Irradiated Human Skin Fibroblasts Research Articles

Exosomal MiR-769-5p Exacerbates Ultraviolet-Induced Bystander Effect by Targeting TGFBR1.

Exosomal microRNAs have been investigated in bystander effect, but it is unclear whether microRNA works in ultraviolet radiation–induced bystander effects (UV-RIBEs) and what the underlying mechanism could be. Exosomes from ultraviolet (UV)–irradiated human skin fibroblasts (HSFs) were isolated and transferred to normal HSFs, followed by the detection of proliferation rate, oxidative damage level, and apoptosis rate. Exosomal miRNAs were evaluated and screened with miRNA sequencing and quantitative reverse transcriptase–polymerase chain reaction method. MiRNA shuttle and bystander photodamage reactions were observed after transfection of miR-769-5p. MiR-769-5p targeting gene transforming growth factor-β1 (TGFBR1), and TGFBR1 mRNA 3′-untranslated region (UTR) was assessed and identified by Western blotting and dual-luciferase reporter assay. Bystander effects were induced after being treated with isolated exosomes from UV-irradiated HSFs. Exosomal miR-769-5p expression was significantly upregulated. Human skin fibroblasts showed lower proliferation, increasing oxidative damage, and faster occurrence of apoptosis after transfection. Exosome-mediated transfer of miR-769-5p was observed. Upregulation of miR-769-5p induced bystander effects, whereas downregulation of miR-769-5p can suppress UV-RIBEs. In addition, miR-769-5p was found to downregulate TGFBR1 gene expression by directly targeting its 3′-UTR. Our results demonstrate that exosome-mediated miR-769-5p transfer could function as an intercellular messenger and exacerbate UV-RIBEs. MiR-769-5p inhibits the expression of TGFBR1 by targeting TGFBR1 mRNA 3′-UTR.

Photochemoprotective effects of Spirulina platensis extract against UVB irradiated human skin fibroblasts

Ultraviolet B (UVB) irradiation is an important cause of a number of harmful side effects such as sunburn, photoaging, pigmentation and skin cancer. Photoprotection, which is used to prevent UVB interaction and to attenuate UVB related damage could be used to prevent the effects of UVB irradiation. Recently, the use of natural products as skin photoprotective agents has been in increasing demand. In this study, we are proposing a panel of in vitro methods for the evaluation of Spirulina platensis extract for its ability to absorb the UVB spectra by determination of its spectrophotometric Solar Protection Factor (SPF), and by attenuating its biological protective effects against UVB-irradiated human skin fibroblast cultures. Our data revealed that S. platensis extract contained high amounts of phenolic compounds (23,465.71 ± 167.72 mg GAE/kg extract) and produced a high degree of absorbance within a wavelength range of 290–320 nm yielding an SPF value of 30.39 ± 3.37 (3 mg/ml), all of which collectively confirmed the photoprotective activity of S. platensis polyphenols. S. platensis extract has no cytotoxicity on UVB irradiation of human skin fibroblasts, peripheral blood mononuclear cells and red blood cells. It was also found to exert antioxidant activities both in vitro and by way of intracellular reactive oxygen species in UVB irradiation fibroblast cells. The induction of interleukin- 6 and -8 secretions by UVB irradiation was dramatically aborted after S. platensis extract treatment. Moreover, the S. platensis extract significantly inhibited tyrosinase activity. Our above results indicated the photochemoprotective effects of S. platensis extract against UVB irradiation in human skin fibroblast cells. Therefore, S. platensis extract could be considered a prospective source of UVB-absorbers with antioxidant capacity. It could also serve as an additive in photoprotective cosmetic formulations which could prevent the harmful effects of UVB radiation.

744 Exosome-mediated miR-4655-3p contributes to UV-radiation induced bystander effects by targeting E2F2

Radiation induced bystander effects(RIBE) refer to the reactions appearing in nonirradiated cells which triggered by the intercellular communication between directly irradiated cells and non-radiated cells. Ultraviolet radiation induced bystander effects(UV-RIBE) are a part of RIBE short of researches. miRNAs are a kind of non-coding RNAs which participate in biological activity by act on target mRNAs. Recent researches imply that exosome-mediated miRNAs play a role in RIBE. HSFs were exposed to 20J/cm2 single UVA radiation or 60mJ/cm2 single UVB radiation. The exosomes were isolated from the culture medium of human skin fibroblasts(HSFs) by differential centrifugation. Three biology targets relevant to photo damage were used to measure the UV-RIBE, including cell proliferation, oxidative damage and apoptosis rate. The miR-4655-3p binding sites of E2F2 mRNA 3’UTR were forecasted by TargetScan and verified by dual luciferase reporter assay. After UV radiation, the expression of miR-4655-3p were significantly higher in irradiated cells and exosomes secreted from irradiated cells, comparing with control group (p<0.05). Exosomes from UV-irradiated HSFs induced UV-RIBE, including decreased cell proliferation, increased oxidative damage and increased apoptosis rate of bystander cells. Up- or down-regulating the expression of miR-4655-3p in exosomes respectively enhanced or weakened UV-RIBE to some extent. All the differences were statistically significant (p<0.05). miR-4655-3p down-regulated E2F2 mRNA expression by directly targeting its 3’UTR. Our outcomes prove that UV radiation could increase the miR-4655-3p expression in irradiated HSFs and their exosomes. Meanwhile, exosomal miR-4655-3p plays a main role in UV-RIBE by directly inhibiting the expression of E2F2 mRNA.

Glycyrrhizic acid (GA) inhibits reactive oxygen Species mediated photodamage by blocking ER stress and MAPK pathway in UV-B irradiated human skin fibroblasts

BackgroundPreviously we have reported that generation of reactive oxygen species is the prime event responsible for calcium mediated activation of PERK-eIF2α-CHOP pathway and apoptosis in UV-B irradiated human skin fibroblasts (Hs68). We have also reported that glycyrrhizic acid (GA) mediates potent photoprotective activity against UV-B – irradiation-induced photodamage in human skin fibroblast. ObjectiveIn the present study, we aimed to investigate the role of GA in preventing oxidative stress mediated unfolded protein response (UPR) and mitogen activated protein kinases (MAPK) pathway. MethodsHuman skin fibroblast (Hs68) cells were exposed to UV-B radiations in lab conditions. Different parameters of UVB induced cellular and molecular changes were analysed using western-blotting, microscopy and flow cytometry. ResultsOur results show that GA has strong photoprotective action against UV-B induced cellular damage. It was observed that: (a) Oxidative disturbances and intracellular Ca2+ imbalance induced by UV-B irradiation was significantly restored by GA treatment; (b) activation of PERK-eIF2α-CHOP and MAPK pathway induced by UV-B was significantly blocked by GA; (c) Loss of mitochondrial membrane potential and apoptosis induced by UV-B were reduced by GA treatment. ConclusionBased on the above findings we conclude GA has a highly significant ROS quenching activity, thereby blocking the cascade of events including release of calcium from ER and subsequent ER stress, MAPK pathway and cellular demise. GA offers highly potent anti photodamage effect and can be exploited for cosmetic or therapeutic purposes.

Effects of unripe apple polyphenols on the expression of matrix metalloproteinase-1 and type-1 procollagen in ultraviolet irradiated human skin fibroblasts

Bioassay-guided isolation of an aqueous ethanolic extract from the unripe fruits of Malus pumila cv. Fuji using matrix metalloproteinase-1 (MMP-1) inhibition and type-1 procollagen inducing assays led to isolation of eight phenolic compounds, reynoutrin (1), quercitrin (2), isoquercitrin (3), phloridzin (4), phloretin-2′-O-xyloglucoside (5), 5-O-p-courmaroylquinic acid (6), chlorogenic acid methyl ester (7), and chlorogenic acid (8). The structures were established on the basis of nuclear magnetic resonance and mass spectroscopic data interpretations. All isolates were evaluated for their inhibitory effects on MMP-1, and compounds 1, 2, and 6 exhibited potent inhibitory effects on MMP-1 with IC50 values of 1.3±0.1, 1.4±0.1, and 3.2±0.6 μM, respectively. When evaluated for their effects on type I procollagen synthesis on human fibroblast cells, compounds 1 and 3 were respectively found to display potent activity for induction of type-1 procollagen by 78.5 and 78.6% at the tested concentration of 12.5 M.

The Photoprotective and Antioxidative Properties of Luteolin are Synergistically Augmented by Tocopherol and Ubiquinone

Ultraviolet radiation induces DNA damage and oxidative stress which can result in skin inflammation, photoaging, and photocarcinogenesis. The flavonoid luteolin that is present in high amounts in the dyers weld, Reseda luteola, is one of the most potent antioxidative plant metabolites and also has ultraviolet-absorbing properties.The aim of this study was to determine whether tocopherol and ubiquinone add synergistic antioxidative values to luteolin. None of the substances showed cytotoxic effects in concentrations from 0.25 to 4 µg/mL. The photoprotective and antioxidant effect of equivalent concentrations of luteolin, tocopherol, and ubiquinone and their combination in a ratio of 4 : 4 : 1 were studied in solar simulator irradiated human skin fibroblasts. Luteolin had a half-maximal radical scavenging concentration of 2 µg/mL, whereas tocopherol and ubiquinone were only effective at higher concentrations. None of the substances showed a phototoxic effect, and only luteolin had a moderate photoprotective effect at 2 µg/mL. The combination of luteolin, tocopherol, and ubiquinone exerted a synergistic radical scavenging effect already at a concentration of 0.25 µg/mL and a complete photoprotection at 2 µg/mL.In summary, our findings suggest that the potent antioxidant and photoprotective effect of flavonoids like luteolin may be further increased by the addition of low concentrations of other antioxidants such as tocopherol and ubiquinone.

Anti‐ageing effects of alpha‐naphthoflavone on normal and UVB‐irradiated human skin fibroblasts

Ageing is a complex and multifactorial process resulting in several functional and aesthetic changes to the skin. We found that α-Naphthoflavone (α-NF) concentration-dependently induced pro-collagen type I protein expression and inhibited MMP-1 protein expression, in both normal and UVB-irradiated cells. SB431542 and SIS3 - inhibitors of TGF-β and Smad3, respectively - significantly alleviate α-NF-caused response of MMP-1 and pro-collagen. LY294002 (PI3K inhibitor) can reverse α-NF-induced ERK, Akt, Smad-3 activation, pro-collagen synthesis and α-NF-suppressed AP-1 activation. PD (ERK inhibitor) was not involved in pro-collagen generation and MMP-1 inhibition. We concluded that α-NF promotes pro-collagen production and inhibits MMP-1 expression via the activation of a PI3K/Akt/Smad-3 pathway in normal and UVB-irradiated human skin fibroblasts, while TGF-β may play an important role in transducing this pathway. These results suggest that α-NF, a natural plant product, has the potential to become a novel anti-ageing skin application.

Radiation-induced bystander signaling pathways in human fibroblasts: A role for interleukin-33 in the signal transmission

The main goal of this study is to elucidate the mechanisms of the signal transmission for radiation-induced bystander response. The NF-κB-dependent gene expression of IL8, IL6, PTGS2/COX2, TNF and IL33 in directly irradiated human skin fibroblasts produced the cytokines and prostaglandin E2 (PGE2) with autocrine/paracrine functions, which further activated signaling pathways and induced NF-κB-dependent gene expression in bystander cells. As a result, bystander cells also started expression and production of interleukin-8, interleukin-6, COX-2-generated PGE2 and interleukin-33 (IL-33) followed by autocrine/paracrine stimulation of the NF-κB and MAPK pathways. A blockage of IL-33 transmitting functions with anti-IL-33 monoclonal antibody added into the culture media decreased NF-κB activation in directly irradiated and bystander cells. On the other hand, the IGF-1-Receptor kinase regulated the PI3K–AKT pathway in both directly irradiated and bystander fibroblasts. A pronounced and prolonged increase in AKT activity after irradiation was a characteristic feature of bystander cells. AKT positively regulated IL-33 protein expression levels. Suppression of the IGF-R1–AKT–IL-33 pathway substantially increased radiation-induced or TRAIL-induced apoptosis in fibroblasts. Taken together, our results demonstrated the early activation of NF-κB-dependent gene expression first in directly irradiated and then bystander fibroblasts, the further modulation of critical proteins, including IL-33, by AKT in bystander cells and late drastic changes in cell survival and in enhanced sensitivity to TRAIL-induced apoptosis after suppression of the IGF-1R–AKT–IL-33 signaling cascade in both directly irradiated and bystander cells.

Extract of Punica granatum inhibits skin photoaging induced by UVB irradiation

Punica granatum (pomegranate) is kind of a fruit consumed fresh or in beverage. It has been widely used in traditional medicine in various parts of the world. In this study, we examined the efficacy of a Punica granatum (PG) extract in protecting skin against UVB-induced damage using cultured human skin fibroblasts. A Korean red PG sample was used, and its effects classified according to if the PG source originated from the rind, seed and fruit. The polyphenol content of PG, which is known to prevent other adverse cutaneous effects of UV irradiation, was measured by GC-MS. The protective effects of PG on UVB-induced skin photoaging were examined by determining the level of procollagen type I and MMP-1 after UVB irradiation. Based on the GC-MS quantitative analysis, catechin, quercetin, kaempferol, and equol were the predominant compounds detected in PG. In the changes of expression of procollagen type I and MMP-1 in UV irradiated human skin fibroblasts treated PG, especially extract prepared from rind, the synthesis of collagen was increased and the expression of MMP-1 was decreased. The major polyphenols in PG, particularly catechin, play a significant role in its photoprotective effects on UVB-induced skin damage.

Ultraviolet B exposure triggers premature senescence in human skin fibroblasts.

Objective To develop a model of ultraviolet B (UVB)-induced premature senescence in human skin fibroblasts (HSF) so as to assess the relationship between stress-induced premature senescence and tumorigenesis. Methods The irradiation dose and frequency were optimized for the induction of prema-ture senescence. HSF were irradiated with UVB of 10 mJ/cm~2 once daily for 5 days, and unirradiated HSFs served as the control. After the last irradiation, cell proliferation was determined by MTT assay on day 3, 4, 5,6 and 7, SA β-Gal staining was performed to evaluate the senescence state of cells on day 3, and RT-PCR to detect the expressions of three senescene-associated genes, including fibronectin (FN), osteonectin (ON) and smooth muscle 22 (SM22) on day 3. Results After five exposures to UVB, HSF showed biological characteris-tics of senescence. As assessed by MTT assay, there was a loss of replicative potential in irradiated cells. The proportion of SA-β-gal-positive cells was 82.0% in UVB-stressed HSFs and 33.7% in the control cells (P < 0.01). The mRNA levels of FN, ON and SM22 were upregulated by 2.7, 2.0 and 2.3 folds respectively in irra-diated HSF compared with the control cells (all P < 0.05). Conclusion A stress-induced premature senes-cence model is established using HSF by repeated exposure to subcytotoxic UVB. Key words: Ultraviolet; Fibroblasts; Cell aging

Linear discriminant analysis-based estimation of the false discovery rate for phosphopeptide identifications.

The development of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has made it possible to characterize phosphopeptides in an increasingly large-scale and high-throughput fashion. However, extracting confident phosphopeptide identifications from the resulting large data sets in a similar high-throughput fashion remains difficult, as does rigorously estimating the false discovery rate (FDR) of a set of phosphopeptide identifications. This article describes a data analysis pipeline designed to address these issues. The first step is to reanalyze phosphopeptide identifications that contain ambiguous assignments for the incorporated phosphate(s) to determine the most likely arrangement of the phosphate(s). The next step is to employ an expectation maximization algorithm to estimate the joint distribution of the peptide scores. A linear discriminant analysis is then performed to determine how to optimally combine peptide scores (in this case, from SEQUEST) into a discriminant score that possesses the maximum discriminating power. Based on this discriminant score, the p- and q-values for each phosphopeptide identification are calculated, and the phosphopeptide identification FDR is then estimated. This data analysis approach was applied to data from a study of irradiated human skin fibroblasts to provide a robust estimate of FDR for phosphopeptides. The Phosphopeptide FDR Estimator software is freely available for download at http://ncrr.pnl.gov/software/.

How Long After Laser Irradiation Should Cellular Responses be Measured to Determine the Laser Effect?

Background: Low level laser therapy (LLLT) is a type of photomodulation, which uses photons to modulate biological activity. This photobiological effect depends on the wavelength, dose, intensity, as well as cell culture conditions. One problem with most of the published data on low level laser irradiation is that most studies do not record the duration between the exposure and the evaluation. It has been established that a single dose of 5 J/cm2 stimulates cell proliferation and mitochondrial activity to normalize cell function and accelerate wound healing. This study investigates the cellular responses of irradiated human skin fibroblasts to establish if the duration after laser irradiation could influence the results. Methods: Normal and wounded human skin fibroblasts were exposed to helium-neon (632.8 nm) laser irradiation using a single dose of 5 J/cm2 on one day or 5 J/cm2 on two consecutive days. The cellular responses were measured 15 min, 1, or 3 h postirradiation. Responses evaluated included the effect on cell migration, changes in mitochondrial activity (cell viability), cell proliferation, and damage or stress caused by the irradiation. Results: The results indicate that a duration of between 1 and 3 h postirradiation is sufficient when measuring the direct effect of laser radiation on cells (cell viability, ATP mitochondrial activity, IL-6 activity, membrane integrity, and DNA damage), however, a longer duration of 24 h is required to demonstrate the indirect effect (cell proliferation and protein expression). Conclusion: This study indicates that the duration of effect should be included as one of the main parameters when reporting on the parameters used in LLLT.

Dermatite séborrhéique

Previously we have reported that generation of reactive oxygen species is the prime event responsible for calcium mediated activation of PERK-eIF2α-CHOP pathway and apoptosis in UV-B irradiated human skin fibroblasts (Hs68). We have also reported that glycyrrhizic acid (GA) mediates potent photoprotective activity against UV-B – irradiation-induced photodamage in human skin fibroblast.In the present study, we aimed to investigate the role of GA in preventing oxidative stress mediated unfolded protein response (UPR) and mitogen activated protein kinases (MAPK) pathway.Human skin fibroblast (Hs68) cells were exposed to UV-B radiations in lab conditions. Different parameters of UVB induced cellular and molecular changes were analysed using western-blotting, microscopy and flow cytometry.Our results show that GA has strong photoprotective action against UV-B induced cellular damage. It was observed that: (a) Oxidative disturbances and intracellular Ca2+ imbalance induced by UV-B irradiation was significantly restored by GA treatment; (b) activation of PERK-eIF2α-CHOP and MAPK pathway induced by UV-B was significantly blocked by GA; (c) Loss of mitochondrial membrane potential and apoptosis induced by UV-B were reduced by GA treatment.Based on the above findings we conclude GA has a highly significant ROS quenching activity, thereby blocking the cascade of events including release of calcium from ER and subsequent ER stress, MAPK pathway and cellular demise. GA offers highly potent anti photodamage effect and can be exploited for cosmetic or therapeutic purposes.

Differences in the Yields of Dicentrics and Reciprocal Translocations Observed in the Chromosomes of Irradiated Human Skin Fibroblasts and Blood Lymphocytes from the Same Healthy Individuals

Plateau-phase human dermal fibroblasts and whole blood G0 lymphocytes obtained from the same three healthy donors were irradiated with different doses of 200 kV X rays. The genomic yields of dicentrics were evaluated in the first postirradiation mitosis. Fluorescence in situ hybridization was used to "paint" the chosen whole chromosomes. The yields of reciprocal translocations were scored for all donors for chromosome 4, and also for the third donor for chromosomes 7 and 9. The amounts of reciprocal translocations were scored in the first as well as in later (4th or 5th) postirradiation cell divisions. The yields of dicentrics involving the painted chromosomes were scored for one donor as well. The yields of dicentrics observed in the lymphocytes and in the skin fibroblasts from the same individual were significantly different. Yields of dicentrics were much lower in fibroblasts than in lymphocytes. This was observed for all doses up to 4 Gy for all three donors studied. Fibroblasts from two individuals were also irradiated with 6 Gy. The interindividual variability was similar in both cell types. The yields of reciprocal translocations in lymphocytes and in fibroblasts were similar. The yields of reciprocal translocations were practically constant when comparing the first mitotic divisions and later divisions. The observed ratio of translocations:dicentrics was higher in fibroblasts than in lymphocytes, where it was about 1 for one donor and about 2 for the other two donors. Significant differences between the yields of dicentrics and translocations as well as between the shapes of their dose responses were observed in lymphocytes and fibroblasts from the same individual. Our experimental data suggest that repair processes with different efficiencies or different repair processes might be active in cells of different types of tissue.

Effect of Dose Rate on the Survival of Irradiated Human Skin Fibroblasts

The survival of cells in density-inhibited, confluent cultures maintained at 37 degrees C was examined following exposure to 137Cs gamma rays at low dose rates (0.023 or 0.153 Gy/h) or to 60Co gamma rays at a single high dose rate (0.70-0.75 Gy/min). Cells from an ataxia telangiectasia (AT) homozygote showed no dose-rate effect, whereas a three- to fivefold increase in D0 was observed for all other cell strains exposed at low dose rates. The magnitude of the dose-rate effect did not differ significantly among cells from persons with hereditary retinoblastoma, basal cell nevus syndrome, or AT-heterozygote compared with normal cell strains, and was not related to the size of the shoulder (extrapolation number) of the survival curve. Furthermore, no differences in the capacity for the repair of potentially lethal damage during confluent holding were observed among these latter cell strains.

COMPARATIVE STUDIES ON THE CORRELATION BETWEEN PYRIMIDINE DIMER FORMATION and TYROSINASE ACTIVITY IN CLOUDMAN S91 MELANOMA CELLS AFTER ULTRAVIOLET‐IRRADIATION

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.

Environmental Wavelengths of Ultraviolet Light Induce Cytoskeletal Damage

The ultraviolet component of sunlight is the major cause of skin cancer and is responsible for accelerating the aging of human skin. It is therefore important to determine the mechanisms by which ultraviolet light alters normal cellular functions. The potential importance of ultraviolet light-induced damage to non-DNA targets has received little attention. Since the cytoskeleton is an important participant in the control of normal cell growth, the microfilaments and microtubules of UV irradiated human skin fibroblasts have been studied using fluorescence microscopy. Polychromatic ultraviolet light, composed of environmentally relevant wavelengths, was found to disrupt the cytoplasmic microtubule complex in a dose dependent manner. The induction of microtubule disassembly did not correlate with the cytotoxicity of ultraviolet light of varying composition.