To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm-2) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.
Blood Platelets/*cytology/*metabolism ; Cell Movement/radiation effects ; Cell Proliferation/radiation effects ; Cells, Cultured ; Collagen/metabolism ; Fibrin/*metabolism ; Fibroblasts/*cytology/metabolism/*radiation effects ; Humans ; Skin/*cytology ; Time Factors ; Ultraviolet Rays/*adverse effects

OBJECTIVETo observe the effect of solar infrared ray (IR) radiation on the expressions of c-Jun and collagens I and III in cultured human skin fibroblasts (HSFs) and explore the molecular mechanism by which IR radiation causes aging of the skin.

METHODSPrimarily cultured HSFs exposed to IR radiation were examined for changes of the cell viability with MTT assay. The mRNA and protein expressions of c-Jun and collagens I and III was detected with real-time quantitative PCR and immunocytochemistry.

RESULTSMTT assay showed that IR irradiation caused inhibition of cell proliferation compared with the control cells. The mRNA and protein expression of collagen I was decreased significantly by IR irradiation with the increase of the irradiation dose (P<0.01). HSFs irradiated by IR for 12 h showed a dose-dependent reduction of the expression of collagen type III mRNA and protein (P<0.05, P<0.01), but the expression increased dose-dependently in response to IR exposure for 24 h (P<0.05 or 0.01). IR irradiation enhanced the mRNA and protein expression of c-Jun in a dose-dependence manner (P<0.05 or 0.01).

CONCLUSIONSIR irradiation can increase the expression of c-Jun, inhibit the expression of collagen I, and cause disturbance in collagen III expression in human skin fibroblasts, which may be one of the mechanism of IR radiation to initiate and promote skin photoaging.

Cell Proliferation ; Cell Survival ; Cells, Cultured ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Fibroblasts ; metabolism ; radiation effects ; Humans ; Infrared Rays ; Proto-Oncogene Proteins c-jun ; metabolism ; RNA, Messenger ; metabolism ; Skin ; cytology ; Skin Aging ; Ultraviolet Rays

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 effect of irradiated human lung fibroblasts (HLFs) on the canonical Wnt/β-catenin signaling pathway in human umbilical cord mesenchymal stem cells (HUMSCs).

METHODSHUMSCs were cultured alone (single group) or co-cultured with HLFs exposed to 5Gy X-rays (co-culture group). The protein levels of GSK-3β, p-GSK-3β, FRAT1 and β-catenin in HUMSCs were examined by Western blotting 3 days after culture or co-culture. WISP-1 protein levels in conditioned medium were examined by ELISA.

RESULTSThe levels of p-GSK3β/GSK3β (0.15 ± 0.05), FRAT1 (0.48 ± 0.07) and β-catenin (0.50 ± 0.07) in co-cultured HUMSCs significantly decreased compared to those in single group (0.55 ± 0.05, 1.16 ± 0.13 and 2.39 ± 0.15, all P<0.05). The supernatant level of WISP-1 in co-culture group was significantly decreased [(602.23 ± 161.47) ng/mL], compared to the single group [(977.77 ± 110.56) ng/mL, P<0.05].

CONCLUSIONIrradiated HLFs attenuate the activation of canonical Wnt/β-catenin signaling pathway in HUMSCs in vitro.

CCN Intercellular Signaling Proteins ; metabolism ; Cells, Cultured ; Coculture Techniques ; Fibroblasts ; cytology ; radiation effects ; Gamma Rays ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Mesenchymal Stromal Cells ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Umbilical Cord ; cytology ; Wnt Signaling Pathway ; X-Rays ; beta Catenin ; metabolism

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 molecular mechanism of dermal damage in heat shock-induced skin aging by observing the expressions of metalloproteinase-1 (MMP-1) and tissue inhibitor of MMP-1 (TIMP-1) in retinoic acid-treated cultured human fibroblasts with heat shock.

METHODSCultured human fibroblasts were treated with tazarotene or all-trans-retinioic acid (at-RA) after heat shock for 30 min in 43 degrees celsius; water bath. Twenty-four hours later, MMP-1 and TIMP-1 contents in the supernatant of the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA).

RESULTSBoth tazarotene and at-RA dose-dependently reduced the expression of MMP-1 and increased the expression of TIMP-1 in cultured human fibroblasts exposed to heat shock, and tazarotene produced stronger effect than at-RA.

CONCLUSIONRetinoic acid can reduce the expression of MMP-1 and increase the expression of TIMP-1 in cultured human fibroblasts, suggesting its therapeutic potential for heat shock-induced skin aging.

Cells, Cultured ; Fibroblasts ; cytology ; metabolism ; Heat-Shock Response ; Humans ; Matrix Metalloproteinase 1 ; genetics ; metabolism ; Nicotinic Acids ; pharmacology ; Skin Aging ; radiation effects ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Tretinoin ; pharmacology

OBJECTIVETo explore the Mechanism of nonablative skin rejuvenation.

METHODThe Kunming mice be used as subjects and divided into three groups (A, B, C). A, B, C groups were irradiated with 1 320 nm cooltouch laser (20 J/cm2) in the skin of left back; B and C groups were irradiated two and three times respectively; the skin of right back of A, B, C groups was adopted as control. The expression of bFGF and TGF-beta1 in the mouse skin was examined by the immunohistochemistry . The fibroblasts were isolated from the foreskin and cultured. One group is a control and other three ones are low, intermediate and high energetic groups respectively. The fibroblasts were irradiated by laser with 15 J/cm2 ,20 J/cm2 and 24 J/cm2 energy for three times. We examined the levels of bFGF and TGF-beta1 by ELISA in 0, 24, 48 and 72 hours.

RESULTSAccording to this research on immunohistochemistry result, there are significant differences in the expression of bFGF and TGF-beta1 between the group irradiated by three times and others (P < 0.01). The number of fibroblasts get increased after being irradiated by laser. The ELISA result indicates that the secretion of bFGF increased in the group of intermediate and high energetic level after laser irradiating and may reach the peak at 24 hours (P < 0.01). The amount of TGF-beta1 secretion, however, seems to get decreased in each group at all energetic levels, and at 24 hours it can reach the top level as well.

CONCLUSIONThe direct influence of laser on the fibroblasts is to promote secretion of bFGF and to inhibit secretion of TGF-beta1, while its influence on the tissue is to promote the secretions of the both. Nonablative skin rejuvenation not only can induce fibroblasts to secrete more bFGF but also induce the blood vessels to release cytokines which stimulate endothelial cell to express more of bFGF and TGF-beta1. Furthermore, fibroblastic proliferation can accelerate by laser's irradiating.

Animals ; Cells, Cultured ; Cosmetic Techniques ; Female ; Fibroblast Growth Factor 2 ; metabolism ; Fibroblasts ; cytology ; radiation effects ; secretion ; Laser Therapy ; Mice ; Mice, Inbred Strains ; Rejuvenation ; Skin ; cytology ; radiation effects ; Transforming Growth Factor beta1 ; metabolism

BACKGROUNDLower fluence of 585-nm flashlamp-pumped pulsed dye laser has been successfully used as a nonablative technique in the treatment of wrinkles. The objective of this study was to evaluate the effect of the pulsed dye laser (585 nm) on the production of collagen and the mRNA expression of collagen related gene in fibroblasts in vitro.

METHODSCultured fibroblasts were treated with a 585-nm flashlamp-pumped pulsed dye laser (fluence 3 J/cm(2), 4 J/cm(2), spot size 7 mm, pulse duration 450 micros). The production of collagen and the mRNA expression of transforming growth factor (TGF)-beta1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen alpha1, alpha2 in fibroblasts were investigated by colorimetry or real time polymerase chain reaction.

RESULTSThe production of collagen was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm(2) (P < 0.001). The mRNA expression of TGF-beta1, SMAD2, SMAD3, SMAD4, SMAD7 and procollagen I was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm(2) (P < 0.001). No significant difference of mRNA expression of SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen was found between controls and fibroblasts treated with pulsed dye laser with a fluence of 4 J/cm(2) (P > 0.05).

CONCLUSIONSLower fluence (3 J/cm(2)) pulsed dye laser increased the collagen production in fibroblasts by up-regulating TGF-beta1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen mRNA expression. These may be the reason it can be effectively used in the treatment of wrinkles.

Analysis of Variance ; Cells, Cultured ; Collagen ; biosynthesis ; Fibroblasts ; cytology ; metabolism ; radiation effects ; Gene Expression ; radiation effects ; Humans ; Lasers ; Procollagen ; genetics ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Smad2 Protein ; genetics ; Smad3 Protein ; genetics ; Smad4 Protein ; genetics ; Smad7 Protein ; genetics ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1

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 investigate the possible effect of exposure to GSM 1,800 MHz radiofrequency electromagnetic fields (RF EMF) on epidermal growth factor (EGF) receptor and its possible interference by noise magnetic fields (MF).

METHODSChinese hamster lung fibroblasts (CHL) were exposed to 1,800 MHz RF EMF (modulated by 217 Hz or 50 Hz, or unmodulated), 2 microT noise MF, and RF EMF combined with 2 microT noise MF for 15 min, respectively. The specific absorption rates (SARs) of RF EMF were 0.1, 0.5, 1.0, 2.0 and 4.0 W/kg. Commercial EGF (1 ng/ml) treatment was used as positive control. EGF receptors on the cell membrane were observed under a laser scanning confocal microscope after indirect immunofluorescence staining.

RESULTSEGF receptor clustering was induced after exposure to GSM 1,800 MHz RF EMF modulated by 217 Hz or 50 Hz MF at SARs of 0.5, 1.0, 2.0, 4.0 W/kg for 15 min as induced by 1 ng/ml EGF, but not at SAR of 0.1 W/kg. And no EGF receptor clustering was found in cells after exposure to unmodulated RF EMF or 2 microT noise MF. In addition, superposition of 2 microT noise MF could inhibit the EGF receptor clustering induced by GSM 1,800 MHz RF EMF.

CONCLUSIONEGF receptor clustering in CHL cells can be induced by GSM 1,800 MHz RF EMF at the lowest SAR of 0.5 W/kg and inhibited by noise MF. The modulation of wave may play an important role in the inducement of receptor clustering after RF exposure.

Animals ; Cell Line ; Cell Membrane ; metabolism ; radiation effects ; Cricetinae ; Cricetulus ; Dose-Response Relationship, Radiation ; Electromagnetic Fields ; Fibroblasts ; metabolism ; radiation effects ; Lung ; cytology ; Radio Waves ; Receptor, Epidermal Growth Factor ; metabolism