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

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

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 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

Ultraviolet light(UV)-sensitive disorders refer to a group of diseases due to damages to the nucleotide excision repair mechanism which cannot effectively repair DNA damage caused by ultraviolet radiation. The inheritance pattern of such diseases, mainly including xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, is autosomal recessive and known to involve 13 genes. As proteins encoded by such genes are involved in DNA repair and transcription pathways. There is overlap between the symptoms of such diseases, and their genotype - phenotype correlations are quite complex. To facilitate genetic and prenatal diagnosis for such diseases, a summary of the research progress is provided, which mainly focused on mutation research and genotype - phenotype correlation studies. We also propose a strategy for their genetic diagnosis based on recent findings of our group.
Biomedical Research ; methods ; trends ; Cockayne Syndrome ; genetics ; DNA Damage ; DNA Repair ; genetics ; Genetic Predisposition to Disease ; genetics ; Humans ; Skin ; metabolism ; pathology ; radiation effects ; Trichothiodystrophy Syndromes ; genetics ; Ultraviolet Rays ; Xeroderma Pigmentosum ; genetics

In the present study, the detrimental effect of beta-emission on pig skin was evaluated. Skin injury was modeled in mini-pigs by exposing the animals to 50 and 100 Gy of beta-emission delivered by 166Ho patches. Clinicopathological and immunohistochemical changes in exposed skin were monitored for 18 weeks after beta-irradiation. Radiation induced desquamation at 2~4 weeks and gradual repair of this damage was evident 6 weeks after irradiation. Changes in basal cell density and skin depth corresponded to clinically relevant changes. Skin thickness began to decrease 1 week after irradiation, and the skin was thinnest 4 weeks after irradiation. Skin thickness increased transiently during recovery from irradiation-induced skin injury, which was evident 6~8 weeks after irradiation. Epidermal expression of nuclear factor-kappa B (NF-kappaB) differed significantly between the untreated and irradiated areas. One week after irradiation, cyclooxygenase-2 (COX-2) expression was mostly limited to the basal cell layer and scattered among these cells. High levels of COX-2 expression were detected throughout the full depth of the skin 4 weeks after irradiation. These findings suggest that NF-kappaB and COX-2 play roles in epidermal cell regeneration following beta-irradiation of mini-pig skin.
Animals ; Cyclooxygenase 2/genetics/*metabolism ; *Holmium ; Male ; NF-kappa B/genetics/*metabolism ; Radiation Injuries, Experimental/metabolism/*veterinary ; Skin/metabolism/*radiation effects ; Swine ; Swine, Miniature

Constructing a bone marrow chimera prior to graft transplantation can induce donor-specific immune tolerance. Mixed chimerism containing hematopoietic cells of both recipient- and donor-origin has advantages attributed from low dose of total body irradiation. In this study, we explored the mechanism of mixed chimerism supplemented with depletion of Natural Killer cells. Mixed chimerism with C57BL/6 bone marrow cells was induced in recipient BALB/c mice which were given 450 cGy of gamma-ray irradiation (n = 16). As revealed by reduced proliferation and cytokine production in mixed leukocyte reaction and ELISpot assay (24.6 vs 265.5), the allo-immune response to bone marrow donor was reduced. Furthermore, the induction of transferable immunological tolerance was confirmed by adoptive transfer and subsequent acceptance of C57BL/6 skin graft (n = 4). CD4+FoxP3+ regulatory T cells were increased in the recipient compartment of the mixed chimera (19.2% --> 33.8%). This suggests that regulatory T cells may be therapeutically used for the induction of graft-specific tolerance by mixed chimerism.
Animals ; Bone Marrow Cells/cytology ; *Bone Marrow Transplantation ; Cell Proliferation ; Chimerism ; Cytokines/metabolism ; Gamma Rays ; Graft Survival ; *Immune Tolerance ; Killer Cells, Natural/immunology/radiation effects ; Leukocytes/immunology/radiation effects ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Models, Animal ; Skin Transplantation ; T-Lymphocytes, Regulatory/cytology/*immunology/metabolism ; Whole-Body Irradiation

OBJECTIVETo study the effect of laminarin polysaccharide (LP) on the activity of matrix metalloproteinase of photoaging skins.

METHODKunming SPF mice were prepared with back hair shaved, and randomly divided into the control group, the model group, the LP low does group (LP-L, 1 mg x kg(-1)), the LP high dose group (LP-H, 5 mg x kg(-1)) and the Vit E (100 mg x kg(-1)) group. They were abdominally injected with drugs twice on a daily basis. Except for the control group, all groups were exposed to ultraviolet rays for 1 hour every day, five times on a weekly basis, with accumulated exposure dose of UVB being 21.60 J x cm(-2) and accumulated exposure dose of UVA being 84.02 J x cm(-2). Eight weeks later, exposed back skins were collected to detect thickness of dermis by HE stain, content of hydroxyproline (Hyp) by chemical colorimetry, and serum MMP-1 and TIMP-1 content by ELISA. In addition, matrix metalloproteinase-1 (MMP-1) mRNA and relative content of tissue inhibitor of metalloproteinase-1 (TIMP1) mRNA was analyzed with Real-time PCR.

RESULTCompared with the model group, the LP-H group could significantly increase the thickness of dermis, skin Hyp content and serum TIMP-1 level, and decrease relative content of MMP-1 mRNA in skin and MMP-1 content in serum.

CONCLUSIONLP can regulate the metabolism of collagen photoaging skins by adjusting the activity of matrix metalloproteinase.

Animals ; Female ; Glucans ; Matrix Metalloproteinase 13 ; biosynthesis ; genetics ; metabolism ; Mice ; Plant Extracts ; chemistry ; pharmacology ; Plants, Medicinal ; chemistry ; Polysaccharides ; chemistry ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Skin Aging ; drug effects ; physiology ; radiation effects ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; metabolism ; Ultraviolet Rays

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) is an enzymatic product of prostaglandin H2 (PGH2) derived from cyclooxygenase (COX)-mediated arachidonic acid metabolism. Despite the high level of 12-HHT present in tissues and bodily fluids, its precise function remains largely unknown. In this study, we found that 12-HHT treatment in HaCaT cells remarkably down-regulated the ultraviolet B (UVB) irradiation-induced synthesis of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cutaneous inflammation. In an approach to identify the down-stream signaling mechanism by which 12-HHT down-regulates UVB-induced IL-6 synthesis in keratinocytes, we observed that 12-HHT inhibits the UVB-stimulated activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB). In addition, we found that 12-HHT markedly up-regulates MAPK phosphatase-1 (MKP-1), a critical negative regulator of p38 MAPK. When MKP-1 was suppressed by siRNA knock-down, the 12-HHT-mediated inhibitory effects on the UVB-stimulated activation of p38 MAPK and NF-kappaB, as well as the production of IL-6, were attenuated in HaCaT cells. Taken together, our results suggest that 12-HHT exerts anti-inflammatory effect via up-regulation of MKP-1, which negatively regulates p38 MAPK and NF-kappaB, thus attenuating IL-6 production in UVB-irradiated HaCaT cells. Considering the critical role of IL-6 in cutaneous inflammation, our findings provide the basis for the application of 12-HHT as a potential anti-inflammatory therapeutic agent in UV-induced skin diseases.
Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Cell Line ; Dual Specificity Phosphatase 1/biosynthesis/genetics ; Enzyme Activation ; Fatty Acids, Unsaturated/*pharmacology ; Humans ; Interleukin-6/*biosynthesis ; Keratinocytes/*metabolism/radiation effects ; NF-kappa B/metabolism ; RNA Interference ; RNA, Small Interfering ; Receptors, Leukotriene B4/genetics ; Signal Transduction/drug effects ; Skin Diseases/drug therapy ; *Ultraviolet Rays ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/metabolism

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.
Adult ; Agrin/genetics ; Antigens, CD44/genetics ; Base Sequence ; DNA Primers/genetics ; Gene Expression/radiation effects ; Glucuronidase/genetics ; Heparan Sulfate Proteoglycans/genetics/*metabolism ; Heparitin Sulfate/metabolism ; Humans ; Male ; Matrix Metalloproteinase 1/genetics ; N-Acetylglucosaminyltransferases/genetics ; RNA, Messenger/genetics/metabolism ; Skin/*metabolism/*radiation effects ; Skin Aging/genetics/physiology ; Syndecan-1/genetics ; Syndecan-4/genetics ; Ultraviolet Rays/*adverse effects ; Young Adult