紫外线对荒漠沙蜥皮肤形态、蜕皮、脂质过氧化和抗氧化酶的影响

为了提高体温,荒漠沙蜥喜好晒太阳的同时增加了紫外线对其皮肤的损伤。本实验研究了不同的紫外线强度(110、300、500、800mJ/cm2)对荒漠沙蜥皮肤形态、蜕皮、脂质过氧化和抗氧化酶的影响。结果显示:皮肤损伤和丙二醛含量的最高峰发生在暴露紫外线300、500、800mJ/cm2后的96、48、24h;SOD活性的最低峰发生在暴露紫外线110、300、500、800mJ/cm2后的24、48、12h;CAT活性在暴露紫外线后立即抑制,然后恢复提高。CAT活性的高低往往伴随皮肤的损伤程度和蜕皮的发生,这表明紫外线对皮肤的损伤与皮肤的脂质过氧化密切相关,CAT是一种主要的抗氧化酶。皮肤的角质层对保护皮肤免受紫外线的损伤也有重要作用。     

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。     

Repeated exposures of human skin equivalent to low doses of ultraviolet-B radiation lead to changes in cellular functions and accumulation of cyclobutane pyrimidine dimers.

Chronic exposure to sunlight may induce skin damage such as photoaging and photocarcinogenesis. These harmful effects are mostly caused by ultraviolet-B (UVB) rays. Yet, less is known about the contribution of low UVB doses to skin damage. The aim of this study was to determine the tissue changes induced by repeated exposure to a suberythemal dose of UVB radiation. Human keratinocytes in monolayer cultures and in skin equivalent were irradiated daily with 8 mJ/cm2 of UVB. Then structural, ultrastructural, and biochemical alterations were evaluated. The results show that exposure to UVB led to a generalized destabilization of the epidermis structure. In irradiated skin equivalents, keratinocytes displayed differentiated morphology and a reduced capacity to proliferate. Ultrastructural analysis revealed, not only unusual aggregation of intermediate filaments, but also disorganized desmosomes and larger mitochondria in basal cells. UVB irradiation also induced the secretion of metalloproteinase-9, which may be responsible for degradation of type IV collagen at the basement membrane. DNA damage analysis showed that both single and repeated exposure to UVB led to formation of (6-4) photoproducts and cyclobutane pyrimidine dimers. Although the (6-4) photoproducts were repaired within 24 h after irradiation, cyclobutane pyrimidine dimers accumulated over the course of the experiment. These studies demonstrate that, even at a suberythemal dose, repeated exposure to UVB causes significant functional and molecular damage to keratinocytes, which might eventually predispose to skin cancer.     

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.     

Development of a peptide antibody specific to human glutathione S-transferase alpha 4-4 (hGSTA4-4) reveals preferential localization in human liver mitochondria

The reactive cellular products generated during the peroxidation of membrane lipids have been implicated as causative agents in a variety of degenerative diseases and aging. In particular, 4-hydroxynon-2-enal (4HNE) is among the most of the produced during lipid peroxidation. In humans and rodent species, the alpha 4 subclass of glutathione S-transferases (mGSTA4-4, rGSTA4-4, hGST-5.8, and hGSTA4-4) exhibits uniquely high glutathione conjugation activity toward 4HNE and other hydroxyalkenals. In human liver, hGSTA4-4-mediated 4HNE conjugation appears to represent the high-affinity pathway for 4HNE detoxification. In the present study, a highly specific polyclonal antibody was developed against hGSTA4-4. Western blotting analysis of human liver subcellular fractions as well as N-terminal sequencing revealed that hGSTA4-4 was localized to mitochondrial fractions, but was not detected in cytosolic fractions. Our results provide evidence that in adult liver, hGSTA4-4 is specifically targeted to the mitochondrion to the apparent exclusion of the cytosol. Targeting of hGSTA4-4 to the mitochondrion holds implications for degenerative diseases associated with oxidative stress that arise from aerobic respiration.     

Potentiation of UVB-induced apoptosis by novel phytosphingosine derivative, tetraacetyl phytosphingosine in HaCaT cell and mouse skin

Inappropriate apoptosis results in the epidermal hyperplasia as in psoriasis and UVB irradiation has been successfully used to treat this kind of skin disorders. Previously, we reported that the novel phytosphingosine derivative, tetraacetyl phytosphingosine (TAPS) induced apoptosis in HaCaT cells. This study examined the effect of UVB irradiation and/or TAPS on the induction of apoptosis in HaCaT. 10 mJ/cm2 of UVB irradiation or 10 microM of TAPS alone exhibited weak cytotoxicity but co-treatment of UVB and TAPS synergistically enhanced the cytotoxicity and apoptosis in HaCaT. The cells treated with UVB and TAPS showed much higher levels of cleaved caspase-3, -8, -9 and Bax than with UVB or TAPS alone, whereas Bcl-2 level was decreased by co-administration of UVB and TAPS. In hairless mice, co-treatment of UVB and TAPS synergistically increased apoptosis, as shown in the HaCaT co-treated with UVB and TAPS. Furthermore, UVB irradiation caused an increase of apoptotic cells in the epidermis and the TAPS-treated mice showed an increase of apoptotic cells in the dermis as well as in the epidermis. These results suggest that the TAPS co-treatment synergistically increases the level of UVB-induced apoptosis via caspase activation by regulating the level of pro-apoptotic Bax and anti-apoptotic Bcl-2.     

The UVB-induced synthesis of vitamin D3 and 1alpha,25-dihydroxyvitamin D3 (calcitriol) in organotypic cultures of keratinocytes: effectiveness of the narrowband Philips TL-01 lamp (311 nm)

Both calcitriol and UVB radiation exert potent antipsoriatic effects. We hypothesize that the therapeutical effect of UVB radiation may be attributed at least in part to UVB-triggered cutaneous synthesis of calcitriol. The optimum wavelength for initiation of the vitamin D(3) pathway was found to be in the range of 300+/-5 nm in vitro and in vivo. The narrowband Philips TL-01 lamp which is commonly used as UVB source for phototherapy of psoriasis has maximum spectral irradiance at around 311 nm which is presumed to be, however, of lesser importance in photochemical activation of the vitamin D(3) pathway. The aim of this study was to compare the vitamin D(3) and calcitriol-inducing potential of UVB from the TL-01 lamp with that of monochromatic UVB at 300+/-2.5 nm and 310+/-2.5 nm in organotypic cultures of keratinocytes supplemented with 25 microM 7-DHC. We found that maximum calcitriol-generating capacity of the TL-01 lamp at 500 mJ/cm(2) and 16 h after irradiation still amounts up to 44% of that found after monochromatic irradiation at 300+/-2.5 nm and 30 mJ/cm(2). Thus, the antipsoriatic effect of UVB emitted from the TL-01 lamp may, at least in part, based on the antiproliferative and prodifferentiative action of newly synthesized calcitriol on epidermal keratinocytes.     

Up-Regulation and redistribution of Bax in ultraviolet B-irradiated melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0-30 mJ/cm2). The levels of p53, Bax, and Bcl-2, determined by immunoblotting, revealed a dose-dependent increase in p53 and Bax, but the level of Bcl-2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl-2. These data suggest that the high constitutional level of Bcl-2 may protect melanocytes from UVB-induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.     

Single exposure of human fibroblasts (WI-38) to a sub-cytotoxic dose of UVB induces premature senescence

In this work, we present a new model of stress-induced premature senescence obtained by exposing human fibroblasts (WI-38) at early passages (passages 2-4) to a single sub-cytotoxic dose of UVB (200 mJ/cm(2)). We show that this treatment leads to the appearance of several biomarkers of senescence such as enlarged and flattened cell morphology, the presence of nuclear heterochromatic foci and beta-galactosidase activity. Furthermore, we demonstrate that a mild ROS production and p53 activation are upstream events required for the induction of premature senescence. Our method represents an alternative in vitro model in photoaging research and could be used to test potential anti-photoaging compounds.     

Oxidative stress mediates cell surface expression of SS-A/Ro antigen on keratinocytes

Exposure to ultraviolet radiation exacerbates the skin lesions of autoimmune diseases, and is known to induce cell surface expression of SS-A/Ro antigen on keratinocytes in vitro. Following up on recent reports on ultraviolet-B (UVB)-induced oxidative stress, we examined the role of oxidative stress in the surface expression of SS-A/Ro antigen on human keratinocytes. First, the exclusive induction by UVB irradiation of the 52-kDa protein (Ro52) but not of the 60-kDa protein (Ro60) of SS-A/Ro antigen was demonstrated by means of indirect immunofluorescence. The surface expression of Ro52 induced by UVB irradiation was concentration-dependently inhibited by N-acetyl-L-cysteine, an antioxidant. Furthermore, surface expression of Ro52 was similarly induced by diamide, a chemical oxidant. We next used Hoechst 33342 staining and the TUNEL assay to demonstrate that a low dose (20 mJ/cm(2)) of UVB did not induce apoptosis but induced the surface expression of Ro52. Moreover, zVAD-fmk, a pan-caspase inhibitor, did not inhibit UVB-induced surface expression of Ro52 even at a high dose (200 mJ/cm(2)) of UVB, which was sufficient to induce apoptosis in keratinocytes in the absence of zVAD-fmk. Taken together, we concluded that UVB-induced surface expression of Ro52 on keratinocytes is mediated by oxidative stress through a pathway other than apoptosis.     

Genetic variation of the cutaneous HPA axis: An analysis of UVB-induced differential responses

Mammalian skin incorporates a local equivalent of the hypothalamic–pituitary–adrenal (HPA) axis that is critical in coordinating homeostatic responses against external noxious stimuli. Ultraviolet radiation B (UVB) is a skin-specific stressor that can activate this cutaneous HPA axis. Since C57BL/6 (B6) and DBA/2J (D2) strains of mice have different predispositions to sensorineural pathway activation, we quantified expression of HPA axis components at the gene and protein levels in skin incubated ex vivo after UVB or sham irradiation. Urocortin mRNA was up-regulated after all doses of UVB with a maximum level at 50 mJ/cm² after 12 h for D2 and at 200 mJ/cm² after 24 h for B6. Proopiomelanocortin mRNA was enhanced after 6 h with the peak after 12 h and at 200 mJ/cm² for both genotypes of mice. ACTH levels in tissue and media increased after 24 h in B6 but not in D2. UVB stimulated β-endorphin expression was higher in D2 than in B6. Melanocortin receptor 2 mRNA was stimulated by UVB in a dose-dependent manner, with a peak at 200 mJ/cm² after 12 h for both strains. The expression of Cyp11a1 mRNA — a key mitochondrial P450 enzyme in steroidogenesis, was stimulated at all doses of UVB irradiation, with the most pronounced effect after 12–24 h. UVB radiation caused, independently of genotype, a dose-dependent increase in corticosterone production in the skin, mainly after 24 h of histoculture. Thus, basal and UVB stimulated expression of the cutaneous HPA axis differs as a function of genotype: D2 responds to UVB earlier and with higher amplitude than B6, while B6 shows prolonged (up to 48 h) stress response to a noxious stimulus such as UVB.     

Ultraviolet radiation-induced photodegradation and 1O2, O2-. production by riboflavin, lumichrome and lumiflavin

Although UVA (320-400 nm) is considered less harmful to skin as compared to UVB (290-320 nm) and UVC (200-290 nm) radiation, certain endogenous chromophores may enhance UVA-induced cutaneous reactions by largely O2-dependent photodynamic reactions. Photodegradation pattern and singlet oxygen (1O2), superoxide anion radical (O2-.) producing capacity of riboflavin (RF), lumiflavin (LF) and lumichrome (LC) were examined to assess their phototoxic potential under UVA. Photolysis of RF upon exposure to UVA, UVB or UVC revealed considerable degradation to LF and LC with a near identical spectral pattern of photodegradation between 250-500 nm. Both LF and LC were stable to UVA (3 J/cm2) and UVB (400 mJ/cm2), whereas RF was photodegraded by 30 and 20%, respectively, under similar irradiation conditions. UVA-sensitized LF and LC respectively, produced nearly 15% higher and 60% lower yield of 1O2 in comparison to RF, whereas, O2-. was generated predominently by RF. Both RF and LF thus appeared to be potential chromophores for evoking deleterious effects of UVA in normal human skin.     

INFLUENCE OF SUCCESSIVE AND COMBINED ULTRAVIOLET A AND B IRRADIATIONS ON MATRIX METALLOELASTASES PRODUCED BY HUMAN DERMAL FIBROBLASTS IN CULTURE

To study the cumulative influence of UV irradiations on skin matrix alterations, human skin fibroblasts were irradiated successively three-fold, at 24h intervals, with UVA (3×5J/cm²), UVB (3×8mJ/cm²), UVA plus UVB (3×5J/cm²and 3×8mJ/cm²) and the levels of 92kDa gelatinase (pro-MMP₉), 72kDa gelatinase (pro-MMP₂) and plasma-membrane elastase type protease were determined, following subsequent 24-h culture in 10% serum-containing medium. UV irradiations had only minor influence (1.4-fold increase for UVB) on secreted levels of pro-MMP₂and decreased the amount of plasma membrane elastase produced by cells. It did however, for UVA and UVB alone, induce a significant increase of 66kDa activated MMP₂production: 2.5- and 1.7-fold respectively. Such enhancement was not observed when combined irradiations were administered. UV exposure possessed a much higher influence on pro-MMP₉secretion by dermal fibroblast enhancing enzyme levels by 2.5-, 6.5- and 5-fold for UVA, UVB and UVA+UVB, respectively.     

UVB irradiation-induced impairment of keratinocytes and adaptive responses to oxidative stress

UVB irradiation of human skin is known to induce pathophysiological processes as oxidative stress and inflammation. HaCaT keratinocytes represent a well-established in vitro model system to investigate the influence of UVB irradiation on cell cultures. It was the aim of these investigations to study the effects of moderate UVB doses on cellular and mitochondrial integrity of HaCaT keratinocytes, biomarkers of oxidative stress and antioxidant protection by superoxide dismutases. F₂-isoprostane concentrations were UVB dose-dependently enhanced reaching a plateau at 50 mJ/cm². Cell viability was reduced and apoptosis was enhanced with increasing UVB doses. The activities of the respiratory chain complexes were practically not altered at lower UVB doses, up to 50 mJ/cm², whereas remarkable decreases, also for the levels of cardiolipin species, were seen at 100 mJ/cm². As an adaptive response to the enhanced oxidative stress, protein levels of MnSOD increased about 3-fold at 50 mJ/cm² and decreased at higher doses. From the data it can be concluded that keratinocytes are sufficiently protected at low UVB doses, whereas higher doses lead to irreversible cell damage.     

Ultraviolet B radiation-induced cell death: critical role of ultraviolet dose in inflammation and lupus autoantigen redistribution

The nuclear self-Ags targeted in systemic lupus erythematosus translocate to the cell membrane of UV-irradiated apoptotic keratinocytes and may represent an important source of self-immunization. It is hard to understand how the noninflammatory milieu accompanying most apoptosis might provoke an immunogenic response leading to autoantibodies. We have found that the precise amount of keratinocyte UV exposure is crucial in determining the rate of apoptosis, the amount of inflammatory cytokine production, and the degree of autoantigen translocation. Low doses of UVB (相似文献   

Importance of amino acid composition to improve skin collagen protein synthesis rates in UV-irradiated mice

Skin collagen metabolism abnormalities induced by ultraviolet (UV) radiation are the major causes of skin photoaging. It has been shown that the one-time exposure of UV irradiation decreases procollagen mRNA expression in dermis and that chronic UV irradiation decreases collagen amounts and induces wrinkle formation. Amino acids are generally known to regulate protein metabolism. Therefore, we investigated the effects of UV irradiation and various orally administered amino acids on skin collagen synthesis rates. Groups of 4-5 male, 8-week-old HR-1 hairless mice were irradiated with UVB (66 mJ/cm2) twice every other day, then fasted for 16 h. The fractional synthesis rate (FSR; %/h) of skin tropocollagen was evaluated by incorporating L-[ring-2H5]-phenylalanine. We confirmed that the FSR of dermal tropocollagen decreased after UVB irradiation. The FSR of dermal tropocollagen was measured 30 min after a single oral administration of amino acids (1 g/kg) to groups of 5-16 UVB-irradiated mice. Branched-chain amino acids (BCAA, 1.34±0.32), arginine (Arg, 1.66±0.39), glutamine (Gln, 1.75±0.60), and proline (Pro, 1.48±0.26) did not increase the FSR of skin tropocollagen compared with distilled water, which was used as a control (1.56±0.30). However, essential amino acids mixtures (BCAA+Arg+Gln, BCAA+Gln, and BCAA+Pro) significantly increased the FSR (2.07±0.58, 2.04±0.54, 2.01±0.50 and 2.07±0.59, respectively). This result suggests that combinations of BCAA and glutamine or proline are important for restoring dermal collagen protein synthesis impaired by UV irradiation.     

Peroxynitrite activates NF-E2-related factor 2/antioxidant response element through the pathway of phosphatidylinositol 3-kinase: the role of nitric oxide synthase in rat glutathione S-transferase A2 induction.

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes through antioxidant response elements (AREs). Our previous study showed that sulfur amino acid deprivation (SAAD) produces peroxides and induces rat glutathione S-transferase A2 (rGSTA2) through NF-E2-related factor 2 (Nrf2)/ARE activation via the pathway of phosphatidylinositol 3-kinase (PI3-kinase). The current study was designed to investigate the role of peroxynitrite in Nrf2/ARE activation and rGSTA2 induction. L-Arginine deficiency or N(G)-nitro-L-arginine methyl ester (L-NAME) reduced peroxide production induced by SAAD in H4IIE cells. Northern and Western blot analyses revealed that the levels of rGSTA2 mRNA and protein were significantly increased 24h after incubation of the cells in SAAD medium, which was inhibited by L-arginine deficiency or L-NAME. Subcellular fractionation and gel shift analyses revealed that SAAD increased the level of nuclear Nrf2 and activated ARE, which were also blocked by L-arginine deficiency or L-NAME. Whereas the exogenous NO donor S-nitroso-N-acetyl-penicillamine (SNAP) alone failed to significantly induce rGSTA2, SNAP enhanced SAAD-inducible rGSTA2 expression, verifying the notion that peroxynitrite derived from NO contributes to rGSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, increased the rGSTA2 mRNA and protein levels in a dose-dependent manner. SIN-1 increased the level of nuclear Nrf2 and activated Nrf2/ARE, which was supershifted by anti-Nrf2 and anti-Maf antibodies. SIN-1 increased the activity of PI3-kinase, as monitored by phosphorylation of Akt. SIN-1-inducible rGSTA2 expression was inhibited by PI3-kinase inhibitors. These results provide evidence that peroxynitrite plays an essential role in nuclear translocation of Nrf2 and ARE activation through the pathway of PI3-kinase and that nitric oxide synthase is involved in the induction of rGSTA2.