Protective effects of a Ficus deltoidea (Mas cotek) extract against UVB-induced photoageing in skin cells

Continuous exposure to ultraviolet (UV) irradiation leads to a variety of skin damage, such as sunburn, pigmentation, premature ageing, and photocarcinogenesis. Various phytochemical extracts have been identified to efficiently protect sun exposed skin from UV induced photodamage. A Ficus deltoidea (Mas cotek) water extract has been widely used for women’s health in Malaysia. In a previous study from this lab, the F. deltoidea extract exhibited strong anti-melanogenic effects towards cultured B16F1 melanoma cells. Additional studies were intended to evaluate the effects of the F. deltoidea extract on antiphotoageing activity using cultured human dermal fibroblasts and immortalised human keratinocytes (HaCaT). Both TNF-α and cyclooxygenase (COX-2) play primary roles in the inflammation process upon UV irradiation and are known to be stimulated by UVB irradiation. Treatment with the F. deltoidea extract dramatically inhibited the UVinduced TNF-α, IL-1α, IL-6, and COX-2 expression. The decreased collagen synthesis of fibroblasts as a result of UVB exposure was restored to a normal level after treatment with the F. deltoidea extract. In addition, the enhanced MMP-1 expression upon UVB irradiation was downregulated by the F. deltoidea extract in a dose-dependent manner. The overall findings indicate that the F. deltoidea extract may exert a protective effect against UVB-induced damage in the skin that is useful for anti-photoageing cosmetic products.     

Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism

Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells.     

Topical tocopherol acetate reduces post-UVB, sunburn-associated erythema, edema, and skin sensitivity in hairless mice.

Exposure of the skin of the back of skh-1 hairless mice to UVB (310 nm peak) irradiation at doses of 0.115-0.23 J/cm2 results after 24-48 h in an erythema which can be quantified using an erythema meter, providing a useful model of sunburn. Application of pure d-alpha-tocopherol acetate, a thick oil, to the skin immediately following the exposure to UVB significantly reduces the increase in erythema index, by 40-55%. At the lower dose (0.115 J/cm2), skin thickness (associated with edematous swelling of the sunburned skin) was measured by a novel non-invasive technique not previously reported for this purpose--magnetic resonance imaging (MRI). In two experiments the UVB-induced increase in skin thickness was significantly reduced at 24 hr by 29 and 54%, and at 48 hr by 26 and 61%. After 8 days the untreated irradiated mouse skin still showed a significant increase in thickness (24%) compared to the untreated unirradiated control, while the treated irradiated control was not significantly thicker than the unexposed control. Skin sensitivity was tested using a modification of the technique of esthesiometry, by observing rapid avoidance responses of the mouse to a pressure of 0.96 g/cm2 exerted by applying to the skin the tip of a nylon esthesiometer fiber extended to 60 mm in length. The untreated irradiated mice were more sensitive (p less than 0.07, Wilcoxon test) than the treated irradiated mice, and also significantly different from the untreated unirradiated control mice (p less than 0.04, Wilcoxon test), but the treated irradiated mice were not significantly differently sensitive when compared to the unirradiated controls (p less than 0.32). Taken together these data indicate that the erythema, edema, and skin sensitivity commonly associated with UVB-induced sunburn are significantly reduced by topical application of tocopherol acetate even after the exposure has occurred. This observation suggests that treatment of sunburn may be possible even after the irradiation has stopped, by a derivative of d-alpha-tocopherol which is stable to autooxidation.     

Wild chrysanthemum extract prevents UVB radiation-induced acute cell death and photoaging

Wild chrysanthemum (Chrysanthemum indicum L.) is traditionally used in folk medicine as an anti-inflammatory agent. It is also used in the southwest plateau region of China to prevent ultraviolet-induced skin damage. However, the role and mechanism by which wild chrysanthemum prevents UV-induced skin damage and photoaging have never been investigated in vitro. In the present study, we found that aqueous extracts from wild chrysanthemum strongly reduced high-dose UVB-induced acute cell death of human immortalized keratinocytic HaCat cells. Wild chrysanthemum extract was also demonstrated to reduce low-dose UVB-induced expression of the photoaging-related matrix metalloproteinases MMP-2 and MMP-9. The ROS level elevated by UVB irradiation was strongly attenuated by wild chrysanthemum extract. Further study revealed that wild chrysanthemum extract reduced UVB-triggered ERK1/2 and p38 MAPK phosphorylation and their protective role, which is partially dependent on inhibiting p38 activation. These results suggest that wild chrysanthemum extract can protect the skin from UVB-induced acute skin damage and photoaging by reducing the intracellular reactive oxygen species (ROS) level and inhibiting p38 MAPK phosphorylation. The present study confirmed the protective role of wild chrysanthemum against UV-induced skin disorders in vitro and indicated the possible mechanism. Further study to identify the active components in wild chrysanthemum extract would be useful for developing new drugs for preventing and treating skin diseases, including skin cancer and photoaging, induced by UV irradiation.     

白藜芦醇对紫外线照射后人角质形成细胞AQP3 表达的影响

目的:探讨白藜芦醇对紫外线照射后人皮肤角质形成细胞水通道蛋白3(AQP3)表达的影响及意义。方法:原代培养人皮肤角质形成细胞,采用UVB(20mJ/cm2,40mJ/cm2)照射角质形成细胞后,立即加入0.1mmol/L的白藜芦醇进行干预。RT-PCR检测照射前后角质形成细胞中AQP3 mRNA的表达量,并用羟胺法、比色法、TBA法检测照射前后细胞超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量。结果:1.UVB照射后角质形成细胞AQP3 mRNA的表达量下降(P<0.05),且UVB照射剂量越大,AQP3 mRNA下降越显著(P<0.05)。2.白藜芦醇能显著增加UVB照射后角质形成细胞SOD和GSH-Px活性,并降低细胞MDA含量(P<0.05)。3.白藜芦醇能显著抑制UVB导致的角质形成细胞AQP3 mRNA下降(P<0.05)。结论:白藜芦醇可能通过抑制UVB导致的AQP3 mRNA下降,及提高氧化酶活性、清除自由基的功能,从而延缓皮肤衰老。     

Activation of T-cell Protein-tyrosine Phosphatase Suppresses Keratinocyte Survival and Proliferation following UVB Irradiation

Chronic exposure to UV radiation can contribute to the development of skin cancer by promoting protein-tyrosine kinase (PTK) signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces protein-tyrosine phosphatase (PTP) inactivation. In the present work, we report that T-cell PTP (TC-PTP) activity is stimulated during the initial response to UVB irradiation, which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. Corroborating with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure, and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer.     

Mutations induced by ultraviolet light

The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms involving oxidized DNA bases. We also discuss the role of DNA damage-tolerant DNA polymerases in UV lesion bypass and mutagenesis.     

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation-induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB-irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0-400 mJ/cm(2) UVB irradiation. UVB at a dose 400 mJ/cm(2) induced the release of various cytokines; interleukin (IL)-1beta, IL-6, IL-8, interferon (IFN)-gamma, granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, and tumor necrosis factor (TNF)-alpha. These results suggest that UVB irradiation-induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.     

Nitric oxide is responsible for oxidative skin injury and modulation of cell proliferation after 24 hours of UVB exposures

Abstract

Nitric oxide (NO) is produced by various mammalian cells and plays a variety of regulatory roles in normal physiology and in pathological processes. This article provides evidence regarding the participation of NO in UVB-induced skin lesions and in the modulation of skin cell proliferation following UVB skin irradiation. Hairless mice were subjected to UVB irradiation for 3 hours and the skin evaluated immediately, 6 and 24 hours postirradiation. The skin lipid peroxidation, and NO levels evaluated by chemiluminescence and inducible nitric oxide synthase (iNOS) and nitrotyrosine immunolabelling increased significantly 24 hours after irradiation and decreased under the treatment with aminoguanidine (AG). On the other hand, cell proliferation markers, PCNA and VEGF showed a strong labelling index when AG was used. The data indicate that NO mediates, at least in part, the lipid peroxidation and protein nitration and also promotes the down regulation of factors involved in cell proliferation. This work shows that the NO plays an important role in the oxidative stress damage and on modulation of cell proliferation pathways in UVB irradiated skin.     

Nitric oxide is responsible for oxidative skin injury and modulation of cell proliferation after 24 hours of UVB exposures

Nitric oxide (NO) is produced by various mammalian cells and plays a variety of regulatory roles in normal physiology and in pathological processes. This article provides evidence regarding the participation of NO in UVB-induced skin lesions and in the modulation of skin cell proliferation following UVB skin irradiation. Hairless mice were subjected to UVB irradiation for 3 hours and the skin evaluated immediately, 6 and 24 hours postirradiation. The skin lipid peroxidation, and NO levels evaluated by chemiluminescence and inducible nitric oxide synthase (iNOS) and nitrotyrosine immunolabelling increased significantly 24 hours after irradiation and decreased under the treatment with aminoguanidine (AG). On the other hand, cell proliferation markers, PCNA and VEGF showed a strong labelling index when AG was used. The data indicate that NO mediates, at least in part, the lipid peroxidation and protein nitration and also promotes the down regulation of factors involved in cell proliferation. This work shows that the NO plays an important role in the oxidative stress damage and on modulation of cell proliferation pathways in UVB irradiated skin.     

Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model

Activation of activator protein-1 (AP-1) and increased expression of cyclooxygenase-2 (COX-2) have been clearly shown to play a functional role in UVB-induced skin tumor promotion. In this study, we examined UVB-induced signal transduction pathways in SKH-1 mouse epidermis leading to increases in COX-2 expression and AP-1 activity. We observed rapid increases in p38 mitogen-activated protein kinase (MAPK) signaling through activation of p38 MAPK and its downstream target, MAPK activated protein kinase-2. UVB also increased phosphatidylinositol 3-kinase (PI3K) signaling as observed through increases in AKT and GSK-3beta phosphorylation. Activation of the p38 MAPK and PI3K pathways results in the phosphorylation of cyclic AMP-responsive element binding protein, which was also observed in UVB-irradiated SKH-1 mice. Topical treatment with SB202190 (a specific inhibitor of p38 MAPK) or LY294002 (a specific inhibitor of PI3K) significantly decreased UVB-induced AP-1 activation by 84% and 68%, respectively, as well as COX-2 expression. Our data show that in mouse epidermis, UVB activation of the p38 MAPK and PI3K pathways leads to AP-1 activation and COX-2 expression.     

Topical application of a selective cyclooxygenase inhibitor suppresses UVB mediated cutaneous inflammation

Ultraviolet B (UVB) radiation causes much of the cutaneous damage after both acute and long-term exposure, and is also the most important etiologic agent in human skin cancer. UVB exposure initially induces an inflammatory response characterized by edema, dermal infiltration of leukocytes, sunburn cell formation, as well as the induction of cyclooxygenase-2 (COX-2) gene expression and subsequent increase in the production and release of prostaglandins. This process of inflammation induced by UVB exposure has been linked to tumor formation. Recently, a specific COX-2 inhibitor, Celecoxib, was developed, which inhibits COX-2-induced inflammation without inhibiting the cytoprotective function of cyclooxygenase-1 (COX-1). The present study compared the effects of topical treatment with Celecoxib (a specific COX-2 inhibitor) and Ibuprofen (a nonspecific COX inhibitor) on the acute UVB-induced cutaneous inflammatory response. We show that the specific inhibition of COX-2 effectively reduced many parameters of UVB-mediated inflammation, including edema, dermal neutrophil infiltration and activation, prostaglandin E2 (PGE2) levels and the formation of sunburn cells. By inhibiting this inflammatory response, topical Celecoxib treatment may ultimately be effective in preventing UVB-induced tumor development in the skin.     

Obesity increases the risk of UV radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling

Obesity has been implicated in several diseases, including cancer; however, the relationship of obesity and susceptibility to ultraviolet (UV) radiation-caused skin diseases has not been investigated. As UV-induced oxidative stress has been implicated in several skin diseases, we assessed the role of obesity on UVB-induced oxidative stress in genetically obese Lep(ob)/Lep(ob) (leptin-deficient) mice. Here, we report that chronic exposure to UVB (120 mJ/cm(2)) resulted in greater oxidative stress in the skin of obese mice in terms of higher levels of H(2)O(2) and NO production, photo-oxidative damage of lipids and proteins, and greater depletion of antioxidant defense enzymes, like glutathione, glutathione peroxidase, and catalase. As UV-induced oxidative stress mediates activation of MAPK and NF-kappaB signaling pathways, we determined the effects of UVB on these pathways in obese mice. Exposure of obese mice to UVB resulted in phosphorylation of ERK1/2, JNK, and p38 proteins of the MAPK family. Compared to wild-type mice, the obese mice exhibited higher levels of phosphorylation of these proteins, greater activation of NF-kappaB/p65, and higher levels of circulating proinflammatory cytokines, including TNF-alpha, IL-1beta and IL-6, on UVB irradiation. Taking these results together, our study suggests for the first time that obesity in mice is associated with greater susceptibility to UVB-induced oxidative stress and therefore may be a risk factor for skin diseases associated with UVB-induced oxidative stress.