Photoprotective and anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo
2006; Elsevier BV; Volume: 47; Issue: 5 Linguagem: Inglês
10.1194/jlr.m500420-jlr200
ISSN1539-7262
AutoresHyeon Ho Kim, Soyun Cho, Serah Lee, Kyu Han Kim, Kwang Hyun Cho, Hee Chul Eun, Jin Ho Chung,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoSkin aging can be attributed to photoaging (extrinsic) and chronological (intrinsic) aging. Photoaging and intrinsic aging are induced by damage to human skin attributable to repeated exposure to ultraviolet (UV) irradiation and to the passage of time, respectively. In our previous report, eicosapentaenoic acid (EPA) was found to inhibit UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Therefore, we investigated the effects of EPA on UV-induced skin damage and intrinsic aging by applying EPA topically to young and aged human skin, respectively. By immunohistochemical analysis and Western blotting, we found that topical application of EPA reduced UV-induced epidermal thickening and inhibited collagen decrease induced by UV light. It was also found that EPA attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation, which is closely related to UV-induced activator protein-1 activation, and by inhibiting JNK and p38 activation. EPA also inhibited UV-induced cyclooxygenase-2 (COX-2) expression without altering COX-1 expression. Moreover, it was found that EPA increased collagen and elastic fibers (tropoelastin and fibrillin-1) expression by increasing transformin growth factor-β expression in aged human skin. Together, these results demonstrate that topical EPA has potential as an anti-skin-aging agent. Skin aging can be attributed to photoaging (extrinsic) and chronological (intrinsic) aging. Photoaging and intrinsic aging are induced by damage to human skin attributable to repeated exposure to ultraviolet (UV) irradiation and to the passage of time, respectively. In our previous report, eicosapentaenoic acid (EPA) was found to inhibit UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Therefore, we investigated the effects of EPA on UV-induced skin damage and intrinsic aging by applying EPA topically to young and aged human skin, respectively. By immunohistochemical analysis and Western blotting, we found that topical application of EPA reduced UV-induced epidermal thickening and inhibited collagen decrease induced by UV light. It was also found that EPA attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation, which is closely related to UV-induced activator protein-1 activation, and by inhibiting JNK and p38 activation. EPA also inhibited UV-induced cyclooxygenase-2 (COX-2) expression without altering COX-1 expression. Moreover, it was found that EPA increased collagen and elastic fibers (tropoelastin and fibrillin-1) expression by increasing transformin growth factor-β expression in aged human skin. Together, these results demonstrate that topical EPA has potential as an anti-skin-aging agent. Skin aging can be attributed to extrinsic aging and intrinsic (chronological) aging and is commonly related to increased wrinkling, sagging, and laxity (1Jenkins G. Molecular mechanisms of skin ageing.Mech. Ageing Dev. 2002; 123: 801-810Crossref PubMed Scopus (399) Google Scholar). Extrinsic aging is generally referred to as photoaging and is caused by repeated exposure to ultraviolet (UV) light. Whereas naturally aged skin is smooth, pale, and finely wrinkled, photoaged skin is coarsely wrinkled and associated with dyspigmentation and telangiectasia (2Gilchrest B.A. Skin aging and photoaging: an overview.J. Am. Acad. Dermatol. 1989; 21: 610-613Abstract Full Text PDF PubMed Scopus (406) Google Scholar). Alterations in collagen, the major structural component of the skin, have been considered to be a cause of skin aging and are observed in naturally aged and photoaged skin (3Fisher G.J. Wang Z.Q. Datta S.C. Varani J. Kang S. Voorhees J.J. Pathophysiology of premature skin aging induced by ultraviolet light.N. Engl. J. Med. 1997; 337: 1419-1428Crossref PubMed Scopus (1181) Google Scholar, 4Varani J. Warner R.L. Gharaee-Kermani M. Phan S.H. Kang S. Chung J.H. Wang Z.Q. Datta S.C. Fisher G.J. Voorhees J.J. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.J. Invest. Dermatol. 2000; 114: 480-486Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar). However, the mechanisms of collagen destruction in aged skin have not been fully clarified. Collagen destruction is, in part, related to the induction of matrix metalloproteinase (MMP) secreted by epidermal keratinocytes and dermal fibroblasts. MMP levels are increased by various stimuli, such as UV light, oxidative stress, and cytokines. UV light rapidly activates activator protein-1 (AP-1) DNA binding and induces MMPs, including MMP-1 (collagenase), stromelysin (MMP-3), and gelatinase (MMP-9) (5Fisher G.J. Datta S.C. Talwar H.S. Wang Z.Q. Varani J. Kang S. Voorhees J.J. Molecular basis of sun-induced premature skin ageing and retinoid antagonism.Nature. 1996; 379: 335-339Crossref PubMed Scopus (1209) Google Scholar). UV-induced MMP-1 expression induces the cleavage of fibrillar collagen (types I and III) at a single site. Once collagen is cleaved by MMP-1, it is further degraded by MMP-3 and MMP-9, which are also increased by UV light exposure (6Sternlicht M.D. Werb Z. How matrix metalloproteinases regulate cell behavior.Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3256) Google Scholar). In human skin, UV-induced AP-1 transcriptional activity that is closely related to MMP expression is limited by c-Jun expression, because c-Fos is constitutively expressed (7Fisher G.J. Talwar H.S. Lin J. Lin P. McPhillips F. Wang Z. Li X. Wan Y. Kang S. Voorhees J.J. Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo.J. Clin. Invest. 1998; 101: 1432-1440Crossref PubMed Scopus (331) Google Scholar). Moreover, whereas c-Fos expression levels in young (18–28 years old) and aged (>80 years old) skin are not different, c-Jun expression is increased more so in aged skin than in young skin (8Chung J.H. Kang S. Varani J. Lin J. Fisher G.J. Voorhees J.J. Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo.J. Invest. Dermatol. 2000; 115: 177-182Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Decreased expression of procollagen is partly mediated by c-Jun expression, which is induced by UV and interferes with procollagen transcription (9Fisher G.J. Datta S. Wang Z. Li X.Y. Quan T. Chung J.H. Kang S. Voorhees J.J. c-Jun-dependent inhibition of cutaneous procollagen transcription following ultraviolet irradiation is reversed by all-trans retinoic acid.J. Clin. Invest. 2000; 106: 663-670Crossref PubMed Scopus (249) Google Scholar). Intrinsic skin aging is largely dependent on genetic factors and is associated with increased fragility and loss of elasticity (10Gilchrest B.A. Age-associated changes in the skin.J. Am. Geriatr. Soc. 1982; 30: 139-143Crossref PubMed Scopus (98) Google Scholar). With increasing age, collagen levels are reduced and MMP secretions increased in sun-protected skin compared with young skin (4Varani J. Warner R.L. Gharaee-Kermani M. Phan S.H. Kang S. Chung J.H. Wang Z.Q. Datta S.C. Fisher G.J. Voorhees J.J. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.J. Invest. Dermatol. 2000; 114: 480-486Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 11Chung J.H. Seo J.Y. Choi H.R. Lee M.K. Youn C.S. Rhie G. Cho K.H. Kim K.H. Park K.C. Eun H.C. Modulation of skin collagen metabolism in aged and photoaged human skin in vivo.J. Invest. Dermatol. 2001; 117: 1218-1224Abstract Full Text Full Text PDF PubMed Google Scholar). Collagen decrease attributable to natural skin aging may arise from its reduced synthesis and increased degradation, with a concomitant increase of MMP expression. Moreover, in aged sun-protected skin, both the number of fibroblasts and their capacity to synthesize procollagen are reduced compared with young skin (4Varani J. Warner R.L. Gharaee-Kermani M. Phan S.H. Kang S. Chung J.H. Wang Z.Q. Datta S.C. Fisher G.J. Voorhees J.J. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.J. Invest. Dermatol. 2000; 114: 480-486Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar). Essential fatty acids are divided into two categories depending on their saturation state. Omega-3 (ω-3) or omega-6 (ω-6) fatty acids have an unsaturated carbon at the third or sixth carbon in the methyl terminus, respectively. The ω-3 fatty acids include α-linolenic acid (18:3), stearidonic acid (18:4), eicosatetraenoic acid (20:4), eicosapentaenoic acid (EPA; 20:5), docosapentaenoic acid (22:5), and docosahexaenoic acid (DHA; 22:6) (12Mirnikjoo B. Brown S.E. Kim H.F. Marangell L.B. Sweatt J.D. Weeber E.J. Protein kinase inhibition by omega-3 fatty acids.J. Biol. Chem. 2001; 276: 10888-10896Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). One of these ω-3 PUFAs, EPA, is abundant in fish oil and is used to treat various diseases, such as inflammatory diseases and cancers. EPA competes with arachidonic acid (AA) for incorporation into cell membrane phospholipids and for the substrate of cyclooxygenase-2 (COX-2) (13Bagga D. Wang L. Farias-Eisner R. Glaspy J.A. Reddy S.T. Differential effects of prostaglandin derived from omega-6 and omega-3 polyunsaturated fatty acids on COX-2 expression and IL-6 secretion.Proc. Natl. Acad. Sci. USA. 2003; 100: 1751-1756Crossref PubMed Scopus (537) Google Scholar). Prostaglandins (PGs) are derived from membrane PUFAs and play important roles in inflammation, immune response, and wound healing (14Herschman H.R. Xie W. Reddy S. Inflammation, reproduction, cancer and all that.... The regulation and role of the inducible prostaglandin synthase.Bioessays. 1995; 17: 1031-1037Crossref PubMed Scopus (82) Google Scholar). In our previous report, we demonstrated that EPA inhibits UV-induced MMP-1 expression in human dermal fibroblasts and that it is mediated by the inhibition of the MEK1/ERK/c-Fos and SEK1/JNK/c-Jun pathways (15Kim H.H. Shin C.M. Park C.H. Kim K.H. Cho K.H. Eun H.C. Chung J.H. Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts.J. Lipid Res. 2005; 46: 1712-1720Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In this study, we investigated whether topical application of EPA prevents UV-induced skin damage and attenuates features of intrinsic aging in human skin in vivo. It was found that topical application of EPA inhibited UV-induced decreases in collagen and that it attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation. We also found that topical application of EPA inhibited not only JNK and p38 activation but also COX-2 expression, which is also induced by UV irradiation. Moreover, we found that topical application of EPA increases the expression of extracellular matrix (ECM) proteins, such as procollagen, tropoelastin, and fibrillin-1, by increasing transformin growth factor-β (TGF-β) expression in aged human skin in vivo. To detect procollagen, monoclonal anti-type I procollagen N-terminal extension peptide (SP1.D8) antibody was obtained from hybridoma culture media (Developmental Studies Hybridoma Bank, Iowa City, IA) and monoclonal anti-human PIC antibody was purchased from Takara (Shiga, Japan). Monoclonal anti-MMP-1 antibody and monoclonal TGF-β1, -β2, and -β3 antibodies were purchased from Oncogene (Boston, MA); monoclonal anti-COX-1 and polyclonal anti-COX-2 antibodies were from Cayman Chemical Co. (Ann Arbor, MI) and Oxford Biomedical Research (Oxford, MI), respectively. Polyclonal anti-p-ERK, anti-t-ERK, anti-p-JNK, anti-t-JNK, anti-p-p38, anti-t-p38, and anti-p-c-Jun antibodies were from Cell Signal Technology (Beverly, MA). Polyclonal anti-β-actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-human tropoelastin antibody and monoclonal anti-human fibrillin-1 antibody were from Elastin Products (Owensville, MO) and Neomarkers (Fremont, CA), respectively. EPA was purchased from Sigma (St. Louis, MO) and dissolved in ethanol-polyethylene glycol (70:30) containing 1% (w/v) tocopherol for topical application to human skin in vivo. To investigate the effects of topical EPA on UV-induced skin damage, young Korean adults (20–30 years old), all volunteers without current or prior skin disease, were enrolled. Young human volunteers (male, average age = 28 years; n = 7) were treated twice (24 h intervals; 24 h after the first treatment, the second treatment was performed) with vehicle or 2% EPA under occlusion. Ethanol-polyethylene glycol (70:30) containing 1% (w/v) tocopherol was used as vehicle. Twenty-four hours after the second treatment, buttock skin was irradiated with UV light [2 minimal erythma doses (MEDs)]. Forty-eight hours after irradiation, buttock skin was obtained by punch biopsy for immunohistochemical analysis and Western blot analysis of procollagen and MMP. To investigate the effects of EPA on UV-induced c-Jun phosphorylation, mitogen-activated protein kinase (MAPK) activation, and COX expression, skin biopsy was performed 6 h after UV irradiation. To investigate the effects of topical EPA on intrinsically aged skin, aged Korean adults (>75 years old), all volunteers without current or prior skin disease, were enrolled. Aged human volunteers (male, average age = 76.5 years; n = 4) were treated with vehicle or 2% EPA for 2 weeks (total of six times; treated on Monday, Wednesday, and Friday) under occlusion. Ethanol-polyethylene glycol (70:30) containing 1% (w/v) tocopherol was used as vehicle. Twenty-four hours after the last treatment, buttock skin was biopsied for immunohistochemical analysis and Western blot analysis. Skin protein was prepared by extracting whole skin biopsied to the upper dermis. This study was conducted according to Declaration of Helsinki principles. All procedures received prior approval from the Institutional Review Board at Seoul National University Hospital, and all subjects provided written informed consent. A Waldmann UV-800 (Waldmann, Villingen-Schwenningen, Germany) phototherapy device and F75/85W/UV21 fluorescent lamp with emmission spectrum between 285 and 350 nm (peak at 310–315 nm) were used as the UV light source, as described previously (16Seo J.Y. Lee S.H. Youn C.S. Choi H.R. Rhie G.E. Cho K.H. Kim K.H. Park K.C. Eun H.C. Chung J.H. Ultraviolet radiation increases tropoelastin mRNA expression in the epidermis of human skin in vivo.J. Invest. Dermatol. 2001; 116: 915-919Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The strength of UV irradiation at the skin surface was measured using a Waldmann UV meter (model 585100). Buttock skin was irradiated with filtered UV light filtered by a Kodacel filter (TA401/407; Kodak, Rochester, NY), and the MED was determined at 24 h after irradiation. MED ranged between 70 and 90 mJ/cm2 for the brown skin of Koreans. Immunohistochemical analyses were performed as described previously (17Son E.D. Lee J.Y. Lee S. Kim M.S. Lee B.G. Chang I.S. Chung J.H. Topical application of 17beta-estradiol increases extracellular matrix protein synthesis by stimulating TGF-beta signaling in aged human skin in vivo.J. Invest. Dermatol. 2005; 124: 1149-1161Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Human skin samples were fixed in 10% formalin for 24 h and embedded in paraffin. Serial sections (4 μm) were mounted onto silane-coated slides (Dako, Glostrup, Denmark). Sections were stained with hematoxylin and eosin and Masson-Trichrome. Acetone-fixed frozen sections were stained with the following primary antibodies in a humidified chamber at 4°C for 18 h: monoclonal anti-procollagen type I (SP1.D8) antibody, monoclonal anti-human PIC antibody, polyclonal rabbit anti-tropoelastin antibody, monoclonal anti-fibrillin-1 antibody, and anti-TGF-β1, -β2, and -β3 antibodies. Control staining was performed with normal rabbit and mouse immunoglobulin, which demonstrated no immunoreactivity (data not shown). Average epidermal thickness was determined using an image-analysis program (BMI plus software; BumMi Universe Co., Ltd.). The ratio of tropoelastin and fibrillin (fiber area to dermis area) was measured from the dermoepidermal junction to the dermis (100 μm depth) at ×400. Western blot analysis of biopsied skin samples was performed as described previously (11Chung J.H. Seo J.Y. Choi H.R. Lee M.K. Youn C.S. Rhie G. Cho K.H. Kim K.H. Park K.C. Eun H.C. Modulation of skin collagen metabolism in aged and photoaged human skin in vivo.J. Invest. Dermatol. 2001; 117: 1218-1224Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, punch-biopsied skin samples were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 20 μg/ml leupeptin, 20 μg/ml aprotinin, 5 mM PMSF, and 1 mM DTT) containing 1% (w/v) Triton X-100. Lysates were centrifuged at 12,000 g for 15 min, and supernatants were collected for Western blot and zymography. Protein concentration of samples was determined by Bradford assay. Equal amount of proteins was loaded onto Tris-glycine gels and then electrophoretically transferred to polyvinylidene difluoride membranes. Membranes were subsequently blocked with 5% skim milk in TBS/T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.05% Tween-20) and incubated with the indicated antibodies. Blotting proteins were visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, England). To assess the gelatinolytic activity of MMP-9, soluble protein was extracted from punch-biopsied skin samples as described above. Equal amounts of protein were subjected to gelatin zymography using zymogram gels containing 10% gelatin, according to the manufacturer’s protocol (Novex, San Diego, CA). After electrophoresis, gels were renatured by incubating in renaturing buffer [50 mM Tris-HCl, pH 7.4, and 2% (v/v) Triton X-100] for 30 min at room temperature. Gels were then incubated in a developing buffer [50 mM Tris-HCl, pH 8.0, 2.5 mM CaCl2, and 0.02% (w/v) sodium azide] for 24 h at 37°C. Proteolytic bands were visualized by staining gels with 0.5% (w/v) Coomassie brilliant blue solution. Statistical analyses were performed using Student’s t-test. P < 0.05 was considered statistically significant. All analyses were performed using Statistical Analysis Software (SAS, Inc., Cary, NC). Results are presented as means ± SEM. Because UV irradiation has been shown to induce epidermal thickening in human skin, we investigated the effects of EPA on UV-induced epidermal thickening and epidermal growth. EPA was applied topically to young human buttock skin, and then skin was treated with UV light (2 MEDs). Forty-eight hours after irradiation, skin was biopsied. Serially sectioned samples were stained with hematoxylin and eosin, as described in Materials and Methods. UV light induced epidermal thickening (Fig. 1) by 214 ± 19.6% (P < 0.05 vs. the UV-untreated control group; n = 7), and topical EPA decreased the epidermal thickness induced by UV treatment by 72 ± 12.6% (P < 0.05 vs. the UV-only-treated group; n = 7). In EPA-only-treated skin, epidermal thickness increased slightly to 130 ± 9% (P < 0.05 vs. the UV-untreated control group; n = 7). Photoaging is caused by repeated UV irradiation, and reductions in collagen expression have been considered to be a cause of wrinkles in photoaged skin. We investigated the effect of EPA on decreased procollagen expression induced by UV light by immunohistochemical analysis and Western blotting in young human skin in vivo. Immunohistochemistry revealed that UV decreased procollagen expression in fibroblasts in the upper dermis and in the dermoepidermal junction. Immunohistochemical results show that EPA increased procollagen staining in UV-untreated skin and that it inhibited the decrease of procollagen staining in UV-treated skin (Fig. 2A). We also found by Western blot analysis that UV light reduced the level of procollagen expression to 18 ± 4.5% (P < 0.05 vs. the UV-untreated control group; n = 7) of UV-untreated control levels (Fig. 2B). EPA restored the level of procollagen that was decreased by UV light to 46 ± 12% (P < 0.05 vs. the UV-only-treated group; n = 7) of UV-untreated control levels, as observed by immunohistochemistry. These results indicate that EPA counteracted the downregulating effects of UV light on procollagen and inhibited the decrease of procollagen. Consistent with the procollagen immunohistochemical profile, collagen fiber staining was increased substantially in EPA-pretreated, UV-irradiated skin compared with vehicle-pretreated, UV-irradiated skin, as revealed by Masson-Trichrome stain (Fig. 2A). These results demonstrate that topical application of EPA inhibited UV-induced decreases of procollagen expression in human skin in vivo. We investigated the effects of EPA on UV-induced MMP-1 and MMP-9 expression by Western blotting and zymography in young human skin in vivo. UV light increased the levels of MMP-1 (P < 0.05 vs. the UV-untreated control group; n = 5) (Fig. 3A) and MMP-9 (P < 0.05 vs. the UV-untreated control group; n = 6) (Fig. 3B). However, EPA significantly inhibited this UV-induced MMP-1 expression by 55 ± 13% (P < 0.05 vs. the UV-only-treated group; n = 5) and MMP-9 expression by 75 ± 7% (P < 0.05 vs. the UV-only-treated group; n = 6). These results show that topical EPA inhibits UV-induced MMP-1 and MMP-9 expression in human skin. AP-1 is closely related to MMPs such as MMP-1 and MMP-9 that induce the decrease of collagen. For the formation of AP-1 complex, Jun proteins form homodimers or heterodimers with Fos protein (18Huang C. Schmid P.C. Ma W.Y. Schmid H.H. Dong Z. Phosphatidylinositol-3 kinase is necessary for 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation and activated protein 1 activation.J. Biol. Chem. 1997; 272: 4187-4194Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The transcriptional activity of AP-1 is also dependent on the degree of phosphorylation of c-Jun and expression of c-Fos as well as their abundance. However, because the level of c-Fos is not altered in photodamaged human skin (8Chung J.H. Kang S. Varani J. Lin J. Fisher G.J. Voorhees J.J. Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo.J. Invest. Dermatol. 2000; 115: 177-182Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), UV-induced c-Jun expression results in decreased collagen (9Fisher G.J. Datta S. Wang Z. Li X.Y. Quan T. Chung J.H. Kang S. Voorhees J.J. c-Jun-dependent inhibition of cutaneous procollagen transcription following ultraviolet irradiation is reversed by all-trans retinoic acid.J. Clin. Invest. 2000; 106: 663-670Crossref PubMed Scopus (249) Google Scholar). Therefore, we investigated the effect of EPA on UV-induced c-Jun phosphorylation, which stabilizes c-Jun protein and then sustains its expression, in young human skin in vivo. UV was found to increase the level of phosphorylated c-Jun (P < 0.05 vs. the UV-untreated control group; n = 6) (Fig. 4), and EPA inhibited UV-induced c-Jun phosphorylation by 79 ± 11% (P < 0.05 vs. the UV-only-treated group; n = 6). These results show that EPA inhibits UV-induced MMP-1 and MMP-9 expression and that this inhibition may be mediated by a reduction in the level of phosphorylated c-Jun, which is known to be closely associated with UV-induced AP-1 activation in human skin. From the results described above, we found that EPA inhibits UV-induced MMP-1 and MMP-9 expression in human skin in vivo, which may be mediated by abolishing c-Jun phosphorylation. Thus, we investigated the effects of EPA on UV-induced MAPK in young human skin in vivo. UV irradiation induced the activation of three MAPKs, ERK, JNK, and p38 (P < 0.05 vs. the UV-untreated control group; n = 6) (Fig. 5). Although EPA did not inhibit UV-induced ERK activation (Fig. 5A), EPA inhibited UV-induced JNK (Fig. 5B) and p38 (Fig. 5C) activation by 54 ± 9% (P < 0.05 vs. the UV-only-treated group; n = 6) and 68 ± 12% (P < 0.05 vs. the UV-only-treated group; n = 6), respectively. Because JNK and p38 are closely related to c-Jun phosphorylation and expression, these findings suggest that the inhibition of UV-induced c-Jun phosphorylation by EPA is mediated by inhibition of the activation of JNK and p38. UV induces COX-2 expression that produces PG from AA in human skin. PG is known to play an important role in MMP expression (19Wahl L.M. Lampel L.L. Regulation of human peripheral blood monocyte collagenase by prostaglandins and anti-inflammatory drugs.Cell. Immunol. 1987; 105: 411-422Crossref PubMed Scopus (60) Google Scholar, 20Pentland A.P. Shapiro S.D. Welgus H.G. Agonist-induced expression of tissue inhibitor of metalloproteinases and metalloproteinases by human macrophages is regulated by endogenous prostaglandin E2 synthesis.J. Invest. Dermatol. 1995; 104: 52-57Abstract Full Text PDF PubMed Scopus (47) Google Scholar, 21Mauviel A. Halcin C. Vasiloudes P. Parks W.C. Kurkinen M. Uitto J. Uncoordinate regulation of collagenase, stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: selective enhancement of collagenase gene expression in human dermal fibroblasts in culture.J. Cell. Biochem. 1994; 54: 465-472Crossref PubMed Scopus (45) Google Scholar) and is also a well-known inhibitor of collagen synthesis (22Clark J.G. Kostal K.M. Marino B.A. Modulation of collagen production following bleomycin-induced pulmonary fibrosis in hamsters. Presence of a factor in lung that increases fibroblast prostaglandin E2 and cAMP and suppresses fibroblast proliferation and collagen production.J. Biol. Chem. 1982; 257: 8098-8105Abstract Full Text PDF PubMed Google Scholar). Therefore, we investigated the effect of EPA on UV-induced COX-2 expression in young human skin in vivo. UV irradiation dramatically induced COX-2 expression (P < 0.05 vs. the UV-untreated control group; n = 4) (Fig. 6A). EPA significantly inhibited UV-induced COX-2 expression by 76 ± 4% (P < 0.05 vs. the UV-treated group; n = 6) in young human skin in vivo (Fig. 6A). However, EPA did not affect COX-1, which is constitutively expressed in human skin (Fig. 6B). This result indicated that EPA inhibited UV-induced COX-2 expression. Histological changes in sun-protected aged human skin (intrinsic aged skin) include reduced collagen levels and reduced expression of tropoelastin and fibrillin-1 (components of elastic fibers) (23Braverman I.M. Fonferko E. Studies in cutaneous aging. I. The elastic fiber network.J. Invest. Dermatol. 1982; 78: 434-443Abstract Full Text PDF PubMed Scopus (398) Google Scholar). We investigated the effect of EPA on collagen and elastic fiber levels in aged human skin in vivo. Compared with vehicle-treated skin, EPA-treated skin demonstrated increased procollagen staining in fibroblasts throughout the dermis as well as denser extracellular staining in the dermoepidermal junction, as shown by PIC and SP1.D8 staining, respectively (Fig. 7A). Consistent with the findings of procollagen immunohistochemistry, collagen staining with Masson-Trichrome also revealed increased collagen fibers throughout the dermis in EPA-treated aged human skin (Fig. 7A). Western blot analysis also showed that EPA increased the level of procollagen by 218 ± 39% (P < 0.05 vs. the vehicle-treated control group; n = 4) (Fig. 7B). In addition to increased collagen expression, EPA increased tropoelastin and fibrillin-1 levels (Fig. 8). Compared with vehicle treatment, EPA treatment substantially increased the intricate network of tropoelastin-immunoreactive elastic fibers in the dermoepidermal junction by 145 ± 22% (P < 0.05 vs. the vehicle-treated control group; n = 4) and fibrillin-1-immunoreactive fibers by 696 ± 138% (P < 0.05 vs. the vehicle-treated control group; n = 4) (Fig. 8A, C). Western blots also revealed that EPA increased the level of tropoelastin and fibrillin-1 by 420 ± 53% (P < 0.05 vs. the vehicle-treated control group; n = 4) and 131 ± 18% (P < 0.05 vs. the vehicle-treated control group; n = 4), respectively (Fig. 8B, D). These results show that topical application of EPA to aged human skin increased procollagen, tropoelastin, and fibrillin-1 expression in the ECM and thus demonstrate that EPA has preventive and therapeutic effects on intrinsic skin aging. Because TGF-β is an important cytokine that induces the synthesis of ECM, we investigated the effect of EPA on the expression of TGF-β1, -β2, and -β3 in aged human skin in vivo (Fig. 9). In vehicle-treated skin, TGF-β1 was weakly expressed in the epidermis and dermis, whereas TGF-β2 and -β3 were stained mainly in the lower epidermis. It was found that topical application of EPA increased not only TGF-β1, which is primarily stained in dermis, but also TGF-β2 and -β3, which are stained mainly in epidermis. These results indicate that topical application of EPA to aged human skin increases ECM expression by stimulating TGF-β signaling. In our previous report, we demonstrated that EPA inhibits UV-induced MMP-1 expression in human dermal fibroblasts (15Kim H.H. Shin C.M. Park C.H. Kim K.H. Cho K.H. Eun H.C. Chung J.H. Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts.J. Lipid Res. 2005; 46: 1712-1720Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and that treating human dermal fibroblasts with EPA inhibited UV-induced AP-1 activation, which ha
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