Ultraviolet B‐induced apoptosis of human skin fibroblasts involves activation of caspase‐8 and ‐3 with increased expression of vimentin

Background: After irradiation with a high dose of ultraviolet B (UVB), cells undergo apoptosis. Caspase‐8 and ‐3 are key mediators of apoptosis in many cells. Vimentin, an important cytoskeleton component, can be cleaved by caspase‐3, ‐6, ‐7 and ‐8. Cell apoptosis is promoted via caspase‐triggered proteolysis of vimentin. In this study, we explored the roles of caspase‐8 and ‐3 and the changes in vimentin expression in UVB‐induced apoptosis of human dermal fibroblasts. Methods: Skin fibroblasts were irradiated with 150 mJ/cm² UVB and cell death was monitored by the 3‐(4,5)‐dimethylthiahiazo(‐z‐y1)‐3,5‐diphenytetrazoliumromide assay and Hoechst staining. Caspase‐8 and ‐3 activities were detected by the caspase activity assay. Vimentin expression was assessed by immunofluorescence and Western blot. Results: Caspase‐8 and ‐3 were activated by 150 mJ/cm² UVB irradiation. Caspase‐8 and ‐3 activities changed in a time‐dependent way after UVB irradiation to induce apoptosis of fibroblasts, and caspase‐8 and ‐3 interacted with each other in this process. However, their substrate, vimentin, showed an enhanced expression over time after UVB irradiation. Conclusions: UVB‐triggered apoptosis of fibroblasts was dependent on the activation of caspase‐8 and ‐3 with an increased expression of vimentin.     

Caspase-3在UVB诱导人皮肤成纤维细胞凋亡中的作用

目的：评价caspase-3在UVB诱导皮肤成纤维细胞凋亡中的作用。方法：皮肤成纤维细胞经150mJ／cm2 UVB照射后,用MTF法检测细胞活性,用Hoechst33258染色法检测细胞凋亡,用抑制剂Z-DEVD-FMK抑制caspase-3活性后,检测凋亡细胞数量。结果：UVB明显抑制成纤维细胞的活性,并导致细胞凋亡,并呈时间-效应关系;加入抑制剂Z-DEVD-FMK抑制了UVB导致的细胞凋亡。结论：Caspase-3在UVB照射诱导皮肤成纤维细胞凋亡中发挥重要作用。     

Dihydroavenanthramide D prevents UV‐irradiated generation of reactive oxygen species and expression of matrix metalloproteinase‐1 and ‐3 in human dermal fibroblasts

Ultraviolet B (UVB) radiation induces photoageing by upregulating the expression of matrix metalloproteinases (MMPs) in human skin cells. Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component in oats. Although anti‐inflammatory, anti‐atherosclerotic and antioxidant effects have been reported, the antiphotoageing effects of DHAvD are yet to be understood. In this study, we investigated the inhibitory effects of DHAvD on UVB‐induced production of reactive oxygen species (ROS) and expression of MMPs, and its molecular mechanism in UVB‐irradiated human dermal fibroblasts. Western blot and real‐time PCR analyses revealed that DHAvD inhibited UVB‐induced MMP‐1 and MMP‐3 expression. It also significantly blocked UVB‐induced ROS generation in fibroblasts. Additionally, DHAvD attenuated UVB‐induced phosphorylation of MAPKs, activation of NF‐κB and AP‐1. DHAvD regulates UVB‐irradiated MMP expression by inhibiting ROS‐mediated MAPK/NF‐κB and AP‐1 activation. DHAvD may be a useful candidate for preventing UV light‐induced skin photoageing.     

UV-induced DNA damage initiates release of MMP-1 in human skin

Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase-1 (MMP-1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet-B (UVB)-irradiated and treated with liposome-encapsulated DNA repair enzymes. The photolyase-mediated DNA repair of epidermal UV damage was associated with a reduction of MMP-1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP-1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP-1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP-1. The MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP-1 release, and enhancing DNA repair can reduce MMP-1 expression in human skin cells and tissue.     

紫外线照射后生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用

目的：明确对UVA及UVB照射后皮肤成纤维细胞生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用。方法：紫外线照射人皮肤成纤维细胞,提取细胞上清液中的微囊泡,利用光散射分析技术鉴定分析微囊泡的大小及数量。将紫外线照射后生成的微囊泡与正常成纤维细胞共孵育,荧光酶标仪定量检测活性氧含量,流式细胞仪检测细胞凋亡率。结果：UVA及UVB照射后皮肤成纤维细胞释放的微囊泡数量及大小明显高于正常成纤维细胞释放的微囊泡。正常纤维细胞、UVA和UVB照射后的成纤维细胞与微囊泡共孵育后活性氧荧光值分别为（52.76±1.4347）、（82.60±4.082）和（85.94±6.264）,凋亡率分别为（3.260±1.732）%,（28.94±2.430）%和（34.48±2.718）%,细胞的氧化损伤和凋亡可被抗氧化剂逆转。结论：急性中长波紫外线照射可诱导皮肤成纤维细胞释放微囊泡进一步介导细胞的氧化损伤和凋亡。     

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 on the expression of marker proteins of gap and adhesion junctions in human epidermis

Aims/background: We aimed to investigate the impact of ultraviolet B (UVB) as well as UVA1 on the epidermal expression of specific markers of gap and adhesion junctions. Methods: Twelve healthy subjects were enrolled in the study. The back of the subjects was irradiated with three MED‐UVB as well as three MED‐UVA1. Twenty‐four hours later, punch biopsies were taken from irradiated and non‐irradiated skin. Immunohistochemical procedures were used for the detection of connexin 43, E‐cadherin, involucrin, Ki‐67 using specific antibodies. Results: Staining intensity of connexin 43 in UVB‐exposed skin was significantly increased when compared with non‐exposed and UVA1‐exposed sites. By contrast, staining intensity of E‐cadherin in UVB‐exposed skin was significantly decreased when compared with non‐exposed and UVA1‐exposed sites. Involucrin and Ki‐67 staining of keratinocytes was significantly increased in UVB‐exposed sites as compared with non‐exposed and UVA1‐irradiated sites. Conclusions: UVB significantly alters the epidermal expression of gap and adhesion junction proteins possibly indicating a role of these proteins in the regulation of UV‐induced inflammation and development and progression of skin cancer.     

中波紫外线对人真皮成纤维细胞中长链非编码RNA表达的影响

目的 对中波紫外线(ultraviolet rays B,UVB)照射后人真皮成纤维细胞(human dermal fibroblasts,HDFs)中差异表达的长链非编码RNA(long non-ucoding RNAs,LncRNA)进行筛选和功能分析.方法 将处于对数生长期8～11代的HDFs分为对照组和UVB ...     

Photoprotective potential of Cordyceps polysaccharides against ultraviolet B radiation-induced DNA damage to human skin cells

Summary Background Ultraviolet (UV) radiation causes DNA damage resulting in photoageing and skin cancer. UVB (290–320 nm) interacts directly with DNA, inducing two major photoproducts: cyclobutane‐pyrimidine dimers (CPDs) and (6‐4) pyrimidine‐pyrimidone photoproducts. Cordyceps sinensis (Berk.) Sacc. is a medicinal fungus with reported anticancer and cytoprotective effects. Objectives To investigate genoprotective effects of polysaccharide‐rich Cordyceps mycelial components against UVB‐induced damage in normal human fibroblast cells. Methods Cultured human fibroblasts (BJ cells) were treated for 30 min and, separately, for 24 h with hot water extract of Cordyceps fungal mycelia or exopolysaccharides. Cells were washed, irradiated with UVB (302 nm), and immediately lysed, after which DNA damage, as strand breaks, was measured using an enzyme‐assisted comet assay that detects CPDs. Results DNA damage in UVB‐irradiated cells was significantly lowered (P < 0·01) with Cordyceps pretreatment. Similar results were seen with 30 min and 24 h pretreatment. Specifically, and in comparison with irradiated cells with no Cordyceps pretreatment, there was a 27% reduction in CPDs in irradiated cells with 24 h pretreatment with 200 μg mL^?1 of the hot water Cordyceps extract, and a 34% reduction with 24 h pretreatment with 200 μg mL^?1 of the exopolysaccharide extract. Conclusions Clear evidence of protection against UVB‐induced CPDs was seen with Cordyceps mycelial extracts. Results indicate that Cordyceps may offer photoprotection and lower the risk of basal cell carcinoma, the main skin cancer caused by CPDs. Further study is needed to identify protective mechanisms.     

茶多酚单体对中波紫外线辐射培养的成纤维细胞氧化损伤的保护机制研究

目的：探讨茶多酚的活性单体表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate，EGCG)对紫外线辐射氧化损伤的保护机制。方法：用722分光光度计测定培养的人皮肤成纤维细胞上清液中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH—Px)的活性，以及脂质过氧化产物丙二醛(MDA)的含量；采用逆转录—聚合酶链反应(RT—PCR)方法测定成纤维细胞合成基质金属蛋白酶1(MMP1)以及其组织抑制因子—1(TIMP—11的mRNA表达水平。结果：EGCG可以减少中波紫外线(UVB）辐射引起的丙二醛沉积，增加抗氧化酶的活性；并且可以抑制UVB诱导的MMP1 mRNA表达，减少胶原蛋白的降解。对TIMP—1的表达则没有观察到显著变化。结论：EGCG可以对UVB辐射损伤的体外培养成纤维细胞起到保护作用。     

Intercellular pathway through hyaluronic acid in UVB‐induced inflammation

Ultraviolet B (UVB) radiation induces inflammation in the skin specifically at the site of exposure. We unexpectedly found that UVB‐induced inflammation was not induced in gp91phox‐depleted mice. To test whether gp91phox is directly involved in UVB‐induced inflammation, neutrophil‐ and hyaluronic acid–depleted mice were also irradiated and examined for their response. Hyaluronic acid–depleted mice showed strongly inhibited UVB‐induced inflammation, but the neutrophil‐depleted mice did not exhibit any suppressed UVB‐induced inflammation. To elucidate the pathway by which UVB irradiation induced inflammation, we examined the expression of nucleotide‐binding domain, leucine‐rich‐containing family, pyrin domain‐containing‐3 (NLRP3) and caspase‐1 in the mouse skin. An increase in the expression of NLRP3 and caspase‐1 was seen following the UVB irradiation of C57BL mice; however, the UVB‐irradiated gp91phox‐knockout (gp91phox^?/?) mice did not have this increase in expression. Furthermore, the plasma IL‐1β level increased after the UVB irradiation in C57BL mice, but there was no change in the gp91phox^?/? mice. These results clearly indicate that nicotinamide adenine dinucleotide phosphate oxidase is activated by gp91phox, which is expressed on the surface in response to the increased expression of hyaluronic acid induced by UVB irradiation, and as result, the generation of reactive oxygen species (ROS) increases. This ROS activate NLRP3, and NLRP3 leads to the production of caspase‐1, which subsequently increases IL‐1β, thereby finally inducing inflammation. It is thought that this system may play an important role in the damage and ageing of skin, and further studies are necessary to confirm these finding.     

Ultraviolet A irradiation stimulates collagenase production in cultured human fibroblasts.

This study was designed to investigate the biochemical mechanisms responsible for the connective tissue changes seen in actinically damaged skin, which is characterized histologically by diminution and ultrastructural alterations of collagen fibrils and deposition of elastotic material in the papillary dermis. We hypothesized that ultraviolet light could stimulate synthesis of interstitial collagenase in the skin, resulting in collagen degradation. Monolayer cultures of human fibroblasts or keratinocytes were irradiated with ultraviolet A (UVA) or ultraviolet B (UVB) radiation and interstitial collagenase or its inhibitor, TIMP (tissue inhibitor of metalloproteinases) assessed in the conditioned medium with Western immunoblots 24 h after irradiation. Northern blot analysis of the irradiated fibroblasts with a cDNA probe representing collagenase was also performed. Cell viability was greater than 90% with all doses of UV radiation studied. A dose-related increase in immunoreactive collagenase was detected in the medium of fibroblasts irradiated with 0-10 J/cm2 of UVA radiation as well as a parallel increase in the collagenase mRNA in the irradiated cells. UVA radiation stimulated collagenase synthesis in both neonatal and adult fibroblasts. TIMP production in UVA-irradiated fibroblasts increased to a lesser degree than did collagenase and its increase did not parallel the increase in collagenase. UVB (0-100 mJ/cm2) did not stimulate collagenase production by fibroblasts. In contrast to the stimulation of collagenase production by fibroblasts, a slight decrease in immunoreactive collagenase was seen in UVA-irradiated keratinocytes. These data suggest that direct stimulation of collagenase synthesis by human skin fibroblasts by UVA radiation may contribute to the connective tissue damage induced by ultraviolet radiation leading to photoaging.     

Isoflavones in aglycone solution enhance ultraviolet B‐induced DNA damage repair efficiency

The isoflavones daidzein and genistein are natural compounds which have anti‐inflammatory and photoprotective activities, and may be effective in the repair of ultraviolet (UV)‐induced photodamage. In this study, an alcoholic solution of aglycone isoflavones with a genistein:daidzein ratio of 1:4 [Rottapharm (RPH)‐aglycone] was examined for its effects on the repair of DNA damage induced by a single dose of UVB irradiation (20 mJ/cm²). For this purpose, human skin cells were first UVB‐irradiated and then treated with RPH‐aglycone. Comet assay analysis was used to estimate the UVB‐induced DNA damage at different time points after treatment by measuring the tail moment parameter. We found that treatment with 10 μmol/L RPH‐aglycone solution resulted in a significantly reduced tail moment at 1 h after treatment, and 34–35% enhancement of damage repair at 4 h after treatment. These results suggest that isoflavone aglycones are protective against UVB‐induced DNA damage.     

Ultraviolet B irradiation increases the expression of trichohyalin‐like 1 protein in human skin xenotransplants

Trichohyalin‐like (TCHHL)1 is a member of the fused‐type S100 protein family. Its function remains unknown, although it has been reported to be expressed in the basal layer of the normal epidermis. The aim of this study was to investigate the effects of ultraviolet B (UVB) irradiation on the expression of TCHHL1 in human skin xenotransplants. Expression of TCHHL1 mRNA was increased in the UVB‐exposed skin 2 days after UVB irradiation. TCHHL1 was immunohistochemically detected in the basal layers after sham irradiation. However, on Day 2 after irradiation, the TCHHL1 signals were spread throughout the basal and spinous layers of the irradiated skin, with increased expression of cytokeratin 14 and a dramatic increase in the number of Ki67‐positive cells observed. These results show that TCHHL1 is a novel protein whose expression can be increased by UVB irradiation. In addition, this study experimentally shows that TCHHL1 is expressed in proliferative keratinocytes.     

过表达p53基因和中波紫外线照射对人皮肤成纤维细胞衰老的影响

目的建立稳定过表达野生型p53基因(wtp53)的人皮肤成纤维细胞系,探讨过表达p53基因及中波紫外线(UVB)照射对人皮肤成纤维细胞(HSF)衰老的影响。方法在脂质体lipofectamine TM 2000介导下,将含有wtp53的真核表达质粒pCMV-p53导入HSF中,经G418筛选抗性克隆,扩大培养,建立转染克隆细胞系。用RT-PCR,实时荧光定量PCR和蛋白印迹法分析目的基因及其蛋白表达。亚毒性剂量UVB照射细胞,以染色法检测SAβ-gal活性,MTT法检测细胞增殖能力。结果成功构建过表达p53的HSF细胞系,转染细胞中存在p53基因及蛋白的稳定过表达。过表达p53的细胞株不论UVB照射与否SAβ-gal活性均未见增高,过表达p53可使HSF的增殖能力减弱,但对UVB诱导的细胞生长停滞的影响并不显著。结论过表达p53不能促进HSF的复制衰老和UVB诱导的早期衰老,过表达p53引起的HSF增殖减弱可能与细胞凋亡有关而非衰老。     

Contrasting effects of an ultraviolet B and an ultraviolet A tanning lamp on interleukin-6, tumour necrosis factor-α and intercellular adhesion molecule-1 expression

BACKGROUND: Recent studies have demonstrated that a tanning lamp emitting predominantly ultraviolet (UV) A induces significant yields of the type of potentially mutagenic DNA damage that are associated with the onset of skin cancer (i.e. cyclobutane pyrimidine dimers). UV-induced immunosuppression is also an important event leading to skin cancer. OBJECTIVES: To the modulation of key immunological molecules following exposure to a broad-spectrum UVB lamp and a predominantly UVA-emitting tanning lamp using model in vitro systems. METHODS: We compared secretion and mRNA expression of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in normal human epidermal keratinocytes, and interferon (IFN)-gamma-induced intracellular adhesion molecule (ICAM)-1 in normal human fibroblasts irradiated in vitro with a broad-spectrum UVB lamp or with a Philips 'Performance' tanning lamp. RESULTS: With broad-spectrum UVB irradiation, upregulation of IL-6 and TNF-alpha mRNA was detected 6 h after irradiation, and a dose-dependent increase of cytokines in the supernatants of irradiated cells was found 24 h after irradiation. In contrast, there was no cytokine secretion and little evidence for mRNA upregulation following exposure to a tanning lamp. When cells were exposed first to broad-spectrum UVB, then the tanning lamp, UVB-induced cytokine secretion was inhibited, although mRNA levels were upregulated to a level close to that observed with UVB alone. By using a Schott WG 320 nm filter to attenuate the level of UVB relative to UVA emitted by the tanning lamp, the inhibition of cytokine secretion was shown to be associated with UVA exposure. Both UV sources inhibited IFN-gamma-induced ICAM-1 mRNA expression in a dose-dependent fashion. By using a Schott WG 335 nm filter, inhibition of ICAM-1 mRNA expression by the tanning lamp was shown to be associated with UVB exposure. CONCLUSIONS: These results suggest that UV sources emitting different levels of UVA and UVB have differential effects on the modulation of different immunoregulatory molecules, and indicate that there are potential interactions between these wavelengths.     

氢气对中波紫外线致皮肤成纤维细胞氧化损伤的影响

【摘要】 目的 探讨氢气对中波紫外线（UVB）致人成纤维细胞氧化损伤的影响。方法 原代培养人成纤维细胞,分组如下：正常对照组,未经任何处理;氢气对照组,富氢培养液处理;UVB照射组;富氢培养液后处理组;富氢培养液预处理组。通过噻唑蓝（MTT）法观察细胞增殖情况,通过化学发光和酶联免疫吸附试验（ELISA）观察超氧化物歧化酶（SOD）、过氧化氢酶（CAT）的活性,丙二醛（MDA）和8异前列腺素2α（8-isoPGE2α）的水平, Western 印迹观察血红素加氧酶-1（HO-1）蛋白的表达情况,采用单因素方差分析进行统计学处理。 结果 给予不同剂量的UVB照射,细胞增殖活性（A490）呈剂量依赖性降低。氢气后处理和预处理组细胞增殖活性明显高于相应UVB组（均P < 0.05）。氢气后、预处理组SOD和CAT活性以及HO-1蛋白表达水平明显高于UVB组（均P < 0.05）,但MDA和8-iso-PGE2α明显低于UVB组（均P < 0.05）。 结论 氢气可以减轻UVB诱发的成纤维细胞氧化损伤。     

Insulin‐like growth factor‐I and UVB photoprotection in human keratinocytes

Ultraviolet radiation (UVR), in particular the UVB spectrum, is a risk factor for skin cancer development. The generation and accumulation of UVB‐induced genetic mutations are fundamental premalignant events. Keratinocyte interactions between other cutaneous cell populations and the surrounding microenvironment determine cell fate and acute photoresponses. In this study, the importance of the insulin‐like growth factor (IGF) system, in particular the insulin‐like growth factor‐I (IGF‐I), on influencing key processes in the keratinocyte acute photoresponse was investigated. Exogenous IGF‐I and other growth factors present in dermal fibroblast‐conditioned media (CM) were found to significantly enhance keratinocyte survival following UVB irradiation in vitro. This pretreatment was also shown to cause a shift in the expression levels of various DNA damage response proteins. Consequently, this was associated with accelerated rates of UVB‐induced cyclobutane pyrimidine dimer removal in these samples. Finally, activation of the IGF system influenced cell cycle progression in UVB‐irradiated keratinocytes. Taken together, these results highlight the importance of the IGF signalling network in initiating the repair of potentially mutagenic DNA damage in human keratinocytes. The dysregulation of these processes may therefore have significant implications in the aetiology of skin cancers and other cutaneous diseases.