UV radiation is known to cause photoaging of the skin and is considered one of the leading cause of developing skin carcinogenesis. Melatonin which has a highly lipophilic molecular structure facilitating penetration of cell membranes and serving as an extra- and intracellular free radical scavenger has been demonstrated to protect photodamage of skin affected by UV exposure. In this study, we have examined the role of melatonin in response to UVB induced photodamaging process, using human skin fibroblasts in vitro. Cell survival curves after UVB irradiation showed dose-dependent decrease. Only 60% of fibroblasts were survived at 140 mJ/cm2 UVB irradiation. By pre-cultivation of cells with melatonin (100 nM), a significant number of cells remained unaffected. After UVB irradiation with 70 mJ/cm2, the level of putrescine was 1.7+/-0.3 fold increased compared to melatonin pre-treated group. In Northern analyses, the transcriptional level of ornithine decarboxylase (ODC) gene expression was increased by UVB irradiation and prohibited by melatonin. These results indicated that melatonin was effectively able to neutralize membrane peroxidation when present in relevant concentration during UVB irradiation and diminishes the UVB-induced increase of polyamine synthesis and ODC gene expression. Collectively, ODC response to UVB induced changes are possibly involves a melatonin or antioxidant sensitive regulatory pathway in normal human skin fibroblast.
Antioxidants/*pharmacology ; Apoptosis/drug effects/radiation effects ; Fibroblasts/*drug effects/*radiation effects ; Human ; Melatonin/*pharmacology ; Ornithine Decarboxylase/biosynthesis/genetics ; Polyamines/*metabolism ; *Ultraviolet Rays

BACKGROUNDWe showed in our previous study that the N-terminal 17-mer peptide of amyloid precursor protein (APP17-mer peptide), an active peptide segment with trophic and antioxidative effects, protects skin fibroblasts against ultraviolet (UV) damage and downregulates matrix metalloproteinase 1 (MMP-1) expression. The aim of the current study was to explore the protective effects of P165, the N-terminal 5-mer peptide analog of amyloid precursor protein that is resistant to enzymolysis, on UVA-induced damage in human dermal fibroblasts (HDFs).

METHODSHDFs were cultured in Dulbecco's modified Eagle's medium without and with P165 (concentrations were 1, 10, and 100 µmol/L). Then, 15 J/cm(2) UVA irradiation was used to obtain the UV-irradiated model. Cell proliferation was analyzed using MTT kit. The collagen type I and MMP-1 contents in cell lysate were determined by enzyme-linked immunosorbent assay (ELISA). Fluorometric assays were performed to detect the formation of intracellular reactive oxygen species (ROS) in the cells.

RESULTSP165 significantly protected the HDFs against UVA-induced cytotoxicity. Compared with the UVA-irradiated control, 1, 10, and 100 µmol/L P165 elevated cell proliferation by 14.98% (P < 0.05), 17.52% (P < 0.01) and 28.34% (P < 0.001), respectively. Simultaneously, 10 and 100 µmol/L P165 increased collagen type I content (both P < 0.05). Moreover, P165 treatment (all concentrations) also markedly suppressed the UVA-induced MMP-1 expression (all P < 0.001). P165 at 1, 10, and 100 µmol/L also reduced UVA-induced ROS generation by 11.27%, 13.69% (both P < 0.05), and 25.48% (P < 0.001), respectively.

CONCLUSIONSP165 could protect the HDFs against UVA-induced photodamage, including cytotoxicity, and MMP-1 generation. Furthermore, it also increased the collagen type I content in the cells. The inhibitory effect on intracellular ROS generation might be involved in these photoprotective effects. Thus, P165 may be a useful candidate in the prevention and treatment of skin photoaging.

Amyloid beta-Protein Precursor ; pharmacology ; Cells, Cultured ; Fibroblasts ; drug effects ; radiation effects ; Humans ; Skin ; cytology ; Ultraviolet Rays

OBJECTIVETo investigate the relationships between skin photoaging and point mutations in mitochondrial DNA (mtDNA) control region for replication of dermal fibroblast.

METHODSCultured dermal fibroblasts were treated by 8-methoxypsora len /ultraviolet A (8-MOP/UVA). mtDNA was extracted by one-step-method and th e PCR products of D-loop and adjacent transcription promoter (DLP(6)) fragment of mtDNA control region for replication were detected by polymerase chain reaction-single strain conformation polymorphism and direct sequencing.

RESULTSAfter treated by 8-MOP/UVA, point mutations of 414 T-->G of DLP(6) fragment of mtDNA control region for replication largely accumulated.

CONCLUSIONAccumulation of point mutations of DLP(6) fragment of mtDNA control region for replication may be closely associated with skin photoaging.

Adult ; Binding Sites ; Cells, Cultured ; DNA Replication ; DNA, Mitochondrial ; drug effects ; radiation effects ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; radiation effects ; Humans ; Male ; Methoxsalen ; pharmacology ; Photosensitizing Agents ; pharmacology ; Point Mutation ; Ultraviolet Rays

BACKGROUND: Heat shock protein 70 (HSP70) exhibits protective effects against ultraviolet (UV)-induced premature skin aging. A standardized extract of Asparagus officinalis stem (EAS) is produced as a novel and unique functional food that induces HSP70 cellular expression. To elucidate the anti-photoaging potencies of EAS, we examined its effects on HSP70 expression levels in UV-B-irradiated normal human dermal fibroblasts (NHDFs). METHODS: NHDFs were treated with 1 mg/mL of EAS or dextrin (vehicle control) prior to UV-B irradiation (20 mJ/cm). After culturing NHDFs for different time periods, HSP70 mRNA and protein levels were analyzed using real-time polymerase chain reaction and western blotting, respectively. RESULTS: UV-B-irradiated NHDFs showed reduced HSP70 mRNA levels after 1-6 h of culture, which were recovered after 24 h of culture. Treatment with EAS alone for 24 h increased HSP70 mRNA levels in the NHDFs, but the increase was not reflected in its protein levels. On the other hand, pretreatment with EAS abolished the UV-B irradiation-induced reduction in HSP70 expression at both mRNA and protein levels. These results suggest that EAS is capable to preserve HSP70 quantity in UV-B-irradiated NHDFs. CONCLUSIONS EAS exhibits anti-photoaging potencies by preventing the reduction in HSP70 expression in UV-irradiated dermal fibroblasts.
Asparagus Plant ; Cells, Cultured ; Female ; Fibroblasts ; drug effects ; radiation effects ; HSP70 Heat-Shock Proteins ; biosynthesis ; Humans ; Middle Aged ; Plant Extracts ; pharmacology ; Polymerase Chain Reaction ; Skin ; drug effects ; radiation effects ; Skin Aging ; drug effects ; radiation effects ; Telomere ; metabolism ; Ultraviolet Rays ; adverse effects

Cordycepin (3'-deoxyadenosine) has been shown to exhibit many pharmacological activities, including anti-cancer, anti-inflammatory, and anti-infection activities. However, the anti-skin photoaging effects of cordycepin have not yet been reported. In the present study, we investigated the inhibitory effects of cordycepin on matrix metalloproteinase-1 (MMP-1) and -3 expressions of the human dermal fibroblast cells. Western blot analysis and real-time PCR revealed cordycepin inhibited UVB-induced MMP-1 and -3 expressions in a dose-dependent manner. UVB strongly activated NF-kappa B activity, which was determined by I kappa B alpha degradation, nuclear localization of p50 and p65 subunit, and NF-kappa B binding activity. However, UVB-induced NF-kappa B activation and MMP expression were completely blocked by cordycepin pretreatment. These findings suggest that cordycepin could prevent UVB-induced MMPs expressions through inhibition of NF-kappa B activation. In conclusion, cordycepin might be used as a potential agent for the prevention and treatment of skin photoaging.
Aging/physiology ; Cells, Cultured ; Deoxyadenosines/*pharmacology ; *Dermis/cytology/drug effects/physiology/radiation effects ; Dose-Response Relationship, Drug ; Enzyme Induction/drug effects ; Fibroblasts/drug effects/metabolism/radiation effects ; Gene Expression Regulation, Enzymologic ; Humans ; Infant, Newborn ; Male ; *Matrix Metalloproteinase 1/antagonists & inhibitors/biosynthesis/genetics/radiation effects ; Matrix Metalloproteinase 3/antagonists & inhibitors/*biosynthesis/genetics/radiation effects ; NF-kappa B/*antagonists & inhibitors/genetics/metabolism ; Skin/physiopathology/radiation effects ; *Ultraviolet Rays

OBJECTIVETo investigate the effects of Heijiangdan Ointment ( HJD) on oxidative stress in (60)Co γ-ray radiation-induced dermatitis in mice.

METHODSFemale Wistar mice with grade 4 radiation dermatitis induced by (60)Co γ-rays were randomly divided into four groups (n=12 per group); the HJD-treated, recombinant human epidermal growth factor (rhEGF)-treated, Trolox-treated, and untreated groups, along with a negative control group. On the 11th and 21st days after treatment, 6 mice in each group were chosen for evaluation. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH) were detected using spectrophotometric methods. The fibroblast mitochondria were observed by transmission electron microscopy (TEM). The expressions of fibroblast growth factor 2 (FGF-2) and transforming growth factor β1 (TGF-β1) were analyzed by western blot.

RESULTSCompared with the untreated group, the levels of SOD, MDA and LDH, on the 11th and 21st days after treatment showed significant difference (P<0.05). TEM analysis indicated that fibroblast mitochondria in the untreated group exhibited swelling and the cristae appeared fractured, while in the HJD group, the swelling of mitochondria was limited and the rough endoplasmic reticulum appeared more relaxed. The expressions of FGF-2 and TGF-β1 increased in the untreated group compared with the negative control group (P<0.05). After treatment, the expression of FGF-2, rhEGF and Trolox in the HJD group were significantly increased compared with the untreated group (P<0.05), or compared with the negative control group (P<0.05). The expression of TGF-β1 showed significant difference between untreated and negative control groups (P<0.05). HJD and Trolox increased the level of TGF-β1 and the difference was marked as compared with the untreated and negative control groups (P<0.05).

CONCLUSIONHJD relieves oxidative stress-induced injury, increases the antioxidant activity, mitigates the fibroblast mitochondrial damage, up-regulates the expression of growth factor, and promotes mitochondrial repair in mice.

Animals ; Biological Products ; pharmacology ; therapeutic use ; Cell Proliferation ; drug effects ; radiation effects ; Cobalt Radioisotopes ; Dermatitis ; complications ; drug therapy ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Fibroblast Growth Factor 2 ; genetics ; metabolism ; Fibroblasts ; drug effects ; pathology ; radiation effects ; Gamma Rays ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Mice ; Mitochondria ; drug effects ; metabolism ; radiation effects ; Ointments ; Oxidative Stress ; drug effects ; radiation effects ; Pharmaceutical Preparations ; Radiation Injuries ; complications ; drug therapy ; pathology ; Superoxide Dismutase ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Up-Regulation ; drug effects ; radiation effects

OBJECTIVETo investigate the effects of quercetin and X-ray on collagen synthesis of cultured human keloid-derived fibroblast and the mechanism.

METHODSCollagen synthesis of cultured human keloid and normal fibroblasts were detected by hydroxyproline colorimetric analysis. Immunocytochemical staining was used to investigate collagen I and III expression. mRNA expression of collagen I and III, and transforming growth factor (TGF)-beta 1 were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR.

RESULTSQuercetin inhibited the collagen synthesis of both keloid and normal fibroblasts in a dose-dependent manner. Immunocytochemical staining indicated that collagen I and III were down-regulated by quercetin and X-ray (P < 0.05), particularly collagen I (P < 0.05). mRNA expression of both collagen I and III in quercetin groups significantly decreased compared with that in control group (P < 0.05), especially in the group treated with both quercetin and X-ray (P < 0.01). mRNA level of TGF-beta 1 gene was down-regulated by quercertin (P < 0.05).

CONCLUSIONSQuercetin will probably be one of the new medicines which could effectively treat keloid. Quercetin combined with X-ray could reduce the dose of radiation.

Adult ; Cells, Cultured ; Collagen Type I ; biosynthesis ; genetics ; radiation effects ; Collagen Type III ; biosynthesis ; genetics ; radiation effects ; Dose-Response Relationship, Drug ; Female ; Fibroblasts ; metabolism ; pathology ; radiation effects ; Humans ; Keloid ; metabolism ; pathology ; Male ; Quercetin ; administration & dosage ; pharmacology ; RNA, Messenger ; biosynthesis ; Skin ; cytology ; Transforming Growth Factor beta1 ; biosynthesis ; genetics ; radiation effects

OBJECTIVETo study the possible induction effect of exposure to 50 Hz magnetic field (MF) on clustering of cell membrane surface receptors for epidermal growth factor (EGF) and tumor necrosis factor (TNF), the starting site of signals of biological effects, and its possible intervention effect.

METHODSLung fibroblasts of Chinese hamster (CHL) were exposed to EGF, TNF, 0.4 mT 50 Hz MF, 0.4 mT noise MF, and 0.4 mT 50 Hz MF combined with 0.4 mT noise MF. Respectively, for different durations, following the treatment, EGF and TNF receptors on the cell membrane were marked by corresponding antibodies with immunohistochemical method, then observed under a confocal microscope.

RESULTSClustering of cell membrane receptors could be induced 5 min after treatment with EGF and TNF, as well as with 50 Hz MF at 0.4 mT, which reached the peak in 15 min. While noise MF with the same intensity did not induce clustering of cell membrane receptors. Superposition of noise MF with the same intensity could inhibit clustering of cell membrane receptors induced by 50 Hz MF.

CONCLUSIONClustering of EGF and TNF receptors on the cell membrane could be induced by 50 Hz MF, suggesting that membrane receptors would be one of the sites where MF signals coupled, and noise MF with the same intensity could inhibit these effects.

Animals ; Cell Line ; Cricetinae ; Electromagnetic Fields ; adverse effects ; Epidermal Growth Factor ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; radiation effects ; Noise ; adverse effects ; Receptor, Epidermal Growth Factor ; metabolism ; Receptors, Cell Surface ; metabolism ; Receptors, Tumor Necrosis Factor ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

BACKGROUNDCathepsin L (CatL) is a cysteine protease with strong matrix degradation activity that contributes to photoaging. Mannose phosphate-independent sorting pathways mediate ultraviolet A (UVA)-induced alternate trafficking of CatL. Little is known about signaling pathways involved in the regulation of UVA-induced CatL expression and activity. This study aims to investigate whether a single UVA irradiation affects CatL expression and activity and whether mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway is involved in the regulation of UVA-induced CatL expression and activity in human dermal fibroblasts (HDFs).

METHODSPrimary HDFs were exposed to UVA. Cell proliferation was determined by a cell counting kit. UVA-induced CatL production and activity were studied with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and fluorimetric assay in cell lysates collected on three consecutive days after irradiation. Time courses of UVA-activated JNK and p38MAPK signaling were examined by Western blotting. Effects of MAPK inhibitors and knockdown of Jun and Fos on UVA-induced CatL expression and activity were investigated by RT-PCR, Western blotting, and fluorimetric assay. Data were analyzed by one-way analysis of variance.

RESULTSUVA significantly increased CatL gene expression, protein abundance, and enzymatic activity for three consecutive days after irradiation (F = 83.11, 56.14, and 71.19, respectively; all P < 0.05). Further investigation demonstrated phosphorylation of JNK and p38MAPK activated by UVA. Importantly, inactivation of JNK pathway significantly decreased UVA-induced CatL expression and activity, which were not affected by p38MAPK inhibition. Moreover, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatL expression and activity.

CONCLUSIONSUVA enhances CatL production and activity in HDFs, probably by activating JNK and downstreaming AP-1. These findings provide a new possible molecular approach for antiphotoaging therapy.

Anthracenes ; pharmacology ; Cathepsin L ; metabolism ; Cells, Cultured ; Child ; Child, Preschool ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Fibroblasts ; cytology ; drug effects ; metabolism ; radiation effects ; Humans ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; drug effects ; radiation effects ; Oncogene Proteins v-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Pyridines ; pharmacology ; Skin ; cytology ; Ultraviolet Rays

OBJECTIVETo investigate the effects of preemptive freezing with different temperature and cross-linking methods on the ultrastructure of collagen membrane and its influence on human fibroblast proliferation.

METHODSBovine collagen type I solution in concentration of 10 g/L was preliminarily frozen at -20 degrees C or - 80 degrees C for 12 hours, and lyophilized at -70 degrees C for 48 hours. The diameter of apertures in collagen membranes prepared with two different preliminary temperatures were observed by scanning electron microscope (SEM) and compared. The preliminary freezing temperature of - 80 degrees C was used for the following study. The apertures of collagen membrane performed with cross-linking glutaraldehyde and ultraviolet (UV) radiation cross-linking with glutaraldehyde (double cross - linking) after preliminary freezing were also compared. The proliferation of human fibroblasts inoculated in above cross-linking collagens were assessed by MTT assay, in terms of absorption value.

RESULTSThe mean diameter of apertures of collagen membrane pre-frozen at -20 degrees C was (172 +/- 374 microm, while that at -80 degrees C was (99 +/- 24) microm. The apertures of collagen membrane were reduced in size after glutaraldehyde cross-linking, while those of double cross-linking showed no change in size. There was obvious difference in absorption value of fibroblasts 8 days after seeding between above two cross-linking methods (1.534 +/- 0.013 for glutaraldehyde cross-linking, 3.778 +/- 0.010 for double cross-linking, P < 0.05).

CONCLUSIONThe collagen membrane after preliminary freezing at - 80 degrees C and double cross-linking with UV radiation and glutaraldehyde may be used as a dermal skeleton substitute.

Animals ; Cattle ; Cells, Cultured ; Coculture Techniques ; Collagen ; ultrastructure ; Cross-Linking Reagents ; chemistry ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; radiation effects ; Freezing ; Glutaral ; chemistry ; Humans ; Skin, Artificial ; Tissue Scaffolds ; Ultraviolet Rays