Salvianolic Acid B Protects Normal Human Dermal Fibroblasts Against Ultraviolet B Irradiation‐Induced Photoaging Through Mitogen‐Activated Protein Kinase and Activator Protein‐1 Pathways

Exposure to ultraviolet (UV) light causes increased matrix metalloproteinase (MMP) activity and decreased collagen synthesis, leading to skin photoaging. Salvianolic acid B (SAB), a polyphenol, was extracted and purified from salvia miltiorrhiza. We assessed effects of SAB on UVB‐induced photoaging and investigated its molecular mechanism of action in UVB‐irradiated normal human dermal fibroblasts. Our results show that SAB significantly inhibited the UVB‐induced expression of metalloproteinases‐1 (MMP‐1) and interleukin‐6 (IL‐6) while promoting the production of type I procollagen and transforming growth factor β1 (TGF‐β1). Moreover, treatment with SAB in the range of 1–100 μg/mL significantly inhibited UVB‐induced extracellular signal‐regulated kinase (ERK), Jun N‐terminal kinase (JNK) and p38 phosphorylation, which resulted in decreasing UVB‐induced phosphorylation of c‐Fos and c‐Jun. These results indicate that SAB downregulates UV‐induced MMP‐1 expression by inhibiting Mitogen‐activated protein kinase (MAPK) signaling pathways and activator protein‐1 (AP‐1) activation. Our results suggest a potential use for SAB in skin photoprotection.     

Ultraviolet radiation attenuates thrombospondin 1 expression via PI3K-Akt activation in human keratinocytes

Thrombospondin 1 (TSP1) is an extracellular glycoprotein and a recognized inhibitor of angiogenesis. Recent studies have demonstrated that UV radiation induces an angiogenic switch, by which it alters the balance between pro- and anti-angiogenic factors in the skin. Here we describe the effects of acute UV exposure on TSP1 expression in human skin epidermis, primary keratinocytes and the epidermal cell line HaCaT. We found that protein and mRNA expressions of TSP1 are significantly reduced in human skin in vivo and in keratinocytes in vitro by a single UV exposure. In human skin and keratinocytes, UV exposure induced the phosphorylation of Akt, a downstream target of the PI3K pathways. Specific inhibitors of PI3K, wortmannin and LY294002, completely blocked Akt activation and UV-induced TSP1 downregulation in keratinocytes. We showed that a specific Akt phosphorylation inhibitor and small interfering RNA-mediated Akt depletion were also blocked by UV-induced TSP1 downregulation in keratinocytes. In conclusion, our findings demonstrate that acute UV exposure downregulates TSP1 expression via PI3K-Akt activation in human keratinocytes. These novel findings may help us understand the regulatory mechanisms of UV-induced skin angiogenesis.     

Prevention of UV radiation-induced premature skin aging in hairless mice by the novel compound Melanocin A

Repetitive exposure of the skin to UV radiation induces various harmful changes, such as thickening, wrinkle formation, inflammation and carcinogenesis. A variety of natural compounds and synthetic compounds have been studied to determine whether they can prevent UV-induced harmful effects. In this study, we investigated the effect of a novel compound, Melanocin A, which was isolated from Eupenicillium shearii F80695, on UV-induced premature skin aging. First, we studied the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)-9 expression in an immortalized human keratinocyte cell line, HaCaT, in vitro. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated the effect of Melanocin A on UV-induced skin changes in hairless mice in vivo. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. Taken together, these results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging.     

Synthesis and ABTS Radical,MMP‐1 Inhibitory Activity of CAPE Analogues

In the photoaging process of skin, the ultraviolet (UV)‐induced reactive oxygen species (ROS) is the key regulator of matrix metalloproteinase (MMPs) expression. In this study, a series of Caffeic acid phenethyl ester (CAPE) analogues were synthesized by conjugating the group VI elements (selenium, sulfur, oxygen)‐containing aliphatic alcohols to polyphenolic acids. Their biological activities were evaluated by in vitro testing of their radical scavenging activity the of ABTS [2,2′‐azinobis‐(3‐ethyl‐benzothiazoline‐6‐sulfonic acid)] radical and inhibitory effect against the matrix metalloproteinase‐1 (MMP‐1) activity of collagen degradation and cytotoxicity of a human dermal fibroblast skin cell. Our results suggest these compounds displayed moderate anti‐free radical, potent MMP‐1 inhibitory, and low cytotoxic activities.     

Phloroglucinol attenuates ultraviolet B radiation-induced matrix metalloproteinase-1 production in human keratinocytes via inhibitory actions against mitogen-activated protein kinases and activator protein-1

Excessive amounts of reactive oxygen species (ROS) induced by ultraviolet (UV) radiation cause skin aging via basement membrane/extracellular matrix degradation resulting from the action of matrix metalloproteinases (MMPs). Recently, phloroglucinol (1,3,5-trihydroxybenzene) was demonstrated to attenuate the cell damage induced by oxidative stress by quenching ROS and stimulating antioxidant systems. In the current study, the effect of phloroglucinol on UVB-induced photoaging was investigated in human HaCaT keratinocytes. Phloroglucinol significantly inhibited the UVB-induced (1) upregulation of MMP-1 mRNA, protein and activity; (2) augmentation of intracellular Ca(2+) levels; (3) phosphorylation of mitogen-activated protein kinases (MAPKs); (4) expression of c-Fos and phospho c-Jun; and (5) enhancement of activator protein-1 (AP-1) binding to the MMP-1 promoter. In addition, the knockdown of MAPKs significantly inhibited UVB-induced MMP-1 expression. The results of this study suggest that phloroglucinol may be useful as a photoprotective compound for the skin.     

Protective Effect of Djulis (Chenopodium formosanum) Extract against UV- and AGEs-Induced Skin Aging via Alleviating Oxidative Stress and Collagen Degradation

Skin aging is a complex process involving photoaging and glycation stress, which share some fundamental pathways and have common mediators. They can cause skin damage and collagen degradation by inducing oxidative stress and the accumulation of reactive oxygen species (ROS). Chenopodium formosanum (CF), also known as Djulis, is a traditional cereal in Taiwan. This study investigated the protection mechanisms of CF extract against ultraviolet (UV) radiation and advanced glycation end products (AGEs)-induced stress. The results indicated that CF extract had strong antioxidant and free radical scavenging effects. It could reduce UV-induced intracellular ROS generation and initiate the antioxidant defense system by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in human skin fibroblasts. CF extract modulated mitogen-activated protein kinase (MAPK) and transformed growth factor-beta (TGF-β) signaling pathways to alleviate oxidative stress-induced skin aging. Moreover, the results revealed that CF extract not only promoted collagen synthesis but also improved aging-induced collagen degradation. CF extract attenuated AGEs-induced ROS production and the upregulation of receptor for AGEs (RAGE). The overall results suggest that CF extract provides an effective anti-aging strategy by preventing skin damage from oxidative stress and collagen loss with potent antioxidant, anti-photoaging, and antiglycation activities.     

Plantamajoside Inhibits UVB and Advanced Glycation End Products‐Induced MMP‐1 Expression by Suppressing the MAPK and NF‐κB Pathways in HaCaT Cells

Photoaging and glycation stress are major causes of skin deterioration. Oxidative stress caused by ultraviolet B (UVB) irradiation can upregulate matrix metalloprotease 1 (MMP‐1), a major enzyme responsible for collagen damage in the skin. Advanced glycation end products (AGEs) accumulate via gradual formation from skin proteins, especially from long‐lived proteins such as dermal elastin and collagen. Plantamajoside (PM), isolated from Plantago asiatica, has various biological effects including anti‐inflammatory and antioxidant effects. In this study, we assessed the protective effects of PM on a human keratinocyte cell line (HaCaT) and primary human dermal fibroblasts (HDF) against stress caused by glyceraldehyde‐induced AGEs (glycer‐AGEs) with UVB irradiation. We found that PM attenuated UVB‐ and‐glycer‐AGEs‐induced MMP‐1 expression in HaCaT and HDF cells and proinflammatory cytokines expression by inhibiting the phosphorylation of mitogen‐activated protein kinases (MAPKs) activated by reactive oxygen species. Specific inhibitors of NF‐κB and MAPKs attenuated the induced expression of MMP‐1. PM also inhibited the phosphorylation of IκBα, and reduced nuclear translocation of NF‐κB in these cells. Furthermore, PM attenuated the upregulation of receptor for AGEs (RAGE) by glycer‐AGEs with UVB irradiation. Therefore, our findings strongly suggest that PM is a promising inhibitor of skin photoaging.     

CD11b+ cells are the major source of oxidative stress in UV radiation-irradiated skin: possible role in photoaging and photocarcinogenesis

Exposure of skin to solar UV radiation induces oxidative stress and suppression of cell-mediated immune responses. These effects are associated with the greater risk of several skin disorders including photoaging and photocarcinogenesis. We have shown that UV-induced infiltrating leukocytes contribute in developing oxidative stress in UV-irradiated skin. The peak period of UV-induced infiltrating leukocytes lies between 48 and 72 h after UV exposure of the skin. In this study we demonstrated that UV (90 mJ/cm2)-induced infiltrating CD11b+ cells in C3H/HeN mice skin were the major source of oxidative stress. Hydrogen peroxide (H2O2) was determined as a marker of oxidative stress. Flow cytometric analysis of viable cells revealed that the number of CD11b+H2O2+ cells were significantly higher (31.8%, P < 0.001) in UV-irradiated skin in comparison with non-UV-exposed skin (0.4%). Intraperitoneal administration of monoclonal antibodies to CD11b (rat IgG2b) to C3H/HeN mice inhibited UVB-induced infiltration of leukocytes, as evidenced by reduction in myeloperoxidase activity (64-80%, P < 0.0005), concomitant with significant reduction in H2O2 production both in epidermis and dermis (66-83%, P < 0.001-0.0005) when compared with the administration of rat IgG2b isotype of anti-CD11b. Furthermore, CD11b+ and CD11b- cell subsets were separated by immunomagnetic cell isolation technique from total epidermal and dermal single cell suspensions obtained 48 h after UV irradiation of the skin and analyzed for H2O2 production. Analytical data revealed that CD11b+ cell population from UV-irradiated skin resulted in significantly higher production of total H2O2 in both epidermis and dermis (87-89%, P < 0.0001) in comparison with CD11b- cell population (11-13% of total H2O2). These data revealed that infiltrating CD11b+ cells were the major source of oxidative stress in UV-irradiated skin and thus may contribute to photoaging and promotion of skin tumor growth within the UV-irradiated skin. Together, these data suggest that reduction in UV-induced skin infiltration of CD11b+ cells may be an alternative and effective strategy to reduce solar UV light-induced oxidative stress-mediated skin disorders including photoaging and photocarcinogenesis.     

Differential activation of ERK 1/2 and JNK in normal human fibroblast-like cells in response to UVC radiation under different oxygen tensions

The mechanisms by which mitogen-activated protein kinases (MAPK) respond to the input of UV-induced signal transduction pathways and the resulting biological functions are not well understood. We investigated whether the level of oxygen tension of culture was responsible for the differential activation of MAPK and different cellular outcomes in UVC-irradiated cells. The intracellular oxidative level of normal human fibroblast-like cells in a normal atmosphere (normoxic, 20% O2) was increased within 30 min after UVC irradiation. When cells were cultured at lower oxygen tension in the presence of an antioxidant N-acetyl-L-cysteine (NAC) or under physiologically hypoxic (5% O2) conditions, the elevation of the oxidative level by UV-irradiation was significantly reduced. Among MAPK, extracellular-signal related kinase (ERK) 1/2 was activated by UV regardless of the oxidative level, while c-Jun N-terminal kinase (JNK) activation was inhibited in NAC-treated and in hypoxic cultures. In addition, in cultures at lower oxygen tension, there was less apoptosis and cell survival was enhanced. These results suggest that UV-induced oxidative stress was responsible for intracellular signaling through the JNK pathway. Furthermore, the balance between ERK1/2 and JNK activities after UV irradiation under different oxygen tensions possibly modified cellular outcome in response to UV.     

Collagen-EGCG Combination Synergistically Prevents UVB-Induced Skin Photoaging in Nude Mice

Ultraviolet (UV) radiation is a major cause of skin photoaging through generating excessive oxidative stress and inflammation. One of the strategies is to use photo-chemoprotectors, such as natural products with antioxidant and anti-inflammatory properties, to protect the skin from photo damage. The present study investigates the photoprotective potentials of topical administration of unhydrolyzed collagen, epigallocatechin gallate (EGCG), and their combination against ultraviolet B (UVB)-induced photoaging in nude mice. It is found that both the solo and combined pretreatments could recover UVB-induced depletion of antioxidative enzymes, including superoxide dismutase and glutathione peroxidase (GSH-Px), as well as an increase of lipid peroxide malondialdehyde and inflammatory tumor necrosis factor-α. Meanwhile, the UVB-stimulated skin collagen degradation is attenuated significantly with drug treatments, which is evidenced by expression analysis of matrix metalloproteinase-1 and hydroxyproline. Additionally, the mouse skin histology shows that the drug-pretreated groups possess decreased epidermis thickness and normal collagen fiber structure of the dermis layer. These results demonstrate that both EGCG and collagen can protect the skin against UVB-induced skin photoaging. Synergistically, the combination of them shows the maximum prevention to skin damage, showing its potential in the application of anti-photoaging formulation products.     

Santamarine Shows Anti-Photoaging Properties via Inhibition of MAPK/AP-1 and Stimulation of TGF-β/Smad Signaling in UVA-Irradiated HDFs

Chronic UVA exposure results in elevated reactive oxygen species in skin which leads to photoaging characterized as upregulated matrix metalloproteinase (MMP)-1 and loss of collagen. Therefore, natural antioxidants are hailed as promising agents to be utilized against photoaging. In the current study, reynosin and santamarine, two known sesquiterpene lactones isolated from Artemisia scoparia, were analyzed for their anti-photoaging properties in UVA-irradiated human dermal fibroblasts (HDFs). Results showed that UVA irradiation (8 J/cm²) upregulated the MMP-1 secretion and expression, and suppressed collagen production, which were significantly reverted by santamarine treatment (10 µM). Although both reynosin and santamarine exhibited ROS scavenging abilities, reynosin failed to significantly diminish UVA-stimulated MMP-1 release. UVA-irradiated HDFs showed increased collagen production when treated with santamarine. As a mechanism to suppress MMP-1, santamarine significantly suppressed the UVA-induced phosphorylation of p38 and JNK and nuclear translocation of p-c-Fos and p-c-Jun. Santamarine promoted collagen I production via relieving the UVA-induced suppression on TGF-β and its downstream activator Smad2/3 complex. Antioxidant properties of santamarine were also shown to arise from stimulating Nrf2-dependent expression of antioxidant enzymes SOD-1 and HO-1 in UVA-irradiated HDFs. In conclusion, santamarine was found to be a promising natural antioxidant with anti-photoaging properties against UVA-induced damages in HDFs.     

A Novel Molecule 3-(1′-Methyltetrahydropyridinyl)-2,4-6-Trihydroxy Acetophenone Alleviates Ultraviolet-B-Induced Photoaging in Human Dermal Fibroblasts and BALB/c Mice

Ultraviolet radiation (UVR) is the major exogenous agent that disturbs tissue homeostasis and hastens the onset of age-related phenotypes (photoaging). Exposure to UV-B radiation promotes apoptosis in human skin cells via induction of Reactive Oxygen Species (ROS)-mediated Endoplasmic Reticulum (ER) stress by activating the PERK-eIF2α-CHOP pathway, which plays a major role in exacerbating skin photoaging. Alleviating the production of ROS and boosting the antioxidant capacity of cells is the foremost therapeutic strategy to avert the repercussions of ultraviolet radiation exposure. In this study, we investigated the role of 3-(1′-methyltetrahydropyridinyl)-2,4-6-trihydroxy acetophenone (IIIM-8) in thwarting the UV-B-induced photoaging. We observed that IIIM-8 ameliorates UV-B-induced oxidative stress, ER stress, Loss of Mitochondrial membrane potential, MAPK activation and Inflammation in irradiated skin cells. Ultraviolet radiation-related damage to fibroblasts within the dermis leads to collagen degradation-the hallmark of photoaging. IIIM-8 substantially restored the synthesis of collagen and prevented its degradation via the downregulation of matrix metalloproteinases. Topical application of IIIM-8 prevented BALB/c mice skin from UV-B-induced leukocyte infiltration, epidermal thickening and disruption of Extracellular matrix components. Implying that IIIM-8 has a strong photoprotective property and has potential to be developed as a topical therapeutic/cosmeceutical agent against UV-B-induced photoaging.     

FORMATION OF PROTEIN-BOUND 3,4-DIHYDROXYPHENYLALANINE IN COLLAGEN TYPES I AND IV EXPOSED TO ULTRAVIOLET LIGHT

Abstract— Collagen was exposed to an ultraviolet (UV) lamp that emitted predominantly in the UVB range. The cross-linking of collagen type I and type IV by UV irradiation was observed. Amino acid analyses revealed that Tyr residues in both collagen types I and IV were decreased by irradiation. In collagen type IV, losses of His and Met residues were also observed. These losses of collagen type IV may be due to the degradation of Trp, which exists in collagen type IV and decreased drastically during UV irradiation. To clarify the mechanism of Tyr modification in both types of collagen, the degradation products of Tyr were analyzed. Dityrosine, which is a dimer of the Tyr residue, could not be detected in the acid hydrolysates of UV-irradiated collagen. However, 3,4-dihydroxyphenylalanine, DOPA, was detected in the hydrolysates using HPLC with an electrochemical detector. The amounts of DOPA in the acid hydrolysates of collagen exposed to UV light for 24 h were approximately 350 pmol/mg protein (collagen type IV) and 80 pmol/mg protein (collagen type I). The DOPA formed may partially contribute to photoaging of the skin.     

Epigallocatechin-3-gallate inhibits photocarcinogenesis through inhibition of angiogenic factors and activation of CD8+ T cells in tumors

There has been considerable interest in the use of botanical supplements to protect skin from the adverse effects of solar UV radiation, including photocarcinogenesis. We and others have shown that topical application of (-)-epigallocatechin-3-gallate (EGCG) from green tea prevents photocarcinogenesis in mice; however, the chemopreventive mechanism of EGCG in an in vivo tumor model is not clearly understood. In this study, UV-B-induced skin tumors with and without treatment of EGCG ( approximately 1 mg/cm(2)) and age-matched skin biopsies from SKH-1 hairless mice were used to identify potential molecular targets of skin cancer prevention by EGCG. These biopsies were analyzed for various biomarkers of angiogenesis and antitumor immune response using immunostaining, Western blotting and gelatinolytic zymography. We report that compared to non-EGCG-treated tumors, topical application of EGCG in UV-induced tumors resulted in inhibition of protein expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9, which play crucial roles in tumor growth and metastasis. In contrast, tissue inhibitor of MMP-1 (TIMP-1), which inhibits MMP activity, was increased in tumors. With respect to the tumor vasculature, EGCG decreased the expression of CD31, a cell surface marker of vascular endothelial cells, and inhibited the expression of vascular endothelial growth factor in tumors, which are essential for angiogenesis. EGCG inhibited proliferating cell nuclear antigen in UV-B-induced tumors as well. Additionally, higher numbers of cytotoxic T lymphocytes (CD8(+) T cells) were detected in EGCG-treated tumors compared with non-EGCG-treated tumors. Together, these in vivo tumor data suggested that inhibition of photocarcinogenesis in mice by EGCG is associated with inhibition of angiogenic factors and induction of antitumor immune reactivity.     

The Injury and Cumulative Effects on Human Skin by UV Exposure from Artificial Fluorescence Emission

The injury and cumulative effects of UV emission from fluorescence lamp were studied. UV intensity from fluorescence lamp was measured, and human skin samples (hips, 10 volunteers) were exposed to low‐dose UV irradiation (three times per week for 13 consecutive weeks). Three groups were examined: control group without UV radiation; low‐dose group with a cumulative dose of 50 J cm^?2 which was equivalent to irradiation of the face during indoor work for 1.5 years; and high‐dose group with 1000 J cm^?2 cumulative dose equivalent to irradiation of the face during outdoor activities for 1 year. Specific indicators were measured before and after UVA irradiation. The findings showed that extending the low‐dose UVA exposure decreased the skin moisture content and increased the transepidermal water loss as well as induced skin color changes (decreased L* value, increased M index). Furthermore, irradiated skin showed an increased thickness of cuticle and epidermis, skin edema, light color and unclear staining collagen fibers in the dermis, and elastic fiber fragmentation. In addition, MMP‐1, p53 and SIRT1 expression was also increased. Long‐term exposure of low‐dose UVA radiation enhanced skin photoaging. The safety of the fluorescent lamp needs our attention.     

Using ultra-weak photon emission to determine the effect of oligomeric proanthocyanidins on oxidative stress of human skin

Exposure of skin to ultraviolet (UV) radiation triggers oxidative stress in skin tissue that can lead to erythema, early skin aging or even cancer. It is suggested that oligomeric proanthocyanidins (OPCs), phytonutrients that belong to the polyphenol family have an anti-oxidant/anti-inflammatory activity on the skin. Measuring ultra-weak photon emission (UPE) is a non-invasive, fairly-sensitive and convenient technique for continuously monitoring oxidative stress. The present study was undertaken to confirm anti-oxidant activity of the specific OPCs cream formulation in human skin by measuring UPE of skin. In the present study 25 healthy female subjects participated. As a baseline measurement of skin, UPE was recorded from the dorsal surface of the subjects’ hands before (spontaneous UPE) and after exposure to UV (UV-induced UPE). The effects of the OPCs cream on spontaneous and UV-induced UPE were measured using a fractionated UV exposure protocol. UV exposure resulted in an increase in UPE from both hands. Repeat UV exposure also resulted in a long-term increase of spontaneous UPE. This is likely due to depletion of anti-oxidant capacity of skin resulting in sensitization of skin to UV. It was assessed by measuring spontaneous UPE at 80 min after each UV exposure. Application of the OPCs cream immediately after UV exposure resulted in a significant (approx. 30%) decrease in UV-induced UPE. Topical OPCs cream application also reduced sensitization of skin to UV following repeated UV exposure (i.e., reduced long-term increase in spontaneous UPE). This study indicates that the specific OPCs cream formulation significantly decreases UV-induced oxidative stress in human skin based on UPE measurement. It therefore suggests that regular use of this OPCs cream might protect skin from harmful effects of UV.     

Nicotinamide Prevents Ultraviolet Radiation-induced Cellular Energy Loss

UV radiation is carcinogenic by causing mutations in the skin and also by suppressing cutaneous antitumor immunity. We previously found nicotinamide (vitamin B3) to be highly effective at reducing UV-induced immunosuppression in human volunteers, with microarray studies on in vivo irradiated human skin suggesting that nicotinamide normalizes subsets of apoptosis, immune function and energy metabolism-related genes that are downregulated by UV exposure. Using human adult low calcium temperature keratinocytes, we further investigated nicotinamide’s effects on cellular energy metabolism. We found that nicotinamide prevented UV-induced cellular ATP loss and protected against UV-induced glycolytic blockade. To determine whether nicotinamide alters the effects of UV-induced oxidative stress posttranslationally, we also measured UV-induced reactive oxygen species (ROS). Nicotinamide had no effect on ROS formation, and at the low UV doses used in these studies, equivalent to ambient daily sun exposure, there was no evidence of apoptosis. Hence, nicotinamide appears to exert its UV protective effects on the skin via its role in cellular energy pathways.