UVB致成纤维细胞损伤及两种中药的保护作用研究

目的观察中波紫外线(UVB)辐射后成纤维细胞(FB)DNA光产物环丁烷嘧啶二聚体(CPD s)产生和清除情况及表没食子儿茶素没食子酸酯(EGCG)和黄芩甙的干预作用。方法以30,60,90 m J/cm2UVB照射FB并予EGCG及黄芩甙干预处理,采用免疫细胞化学法在照光后不同时间检测CPD s的产生和清除情况。结果细胞损伤程度随照光剂量加大而加重;30 m J/cm2UVB照射后细胞CPD s生成量在辐射后1 h左右达到高峰,同时细胞也开始清除CP-D s,辐射后4 h内清除速率较快,4 h后清除速率逐渐降低,至24 h基本清除CPD s;EGCG和黄芩甙处理UVB辐射的细胞CPD s少于单纯照光组(P<0.05)。结论UVB辐射可以导致FB的DNA损伤而产生光产物CPD s;细胞损伤程度显示剂量依赖性;细胞自身有修复能力;EGCG和黄芩甙均可降低UVB辐射所致的光产物水平。     

Baicalin protects human fibroblasts against ultraviolet B-induced cyclobutane pyrimidine dimers formation

Exposure to ultraviolet B (UVB) irradiation is a major risk factor for the development of skin cancer. Therefore, it is important to identify agents that can offer protection against UVB-caused DNA damage. Photocarcinogenesis is caused largely by mutations at the sites of incorrectly repaired DNA photoproducts, of which the most common are the cyclobutane pyrimidine dimers (CPDs). In this study, a DNA damage model of UVB irradiation-induced fibroblasts was established. The immunocytochemical staining, immuno dot blotting and Western blotting were employed in the study. We demonstrated that pre-treatment of fibroblasts with Baicalin dose-dependently reduced the amount of UVB-generated CPDs. Compared with UVB irradiated cells, UVB-induced p53 accumulation was less pronounced in Baicalin-treated cells. Taken together, these results suggest that Baicalin prevent CPDs formation induced by UVB. Baicalin is therefore a promising protective substance against UVB radiation.     

Aged human skin removes UVB-induced pyrimidine dimers from the epidermis more slowly than younger adult skin in vivo

Although many studies have been reported on the repair of ultraviolet light (UV)-induced cyclobutane-type pyrimidine dimers (CPDs) in DNA, the effects of aging on the removal of UV-induced CPDs from the human skin epidermis in vivo remains uncertain. Therefore, we employed immunoblotting and immunohistochemical methods using monoclonal antibodies (TDM-2) to CPDs to study age-related differences in the time required for the in vivo removal of UVB-induced CPDs. The flexure surfaces of the upper arms of five young men were exposed to UVB light at a fluence of 35 and 700 mJ/cm², and four older men were also irradiated with the same doses of UVB mentioned above. Each area of skin was biopsied before and immediately after irradiation, and at 4, 24 h, 2 and 4 days after irradiation in the younger group; and before and immediately after irradiation, and at 24 h, 4, 7, and 14 days after irradiation in the older group. A total of 108 DNA samples were taken from the epidermis of 108 biopsied specimens. These samples were immunoblotted using TDM-2 and the intensities of the immunoprecipitates were measured by photodensitometer. Our results show that the CPDs had been removed from the epidermis at 4 days after irradiation at either dose in the younger group, and between 7–14 days after irradiation in the aged group. The results of our immunohistochemical studies were consistent with those of our immunoblotting studies, and indicated that basal cells repair CPDs more quickly than prickle cells in the epidermis except the amounts at 24 h after UVB irradiation, and that the CPDs were removed by epidermal turnover after the nucleotide excision repair (NER). Our results showed age-associated decline in the NER in vivo, indicating high risk of UV-associated skin cancer.     

Polydeoxyribonucleotide promotes cyclobutane pyrimidine dimer repair in UVB-exposed dermal fibroblasts

BACKGROUND: DNA is the main cellular chromophore for ultraviolet B (UVB). Its absorption leads to the generation of typical photoproducts. The most frequent types (about 80%) are cyclobutane pyrimidine dimers (CPDs). Several studies have suggested that treatment with deoxyribonucleosides can protect some cell types from DNA damage. The aim of this work was to evaluate the ability of the polydeoxyribonucleotide (PDRN) to protect human dermal fibroblasts from UVB-induced DNA damage. METHODS: Human dermal fibroblasts were irradiated with 600 mJ/cm(2) of UVB radiation. Cells were analyzed at increasing time points from irradiation to study the recovery from UVB-induced DNA photodamage. Damage repair was subsequently assessed by immunocytochemical analysis of CPDs levels and by measurement of p53 protein expression. RESULTS: The extracellular addition of 100 microg/ml PDRN immediately after irradiation caused a strong activation of p53 protein in the first 24 h. This signal was accompanied by an increase in CPDs repair rates at early time points of recovery. CONCLUSIONS: The addition of PDRN to the culture medium supports CPDs repair probably providing a faster supply of precursors for the deoxyribonucleotide triphosphates pool necessary to UVB-damaged cells. This condition could promote the action of the salvage pathway, thereby accelerating DNA repair, but other inducible responses linked to increased p53 expression could be involved.     

表没食子儿茶素没食子酸酯对中波紫外线诱导人原代表皮黑素细胞光损伤干预作用的研究

目的:观察中波紫外线(UVB)诱导人原代表皮黑素细胞光产物的形成和清除情况,以及表没食子儿茶素没食子酸酯(EGCG)的干预作用.方法:选择10、20、40、80、100 mg/L 5种浓度EGCG作用于体外培养的人表皮黑素细胞72 h,测定细胞增殖活性.采用免疫斑点印迹技术在30 mJ/cm2UVB照射及EGCG干预下.分别取照射后0.5 h和24 h检测环丁烷嘧啶二聚体(CPDs)的产生和清除量.结果:低浓度(＜20 mg/L)EGCG能促进黑素细胞增殖.UVB照射后黑素细胞内光产物形成较快,但清除缓慢.EGCG对UVB诱导光产物的形成无明显影响,但能加速光产物的清除(P＜0.05).结论:EGCG能加速UVB照射后黑素细胞内光产物的清除.     

A clinical trial and molecular study of photoadaptation in vitiligo

Background Photoadaptation to ultraviolet (UV) B phototherapy is due to both pigmentary and nonpigmentary influences. Objectives To measure photoadaptation in vitiliginous skin and to compare it with normal pigmented skin. Methods Seventeen patients with Fitzpatrick skin phototypes III–VI with vitiligo received six to nine UVB treatments, two to three times weekly. Minimal erythema dose (MED) testing was done at baseline and after all treatments; the percentage change in MED was analysed as a measure of photoadaptation. The percentage decrease in cyclobutane pyrimidine dimers (CPDs) over 24 h after a single exposure of 1 MED was analysed on vitiliginous and normal skin. Results The mean ± SD percentage change in MED from before to after treatments was: treated vitiliginous skin 28·5 ± 39·9% (P = 0·015), treated normal skin 35·9 ± 49·9% (P = 0·015), untreated vitiliginous skin 11·9 ± 22·6% (P =0·070), untreated normal skin 25·1 ± 41·3% (P = 0·041). Of these patients, two‐thirds had a positive percentage change in MED (photoadaptation). The mean amount of CPDs induced per megabase of DNA immediately after exposure was significantly higher in vitiliginous skin. The mean ± SD percentage decrease in CPDs (rate of repair) in 24 h was 35·7 ± 26·8% in vitiliginous skin (P = 0·027) and 46·2 ± 19·5% in normally pigmented skin (P = 0·001); no difference was noted in the repair in vitiliginous skin compared with normal skin (P = 0·4). Conclusions Photoadaptation in vitiliginous and normal skin was observed in two‐thirds of patients. Vitiliginous skin had significantly more CPDs following UVB exposure; the rate of repair of UVB‐induced DNA damage was equivalent to that in normal skin.     

Higher cyclobutane pyrimidine dimer and (6-4) photoproduct yields in epidermis of normal humans with increased sensitivity to ultraviolet B radiation

Cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct induced in the epidermis of five Japanese volunteers exposed to ultraviolet (UVB) radiation were measured with monoclonal antibodies specific for each photoproduct. The volunteers comprised two individuals who are sensitive to solar irradiation (low minimal erythema dose [MED]) and three who are less sensitive. The yields of CPD and (6-4) photoproduct were within similar ranges after 1 MED or 3 MED doses. The yields of both photoproducts after the same dose of irradiation (120 mJ/cm²) were higher in UV-sensitive individuals than in less sensitive individuals. By 24 h after irradiation, an average of 60% of CPD had been removed after the 1 MED dose, 27% after the 3 MED dose and 34% after 120 mJ/cm². The (6-4) photoproduct was removed within 24 h, independently of the dose of UVB tested. These data suggest that DNA photoproducts participate in initiating UVB-induced erythema, and partially explain why individuals with higher sensitivity to UVB have a higher risk of UV-induced skin cancer.     

Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin

Abstract: Solar ultraviolet (UV) radiation, particularly its UVB (290–320 nm) component, is the primary cause of many adverse biological effects including photoageing and skin cancer. UVB radiation causes DNA damage, protein oxidation and induces matrix metalloproteinases (MMPs). Photochemoprevention via the use of botanical antioxidants in affording protection to human skin against UVB damage is receiving increasing attention. Pomegranate, from the tree Punica granatum, contains anthocyanins and hydrolysable tannins and possesses strong antioxidant and anti‐tumor‐promoting properties. In this study, we determined the effect of pomegranate‐derived products – POMx juice, POMx extract and pomegranate oil (POMo) – against UVB‐mediated damage using reconstituted human skin (EpiDerm^TM FT‐200). EpiDerm was treated with POMx juice (1–2 μl/0.1 ml/well), POMx extract (5–10 μg/0.1 ml/well) and POMo (1–2 μl/0.1 ml/well) for 1 h prior to UVB (60 mJ/cm²) irradiation and was harvested 12 h post‐UVB to assess protein oxidation, markers of DNA damage and photoageing by Western blot analysis and immunohistochemistry. Pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) cyclobutane pyrimidine dimers (CPD), (ii) 8‐dihydro‐2′‐deoxyguanosine (8‐OHdG), (iii) protein oxidation and (iv) proliferating cell nuclear antigen (PCNA) protein expression. We also found that pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) collagenase (MMP‐1), (ii) gelatinase (MMP‐2, MMP‐9), (iii) stromelysin (MMP‐3), (iv) marilysin (MMP‐7), (v) elastase (MMP‐12) and (vi) tropoelastin. Gelatin zymography revealed that pomegranate‐derived products inhibited UVB‐induced MMP‐2 and MMP‐9 activities. Pomegranate‐derived products also caused a decrease in UVB‐induced protein expression of c‐Fos and phosphorylation of c‐Jun. Collectively, these results suggest that all three pomegranate‐derived products may be useful against UVB‐induced damage to human skin.     

Induction and repair of UVB-induced cyclobutane pyrimidine dimers and (6-4) photoproducts in organ-cultured normal human skin

Summary To examine the induction and repair of UV-induced DNA damage, indirect immunofluorescence was performed on UVB-irradiated organ-cultured normal human skin using monoclonal antibodies specific for either cyclobutane pyrimidine dimers or (6-4) photoproducts. Nuclear immunofluorescence of cyclobutane pyrimidine dimers and (6-4) photoproducts were observed in a dosedependent manner after UVB irradiation. The intensity of nuclear immunofluorescence of the upper epidermal layers was stronger and clearer than that of the lower epidermal layers. DNA repair time-course studies showed that both types of DNA damage could be repaired within 24 h after UVB irradiation.     

Mitigation of acute ultraviolet B radiation-mediated damages by baicalin in mouse skin

Background: Solar ultraviolet (UV) irradiation, in particular UVB with a wavelength range between 290 and 320 nm, induces different hazardous effects on the skin, including sunburn, photoaging and cancer. Protection against sun-induced damage is therefore a highly desirable goal. Chemoprevention is being investigated as a potential approach for the management of UV damages including skin cancer.
Aim: In this study, to determine the relevance of our in vitro findings to in vivo situations, we assessed the effects of baicalin on UVB-mediated damages in mice skin.
Methods: Balb/C hairless mice were topically pretreated (24 h before UVB) or post-treated (5 min after UVB) with baicalin (1 mg/cm² skin area/mouse/100 μl acetone) and were exposed to UVB 24 h later (180 mJ/cm²). The animals were sacrificed 1 and 24 h after the UVB exposure. Skin edema, histopathology changes, hydrogen peroxide (H₂O₂) and cyclobutane pyrimidine dimers (CPDs)-positive cells were assessed to determine the UVB-induced photodamage.
Results: Our data demonstrated that a topical application of baicalin, either as a pretreatment or as a post-treatment, resulted in a significant decrease in UVB mediated increases in skin edema, skin hyperplasia and infiltration of leukocytes. Further, baicalin treatments (pre and post) also resulted in a significant decrease in UVB mediated (1) generation of H₂O₂ and (2) formation of DNA photolesions: CPDs.
Conclusion: Based on these data, we suggest that baicalin could be developed as an agent for the management of conditions elicited by UV exposure including skin cancer.     

High levels of 8-hydroxy-2'-deoxyguanosine appear in normal human epidermis after a single dose of ultraviolet radiation

Major photoproducts induced by carcinogenic ultraviolet (UV) radiation are the cylobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4 PPs). 8-Hydroxy-2 -deoxyguanosine (8-OHdG) is also a DNA base-modified product generated by reactive oxygen species in conditions of ultraviolet stress, Although UVB-induced CPDs and 6-4 PPs have been investigated in animal and human skin, little is known about the role of 8-OHdG in UVB-induced human skin damage or carcinogenesis. Normal human skin from three volunteers was exposed to UV radiation, and the time course of induction and removal of 8-OHdG was examined by immunohistochemical analysis with catalysed signal amplification on formalin-fixed paraffin sections. Formation of CPDs and 6-4 PPs was also examined by immunostaining on the same skin specimens. Control epidermis with no exposure to UV radiation showed little nuclear staining of 8-OHdG, but an increased level of 8-OHdG was clearly observed in epidermis biopsied after irradiation. Induced 8-OHdG can rapidly be removed from nucleus during the first 24-48 h, as the staining intensity diminished gradually, almost reaching the control level by 72-96 h after irradiation. Staining for CPDs or 6-4 PPs revealed induction of these photoproducts in human skin, although 6-4 PP-positive cells disappeared more rapidly than those that stained for CPDs or 8-OHdG. Together with protective effect of antioxidants, our results indicate that not only CPDs and 6-4 PPs but also 8-OHdG may play a significant part in UV carcinogenesis.     

MiR-23a regulates DNA damage repair and apoptosis in UVB-irradiated HaCaT cells

BackgroundMiRNAs remain at a constant level under physiological conditions. However, how the expression of miRNAs is regulated and what are the roles of miRNAs in response to UVB damage to skin cells is still not fully understood. In our preliminary study, we observed that miR-23a was upregulated following a treatment with a DNA repair agent and UVB exposure.ObjectiveTo investigate the regulation and function of miR-23a in response to UVB-induced injury in human keratinocyte cell line (HaCaT) cells.MethodsThe changes in expression of miR-23a after UVB irradiation of HaCaT cells were measured by qRT-PCR. The level of miR-23a expression was also modulated by transfecting with a miR-23a mimic or an inhibitor. Cell viability was assessed by the CCK-8 assay. Immunofluorescence staining and Southwestern dot blotting were used to detect the levels of cyclobutane pyrimidine dimers (CPDs). Flow cytometry, Hoechst staining, and measurements of caspase-3 activity were employed to measure the incidence of apoptosis. The mRNA and protein expression levels of genes related to DNA reparation and apoptosis, such as topoisomerase-1, caspase-7, and STK4, were analyzed by qRT-PCR and Western blotting, respectively.ResultsMiR-23a expression was remarkably up-regulated at 4 h and 24 h after the UVB irradiation of HaCaT cells. UVB-induced apoptosis was increased by down-regulation of miR-23a. UVB-induced removal of CPDs was accelerated by miR-23a up-regulation and delayed by miR-23a down-regulation. Forced over-expression of miR-23a decreased the expression of UVB-induced topoisomerase-1\caspase7\STK4 at both the mRNA and protein levels, and these effects were reversed by down-regulation of miR-23a.ConclusionThe protection of HaCaT cells against UVB damage is afforded by miR-23a through regulation of topoisomerase-1\caspase7\STK4, and this miRNA may be a novel therapeutic target in skin diseases related to UVB radiation.     

UVA does not photoreactivate pyrimidine dimers in cultured human fibroblasts

Abstract Pyrimidine dimers were induced in duplicates of cultured human skin fibroblasts by irradiation with various doses of UVB radiation. Subsequently, one set of cells was further exposed to either 5 or 10 J/cm² of UVA radiation to assess the photoreactivating activity of this spectral range in a human cell system. Following irradiation, pyrimidine dimers were quantified in all cells by determining the number of endonuclease-sensitive sites (ESS). No difference in the yield of ESS was observed between cells which had been irradiated with UVB only as compared to cells which subsequently had been exposed to 5 or 10 J/cm² UVA. In contrast, subsequent exposure of UVB-irradiated cells of Monodelphis domestica to 10 J/cm² UVA resulted in an almost 50% reduction of UVB-induced pyrimidine dimers. These data indicate that UVA does not induce photoenzymatic repair in human fibroblasts.     

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.     

Loss of INK4a/Arf gene enhances ultraviolet radiation‐induced cutaneous tumor development

The CDKN2A locus encodes for tumor suppressor genes p16^INK4a and p14^Arf which are frequently inactivated in human skin tumors. The purpose of this study was to determine the relationship between loss of INK4a/Arf activity and inflammation in the development of ultraviolet (UV) radiation‐induced skin tumors. Panels of INK4a/Arf^‐/? mice and wild‐type (WT) mice were treated with a single dose of UVB (200 mJ/cm²). For long‐term studies, these mice were irradiated with UVB (200 mJ/cm²) three times weekly for 30 weeks. At the end of the experiment, tissues were harvested from mice and assayed for inflammatory biomarkers and cytokines. A single dose of UVB resulted in a significant increase in reactive oxygen species (ROS) and 8‐dihydroxyguanosine (8‐oxo‐dG) lesions in INK4a/Arf^?/? mice compared to WT mice. When subjected to chronic UVB, we found that 100% of INK4a/Arf^?/‐ mice had tumors, whereas there were no tumors in WT controls after 24 weeks of UVB exposure. The increase in tumor development correlated with a significant increase in nuclear factor (NF)‐κB, cyclooxygenase‐2 (COX‐2), prostaglandin E₂ (PGE₂) and its receptors both in UVB‐exposed skin and in the tumors. A significant increase was seen in inflammatory cytokines in skin samples of INK4a/Arf^‐/‐ mice following treatment with chronic UVB radiation. Furthermore, significantly more CD11b⁺Gr1⁺ myeloid cells were present in UVB‐exposed INK4a/Arf^‐/‐ mice compared to WT mice. Our data indicate that by targeting UVB‐induced inflammation, it may be possible to prevent UVB‐induced skin tumors in individuals that carry CDKN2A mutation.     

Narrowband ultraviolet B radiation suppresses contact hypersensitivity

Background/purpose: A main mechanism responsible for the efficacy of narrowband ultraviolet (UV)B is thought to be the induction of apoptosis in pathogenetically relevant cells. Narrowband UVB therapy, however, generally induces a relatively long remission period. Recently, evidence that UVB radiation induces regulatory T (Treg) cells was reported. Based on these findings, we examined whether narrowband UVB suppresses contact hypersensitivity (CHS) by inducing Treg cells. Methods: The shaved abdomens of C3H/HeN mice were irradiated with broadband or narrowband UVB. CHS was defined as an ear‐swelling response. To examine whether tolerance can be induced by adoptive transfer, lymph node cells from UVB‐irradiated mice were injected into naïve mice before sensitization and CHS challenge. Results: Narrowband UVB exposure dose dependently suppressed CHS. Significant suppression was observed at doses between 1000 and 3000 mJ/cm² (P<0.05). The suppressive effect achieved with 1000 mJ/cm² narrowband UVB was very similar to the effect achieved with 100 mJ/cm² broadband UVB. The suppressive effects on CHS were transferred to naïve mice by the injection of lymph node cells from tolerant mice. Conclusion: Narrowband UVB induced local and systemic suppression of CHS. In addition, narrowband UVB induces tolerance to CHS and the suppressive effects were transferable to naïve mice.