damage to collagen fibers and an excessive deposition
Molecules 2016, 21, 1213; doi:10.3390/molecules21091213
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abnormal elastic and reduces reduces of abnormal elasticfibers fibers[4,5]. [4,5].Finally, Finally,this thistype typeofofexposure exposureincreases increases epidermal epidermal thickness and skin elasticity, elasticity,causing causingskin skinaging agingand anddamage damage[6–8]. [6–8]. skin Chronic UV UV exposure exposure causes causes serious serious skin skin injuries injuries by by generating generating oxidative oxidative stress, stress, inflammation, inflammation, Chronic and DNA DNA lesions lesions [9–11]. [9–11]. UV exposure increases the generation generation of reactive reactive oxygen oxygen species species (ROS) (ROS) and causingperoxidation peroxidationof ofthe thecell cellmembrane, membrane,subsequently subsequentlyleading leadingto tothe thedamage damageof of keratinocytes keratinocytesand and causing fibroblasts. ROS triggers triggers expression expression of of matrix matrix metalloproteins metalloproteins (MMPs) (MMPs) and and genes, genes, such such as as MMP-1 MMP-1 fibroblasts. and MMP-3, MMP-3, causing causing photodamage, photodamage, including including wrinkle wrinkle formation, formation, solar solar elastosis, elastosis, and and extracellular extracellular and matrix (ECM) (ECM) degradation degradation such such as as collagen, collagen, elastin, elastin, and and proteoglycan, proteoglycan, which which maintain maintain the the cell cell and and matrix skin structures structures [12,13]. [12,13]. UV UV irradiation irradiation upregulates upregulates inflammation-related inflammation-related genes, genes, including including interleukin interleukin skin (IL)-1 and and -6 -6 receptors; receptors; and and peroxisome peroxisome proliferator-activated proliferator-activated receptor receptor gamma, gamma, and and cause cause release release (IL)-1 of proinflammatory proinflammatory cytokines cytokines such such as as IL-1, IL-1, IL-6 IL-6 and andTNF-α. TNF-α. Furthermore, Furthermore, UV UV also also increases increases the the of prostaglandinEE22 concentration concentration and and generating generating nitric nitric oxide oxide(NO) (NO)[9,14]. [9,14]. The The overexpression overexpression of ofinducible inducible prostaglandin NO synthase synthase (iNOS) (iNOS) and and translocation translocation of of nuclear nuclear factor factor kappaB kappaB (NF-κB) (NF-κB) into into the the nucleus nucleus have have been been NO reportedto totrigger triggerdownstream downstreamsignaling signalingtransduction, transduction,thus thuscausing causingskin skininflammation inflammation[15,16]. [15,16]. reported Ergostatrien-3β-ol (EK100) (EK100) was was isolated isolated from from the the submerged submerged whole whole broth broth of of Antrodia Antrodiacamphorata camphorata Ergostatrien-3β-ol (A. camphorata), which is a widely used herb. Because of its rarity and difficult cultivation, A. camphorata (A. camphorata), which is a widely used herb. Because of its rarity and difficult cultivation, A. camphorata is aa highly highly valued valued polypore polypore mushroom that is native only to Taiwan [17,18]. The fruiting fruiting body body and and is culturedmycelia mycelia composed of acids, fatty lignans, acids, sesquiterpenes, lignans, sesquiterpenes, and triterpenoids [19]. cultured areare composed of fatty and triterpenoids [19]. Furthermore, Furthermore, thesubmerged mycelia from submerged is used health food [20]. and The nutraceuticals The the mycelia from culture is used inculture health food andin nutraceuticals fermented [20]. cultured fermented cultured broth has antiinflammatory, shown antioxidant,vasodilatory, antiinflammatory, vasodilatory, and hepatoprotective broth has shown antioxidant, and hepatoprotective properties [21–23]. properties [21–23]. EK100, aof major component whole of the broth submerged whole brothwas of A. camphorata, was EK100, a major component the submerged of A. camphorata, reported to have reported to have antidiabetic and dyslipidemic activities in mice high-fat-diet-fed mice EK100 [17]. Inexhibited addition, antidiabetic and dyslipidemic activities in high-fat-diet-fed [17]. In addition, EK100 exhibited antiinflammatory effects on λ-carrageenan-induced paw edema in mice [24]. antiinflammatory effects on λ-carrageenan-induced paw edema in mice [24]. Compoundsor ornatural naturalproducts productssuch suchas aspolyphenols polyphenolsand andanthocyanin anthocyaninthat thatexhibit exhibitan anantioxidant antioxidant Compounds or antiinflammatory antiinflammatory activity activity or or inhibit inhibit MMPs MMPs expressions expressions have have been been shown shown to to protect protect the the skin skin or against photoaging and EK100 with strong antioxidant and against andphotocarcinogenesis photocarcinogenesis[1,25,26]. [1,25,26].Therefore, Therefore, EK100 with strong antioxidant antiinflammation activity may be be a desirable candidate for and antiinflammation activity may a desirable candidatefor forfurther furtherdevelopment developmentas as aa treatment for UV-inducedskin skininflammation inflammationand anddamage. damage. In In this this study, study,we wefocused focusedon onantiphotodamage antiphotodamageactivity activity of of UV-induced EK100, particularly its antiinflammatory and antiphotoaging activities and the associated mechanisms EK100, particularly its antiinflammatory and antiphotoaging activities and the associated mechanisms on hairless hairlessmouse mouseskin. skin. on 2. Results 2. Results 2.1. 2.1. Body Body Weight Weight of of the the Mice Mice with with Topical TopicalApplication ApplicationofofEK100 EK100 All All animals animals in in each each group group had hadaasimilar similarmean meanbody bodyweight weightat atbaseline. baseline. The The body body weight weight of of the the mice in the five groups was not significantly different after the 10-week treatment (Figure 1). mice in the five groups was not significantly different after the 10-week treatment (Figure 1).
Figure 1. Body weight of the mice in the five groups. Figure 1. Body weight of the mice in the five groups.
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2.2. Topical Application of EK100 Significantly Prevented UVB-Induced Skin Erythema and Damage 2.2. Topical Application of EK100 Significantly Prevented UVB-Induced Skin Erythema and Damage Skin erythema (a* values) indicates the extent of inflammation. In Figure 2, the results indicate Molecules 2016, 21, 1213 3 of 14 Skin erythema (a* values) indicates the extent of inflammation. In Figure 2, the results indicate that UVB irradiation significantly increased the a* values of the mouse skin in the 8th, 9th, and 10th that UVB irradiation significantly increased Prevented the a* values of the mouseErythema skin in the 9th, and 10th 2.2. Topical Application of EK100 the Significantly UVB-Induced and8th, Damage week. EK100 treatment reduced a* values in the 9th, and 10thSkin week. The results suggested that week. EK100 treatment reduced the a* values in the 9th, and 10th week. The results suggested that topical application of EK100 inhibited UVB-induced skin erythema and inflammation. Skin erythema (a* values) indicates the extent of inflammation. In Figure 2, the results indicate topical application of EK100 inhibited UVB-induced skin erythema and inflammation. that UVB irradiation significantly increased the a* values of the mouse skin in the 8th, 9th, and 10th week. EK100 treatment reduced the a* values in the 9th, and 10th week. The results suggested that topical application of EK100 inhibited UVB-induced skin erythema and inflammation.
Figure 2. Effect of EK100 on a* values in chronic UVB-irradiated hairless mice. UVB irradiation Figure 2. Effect of EK100 on a* values in chronic UVB-irradiated hairless mice. UVB irradiation significantly increased the a* values of the mouse skin in the 8th, 9th, and 10th weeks; however, significantly increased the a* values of the mouse skin in the 8th, 9th, and 10th weeks; however, EK100 a–d: Values Figure 2. Effect reduced of EK100the on a* a* values values in in the chronic mice. not UVBfollowed irradiation EK100 treatment 9th, UVB-irradiated and a–d 10th week.hairless by a treatment reduced the a* values in the 9th, and 10th week. : Values not followed by a common letter significantly the a* values of (p the< mouse common letter increased are significantly different 0.05). skin in the 8th, 9th, and 10th weeks; however, are significantly different (p
