UVA-irradiated Human Dermal Fibroblasts Research Articles

Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts

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.

Licochalcone A activates Nrf2 in vitro and contributes to licorice extract-induced lowered cutaneous oxidative stress in vivo.

The retrochalcone licochalcone A (LicA) has previously been shown to possess antimicrobial and anti-inflammatory properties. In this study, we focused on pathways responsible for the antioxidative properties of LicA. In vitro, LicA protected from oxidative stress mediated by reactive oxygen species (ROS) by activating the expression of cytoprotective phase II enzymes. LicA induced nuclear translocation of NF-E2-related factor 2 (Nrf2) in primary human fibroblasts and elevated the expression of the cytoprotective and anti-inflammatory enzymes heme oxygenase 1 and glutamate-cysteine ligase modifier subunit. LicA-treated cells displayed a higher ratio of reduced to oxidized glutathione and decreased concentrations of ROS in UVA-irradiated human dermal fibroblasts, as well as in activated neutrophils. In vivo, ultraweak photon emission analysis of skin treated with LicA-rich licorice extract revealed a significantly lowered UVA-induced luminescence, indicative for a decrease in oxidative processes. We conclude from these data that topical application of licorice extract is a promising approach to induce Nrf2-dependent cytoprotection in human skin.

The chromene sargachromanol E inhibits ultraviolet A-induced ageing of skin in human dermal fibroblasts.

Skin ageing is influenced by environmental factors such as ultraviolet (UV) radiation. The effects of UV radiation on skin functions should be investigated using human in vitro models to understand the mechanisms of skin ageing. Additionally, marine algae provide a valuable source for identifying and extracting biologically active substances. In this study, sargachromanol E was isolated from a marine brown alga, Sargassum horneri, and its inhibitory effect on skin ageing was investigated using UVA-irradiated dermal fibroblasts. Formation of intracellular reactive oxygen species (ROS), lipid peroxidation and protein oxidation induced by UVA irradiation were investigated in UVA-irradiated human dermal fibroblasts. The levels of matrix metalloproteinases (MMPs) were determined by reverse-transcriptase polymerase chain reaction and Western blot analysis. Sargachromanol E did not exhibit any significant cytotoxicity or phototoxicity in UVA-exposed dermal fibroblasts. Additionally, sargachromanol E suppressed intracellular formation of ROS, membrane protein oxidation, lipid peroxidation and expression of collagenases such as MMP-1, MMP-2 and MMP-9, all of which are caused by UVA exposure. It was further found that these inhibitions were related to an increase in the expression of the tissue inhibitor of metalloproteinase (TIMP) genes, TIMP1 and TIMP2. Moreover, we have shown that the transcriptional activation of activator protein 1 (AP-1) signalling caused by UVA irradiation was inhibited by treatment with sargachromanol E. This study suggests that UVA irradiation modulates MMP expression via the transcriptional activation of AP-1 signalling, whereas treatment with sargachromanol E protected cell damage caused by UVA irradiation.

Protective Effects of Fermented Citrus Unshiu Peel Extract against Ultraviolet‐A‐induced Photoageing in Human Dermal Fibrobolasts

The aqueous extracts of Citrus unshiu peel containing flavonoid glycosides was used as co-substrate with Schizophyllum commune mycelia producing β-glucosidase and its biological activities were studied. β-glucosidase-produced S. commune mycelia converted the glycosides (narirutin and hesperidin) into aglycones (naringenin and hesperetin). The photoprotective potential of fermented C. unshiu peel extract with S. commune (S-CPE) was tested in human dermal fibroblasts (HDFs) exposed to UVA. It was revealed that S-CPE had an inhibitory effect on human interstitial collagenase (matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDFs. The treatment of UVA-irradiated HDFs with S-CPE resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA. The UVA irradiation raised the proportion of senescence-associated β-galactosidase (SA-β-gal) positive cells in comparison with the normal control group. The treatment of UVA-irradiated HDFs with S-CPE was shown to decrease the level of SA-β-gal (by approximately 45% at an S-CPE concentration 0.1%, w/v) compared with the UVA-irradiated HDFs. It was found that S-CPE containing hesperetin has notable collagen biosynthetic activity for fibroblasts, indicating that S-CPE can be promising cosmetic ingredients.

MiR-155 Negatively Regulates c-Jun Expression at the Post-transcriptional Level in Human Dermal Fibroblasts in vitro: Implications in UVA Irradiation-induced Photoaging

Objective: C-Jun plays a critical role in ultraviolet A (UVA) irradiation-induced photoaging. The exact mechanisms by which UVA irradiation up-regulates c-Jun expression in human dermal fibroblasts (HDFs) are still not completely understood. We undertook this study to investigate whether microRNA-155 (miR-155) directly regulates the expression of c-Jun in HDFs in vitro. Methods: Expression of c-Jun mRNA and protein and miR-155 in UVA-irradiated HDFs were detected using quantitative real-time RT-PCR and Western blotting. Luciferase reporter assays were performed to examine whether a miR-155 binding site in the 3′-untranslated region (3′-UTR) of the c-Jun gene is responsible for miR-155-mediated c-Jun regulation in HEK293A cells, and expression of c-Jun mRNA and protein in UVA non-exposed and exposed HDFs trasfected with a miR-155 mimic or a miR-155 inhibitor was detected by quantitative real-time RT-PCR and Western blotting. Results: Expression of miR-155 was markedly reduced and that of c-Jun mRNA and protein was significantly up-regulated in UVA-irradiated HDFs. Luciferase reporter assays indicated that c-Jun is a direct target of miR-155 in HEK293A cells. In both UVA non-exposed and exposed HDFs, miR-155 mimic decreased c-Jun protein levels, while miR-155 inhibitor increased c-Jun protein levels, but both had no effect on c-Jun mRNA expression, which suggest that miR-155-induced c-Jun inhibition occurs at the post-transcriptional level. Conclusions: Our results demonstrate that miR-155 directly controls c-Jun expression in HDFs at the post-transcriptional level and might function as a protective miRNA in HDFs.

Matrix Metalloproteinase-1 Inhibitory Activities of Morinda citrifolia Seed Extract and Its Constituents in UVA-Irradiated Human Dermal Fibroblasts

The objective of this study was to examine whether a 50% ethanolic extract (MCS-ext) of the seeds of Morinda citrifolia (noni) and its constituents have matrix metalloproteinase-1 (MMP-1) inhibitory activity in UVA-irradiated normal human dermal fibroblasts (NHDFs). The MCS-ext (10 μg/mL) inhibited MMP-1 secretion from UVA-irradiated NHDFs, without cytotoxic effects, at 48 h after UV exposure. The ethyl acetate-soluble fraction of MCS-ext was the most potent inhibitor of MMP-1 secretion. Among the constituents of the fraction, a lignan, 3,3'-bisdemethylpinoresinol (1), inhibited the MMP-1 secretion at a concentration of 0.3 μM without cytotoxic effects. Furthermore, 1 (0.3 μM) reduced the level of intracellular MMP-1 expression. Other constituents, namely americanin A (2), quercetin (3) and ursolic acid (4), were inactive. To elucidate inhibition mechanisms of MMP-1 expression and secretion, the effect of 1 on mitogen-activated protein kinases (MAPKs) phosphorylation was examined. Western blot analysis revealed that 1 (0.3 μM) reduced the phosphorylations of p38 and c-Jun-N-terminal kinase (JNK). These results suggested that 1 suppresses intracellular MMP-1 expression, and consequent secretion from UVA-irradiated NHDFs, by down-regulation of MAPKs phosphorylation.

Role of NF-κB–p53 crosstalk in ultraviolet A-induced cell death and G1 arrest in human dermal fibroblasts

Photoaging is the premature aging of the skin caused by repeated exposure to sunlight and is characterized by a depletion of the dermal extracellular matrix. This depletion is due to the loss of fibroblast cells and their multiple functions. UVA was revealed as a major inducer of photoaging in various clinical studies. As UVA photons have long wavelength spectra, UVA penetrates deeper into the dermis than UVB and UVC, leading to the induction of cell death, the destruction of the dermal extracellular matrix through the induction of matrix metalloproteinase expression, and the repression of collagen expression. However, the exact effects of UVA on the skin remain a matter of debate. Here, we assess cell cycle stage to demonstrate that NF-κB-p53 crosstalk induces apoptosis and growth arrest in UVA-irradiated human dermal fibroblasts. In addition, UVA irradiation led to an increase of NF-κB-HDAC1 complexes, which in turn repressed cyclin D1 expression in UVA-irradiated human dermal fibroblasts. We provide direct evidence that UVA irradiation induces changes in the p53-dependent NF-κB complex that lead to growth arrest and apoptosis through the repression of cyclin D1. These studies uncovered that NF-κB-p53 crosstalk is a key regulator of UVA-dependent growth arrest and apoptosis.

Biological activities of fructus arctii fermented with the basidiomycete Grifola frondosa

Fructus arctii extract containing phenolic glycosides was cultured with Grifola frondosa mycelia to produce β-glucosidase and its biological activities were studied. This β-glucosidase converted the glycosides (arctiin and caffeic acid derivatives) into aglycones (arctigenin and caffeic acid). Fermented Fructus arctii extract (G-FAE) with G. frondosa had antioxidant and 5-lipoxygenase inhibitory activities. The photoprotective potential of G-FAE was tested in human dermal fibroblasts (HDF) exposed to ultra-violet A (UVA). It was revealed that G-FAE had an inhibitory effect on human interstitial collagenase (matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDF. The treatment of UVA-irradiated HDF with G-FAE resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA. G-FAE also showed notable stimulation of collagen biosynthetic activity for fibroblasts. These diverse functionalities suggest that G-FAE could be a promising cosmetic ingredient.

대황 추출물의 항산화 활성 및 MMP-1 저해 활성

Rheum undulatum L. has been commonly used as a cure for hematemesis, dropsy, and haematuria in the Oriental medicine for a long time. The main constituents of R. undulatum are chrysophanol and emodin, which are an antioxidative substance that has an anthraquinone structure. In the present study, to develop a new anti-aging agent, we examined the antioxidant activity and the inhibitory effect of the R. undulatum extract on the synthesis of MMP-1 in UVA-irradiated human dermal fibroblasts and MMP-1 activity. The R. undulatum extract was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and superoxide radicals in the xanthine/ xanthine oxidase system by a dose-dependent manner, respectively. UVA-induced MMP-1 expression was reduced about 79.5% by 1 /ml of the R. undulatum extract and also inhibited MMP-1 activity in a dose-dependent manner. In conclusion, it was observed that the R. undulatum extract has the antioxidant activity, regulation of UVA-induced MMP-1 production, and inhibition of MMP-1 activity. Therefore, these results suggest that the R. undulatum extract can be developed as a new anti-aging component of cosmetics.

Anti-oxidative and photo-protective effects of coumarins isolated fromFraxinus chinensis

Free radicals and reactive oxygen species (ROS), which are generated by UV irradiation, may cause serious injury to skin cell membranes, DNA and functional proteins. In addition, these agents stimulate the expressions of matrix metalloproteinases (MMPs), which can degrade most components of the extracellular matrix (ECM), including collagen. In order to develop new anti-photoaging agents, five major components from the extract of Fraxinus chinensis extract (FCE) were identified. Two of the major components of FCE were found to be esculin (11.2%) and esculetin (1.9%). FCE (IC50: 50.0 microg/mL 1, 1-diphenyl-2-picrylhydrazyl (DPPH); 19.8 microg/mL, superoxide anion radical) and esculetin (IC50: 2.1 microg/mL DPPH; 0.6 microg/mL, superoxide anion radical) showed strong antioxidative activities. Of the compounds tested, esculetin showed the strongest scavenging activity against DPPH radicals, followed by superoxide anions from the xanthine/xanthine oxidase system. The intracellular ROS scavenging activity showed that oxidation of 5-(6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was effectively inhibited by esculetin, with potent free radical scavenging activity was also shown in UVB-irradiated human dermal fibroblasts (HDFs). Moreover, treatment of UVA-irradiated HDFs with esculetin resulted in dose-dependent decreases in the expression levels of MMP-1 mRNA and protein. From these results, FCE and one of its components, esculetin, were predicted to be potentially useful as ingredients in cosmetics for protecting against photoaging.

Antioxidative Activity of the Hydrolytic Enzyme Treated Sorbus commixta Hedl. and its Inhibitory Effect on Matrix Metallopro- teinase- 1 in UV Irradiated Human Dermal Fibroblasts

Superoxide radical scavenging activity and DPPH radical scavenging activity were assessed in order to evaluate the antioxidant effect of the Sorbus commixta Hedl. extract (SCoE). SCoE was also treated with several carbohydrate-hydrolytic enzymes that significantly increased the total phenol and flavonoid composition of SCoE. The enzymatically treated SCoE was then assessed for antioxidative activity. The most efficient radical scavenging activity was observed when SCoE was treated with -glucanase. The radical scavenging activity of beta-glucanase-treated SCoE (beta-GSCoE) enhanced the viability of human dermal fibroblasts (HDFs) exposed to ultraviolet (UV) light. The intracellular reactive oxygen species (ROS) scavenging activity of beta-GSCoE was assessed using UVB (20 mJ/cm2)-irradiated HDFs. UVB irradiation increased dichlorofluorescein (DCF) fluorescence, which was measured by a 5-(6-)chloromethyl-2',7'- dichlorodihydrofluorescein diacetate (CM-H2DCFDA). DCF-fluorescence was significantly decreased in the beta-GSCoE-containing culture medium, suggesting that beta-GSCoE scavenges free radicals. The protective effect was further verified by assessing the expression of matrix metalloproteinase-1 (MMP-1) in UVA-irradiated HDFs. The treatment of UVA-irradiated HDFs with beta-GSCoE resulted in a dose-dependent decrease in the expression level of MMP-1 protein and mRNA. These results suggest that beta-GSCoE may mitigate the effects of photoaging in skin by reducing UV-induced adverse skin reactions.

IL-1 receptor antagonist attenuates MAP kinase/AP-1 activation and MMP1 expression in UVA-irradiated human fibroblasts induced by culture medium from UVB-irradiated human skin keratinocytes

Solar UV light comprises UVB wavelengths (290-320 nm) and UVA wavelengths (320-400 nm). UVB radiation reaches the epidermis and, to a lesser extent, the upper part of the dermis, while UVA radiation penetrates more deeply into human skin. Existing studies have demonstrated that UV-irradiated epidermal keratinocytes release cytokines that indirectly promote MMP-1 production in dermal fibroblasts. In this study, we first investigated the effect of IL-1 on MAPK activity, c-Jun and c-Fos mRNA expression, and MMP-1 and MMP-2 production in UVA-irradiated human dermal fibroblasts. The results showed that UVA irradiation dose-dependently increased MMP-1 but not MMP-2 production in human skin fibroblasts. IL-1alpha and IL-1beta promoted MMP-1 but not MMP-2 production in UVA-irradiated fibroblasts. Both IL-1alpha and IL-1beta activated MAP kinase, significantly elevating c-Jun and c-Fos mRNA expression. We then investigated the indirect effect of UVB-irradiated keratinocyte culture medium on MMP-1 production in UVA-irradiated primary cultured human dermal fibroblasts and the effect of IL-1Ra. The results showed that cell culture medium from UVB-irradiated keratinocytes increased MMP-1 production in UVA-irradiated fibroblasts, and IL-1Ra dose-dependently inhibited MMP-1 production. IL-1Ra dose-dependently inhibited c-Jun mRNA expression of fibroblasts with no significant effect on c-Fos mRNA expression. These results demonstrate that UVB-irradiated keratinocytes promoted MMP-1 production in UVA-irradiated fibroblasts in a paracrine manner while IL-1Ra reduced MMP-1 production through inhibiting c-Jun mRNA expression. Collectively, our data suggest that IL-1 plays an important role in the dermal collagen degradation associated with UV-induced premature aging of the skin and IL-1Ra may be applied for the prevention and treatment of photoaging.

Production of exopolysaccharide from mycelial culture of Grifola frondosa and its inhibitory effect on matrix metalloproteinase-1 expression in UV-irradiated human dermal fibroblasts

Exopolysaccharide (EPS) was prepared by submerged mycelial culture of a newly isolated mushroom Grifola frondosa HB0071 in a 5-l stirred-tank fermenter. This fungus produced a high concentration of biomass (24.8 g l −1 at day 4), thereby achieving high EPS concentration (7.2 g l −1 at day 4). EPS was proven to be a proteoglycan consisting of 85.6% carbohydrates (mostly glucose) and 7.3% proteins with a molecular weight of 1.0 × 10 6 Da. The photoprotective potential of EPS was tested in human dermal fibroblasts (HDF) exposed to ultraviolet-A (UVA) light. It was revealed that EPS had an inhibitory effect on human interstitial collagenase (matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDF without any significant cytotoxicity. The treatment of UVA-irradiated HDF with EPS resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA (by maximum 61.1% at an EPS concentration 250 μg ml −1). These results suggest that EPS obtained from mycelial culture of G. frondosa HB0071 may contribute to inhibitory action in photoaging skin by reducing the MMP 1-related matrix degradation system.