低剂量中波紫外线照射Nrf2基因缺陷小鼠后诱导晒伤反应的实验研究

[目的]观察Nrf2-Keap1通路在保护皮肤免受中波紫外线(UVB)影响中的作用.[方法]8周龄雌性野生型(即Nrf2+/+组)和Nrf2-/-小鼠,每组8只,分别用200 mJ/cm2 UVB(FL120SE灯)照射小鼠耳朵背侧,用刻度计分别测量UVB照射前和照射后1、2、4、7、9、11、14 d小鼠耳朵厚度,记录照射前后两组鼠耳皮肤镜下形态,36 h取各组鼠耳标本进行HE染色及TUNEL实验,记录结果并进行统计学分析.[结果]Nrf2-/-组小鼠耳平均厚度为(0.49±0.22) cm,Nrf2+/+组为(0.25±0.03) cm,经秩和检验,差异有统计学意义(P＜0.01).UVB照射小鼠后36 h组织学检查,Nrf2-/-组真皮水肿和表皮坏死,淋巴细胞浸润和真皮血管扩张更显著;表皮晒伤细胞数[(17.0±3.9)/视野]也较Nrf2+/+组[(5.0±1.7)/视野]明显,两组差异有统计学意义(P＜ 0.01);Nrf2-/-组TUNEL阳性细胞数约是Nrf2+/+组的5倍.单一UVB照射Nrf2-/-小鼠较Nrf2+/+小鼠发生更强烈而持久的晒伤反应.[结论]Nrf2-Keap1通路可保护皮肤免受UVB的影响,包括由于UVB照射引起的细胞凋亡和氧化损伤.     

Sesamol inhibits UVB-induced ROS generation and subsequent oxidative damage in cultured human skin dermal fibroblasts

The exposure of cells to ultraviolet B radiation (UVB) can induce the production of reactive oxygen species (ROS) which damage cellular components. Free radical scavengers and antioxidants can interfere with the production of ROS. We studied cytotoxicity, intracellular ROS levels, lipid peroxidation, antioxidant status and oxidative DNA damage in cultured human skin dermal fibroblast adult cells (HDFa) exposed to UVB in the presence of sesamol, a natural phenolic compound. The levels of cytotoxicity, intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptotic morphological changes were significantly increased in UVB irradiated HDFa cells. We also observed that the activities of enzymatic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) and the levels of non-enzymatic antioxidant status (GSH) were significantly decreased in UVB irradiated cells. On the other hand, sesamol pretreatment significantly decreased cytotoxicity, intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptotic morphological changes in sesamol-pretreated and UVB-irradiated HDFa cells. We have also observed increased enzymatic and non-enzymatic antioxidants status in sesamol plus UVB-irradiated cells. Among the different doses tested, 80 μM of sesamol shows maximum protection for UVB-induced oxidative damage. In conclusion, UVB-induced ROS formation, cell fatality, lipid peroxidation, antioxidant depletion and oxidative DNA damage in HDFa cells is inhibited by sesamol, which, probably through its ROS scavenging activity.     

Prunella vulgaris extract and rosmarinic acid prevent UVB-induced DNA damage and oxidative stress in HaCaT keratinocytes

Solar radiation is a very important exogenous factor in skin pathogenesis and can lead to the development of a number of skin disorders. UVB irradiation is known to induce oxidative stress, inflammation and especially DNA lesions in exposed cells. It is important, therefore, to identify agents that can offer protection against UVB-caused skin damage. Natural compounds have been studied for their possible ability to control/modulate various lifestyle-related diseases. The application of plant compounds/extracts with screening, antioxidant and anti-inflammatory activities may also successfully protect the skin against UV-caused injury. We assessed the potency of Prunella vulgaris extract (PVE) and its main phenolic acid component, rosmarinic acid (RA), to suppress UVB-induced (295–315 nm) alterations to human keratinocytes HaCaT using a solar simulator. Pre- and post-treatment of HaCaT cells with PVE (5–50 mg/l) and RA (0.18–1.8 mg/l) reduced breakage together with the apoptotic process. PVE and RA also significantly eliminated ROS production and diminished IL-6 release. Taken together, both PVE and RA prevent UVB-caused injury to keratinocytes. However their efficacy needs to be demonstrated in vivo.     

Nrf2激活剂防御紫外线致皮肤氧化应激损伤的研究

核因子E2相关因子2(Nrf2)激活剂作为一种新型的光保护剂,能有效抵御UV照射对皮肤的损伤。天然Nrf2激活剂可提取自茶多酚、姜黄素、莱菔硫烷、白藜芦醇、丹参酮、小白菊等,为以Nrf2信号通路为靶点治疗氧化应激损伤皮肤病提供了广阔前景。     

Expression of the human Cathepsin L inhibitor hurpin in mice: skin alterations and increased carcinogenesis

The serine protease inhibitor (serpin) hurpin (serpin B13) is a cross class-specific inhibitor of the cysteine protease Cathepsin (Cat) L. Cat L is involved in lysosomal protein degradation, hair follicle morphogenesis, epidermal differentiation and epitope generation of antigens. Hurpin is a 44 kDa protein which is expressed predominantly in epidermal cells. In psoriatic skin samples, hurpin was strongly overexpressed when compared with normal skin. Keratinocytes overexpressing hurpin showed increased resistance towards UVB-induced apoptosis. To further analyse the functional importance of this inhibitor, we have generated transgenic mice with deregulated Cat L activity by expressing human hurpin in addition to the endogenous mouse inhibitor. The three independent transgenic lines generated were characterized by identical effects excluding insertional phenotypes. Macroscopically, mice expressing human hurpin are characterized by abnormal abdominal fur. The number of apoptotic cells and caspase-3 positive cells was reduced after UV-irradiation in transgenic animals compared with wild-type mice. Interestingly, after chemical carcinogenesis, transgenic mice showed an increased susceptibility to develop skin cancer. Array analysis of gene expression revealed distinct differences between wild-type and hurpin-transgenic mice. Among others, differentially expressed genes are related to antigen presentation and angiogenesis. These results suggest an important role of Cat L regulation by hurpin which might be of clinical relevance in human skin diseases.     

Effect of glycolic acid on UVB-induced skin damage and inflammation in guinea pigs

OBJECTIVES: Recently the use of glycolic-acid-containing cosmetics has received increased public interest in their supposed ability to reduce wrinkles, roughness, age spots and other skin damage. However, the safety of such products when used excessively or chronically, especially by photosensitive people, is being questioned. The purpose of this study was to examine the effects of glycolic acid alone or in combination with UVB on skin damage and inflammatory response. METHOD: Guinea pigs were treated with glycolic acid (from 1 to 7 mg/cm(2)) alone or in combination with UVB (0.4 or 3 J/cm(2)) for 14 days. Skin damage was evaluated by scoring the skin irritation value by the method of Draize and by histopathological observations. Cyclooxygenase 2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production were also assessed. RESULTS: Glycolic acid caused an increase in the level of skin damage in a dose- and time-dependent manner. Lower doses (1 and 3 mg/cm(2)) of glycolic acid mostly caused erythema and eschar, and these consequently formed scales, whereas higher doses (5 and 7 mg/cm(2)) of glycolic acid caused redness, edema and necrotic ulceration. Glycolic acid also increased the thickness of the epidermal layer, reduced the organization of the stratum corneum and eventually destroyed some parts of the epidermal layer at 7 mg/cm(2). UVB (0.4 and 3 J/cm(2)) caused redness and edema as well as reduced the integrity of the stratum corneum. Glycolic acid enhanced the UVB-induced skin damage. The magnitude of the damage caused by combined UVB and glycolic acid treatment was much greater than that caused by glycolic acid or UVB alone. Moreover, partial destruction of the epidermal layer was observed in skin treated with 3 J/cm(2) UVB and 3 mg/cm(2) glycolic acid. However, glycolic acid did not change the basal and UVB-induced PGE(2) production and COX-2 protein expression. CONCLUSION: These results show that glycolic acid causes skin damage in a dose- and time-dependent manner and that it enhances UVB-induced skin damage without accompanying PGE(2) production or COX-2 protein expression. Therefore, caution should be exercised by those using glycolic acid on a chronic basis or excessively. Moreover, those with photosensitive skins and those more exposed to the sun should be particularly careful.     

UV-induced DNA damage in carcinogenesis and its repair

The purpose of this study is to ask what kind of DNA damage is involved in UV carcinogenesis. Firstly, ras gene alterations were analyzed in UV-induced mice skin cancers. Five types of base changes resulting in activated ras were detected in nine UV-induced skin cancers. Unexpectedly, transversions predominated, whereas previous findings using shuttle vectors indicated that UVC predominantly causes transition-type mutations, which implies the involvement of DNA damage other than dimers in UV carcinogenesis in vivo, in the presence of endogenous photosensitizers. Secondly, we detected mutations both in p53 and ras of skin cancers from patients with xeroderma pigmentosum (XP). Fifty percent of non-melanoma-skin cancers (NMSCs) from XP patients had mutations in p53. The mutation occurred preferentially at CC sites and transitions predominated for p53, whereas ras mutations were far less frequent over the same samples, indicating that DNA damage caused by sunlight rarely hits the crucial sites of ras. Lastly, p53 mutations on NMSCs were compared between sun-exposed area and non/less sun-exposed area. The frequency of p53 mutations between these two groups were almost comparable. However, 67% had the transition at dipyrimidine sites in NMSCs from sun-exposed area, whereas only 20% had the same type of mutations from non/less exposed area (P<0.05).     

The role of oxidative DNA damage in human arsenic carcinogenesis: detection of 8-hydroxy-2'-deoxyguanosine in arsenic-related Bowen's disease.

Arsenic is widely distributed in nature in the form of either metalloids or chemical compounds, which cause a variety of pathologic conditions including cutaneous and visceral malignancies. Recently, reactive oxygen species have been hypothesized to be one of the causes of arsenic-induced carcinogenesis. 8-Hydroxy-2'-deoxyguanosine is one of the major reactive oxygen species-induced DNA base-modified products that is widely accepted as a sensitive marker of oxidative DNA damage. We studied the presence of 8-hydroxy-2'-deoxyguanosine by immunohistochemistry using N45.1 monoclonal antibody in 28 cases of arsenic-related skin neoplasms and arsenic keratosis as well as in 11 cases of arsenic-unrelated Bowen's diseases. The frequency of 8-hydroxy-2'-deoxyguanosine positive cases was significantly higher in arsenic-related skin neoplasms (22 of 28; 78%) than in arsenic-unrelated Bowen's disease (one of 11; 9%) (p < 0.001 by chi2 test). 8-Hydroxy-2'-deoxyguanosine was also detected in normal tissue adjacent to the arsenic-related Bowen's disease lesions. Furthermore, arsenic was detected by neutron activation analysis in the deparaffined skin tumor samples of arsenic-related disease (four of five; 80%), whereas arsenic was not detected in control samples. Our results strongly suggest the involvement of reactive oxygen species in arsenic-induced human skin cancer. Key word: neutron activation analysis.     

Photoprotection against the UVB-induced oxidative stress and epidermal damage in mice using leaves of three different varieties of Lepidium meyenii (maca)

Background Skin exposure to ultraviolet (UV) B radiation leads to epidermal damage and generation of reactive oxygen species. The photoprotective effect of extracts of three varieties of leaves (red, yellow, and black) from maca (Lepidium meyenii), a plant from the Peruvian highlands, was assessed in mouse skin exposed to UVB radiation. Materials and methods The hydroalcoholic extracts of three varieties of maca leaves were applied topically to the dorsal skin of young–adult male mice prior to exposition to UVB radiation. Results The three varieties had UVA/UVB absorptive properties and presented antioxidant activity, being highest with red maca, followed by black and yellow maca. The three varieties of maca leaves prevented the development of sunburn cells, epidermal hyperplasia, leukocytic infiltration, and other alterations produced by UVB radiation. Mice treated with black maca showed the highest superoxide dismutase levels, and mice treated with black and yellow maca showed higher catalase levels in skin, whereas red maca protected the skin and liver against significant increases in the lipid peroxidation activity observed in the unprotected animals. Conclusion The presence of significant antioxidant activity and the inhibition of lipid peroxidation suggest that the observed protection could be partly attributable to this mechanism.     

Skin organ culture as a model to study oxidative stress, inflammation and structural alterations associated with UVB-induced photodamage

Background: Ultraviolet (UV) irradiation is a major cause of skin damage, of long‐term alteration of skin metabolism, homoeostasis and physical structure. The analysis of UV‐induced pathogenic processes requires in vitro models allowing biochemical studies, and appropriate for the development of novel, accurate diagnosis methods based on non‐invasive procedures. Objectives: This work was aimed to reproduce the effects of UVB on whole‐skin explants ex vivo and to study underlying biochemical mechanisms, especially in correlation with skin autofluorescence. Methods: Human skin organ cultures were irradiated with UVB and subjected to enzyme assays, Western blots, solid‐phase ELISA, HPLC and fluorescence measurements. Results: UVB irradiation was found to enhance ROS production, to deplete the pool of low‐molecular‐weight antioxidants and to decrease the overall antioxidant capacity in the epidermis, in a manner dependent on xanthine‐oxidase activity. Epidermal cell proliferation and mitochondrial activity were transiently stimulated. IκB‐α was degraded, and the secretion of inflammatory cytokines was drastically increased. Inducible nitric oxide synthase activity was increased in non‐irradiated controls, probably due to the mechanical stress of skin excision, and this phenomenon was suppressed by UVB. Autofluorescence measurements revealed alterations of dermal protein crosslinks following UVB irradiation. Conclusions: Skin organ culture proved to be an integrated model appropriate for in vitro analysis of UVB biologic effects and their correlations, and for the study of non‐invasive diagnostic methods in cellular and molecular terms.     

Protection against endogenous and UVB-induced oxidative damage in stratum corneum lipids by an antioxidant-containing cosmetic formulation

A 16-week human clinical study was carried out to determine the ability of antioxidants in a cosmetic vehicle to inhibit the induction of lipid peroxidation in stratum corneum lipids. The study consisted of a twice daily application of material for 12 weeks followed by a 4-week regression phase. Stratum corneum lipids were collected and then exposed to 500 mJ/cm2 of ultraviolet B (UVB) radiation in order to avoid excessive erythemal damage to the subjects. Lipid peroxides were assayed by a methylene blue derivative assay and expressed per unit area of skin. During the treatment period, decreases in the level of lipid peroxides were observed on the sites treated with the compositions containing antioxidants, as compared to the untreated sites, and expressed as percent differences. Decreases were observed in endogenous as well as UV-induced lipid peroxides followed by a return to baseline levels. These results demonstrate that antioxidants in a topical cosmetic formulation were effective in protecting human stratum corneum lipids against endogenous oxidation or if challenged by 500 mJ/cm2 UVB.     

Increased oxidative damage to fibroblasts in skin with and without lesions in psoriasis

Differences in oxidative damage, as measured by an increase in the carbonylation of macromolecules, were determined in situ with skin biopsies from psoriatic patients and controls. High levels of carbonyl residues were consistently detected in the dermis and never in the epidermis of sections of these skin biopsy samples. The dermis of psoriatic skin without lesions had a higher level of carbonylation than the dermis of normal skin. In this study, we found that there was more oxidative damage in cultured fibroblasts prepared from skin with and without lesions from psoriasis patients than in normal fibroblasts from the skin of age-matched controls. The extent of protein carbonylation in cell extracts was determined by immunoblotting, using an antidinitrophenylhydrazone antibody, and in intact cells was determined by immunocytochemical analysis with the same antibody. The higher level of carbonylation detected was used here as a measure of oxidative stress, and showed that some oxidative damage occurred before the appearance of typical psoriatic plaques. These results suggest that fibroblasts are affected before the onset of psoriasis and that this damage is independent of any inflammatory infiltrate.     

Variations in excision repair of UVB-induced pyrimidine dimers in DNA of human skin in situ

The excision repair kinetics of UVB (280-320 nm)*-induced pyrimidine dimers in DNA of human skin in situ was determined for seventeen volunteers using a dimer-specific endonuclease from Micrococcus luteus in conjunction with agarose gel electrophoresis. Removal of pyrimidine dimers from human skin could be detected within 6 h after irradiation and the average half-life for removal of pyrimidine dimers was 11.0 h (+/- 4.3 h). However, there was significant inter-individual variability of repair as indicated by a half-life coefficient of variation of 38%.     

Fibulin-2 and fibulin-5 alterations in tsk mice associated with disorganized hypodermal elastic fibers and skin tethering

The Tight skin (Tsk) mouse is an important model of skin fibrosis that occurs in systemic sclerosis. These mice develop skin tethering and thickening associated with expression of a mutant fibrillin-1 gene. We show that Tsk fibrillin-1 leads to marked alterations in elastic fibers of the hypodermis of Tsk animals. In Tsk mice, a prominent elastic fiber layer found normally at the interface between hypodermal muscle and connective tissue was absent from an early age. The lack of elastic fibers at the hypodermal muscle-connective tissue (M-CT) interface was associated with a loss of staining for fibulin-5 in the same region. These mice also formed disorganized elastic fibers throughout hypodermal connective tissue as they aged. The increased elastic fibers in Tsk hypodermal connective tissue was associated with increased fibrillin-1 and fibulin-2 matrices. These results suggest that Tsk fibrillin-1 causes skin tethering by altering matrix protein composition in Tsk hypodermal connective tissues. The closely parallel alterations in elastogenesis associated with increased fibulin-2 in hypodermal connective tissues and decreased fibulin-5 at the hypodermal M-CT interface suggest that these proteins mediate the effect of Tsk-fibrillin-1 on elastogenesis.     

Sirolimus reduces the incidence and progression of UVB-induced skin cancer in SKH mice even with co-administration of cyclosporine A

Transplant immunosuppressants have been implicated in the increased incidence of non-melanoma skin cancer in transplant recipients, most of whom harbor considerable UVB-induced DNA damage in their skin prior to transplantation. This study was designed to evaluate the effects of two commonly used immunosuppressive drugs, cyclosporine A (CsA) and sirolimus (SRL), on the development and progression of UVB-induced non-melanoma skin cancer. SKH-1 hairless mice were exposed to UVB alone for 15 weeks, and then were treated with CsA, SRL, or CsA+SRL for 9 weeks following cessation of UVB treatment. Compared with vehicle, CsA treatment resulted in enhanced tumor size and progression. In contrast, mice treated with SRL or CsA+SRL had decreased tumor multiplicity, size, and progression compared with vehicle-treated mice. CsA, but not SRL or combined treatment, increased dermal mast cell numbers and TGF-beta1 levels in the skin. These findings demonstrate that specific immunosuppressive agents differentially alter the cutaneous tumor microenvironment, which in turn may contribute to enhanced development of UVB-induced skin cancer in transplant recipients. Furthermore, these results suggest that CsA alone causes enhanced growth and progression of skin cancer, whereas co-administration of SRL with CsA causes the opposite effect. JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub     

Decreased incidence of papillomas in mice with impaired EGFR function during multi-stage skin carcinogenesis

Genetically modified mouse lines revealed that the epidermal growth factor receptor (EGFR) is essential for the development and homoeostasis of the epidermis and hair follicles. However, more detailed studies have been precluded by the shortened lifespan of Egfr knockout mice. We employed the mouse line Wa5 (carrying a point mutation resulting in the expression of a dominant negative receptor) to analyse the impact of significantly reduced EGFR signalling during multi-stage chemical skin carcinogenesis. Seven-week-old Wa5 females and control littermates received a single application of 7,12-dimethylbenz(a)anthracene followed by multiple applications of 12-O-tetradecanoylphorbol-13-acetate for 26 weeks. Wa5 mice remained free of papillomas for a longer time and developed significantly fewer tumors than control littermates. In contrast, the mean tumor size was not different between groups. The present data indicate that EGFR signalling contributes to tumor growth during multi-stage chemical carcinogenesis of the skin in mice possibly by acting as a survival factor for skin tumor cells.     

Impaired contact hypersensitivity reaction and reduced production of vascular endothelial growth factor in tumor necrosis factor-alpha gene-deficient mice

Tumor necrosis factor (TNF)-alpha is an important proinflammatory cytokine in contact hypersensitivity (CHS) reactions. Previous efforts to assay CHS in TNF-alpha gene-deficient (-/-) mice have demonstrated a significant reduction in ear skin weight at 24 h following challenge by oxazolone, although the mechanisms of this suppression have not been examined. To further characterize the impaired CHS during evolution of the elicitation phase in TNF-alpha -/- mice and to clarify its mechanisms, focusing on the roles of TNF-alpha and vascular endothelial growth factor (VEGF), we used an established method of CHS assay-sensitization and challenge by trinitrochlorobenzene (TNCB)- in TNF-alpha -/- and wild-type mice. We compared the histopathology of the sequential evolution of CHS between the two groups of mice and assessed both the extent of inflammatory cell infiltration and the degree of dilatation in dermal vessels labeled with CD31. We quantified the production of VEGF in the epidermis at specific time points by using a murine VEGF ELISA kit. The CHS reaction was markedly suppressed in TNF-alpha -/- mice at all time points of the elicitation phase. Histologically, in TNF-alpha -/- mice we observed diminished vascular permeability, reduced numbers of infiltrating inflammatory cells, neutrophils at 12 h, mononuclear cells and eosinophils at 24 h, and a decreased area of dilatation of vessels labeled with CD31. The level of epidermal VEGF in wild type mice increased rapidly after challenge and peaked at 24 h, paralleling the peak of ear swelling. In contrast, the peak level of epidermal VEGF in TNF-alpha -/- mice was significantly reduced. These results suggest that TNF-alpha plays an enhancing role in the elicitation phase of the CHS reaction. Diminished degrees of vascular permeability, dilatation of CD31+ vessels, and inflammatory cell infiltration in TNF-alpha -/- mice are likely to be the result of a lack of TNF-alpha and reduced production of epidermal VEGF.