Transepidermal water loss after photodynamic therapy, UVB radiation and topical corticosteroid is independent of inflammation

Background/purpose: Photodynamic therapy (PDT) and ultraviolet radiation cause an inflammatory reaction of the skin. This may lead to disruption of the skin barrier function. We examined the acute effect of PDT and short-wave ultraviolet radiation (UVB) on the barrier function of the epidermis. Furthermore, we explored the effect on the skin barrier of topical corticosteroid previous to UVB exposure.
Methods: Eight patients with acne vulgaris of the face were treated with PDT two times, and eight patients were left untreated. The transepidermal water loss (TEWL) was measured before treatment and at three control visits after PDT over 12 weeks. Twenty healthy volunteers were irradiated with UVB in two areas on the back. One area was treated with topical high-potency corticosteroid before irradiation. TEWL was measured before, 15 min and 24 h after UVB exposure.
Results: TEWL did not differ significantly between the various visits in each acne group and no significant difference in the TEWL between PDT-treated patients and the control group was found at any visit ( P >0.05). TEWL was not significantly altered 15 min and 24 h after a single exposure to UVB and no positive relationship was found between UV-induced erythema and change in TEWL ( P >0.05). Application of topical corticosteroid before UVB did not affect the skin barrier function significantly ( P >0.05).
Conclusion: Neither PDT nor a single exposure to UVB damaged the permeability barrier function of the skin and no effect on the TEWL was found when topical corticosteroid was applied previous to UVB irradiation.     

Augmentation of sebaceous lipogenesis by an ethanol extract of Grifola frondosa (Maitake mushroom) in hamsters in vivo and in vitro

Abstract:  Grifola frondosa (Maitake mushroom) is an edible and medicinal mushroom with versatile effects such as antitumor and immunomodulating actions. Here, we demonstrated that an ethanol extract of G. frondosa fruiting body (Maitake extract) augmented intracellular lipid droplet formation and the production of triacylglycerols (TG), a major component of sebum, along with the activation of diacylglycerol acyltransferase, a rate-limiting enzyme of TG synthesis in cultured hamster sebocytes. The topical treatment of Maitake extract on the skin of hamster auricles augmented sebum accumulation in sebaceous glands and ducts. However, in comparison with the Maitake extract, another ethanol extract prepared from Agaricus blazei Murill showed less activity in sebaceous lipogenesis in hamsters in vivo and in vitro . These results provide novel evidence that Maitake extract augments sebaceous lipogenesis in hamsters in vivo and in vitro . Thus, Maitake extract is likely to be a unique agent leading to the remission of dry skin.     

Agents that cause enlargement of sebaceous glands in hairless mice. II. Ultraviolet radiation

Summary We have developed a murine model to measure the effects on sebaceous glands of ultraviolet (UV) radiation. Hairless mice were irradiated with Westinghouse FS-40 tubes filtered to attenuate the radiation below 290 nm. Emission was mainly in the UVB range (peak, 313 nm). Single and multiple exposures were given with fractions or multiples of one minimal erythemal dose (MED). Biopsies, fixed for light microscopy, were stained with H & E. Under high power, sebocytes of 30 glands per specimen were counted and the means determined. A single exposure of 1 MED caused a significant increase in sebocyte count, as did thrice-weekly exposures to 0.5 MED for 3 weeks. One 3-MED exposure produced sebocyte necrosis, yet 30 exposures at 4 MED failed to ablate the glands. In both cases there was rebound enlargement which had not returned to control levels by the end of the studies (24–30 weeks). Prolonged irradiation produced maximum enlargement in a few weeks. Thus, in a manner similar to other skin components, the response of sebaceous glands to UV radiation is one of hyperplasia.     

Ultraviolet B radiation regulates cysteine‐rich protein 1 in human keratinocytes

Background: Cysteine‐rich protein 1 (CRP1) is a growth‐inhibitory cytoskeletal protein that is induced by ultraviolet (UV) C radiation radiation in fibroblasts. Our aim was to investigate the effects of UV radiation on CRP1 in keratinocytes, the main cell type subjected to UV radiation in the human body. Methods: The effects of physiologically relevant doses of UVB radiation on CRP1 protein levels were studied in cultured primary keratinocytes and transformed cell lines (HaCaT, A‐431) by immunoblotting. UVB‐induced keratinocyte apoptosis was assessed by flow cytometry and monitoring caspase activity. Expression of CRP1 in human skin in vivo was studied by immunohistochemistry in samples of normal skin, actinic keratosis (AK) representing UV‐damaged skin and squamous cell carcinoma (SCC), a UV‐induced skin cancer. Results: CRP1 expression increased by UVB radiation in primary but not in immortalized keratinocytes. Upon high, apoptosis‐inducing doses of UV radiation, CRP1 was cleaved in a caspase‐dependent manner. In normal skin, CRP1 was expressed in smooth muscle cells, vasculature, sweat glands, sebaceous glands and hair root sheath, but very little CRP1 was present in keratinocytes. CRP1 expression was elevated in basal cells in AK but not in SCC. Conclusion: CRP1 expression is regulated by UVB in human keratinocytes, suggesting a role for CRP1 in the phototoxic responses of human skin.     

Predominant effects of Polypodium leucotomos on membrane integrity,lipid peroxidation,and expression of elastin and matrixmetalloproteinase-1 in ultraviolet radiation exposed fibroblasts,and keratinocytes

BACKGROUND: Polypodium leucotomos has been reported to have antioxidant, anti-inflammatory and photoprotective properties. Exposure of skin to ultraviolet (UV) radiation can lead to deposition of excessive elastotic material, reduction in collagen, and increased expression of matrix metalloproteinases (MMPs). OBJECTIVE: The goal of this research was to determine the effects of P. leucotomos in the absence or presence of UVA or UVB radiation on membrane damage, lipid peroxidation, and expression of elastin and MMP-1 in fibroblasts and keratinocytes, respectively. METHODS: Fibroblasts and keratinocytes, respectively, were irradiated by a single exposure to UVA (0.6, 1.8 or 3.6 J) or UVB radiation (0.75, 2.5 or 7.5 mJ), and then incubated with, or without, P. leucotomos (0.01, 0.1 and 1%) and examined for membrane damage, lipid peroxidation, expression of elastin (protein levels) and MMP-1 (protein levels or MMP-1 promoter activity). RESULTS: UV radiation did not significantly alter membrane integrity, lipid peroxidation or MMP-1 expression, but increased elastin expression. P. leucotomos significantly improved membrane integrity, inhibited lipid peroxidation, increased elastin expression, and inhibited MMP-1 expression in both fibroblasts, and keratinocytes. The effects of P. leucotomos predominated in the presence of UVA or UVB in both fibroblasts and keratinocytes, respectively, with the exception of inhibition of MMP-1 protein levels in fibroblasts only in combination with UV radiation. CONCLUSION: Lower concentration of P. leucotomos (lower than 0.1%), may be beneficial in preventing photoaging by improving membrane integrity and inhibiting MMP-1, without increasing elastin expression. Higher concentration (greater than 0.1%) of P. leucotomos may reverse the loss of normal elastic fibers associated with intrinsic aging.     

A citrus polymethoxy flavonoid, nobiletin inhibits sebum production and sebocyte proliferation, and augments sebum excretion in hamsters

Acne vulgaris is characterized by excess sebum production, and apart from all-trans retinoic acid (atRA) or 13-cis retinoic acid (13-cisRA), there are few effective agents for acne therapy that directly suppresses sebaceous lipogenesis. In this study, we demonstrated that topical application of a citrus polymethoxy flavonoid, nobiletin, to hamster auricles decreased skin surface triacylglycerols (TG) level and the size of sebaceous glands along with inhibition of diacylglycerol acyltransferase (DGAT)-dependent TG synthesis and sebocyte proliferation. The inhibitory actions were similar to that observed with atRA and 13-cisRA in hamster sebocytes. The antilipogenic and antiproliferative actions of nobiletin were also reproduced in UVB (5.4 kJ/m2)-irradiated hamsters, which showed aberrant enhancement of sebum accumulation and sebaceous enlargement. Furthermore, nobiletin, but not 13-cisRA, augmented sebum excretion along with increases in intracellular cAMP level, protein kinase A (PKA) activation, and apoptosis-independent phosphatidylserine (PS) externalization in cell membrane. These phenomena were reproduced by forskolin and inhibited by a PKA inhibitor, H-89. These results provide early evidence that nobiletin is an effective candidate for acne therapy through mechanisms that include the inhibition of DGAT-dependent TG synthesis and sebocyte proliferation, and the progression of apoptosis-independent and PS-externalization-dependent sebum excretion by PKA activation.     

Permeability barrier function of skin exposed to ionizing radiation

OBJECTIVE: To characterize the epidermal permeability barrier function of skin during exposure to ionizing radiation. DESIGN: A prospective cohort study. SETTING: University hospital medical center. PATIENTS: Fifteen women receiving local radiation therapy (5000-6000 rad [50-60 Gy]) following breast-conserving surgery for breast cancer. MAIN OUTCOME MEASURES: Clinical symptoms and transepidermal water loss (TEWL). RESULTS: Epidermal permeability barrier function is impaired in patients who exhibit clinical signs of radiation dermatitis. The functional damage to the stratum corneum induced by ionizing radiation occurs with a delayed course, starting within a mean period of 11 days and reaching maximal values after a mean period of 27 days (range, 13-75 days). The onset of TEWL increase precedes the onset of radiation dermatitis and the maximal TEWL measurements precede the peak of skin changes. Patients with an early onset of TEWL increase show a longer duration of skin symptoms. CONCLUSIONS: Skin changes caused by radiation dermatitis are associated with an increase in TEWL. The barrier impairment is comparable to the changes observed with UV radiation exposure but exhibits an even more delayed course. Our results suggest that preservation of the epidermal permeability barrier function by topical treatment may ameliorate radiation dermatitis.     

Control of human sebocyte proliferation in vitro by testosterone and 5-alpha-dihydrotestosterone is dependent on the localization of the sebaceous glands.

Androgens stimulate the activity of sebaceous glands in vivo. In this study the in vitro effect of androgens on the proliferation of cultured human sebocytes derived from facial and non-facial skin were assessed. Human sebocytes from sebaceous glands isolated from the face and the upper and lower legs of five individuals were cultured in vitro with or without testosterone or 5-alpha-dihydrotestosterone (5 alpha-DHT) at different concentrations (10(-11)-10(-5) M). Cell proliferation was assessed in 96-well culture plates using a fluorometric assay. Testosterone and 5 alpha-DHT stimulated the proliferation of human facial sebocytes in a significant dose-dependent manner. In our system 5 alpha-DHT exhibited the strongest effect; on the contrary, the proliferation of non-facial sebocytes was inhibited by testosterone, whereas 5 alpha-DHT enhanced their growth. The stimulatory effect of 5 alpha-DHT was more prominent on facial than on non-facial sebocytes. These results provide first evidence that the effect of testosterone and 5 alpha-DHT on the proliferation of cultured human sebocytes may depend on the localization of the sebaceous glands at different skin regions.     

Extract of Punica granatum inhibits skin photoaging induced by UVB irradiation

Background Punica granatum (pomegranate) is kind of a fruit consumed fresh or in beverage. It has been widely used in traditional medicine in various parts of the world. In this study, we examined the efficacy of a Punica granatum (PG) extract in protecting skin against UVB‐induced damage using cultured human skin fibroblasts. Methods A Korean red PG sample was used, and its effects classified according to if the PG source originated from the rind, seed and fruit. The polyphenol content of PG, which is known to prevent other adverse cutaneous effects of UV irradiation, was measured by GC‐MS. The protective effects of PG on UVB‐induced skin photoaging were examined by determining the level of procollagen type I and MMP‐1 after UVB irradiation. Results Based on the GC‐MS quantitative analysis, catechin, quercetin, kaempferol, and equol were the predominant compounds detected in PG. In the changes of expression of procollagen type I and MMP‐1 in UV irradiated human skin fibroblasts treated PG, especially extract prepared from rind, the synthesis of collagen was increased and the expression of MMP‐1 was decreased. Conclusion The major polyphenols in PG, particularly catechin, play a significant role in its photoprotective effects on UVB‐induced skin damage.     

Impact of ultraviolet radiation and ozone on the transepidermal water loss as a function of skin temperature in hairless mice

Exposure to ultraviolet radiation or ozone leads to skin damage including oxidation of skin biomolecules, as well as to depletion of constitutive antioxidants. The highly organized stratum corneum forming the main barrier against most xenobiotics is particularly susceptible to such damage and possible barrier perturbation may be the consequence. Whereas ample evidence exists for an increased permeability for different solutes including water after exposure to ultraviolet radiation, such an effect has not yet been reported for ozone. This study reports on the effect of such oxidative stressors using the hairless mouse as the skin model and measuring temperature-controlled transepidermal water loss (TEWL) as an indicator for skin barrier integrity. First, a strong dependency of the TEWL on skin temperature was observed, an effect that was clearly more pronounced than that found in man. Given this temperature dependency in untreated animals, we proceeded to determine the effects of both ultraviolet radiation and ozone on TEWL over a relevant physiological skin temperature range. Solar-simulated ultraviolet radiation (0.75-3 minimal erythemal dose) resulted in a delayed and dose-dependent skin barrier disruption over the entire temperature range investigated. Conversely, daily ozone exposure at 2 ppm for 1 week, however, did not significantly alter TEWL up to 72 h after the last exposure. The results demonstrate a differential response of the epidermis to two environmental stressors associated with oxidative damage; they suggest that chronic ozone exposure at relevant environmental levels does not lead to a detectable skin barrier defect, while solar UV exposure was demonstrated to increase epidermal water loss. Furthermore, experimental evidence clearly suggests that future studies applying TEWL measurements in animal models should be performed under carefully controlled skin temperature conditions.     

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.     

Skin damage and mitochondrial dysfunction after acute ultraviolet B irradiation: relationship with nitric oxide production

BACKGROUND: Ultraviolet (UV) radiation is the main environmental carcinogen. It is able to induce injury in the keratinocytes, which triggers mechanisms in order to protect the skin against molecular alterations that may lead to the development of skin cancer. UVB is capable of producing genotoxic damage, directly or indirectly through reactive oxygen species, inducing DNA alterations and mutations. UVB radiation has also been associated with the generation of nitric oxide (NO), which is able to induce many physiological and physiopathological processes. The aim of the current study was to investigate the effect of UVB irradiation in hairless mice skin. METHODS: We evaluated the effect of an acute dose (200 mJ/cm(2)) of UVB irradiation correlating with histological alterations, nitric oxide synthase expression and activity, mitochondrial respiratory function, superoxide anion production and lipid peroxidation, 0, 6, 17 and 24 h post-irradiation treatment. RESULTS: Morphological analysis showed disruption of the epidermal stratum corneum and basale after UVB irradiation. The results indicated that skin UVB irradiation was associated with an increased cytosolic inducible nitric oxide synthase (iNOS) expression, inversely related to lipid peroxidation processes. An increase in mitochondrial superoxide anion (O(2) (*-)) and NO production 17 h post-irradiation was correlated with a mitochondrial dysfunction, all of them integrating the skin response to acute UVB irradiation. CONCLUSIONS: UVB irradiation of the skin produces morphological alterations as a consequence of the induction of molecular mechanisms associated with mitochondrial respiratory dysfunction and O(2) (*-) production, probably mediated by the increased mitochondrial NO production. On the other hand lipid peroxidation decrease inversely correlates with cytosolic iNOS expression, suggesting a protective role for the inflammatory response.     

Protective effect of dl-alpha-tocopherol on the cytotoxicity of ultraviolet B against human skin fibroblasts in vitro

The effect of dl-alpha-tocopherol on ultraviolet light, 280-320 nm (UVB)-induced damage of human skin fibroblasts was studied by measuring the colony-forming ability, unscheduled DNA synthesis (UDS) and malondialdehyde (MDA) production. Regarding the cell toxicity, the values of the mean lethal dose (D0) of UV in fibroblast strains from 5 normal subjects were examined. D0 increased dose-dependently when the cells were cultured in the presence of dl-alpha-tocopherol at the concentration of 10-1000 micrograms/ml. UDS induced by 500 J/m2 UVB irradiation was not altered by treatment of 100 micrograms/ml dl-alpha-tocopherol. MDA did not increase after 500 J/m2 UVB irradiation in the fibroblasts cultured with 100 micrograms/ml dl-alpha-tocopherol, while MDA in the fibroblasts cultured without dl-alpha-tocopherol increased after irradiation. These results suggest that dl-alpha-tocopherol protects human skin fibroblasts against the cytotoxic effect of UVB, and its mechanism seems to be related to inhibition of UV-induced lipid peroxidation or to the antioxidation effect of dl-alpha-tocopherol.     

Molecular events associated with apoptosis and proliferation induced by ultraviolet-B radiation in the skin of hairless mice

BACKGROUND: It is recognized that UV radiation produced apoptotic cells (sun burn cells) in the epidermis of mice. However, the relationship between apoptosis and cell proliferation after UV exposure in the skin of hairless mice are still unclear. OBJECTIVE: To investigate the effects of ultraviolet (UV) radiation on molecular events associated with apoptosis and proliferation in SKH1-hr mouse skin. METHODS: Mice were irradiated with daily UVB exposure of 0.1 or 0.25 J/cm(2) for 14 days. The skin tissues were analyzed at 2 and 24 h after the end irradiation for the presence of apoptotic cells and Bromodeoxyuridine (BrdU)-positive cells. We measured the expression of p53, p21, bcl-2, bax and E2F-1. RESULTS: The results indicated that UVB irradiation caused to increase apoptotic cells in the epidermis of mice. The expression of p53 and p21 was increased at 2 and 24 h after irradiation compared with the control. UV radiation induced high levels of bax at 2 and 24 h after irradiation with a concomitant decrease in bcl-2 expression. The expression of E2F-1 in the skin was also increased at 2 and 24 h after irradiation. Coinciding with these changes, BrdU positive cells increased at 2 and 24 h after UVB exposure at the epidermis of hairless mice, which observed the apoptotic expression. CONCLUSION: These results suggest that UVB irradiation of mouse skin induces apoptosis and is mediated by the p53/p21/E2F-1/bax pathway and that the dead cells are replaced by hyperproliferative cells, leading to epidermal hyperplasia.     

中波紫外线对人角质形成细胞iNOS的作用

目的观察中波紫外线(UVB)照射对体外培养正常人角质形成细胞(HKC)中诱导型一氧化氮合成酶(iNOS)的作用,探讨紫外线引起皮肤细胞急性损伤的发病机制。方法体外培养HKC,用免疫荧光染色和蛋白印迹技术检测照射后不同时间细胞内iNOS的水平。结果HKC经UVB照射后,免疫荧光法检测到细胞膜胞浆侧有iNOS,蛋白印迹法检测到细胞提取总蛋白中分子量为130kDa的蛋白条带可以被抗iNOS多克隆抗体所识别,二者结果相符合。结论UVB照射可诱导体外培养的HKC在胞浆内产生iNOS,iNOS蛋白的表达可能与紫外线引起的皮肤急性炎症反应有关。     

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters sebaceous gland cell differentiation in vitro

Chloracne is a characteristic marker of intoxication by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or related compounds. Decreased lipogenesis is a prominent clinical sign in this disease. However, the activity of dioxins on human sebaceous glands is still unclear. In this study, the effects of TCDD on sebaceous gland differentiation were studied both in human skin samples maintained ex vivo and in cultured SZ95 sebocytes. Aryl hydrocarbon receptor (AhR) protein expression, the receptor for dioxin, was detected in SZ95 sebocytes. Its expression was markedly inhibited by TCDD. Furthermore, we detected a reduced release of neutral lipids (10(-10) -10(-8) M; P<0.001) and decreased expression of epithelial membrane antigen and keratin 7, all of which are specific markers of sebaceous differentiation. Markedly, increased expression of the keratinocyte differentiation marker keratin 10 and of peroxisome proliferators-activated receptor-δ was assessed in SZ95 sebocytes treated with TCDD. To corroborate these in vitro data, an ex vivo sebaceous gland-rich skin culture model was investigated. Obvious shrinkage of sebaceous glands with sebaceous duct hyperplasia and increased expression of keratin 10 in the atrophic sebaceous glands were observed on the 5th day of TCDD treatment. In conclusion, TCDD affects the differentiation of sebaceous gland cells probably by switching human sebaceous into keratinocyte-like differentiation. In addition and together with the results of a parallel study (J Dermatol Sci 58, 2010, 211), we provide evidence that TCDD effects on human sebocytes are mediated through the AhR signalling pathway.