1. Introduction
Skin is the external part of the human body and plays a crucial role in protection from the exogenous invasion of pathogenic factors or pollutants. Long-term exposure to ultraviolet (UV) irradiation induces visible signs of aging, known as photoaging, which include characteristics such as wrinkles, pigmentation, sagging, and inflammation. Exposure to solar light induces oxidative stress and inflammation in the skin, resulting in the activation of aging-related pathways and causing skin aging and damage [
1]. The extracellular matrix (ECM) is abundant in the dermis and is responsible for maintaining the skin structure and resilience. The major components of the ECM are the Type I and III collagen, along with small amounts of elastin and glycoproteins [
2]. Matrix metalloproteinases (MMPs) are responsible for the degradation of the ECM, whereas tissue inhibitors of metalloproteinases (TIMPs) may inhibit the activity of MMPs [
3]. In addition, many factors, such as inflammation and reactive oxygen species (ROS), may cause ECM degradation. Regulation of collagen gene expression is related to the physiology and pathology of skin aging and disorders [
4].
UV exposure triggers two transcription factor pathways, namely the activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) families, which modulate the expression of various UV responses [
5]. Inhibitor κB (IκB) generally binds with NF-κB in the cytoplasm to create an inactive complex; however, UV exposure or ROS promotes IκB ubiquitination, triggering NF-κB translocation into the nucleus and causing inflammation [
6]. NF-κB activation stimulates MMP-1 production, which degrades collagen in the dermis and results in wrinkle formation. UV irradiation upregulates various cytokines, such as interleukins (ILs), inducible nitric oxide synthase (i-NOS), and cyclooxygenase (COX)-2, and this consequently stimulates inflammation and results in skin sunburn and erythema. IL and NF-κB upregulate COX-2 expression, causing photodamage of the skin [
7].
The seeds of
Sesamum indicum Linn. (
Pedaliaceae) have been used as a seasoning and cooking oil in the eastern part of Asia. They contain abundant lignans, such as sesamol, sesamin, and seamolin, which have been reported to exhibit an excellent antioxidative activity [
8,
9]. Sesamin oil was reported to promote wound healing and have antioxidative, anti-inflammatory, and hypolipemia activities [
10,
11,
12]. Our previous study demonstrated that sesamol inhibited melanin synthesis in B16F10 cells and C57BL/6 mice [
13,
14]. Sesamin has also been reported to exhibit antioxidative, anti-inflammatory, and antinociceptive activities [
15,
16,
17]. It scavenges ROS and nitric oxides (NO) and inhibits proinflammatory cytokines production, protecting the liver from injury in rats [
18]. Sesamin elevated tocotrienol contents in the skin to reduce sunburn and the incidence of tumors [
19]. We hypothesized that sesamin, with its potent antioxidative and anti-inflammatory activities, may exhibit benefits for the skin photodamage. The aim of the present study was to investigate the activity and mechanism of sesamin against photodamage and photoinflammation induced by UVB irradiation in human skin fibroblasts (Hs68 cells) and hairless mouse skin.
2. Materials and Methods
2.1. Materials
All reagents and chemicals used in the present study were the reagent grade. Reagents used in the cell culture, including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin, were obtained from Gibco, Invitrogen (Carlsbad, CA, USA). Albumin (from bovine serum), 2′,7′-dichlorofluorescin diacetate (DCFDA), dimethyl sulfoxide, leupeptin, paraformaldehyde, and phenylmethylsulfonyl fluoride were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Sodium dodecyl sulfate (SDS), Tris, and Tween 20 were obtained from the USB Corporation (Cleveland, OH, USA). The ECL western blotting detection reagent was obtained from Amersham Biosciences (Little Chalfont, England). MAP kinase inhibitors, including c-Jun N-terminal kinases (JNK) inhibitor II, PD98059, and SB203580, were obtained from Calbiochem (Darmstadt, Germany).
2.2. Cell Culture and UV Irradiation
Hs68 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan), seeded in DMEM with 10% FBS and 100 U/mL penicillin/streptomycin, and incubated at 37 °C with 5% CO
2. A CL-1000M UV crosslinker with two UV lamps (peak wavelength at 302 nm) was applied for UV irradiation (UVP, Upland, CA, USA). Cells covered with PBS were exposed to 80 mJ/cm
2 UVB for the DCFDA assay and 40 mJ/cm
2 UVB for other experiments as described previously [
20].
2.3. Detection of ROS Production
A DCFDA fluorescence assay was performed to study the intracellular ROS levels in Hs68 cells after UVB exposure as described previously [
20]. Briefly, the cells were treated with various concentrations of sesamin after UVB irradiation. DCFDA was added to the cells after which the cells were incubated for 30 min. Fluorescence was detected at an emission wavelength of 520 nm and excitation wavelength of 488 nm using a microplate reader (Thermo Electron Corporation, Vantaa, Finland), and photos were captured using a fluorescence microscope (Leica DMIL, Wetzlar, Germany).
2.4. Western Blot Analysis
The protein expression of sesamin in Hs68 cells after UVB irradiation was assayed through western blotting [
20]. Fibroblasts were harvested and lysed using the lysis buffer. The proteins were separated by gel electrophoresis on an SDS-polyacrylamide gel and recognized by specific antibodies. The proteins were probed with antibodies after transferring to polyvinylidene difluoride membranes. An ECL western blotting detection system (Fujifilm, LAS-4000, Tokyo, Japan) was used to detect the proteins, and the densities were measured using a densitometric program (MultiGauge V2.2, Fuji Pharma, Tokyo, Japan).
2.5. Measuring Total Collagen Synthesis in Fibroblasts
The total collagen synthesis in fibroblasts was detected using a Sircol collagen detection kit [
21]. After UVB exposure and the sesamin treatment, the cell culture medium was used to measure the collagen content. Isolation and concentration reagents were mixed with the culture medium and incubated. Finally, the Sircol dye was added to the sample. After centrifugation, the washing reagent was mixed with the pellets, and the sample was centrifuged again. An alkali reagent was added to dissolve the precipitate, and the absorbance was detected at 555 nm.
2.6. Immunofluorescence Staining
Hs68 cells were seeded on coverslips and treated with UVB irradiation. Subsequently, sesamin at various concentrations was added to the cells after which the cells were incubated overnight. The cells were incubated with primary and Alexa Fluor 488 antirabbit IgG secondary antibodies (Invitrogen, Waltham, MA, USA). Finally, the cells on the coverslip were stained with the ProLong Gold antifade reagent (Thermo Fisher Scientific Inc, Waltham, MA, USA) and examined under a fluorescence microscope [
22].
2.7. Effect of Sesamin Treatment on Photodamage in Hairless Mice
2.7.1. Animals
Five-week-old female BALB/cAnN.Cg-Foxn1nu/CrlNarl mice were obtained from the National Laboratory Animal Center in Taipei, Taiwan. Animals were kept in the animal center of China Medical University and allowed to accommodate to the environment for one week. Protocols for animal experiments were approved (104-240-B) by the Institutional Animal Use and Care Committee of China Medical University.
2.7.2. Experimental Design
The mice were randomly divided into the following five groups: Non-UVB irradiation or nonsesamin treatment (normal), UVB-irradiated, vehicle-treated and UVB-irradiated (vehicle), UVB-irradiated and 50-μM-sesamin-treated (UVB + 50 μM sesamin), and UVB-irradiated and 200-μM-sesamin-treated (UVB + 200 μM sesamin) groups. Hairless mice were exposed to gradient doses of UVB irradiation as described previously [
20]. PEG400 (50 μL) was topically applied on the dorsal skin after UVB exposure in the vehicle-treated group daily, and 50 μL of 50 and 200 μM sesamin was applied to the appropriate sesamin groups. At the end of the experiment, the exposed areas were excised and then immersed in 10% formaldehyde in the PBS. The slides were mounted with a coverslip, stained in hematoxylin and eosin or Masson’s trichrome, and examined under a microscope or applied for immunohistological analysis as previously described [
23].
2.7.3. Detection of Erythema (a* Value) and Transepidermal Water Loss of Mice Skin
The effect of sesamin on UVB-induced erythema was detected by using a spectrocolorimeter (SCM-108, Laiko company, Tokyo, Japan), and the transepidermal water loss (TEWL) on the dorsal skin of hairless mice was measured in the 10th week by using a Tewameter TM 300 (Courage + Khazaka electronic GmbH, Cologne, Germany) as previously described [
24].
2.7.4. Immunohistological Analysis
The skin samples were incubated with primary antibodies for MMP-1, IL-6, NF-κB, and i-NOS. After being washed with PBS twice, the skin slides were incubated with the secondary antibody. The samples were examined under a microscope.
2.8. Statistical Analysis
Data are presented as mean ± standard deviation from at least triplicate independent experiments in vitro study. Statistically significant differences between the groups were determined using the Student’s t-test or ANOVA. p < 0.05 was considered to be significant.
4. Discussion
Oxidative stress is one of the major factors for aging, and the biochemical reaction associated with normal metabolic processes often produces free radicals and ROS [
27,
28]. UV irradiation promotes ROS generation and triggers aging-related signal transduction, resulting in skin sagging, rough skin, hyperpigmentation, and skin cancers [
29]. Many reports have demonstrated that natural products with antioxidants exhibit anti-inflammatory and antiphotoaging activities [
30,
31,
32]. Sesame and its major active component, sesamin, were reported to exhibit free-radical-scavenging and antioxidative activities [
33,
34]. In the present study, sesamin inhibited UVB-induced ROS formation in skin fibroblasts; therefore, it may be used as a photoprotective agent.
Long-term exposure to UV causes collagen degradation in the dermis, leading to photodamage. UV exposure induces the skin damage by promoting collagen degradation or collagen synthesis inhibition through the regulation of the transforming growth factor (TGF)-β/Smad pathway. MMP-1 degrades the collagen fiber bundles, and, subsequently, MMP-3 promotes the activity of MMP-1, further degrading collagen to fragments. MMP-9 can further degrade the MMP-1 cleaved collagen [
35,
36]. The C-terminal domain and N-terminal domain of TIMPs can conjugate with the active site of MMPs and inhibit them, resulting in the inhibition of collagen damage [
37]. The results of this study show that the sesamin treatment could block UVB-induced collagen degradation by inhibiting MMP-1, -3, and -9 expressions in human skin fibroblasts. Furthermore, sesamin elevated the expression of TIMP-1. In the animal study, results demonstrate that sesamin exhibited potent antioxidant activity and ameliorated wrinkle formation induced by chronic UVB exposure.
UV irradiation increased intracellular oxidative stress and induced the phosphorylation of MAP kinases, resulting in the transcription factor AP-1 translocating into the nucleus to trigger secretion of MMPs [
38]. Sesamin inhibited UVB-induced AP-1 and the phosphorylation of ERK, JNK, and p38 proteins. Moreover, UV irradiation inhibited collagen biosynthesis in fibroblasts. TGF-β is a multifunction regulator and modulates the growth, differentiation, apoptosis, migration, adhesion, and immune response of cells [
39,
40]. The TGF-β pathway is the major pathway of type Ⅰ procollagen synthesis. TGF-β activates the combination of downstream proteins, Smad2, and Smad3, which bind with Smad4 to form a complex [
41]. The Smad complex enters the nucleus to synthesize the type Ⅰ procollagen. UV irradiation activates Smad7 to inhibit the TGF-β receptor and block signal transduction, leading to the inhibition of collagen synthesis [
42]. Our results suggest that sesamin inhibited Smad7 overexpression and increased Smad3 expression to increase the content of total collagen.
Overexposure to UV irradiation triggered the activation of the MAP kinase pathway and of NF-ĸB, resulting in COX-2 and i-NOS protein expression and then causing skin erythema and inflammation. The results of this study indicate that UVB upregulated COX-2 and i-NOS protein expression and NF-ĸB translocation in human skin fibroblasts, whereas sesamin inhibited these effects. The results signify that treatment with sesamin ameliorated UVB-induced skin inflammation. A previous study reported that sesamin inhibited inflammation of neurons in rats with intracerebral hemorrhage by suppressing ERK and p38 activation [
43]. Sesamin protected neurons from lipopolysaccharides damage by inhibiting the p38 MAP kinase pathway and NF-κB activation [
15]. Additionally, cotreatment with the JNK or p38 inhibitor and sesamin significantly reduced the protein expression of COX-2; thus, sesamin may inhibit COX-2 expression through the JNK and p38 pathways, resulting in anti-inflammation. UVB can activate the p38 pathway to induce skin inflammation and can even lead to cancer [
44,
45,
46].
Chronic exposure to UV causes photodamaging of the skin and induces various skin disorders, including sunburn, dryness, wrinkles, and skin cancer [
47]. UV light also can induce oxidative stress and inflammation of the skin. After UVB exposure for two and four weeks, the a* value was increased and then significantly increased at 10 weeks, indicating inflammation of the mouse skin. The sesamin treatment reversed UVB-induced skin erythema and inflammation. The skin is a barrier that protects the body from deleterious factors in the environment and prevents water loss, in addition to maintaining the homeostasis of water and electrolytes [
48,
49]. TEWL may be an index for the barrier function of the skin. TEWL increases when the stratum corneum is damaged. In this study, long-term UVB exposure caused the TEWL to increase; however, TEWL was not significantly changed after the sesamin treatment for 10 weeks, indicating that sesamin did not cause skin toxicity.
UV exposure induces degradation of ECM in the dermis structural and compositional remodeling in dermis, resulting in the wrinkle and sagging of the skin [
50]. In the evaluation of wrinkle formation, chronic exposure to cumulative UVB for 10 weeks caused significant wrinkle formation on mice dorsal skin. However, the topical application of sesamin reduced UVB-induced wrinkles on the skin. In addition, UVB-induced epidermal thickness decreased significantly after the sesamin treatment. After UVB exposure, brown granulates were present in the epidermis, indicating immune cell infiltration in the skin. Sesamin reduced the infiltration of leukocytes in the skin. The results of this study indicate that sesamin inhibited UVB-induced skin inflammation. UVB decreased collagen in the dermis, and the vehicle slightly increased the content of collagen. Sesamin significantly increased the content and density of collagen in the dermis, which may reduce UV-induced skin damage. UV irradiation caused hyperplasia of the epidermis, reduction of collagen content, and denaturation of elastin, leading to skin photoaging. Sesamin ameliorated skin hyperplasia and collagen degradation.