UVB-induced Premature Senescence Research Articles

Melatonin inhibits senescence-associated melanin pigmentation through the p53-TYR pathway in human primary melanocytes and the skin of C57BL/6 J mice after UVB irradiation

UVB exposure accelerates skin aging and pigmentation. Melatonin effectively regulates tyrosinase (TYR) activity and aging. The purpose of this study was to determine the association between premature senescence and pigmentation, and the mechanism of melanin synthesis effected by melatonin. Primary melanocytes were extracted and identified from the male foreskin. To inhibit TYR expression, primary melanocytes were transduced with the lentivirus pLKD-CMV-EGFP-2A-Puro-U6-TYR. The wild-type TYR(+/+) and TYR(–/–) or TYR(+/–) knockout C57BL/6 J mice were used to determine the role of TYR on melanin synthesis in vivo. Results showed that UVB-induced melanin synthesis is dependent on TYR in primary melanocytes and mice. Furthermore, in primary melanocytes pretreated with Nutlin-3 or PFT-α to up or downregulate p53, results showed that premature senescence and melanin synthesis increased in primary melanocytes after UVB irradiation at 80 mJ/cm2, and further increased after being treated with Nutlin-3, while significantly decreased with PFT-α. In addition, melatonin inhibited UVB-induced premature senescence associated with inactivation of p53 and phosphorylation of p53 on Ser15 (ser-15), a decrease of melanin synthesis accompanied by reduced TYR expression. Moreover, skin erythema and pigmentation induced by UVB were reduced in the dorsal and ear skin of mice topically pretreated with 2.5% melatonin. These indicate that melatonin inhibits UVB-induced senescence-associated pigmentation via the p53-TYR pathway in primary melanocytes and prevents pigmentation obviously in the dorsal and ear skin of C57BL/6 J mice after UVB irradiation.Key messagesP53 links UVB irradiation-induced senescence and senescence-associated pigmentation and regulates TYR in primary melanocytes after UVB irradiation.Melatonin inhibits senescence-associated pigmentation through the p53-TYR pathway in primary melanocytes. Melatonin prevents skin erythema and melanin pigmentation induced by UVB irradiation in the dorsal and ear skin of C57BL/6J mice.

Senescence Induced by UVB in Keratinocytes Impairs Amino Acids Balance

Skin is one of the most exposed organs to external stress. Namely, UV rays are the most harmful stress that could induce important damage leading to skin aging and cancers. At the cellular level, senescence is observed in several skin cell types and contributes to skin aging. However, the origin of skin senescent cells is still unclear but is probably related to exposure to stresses. In this work, we developed an invitro model of UVB-induced premature senescence in normal human epidermal keratinocytes. UVB-induced senescent keratinocytes display a common senescent phenotype resulting in an irreversible cell cycle arrest, an increase in the proportion of senescence-associated β-galactosidase‒positive cells, unrepaired DNA damage, and a long-term DNA damage response activation. Moreover, UVB-induced senescent keratinocytes secrete senescence-associated secretory phenotype factors that influence cutaneous squamous cell carcinoma cell migration. Finally, a global transcriptomic study highlighted that senescent keratinocytes present a decrease in the expression of several amino acid transporters, which is associated with reduced intracellular levels of glycine, alanine, and leucine. Interestingly, the chemical inhibition of the glycine transporter SLC6A9/Glyt1 triggers senescence features.

Stilbene glucoside inhibits ultraviolet radiation B-induced photoaging in human skin fibroblasts

To study the effects of tetrahydroxy stilbene-2-O-β-D-glucoside (TSG) on stress-induced premature senescence of human skin fibroblasts (HSF) exposed to ultraviolet radiation B (UVB) and its possible mechanism. HSFs were repeatedly exposed to UVB at a subcytotoxic level. TSG treatment (0.02, 0.10 and 0.50 mmol/L) was given immediately after each irradiation. The HSFs were divided into six groups:blank control group, model group, UVB+0.02 mmol/L TSG group, UVB+0.10 mmol/L TSG group, UVB+0.50 mmol/L TSG and TSG group (0.50 mmol/L). Cell counting kit-8 (CCK-8) was used to evaluate the proliferative activity of cells; senescence-associated-β-galactosidase (SA-β-gal) staining was performed to estimate the degree of premature senescence in cells; TBA method and WST-1 method were used to detect intracellular malondialdehyde (MDA) and superoxide dismutase (SOD) activities; and ELISA was applied to quantify the secretion level of matrix metalloproteinase1 (MMP-1) in cultured supernatant. Compared with the blank control group, the proliferative activity and SOD level in the model group decreased (P<0.05), while the percentage of SA-β-gal-positive cells, MDA and MMP-1 levels increased (P<0.05 or P<0.01). Compared with the model group, the proliferative activity and SOD level increased in UVB+TSG groups (all P<0.05), and the percentage of SA-β-gal-positive cells, MDA and MMP-1 levels decreased (P<0.05 or P<0.01). TSG can inhibit UVB-induced premature senescence of HSF, which may be related to the inhibition of oxidative stress and high expression of MMP-1.

Astragaloside exerts anti-photoaging effects in UVB-induced premature senescence of rat dermal fibroblasts through enhanced autophagy

BackgroundAstragalus membranaceus is a fundamental herb in Traditional Chinese Medicine and has attracted significant attention due to its anti-inflammatory, and longevity effects. However, its anti-photoaging property remains to be defined. Autophagy plays important roles in regulating cell homeostasis and aging processes. Whether regulation of autophagy could be an efficient way for anti-photoaging is still unclear. ObjectiveTo investigate the effects and the possible mechanism of astragaloside on anti-photoaging in UVB-induced photoaging cell model. MethodsPrimary rat dermal fibroblasts were prepared by repeated exposures to UVB irradiation. The expression levels of cytokines and signal molecules were determined by RT-PCR and western blot. SA-β-gal staining was performed to indicate senescence level. Intracellular reactive oxygen species and mitochondrial membrane potential were monitored by fluorescent probes DCFH-DA and JC-1. The cell viability was determined using Cell Counting Kit-8. ResultsAstragaloside increases the expression of collagen-I (Col1) downregulated by UVB. UVB-induced oxidative stress and photoaging could be inhibited by astragaloside. The degradation of Col1 caused by UVB irradiation through activated ERK and p38 signals could be suppressed by astragaloside. Importantly, autophagy was induced by astragaloside. Col1 could be further accumulated by chloroquine but decreased by 3-methyladenine in photoaged cell after treatment of astragaloside. ConclusionAutophagy play essential roles, at least partially, in modulating the formation and degradation of Col1 in photoaging cell model. Astragaloside increases the accumulation of Col1 and protects UVB-induced photoaging cells through not only ERK and p38 inhibition but also autophagy activation, indicating the potential application of astragaloside for anti-photoaging therapy.

1115 Long noncoding RNA associated-competing endogenous RNAs in UVB-induced premature senescence of human dermal fibroblasts

1115 Long noncoding RNA associated-competing endogenous RNAs in UVB-induced premature senescence of human dermal fibroblasts

Adiponectin Suppresses UVB-Induced Premature Senescence and hBD2 Overexpression in Human Keratinocytes.

Recent studies have revealed that adiponectin can suppress cellular inflammatory signaling pathways. This study aimed to elucidate the effect of adiponectin on the unregulated production of hBD2 in UVB-induced premature senescent keratinocytes. We constructed an in vitro model of premature senescent keratinocytes through repeated exposure to low energy UVB. After repeated low energy UVB exposure, there was significant generation of reactive oxygen species (ROS) and induction of senescence-associated markers, including senescence associated beta-galactosidase activity and expression of p16INK4a and histone H2AX. In addition, the present clinical study showed higher expression of hBD2 in sun-exposed skin of elderly group, and the overexpression of hBD2 was observed by c-Fos activation in vitro. Adiponectin has the ability to scavenge ROS and consequently inhibit MAPKs and SA-markers in UVB-exposed keratinocytes. An inhibitor study demonstrated that adiponectin downregulated hBD2 mRNA expression through suppression of the AP-1 transcription factor components c-Fos via inactivation of p38 MAPK. Collectively, the dysregulated production of hBD2 by the induction of oxidative stress was attenuated by adiponectin through the suppression of p38 and JNK/SAPK MAPK signaling in UVB-mediated premature senescent inducible conditions. These results suggest the feasibility of adiponectin as an anti-photoaging and anti-inflammatory agent in the skin.

MiR-23a-depressed autophagy is a participant in PUVA- and UVB-induced premature senescence.

Autophagy is a cellular catabolic mechanism that is activated in response to stress conditions, including ultraviolet (UV) irradiation, starvation, and misfolded protein accumulation. Abnormalities in autophagy are associated with several pathologies, including aging and cancer. Furthermore, recent studies have demonstrated that microRNAs (miRNAs) are potent modulators of the autophagy pathway. As a result, the current study aims to elucidate the role of the autophagy-related miRNA miR-23ain the process of photoaging. Experiments demonstrated that the antagomir-mediated inactivation of miR-23a resulted in the stimulation of PUVA- and UVB-depressed autophagy flux and protected human fibroblasts from premature senescence. Furthermore, AMBRA1 was identified as a miR-23a target. AMBRA1 cellular levels increased following the introduction of miR-23a antagomirs. And a bioinformatics analysis revealed that the AMBRA1 3′ UTR contains functional miR-23a responsive sequences. Finally, it was also demonstrated that both AMBRA1 overexpression and Rapamycin treatment were both able to rescue fibroblasts from PUVA and UVB irradiation-induced autophagy inhibition, but that these effects could also be mitigated by miR-23a overexpression. Therefore, this study concludes that miR-23a-regulated autophagy is a novel and important regulator of ultraviolet-induced premature senescence and AMBRA1 is a rate-limiting miRNA target in this pathway.

ALA-PDT elicits oxidative damage and apoptosis in UVB-induced premature senescence of human skin fibroblasts

Background5-Aminolevulinic acid photodynamic therapy (ALA-PDT) has been used for the treatment of skin photoaging. It can significantly improve the appearance of fine lines, dotted pigmentation, and roughness of photoaged skin. However, the mechanisms by which ALA-PDT yields rejuvenating effects on photoaged skin have not been well elucidated. Thus, in this study we explored the effects of ALA-PDT in photoaged fibroblasts. MethodsWe established a stress-induced premature senescence (SIPS) model by repeated exposures of human dermal fibroblasts (HDFs) to ultraviolet B (UVB) irradiation. Cells were irradiated by red light laser at 635nm wavelength (50mW/cm2). Intracellular protoporphyrin IX (PpIX) was detected by confocal microscopy. Intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) change were detected by fluorescence microscopy and flow cytometry. Morphological changes were observed by optical microscopy. Proliferative activity was measured by a cell counting kit-8 (CCK-8). Cell apoptosis was detected by fluorescence microscopy using Hoechst staining and flow cytometry using annexin V/propidium Iodide double staining. ResultsIntracellular PpIX fluorescence in UVB-induced premature senescent HDFs (UVB-SIPS-HDFs) reached the highest intensity after incubation with 1.00mmol/L ALA for 6h (P<0.05). Compared with control group, intracellular ROS level, MMP, and apoptotic rate were increased (P<0.05) and proliferative activity was decreased (P<0.05) in UVB-SIPS-HDFs treated with ALA-PDT, which were positively correlated to ALA incubation time and red light laser dose. ConclusionOur study demonstrated that ALA-PDT elicits oxidative damage and apoptosis in photoaged fibroblasts in vitro, which may be the basis for the rejuvenating effects on photoaged skin.

Salidroside protects against premature senescence induced by ultraviolet B irradiation in human dermal fibroblasts.

Salidroside, the predominant component of a Chinese herbal medicine, Rhodiola rosea L., becomes an attractive bio-agent due to its multifunction. Although it is well proposed that this herbal medicine may have photoprotective effect according to the folk hearsay, the direct supportive experimental evidences linking the drug with skin ageing have rarely been reported so far. The study was conducted to investigate the photoprotective role of salidrosdie and its related mechanisms in vitro. First, a premature senescence model induced by UVB irradiation (250 mJ cm(-2)) in human dermal fibroblasts (HDFs) was established, and senescent phenotypes were evaluated by cell morphology, cell proliferation, senescence-associated beta-galactosidase (SA-β-gal) activity and cell cycle distribution. Then the photoprotective effect of salidroside was investigated. Cells were pre-treated with various doses of salidroside (1, 5 and 10 μM) followed by the sublethal dosage of UVB exposure and then were harvested for various detections, including senescence-associated phenotypes and molecules, alteration of oxidative stress, matrix metalloproteinase-1 (MMP-1) secretion and inflammatory response. Pre-treatment of salidroside dose dependently reversed the senescent state of HDFs induced by UVB as evidenced by elevated cell viability, decreased SA-β-gal activity and relieving of G1/G0 cell cycle arrest. UVB-induced increased protein expression of cyclin-dependent kinase (CDK) inhibitors p21(WAF) (1) and p16(INK) (4) was also repressed by salidrosdie treatment in a dose-dependent manner. Meanwhile, the increment of malondialdehyde (MDA) level in UVB-irradiated HDFs was inhibited upon salidroside treatment. Additionally, salidroside significantly attenuated UVB-induced synthesis of MMP-1 as well as the production of IL-6 and TNF-α in HDFs. Our data provided the evidences for the protective role of salidroside against UVB-induced premature senescence in HDFs probably via its anti-oxidative property and inhibition on production of MMP-1 and pro-inflammatory cytokines, which indicated its potential utilization as an active ingredient in the preparation of photoprotective formulation.

Molecular Insights into SIRT1 Protection Against UVB-Induced Skin Fibroblast Senescence by Suppression of Oxidative Stress and p53 Acetylation.

Stresses, such as exposure to ultraviolet radiation and those associated with aging, are known to cause premature cellular senescence that is characterized by growth arrest and morphological and gene expression changes. This study was designed to investigate the protective effect of Sirtuin1 (SIRT1) on the UVB-induced premature senescence. Under in vitro experimental conditions, exposure to a subcytotoxic dose of UVB enhanced human skin fibroblasts senescence, as characterized by increased β-galactosidase activity and increased levels of senescence-associated proteins. However, adenovirus-mediated SIRT1 overexpression significantly protected fibroblasts from UVB-induced cellular deterioration. Exposure to UVB-induced cell senescence was associated with oxidative stress and p38 mitogen-activated protein kinase activation. Molecular analysis demonstrated that deacetylation of Forkhead box O3α (FOXO3α) by SIRT1 changed the transcriptional activity of FOXO3α and increased resistance to the oxidative stress. In addition, SIRT1 suppressed UVB-induced p53 acetylation and its transcriptional activity, which directly affected the cell cycle arrest induced by UVB. Further study demonstrated that SIRT1 activation inhibited cell senescence in the skin of the HR1 hairless mouse exposed to UVB. The study identifies a new role for SIRT1 in the UVB-induced senescence of skin fibroblats and provides a potential target for skin protection through molecuar insights into the mechanisms responsible for UVB-induced photoaging.

Comments on “Anti-photoaging potential of Botulinum Toxin Type A in UVB-induced premature senescence of human dermal fibroblasts in vitro through decreasing senescence-related proteins”

Comments on “Anti-photoaging potential of Botulinum Toxin Type A in UVB-induced premature senescence of human dermal fibroblasts in vitro through decreasing senescence-related proteins”

Response to Comments on “Anti-photoaging potential of Botulinum Toxin Type A in UVB-induced premature senescence of human dermal fibroblasts in vitro through decreasing senescence-related proteins”

Response to Comments on “Anti-photoaging potential of Botulinum Toxin Type A in UVB-induced premature senescence of human dermal fibroblasts in vitro through decreasing senescence-related proteins”

Influence of RNA interference in p53 gene on the expressions of genes involved in ultraviolet B-induced premature senescence and photocarcinogenesis in human skin fibroblasts

Objective To evaluate the effect of RNA interference in p53 gene on the expressions of genes involved in ultraviolet B (UVB)-induced premature senescence and photocarcinogenesis in human skin fibroblasts (HSFs).Methods A previously established HSF cell clone with repressed expression of p53,which was named as HSF-p53,was cultured and irradiated with a subcytotoxic dose (10 mJ/cm2) of UVB once a day for five consecutive days.The HSFs with normal expression of p53 served as the control.Subsequently,β-galactosidase (SA-β-gal)-staining was performed to estimate the degree of senescence,quantitative real-time PCR array was performed to determine the mRNA expressions of photocarcinogenesis-and senescence-associated genes,including p53,p21,p19,p16,pRb,fibronectin,osteonectin,smooth muscle 22 (SM22),bax,bcl-2,hypoxia-inducible factor-1 α (HIF-1α),vascular endothelial growth factor(VEGF),and human double minute-2 (hdm2).Statistical analysis was carried out by Student's t test using the software SPSS 10.0.Results The percentage of SA-β-gal-positive cells in irradiated HSF-p53 was 19.70％ ± 0.85％,significantly higher than that in unirradiated HSF-p53 (12.77％ ± 0.81％,t =6.45,P ＜ 0.05),but lower than that in irradiated control HSFs (50.48％ ± 5.30％,t =7.86,P ＜ 0.05),and similar to that in unirradiated control HSFs (18.50％ ± 0.45％,t =2.57,P ＞ 0.05).Compared with the control HSFs,the HSF-p53 showed decreased expressions of p21,p19,fibronectin,osteonectin,SM22 and bax genes (all P ＜ 0.05),but increased expressions of bcl-2,HIF-1α,VEGF and hdm2 genes (all P ＜ 0.05),and a similar expression of p16 gene (P ＞ 0.05); the repeated UVB radiation significantly promoted the expressions of p16 and pRb genes (both P ＜ 0.05),but had no obvious effect on the expressions of the other genes in HSF-p53 compared with unirradiated HSF-p53 (all P ＞ 0.05).Conclusions The inhibition of p53 expression may decelerate the UVB-induced premature senescence in HSFs,which may be involved in the p53-dependent tumor suppression. Key words: Genes, p53; RNA interference; Aging, premature; Ultraviolet rays; Fibroblasts

Mangiferin inhibits ultraviolet B-induced premature senescence in human fibroblasts: an experiment study

Objective To clarify whether mangiferin inhibits stress-induced premature senescence (SIPS) in human diploid fibroblasts (HDFs) induced by repeated exposure to a sub-cytotoxic dose of ultraviolet B (UVB),and to investigate the mechanism underlying the effect of UVB.Methods HDFs were isolated from the circumcised foreskin of healthy males,and subjected to a primary culture and 6-12 passages of subculture.Then,the HDFs were divided into six groups,i.e.blank control group receiving no treatment,UVB-SIPS group receiving UVB irradiation only,three combination groups receiving UVB irradiation and post-irradiation treatment with mangiferin at 1.0,2.0 and 4.0 mg/L respectively,and mangiferin group treated with mangiferin 4.0 mg/L only.Irradiation was performed once a day for five consecutive days,and mangiferin treatment was given immediately after each irradiation.Cell counting kit-8 (CCK-8) was used to evaluate the proliferative activity of cells,and β-galactosidase (SA-β-Gal) staining to estimate the degree of premature senescence in cells,flow cytometry to detect cell cycle,Western blot to quantify the protein expressions of senescence-related proteins p53,p21 and p16,real time-PCR to determine the mRNA expression levels of matrix metalloproteinase (MMP1),MMP3,collagen types Ⅰ and Ⅲ,p53,p21 and p16,at 72 hours after the last irradiation.One-way analysis of variance was used for statistical analysis.Results The proliferative activity (expressed as the absorbance value at 450 nm) of HDFs was 0.322 9 ± 0.011 3,0.336 1 ± 0.016 3,0.342 6 ± 0.014 4 and 0.288 2 ± 0.020 7 respectively in the UVB + mangiferin 1.0 mg/L group,UVB + mangiferin 2.0 mg/L group,UVB + mangiferin 4.0 mg/L group,and UVB-SIPS group respectively (F =110.08,P ＜ 0.05),with the percentage of SA-β-Gal-positive cells being (88.83 ± 4.54)％,(46.33 ± 5.51)％,(32.17 ± 6.05)％ and (93.67 ± 3.75)％ respectively (F =283.54,P ＜ 0.05),and the proportion of cells in the G1 phase being (72.19 ± 3.42)％,(60.99 ± 2.70)％,(49.80 ± 2.10)％ and (82.09 ± 0.89)％ respectively (F =156.01,P ＜ 0.05).Compared with the UVB-SIPS group,the three combination groups showed significantly decreased expression levels of MMP1 and MMP3 mRNAs (F =69.41,106.41,respectively,both P ＜ 0.05) as well as p53,p21 and p16 mRNAs (F =265.60,151.82,329.85,respectively,all P ＜ 0.05) and proteins (F =160.51,158.53,75.38,respectively,all P ＜ 0.05),but increased expression levels of COL1a1 and COL3a1 mRNAs (F =66.41,46.81,respectively,both P ＜ 0.05).In these combination groups,the changes in the above parameters were dependent on the concentration of mangiferin to a degree.Conclusion Mangiferin may inhibit UVB-induced premature senescence in HDFs via downregulating the expressions of p53,p21 and p16 genes. Key words: Ultraviolet rays; Fibroblasts; Mangiferin; Cell aging

Anti-photoaging potential of Botulinum Toxin Type A in UVB-induced premature senescence of human dermal fibroblasts in vitro through decreasing senescence-related proteins

This study was aimed to evaluate the anti-photoaging effects of Botulinum Toxin Type A (BoNTA) in Ultraviolet B-induced premature senescence (UVB-SIPS) of human dermal fibroblasts (HDFs) in vitro and the underlying mechanism. We established a stress-induced premature senescence model by repeated subcytotoxic exposures to Ultraviolet B (UVB) irradiation. The aging condition was determined by cytochemical staining of senescence-associated β-galactosidase (SA-β-gal). The tumor suppressor and senescence-associated protein levels of p16INK-4a, p21WAF-1, and p53 were estimated by Western blotting. The G1 phase cell growth arrest was analyzed by flow cytometry. The mRNA expressions of p16, p21, p53, COL1a1, COL3a1, MMP1, and MMP3 were determined by real-time PCR. The level of Col-1, Col-3, MMP-1, and MMP-3 were determined by ELISA. Compared with the UVB-irradiated group, we found that the irradiated fibroblasts additionally treated with BoNTA demonstrated a decrease in the expression of SA-β-gal, a decrease in the level of tumor suppressor and senescence-associated proteins, a decrease in the G1 phase cell proportion, an increase in the production of Col-1 and Col-3, and a decrease in the secretion of MMP-1 and MMP-3, in a dose-dependent manner. Taken together, these results indicate that BoNTA significantly antagonizes premature senescence induced by UVB in HDFs in vitro, therefore potential of intradermal BoNTA injection as anti-photoaging treatment still remains a question.

Effects of sirolimus on ultraviolet B irradiation-induced premature senescence of skin fibroblasts

Objective To observe the effect of sirolimus,an autophagy enhancer,on premature senescence in fibroblasts induced by repeated exposure to a subtoxic dose of ultraviolet B (UVB).Methods Skin fibroblasts from foreskin tissue of healthy adolescents were classified into six groups:control group cultured in Dulbecco's modified Eagles' medium (DMEM) containing 1％ calf serum,UVB group receiving UVB irradiation only,sirolimus group treated with sirolimus of 10 mg/L (added after daily exchange of culture medium),and three combined groups receiving UVB irradiation immediately followed by overnight treatment with sirolimus of 0.1,1.0 and 10.0 mg/L respectively.UVB irradiation was given at a dose of 10 mJ/cm2 once a day for five successive days.After five days of treatment,cell counting kit-8 (CCK-8) was used to evaluate cell viability,β-galactosidase staining to detect senescent ceils,Western blot to quantify the expressions of p53,LC3-B and beclin 1 in these fibroblasts.Autophagy level was determined by acridine orange staining followed by fluorescence microscopy and transmission electron microscopy.Data were processed by the SPSS 16.0 software,and statistical analysis was done by one-way analysis of variance,t test and least significance difference.Results Sirolimus significantly increased the proliferative activity of fibroblasts in a dose-dependent manner,with the absorbance value at 450 nm being 0.27 ± 0.02,0.36 ± 0.04 and 0.39 ± 0.04 for fibroblasts irradiated with UVB followed by treatment with sirolimus of 0.1,1.0 and 10 mg/L respectively,compared to 0.26 + 0.01 for fibroblasts irradiated with UVB only (all P ＜ 0.05).Significant differences were also observed between the fibroblasts irradiated with UVB followed by treatment with sirolimus of 0.1,1.0 and 10 mg/L and those irradiated with UVB only in the percentage of β-galactosidasepositive fibroblasts (92.50％ ± 0.34％,42.40％ ± 0.53％ and 6.20％ ± 0.39％ vs.95.10％ ± 0.32％,all P ＜ 0.05)and intracellular intensity of acridine orange-induced fluorescence (36.43 ± 0.24,45.25 ± 0.33 and 48.69 ± 0.37 vs.33.99 ± 0.32,all P ＜ 0.05).Moreover,the expressions of p53,LC3-B and beclin 1 in the three combined groups differed significantly from those in the UVB group (all P ＜ 0.05).Conclusion Sirolimus can inhibit UVBinduced premature senescence likely via upregulation of autophagy in fibroblasts. Key words: Fibroblasts; Cell aging; Ultraviolet rays; Sirolimus; Autophagy

Elevated miR-34c-5p Mediates Dermal Fibroblast Senescence by Ultraviolet Irradiation

Previous studies showed that several miRNAs can regulate pathways involved in UVB-induced premature senescence and response to ultraviolet irradiation. It has also been reported that miR-34c-5p may be involved in senescence-related mechanisms. We propose that miR-34c-5p may play a crucial role in senescence of normal human primary dermal fibroblasts. Here, we explored the roles of miR-34c-5p in UVB-induced premature senescence on dermal fibroblasts. MiR-34c-5p expression was increased in dermal fibroblasts after repeated subcytotoxic UVB treatments. Underexpression of miR-34c-5p in dermal fibroblasts led to a marked delay of many senescent phenotypes induced by repeated UVB treatments. Furthermore, underexpression of miR-34c-5p in dermal fibroblasts can antagonize the alteration of G1-arrested fibroblasts. Moreover, E2F3, which can inactivate p53 pathway and play a role in cell cycle progression, is a down-stream target of miR-34c-5p. Forced down-expression of miR-34c-5p decreased the expression of UVB-SIPS induced P21 and P53 at both mRNA and protein levels. Our data demonstrated that down-regulation of miR-34c-5p can protect human primary dermal fibroblasts from UVB-induced premature senescence via regulations of some senescence-related molecules.

Ligand-activated PPARδ inhibits UVB-induced senescence of human keratinocytes via PTEN-mediated inhibition of superoxide production

UV radiation-mediated photodamage to the skin has been implicated in premature aging and photoaging-related skin cancer and melanoma. Little is known about the cellular events that underlie premature senescence, or how to impede these events. In the present study we demonstrate that PPARδ (peroxisome-proliferator-activated receptor δ) regulates UVB-induced premature senescence of normal keratinocytes. Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly attenuated UVB-mediated generation of ROS (reactive oxygen species) and suppressed senescence of human keratinocytes. Ligand-activated PPARδ up-regulated the expression of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and suppressed the PI3K (phosphatidylinositol 3-kinase)/Akt pathway. Concomitantly, translocation of Rac1 to the plasma membrane, which leads to the activation of NADPH oxidases and generation of ROS, was significantly attenuated. siRNA (small interfering RNA)-mediated knockdown of PTEN abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt/Rac1 signalling and on generation of ROS in keratinocytes exposed to UVB. Finally, when HR-1 hairless mice were treated with GW501516 before exposure to UVB, the number of senescent cells in the skin was significantly reduced. Thus ligand-activated PPARδ confers resistance to UVB-induced cellular senescence by up-regulating PTEN and thereby modulating PI3K/Akt/Rac1 signalling to reduce ROS generation in keratinocytes.

Effects of ginsenoside and Lycium barbarum polysaccharide on UVB irradiation-induced premature senescence of skin fibroblasts.

Objective To observe the expressions of senescence-associated biomarkers in fibroblasts after repeated exposures to subtoxic doses of ultraviolet B (UVB), and to study the effect of ginsenoside Rb1 and Rg1 as well as Lycium barbarum polysaccharide on the UVB-induced premature senescence and on the expressions of senescence-associated signals including pl6, p21 and pS3. Methods Skin fibroblasts were classified into 8 groups to receive pretreatment with traditional Chinese medicine (TCM) monomers only, UVB irradiation only, no treatment, or both pretreatment and irradiation. UVB was given successively at a dose of 15 mJ/cm~2 for 10 times, and the concentration of three monomers was 50 mg/L. After 5 days of treatment, light microscopy was used to observe the morphology of fibroblasts, transmission electron microscopy to study the cell ultrastructure,β-galactosidase histochemical staining to detect senescent cells, flow cytometry to analyze cell cycle, and RT-PCR to measure the mRNA expressions of p16, p21 and p53 in these skin fibroblasts. Results None of the 3 monomers had any effect on cell morphology, P-galactosidase activity, cell cycle or the mRNA expression of p53, p21 and pl6 in skin fibroblasts. After UVB irradiation, some changes occurred to cell morphology and ultrastructure; 91.5% of the cells were stained positively for P-galactosidase. The proportion of cells in G1 phase was 88.63% ± 4.67% in irradiated fibroblasts, significantly different from that in untreated controls (49.18% ± 5.53%, P< 0.05) and that in irradiated fibroblasts pretreated with ginsenoside Rbl and Rgl as well as Lycium barbarum polysaccharide (71.04% ± 1.64%, 70.38% ± 2.58%, 80.09% ± 3.46%, all P < 0.05). Compared with untreated fibroblasts, the mRNA expression of p53, p21 and pl6 significantly increased in irradiated fibroblasts (P < 0.05), however, the induced increase in the mRNA expression of pl6 was inhibited by all the three monomers (all P< 0.05), that of p2l by ginsenoside Rb1 and Rg1 (P< 0.05), and that of p53 by ginsenoside Rbl and Lycium barbarum polysaccharide (both P < 0.05). Conclusions Ginsenoside Rbl, Rgl and lycium barbarum polysaccharide can inhibit UVB-induced premature senescence, which may be associated with the down-regulation of mRNA expressions of pl6, p21 and p53. Key words: Fibroblasts; Cell aging; Ultraviolet rays; GINSENOSIDE; Lycium barbarum polysaccharide

Identification of potential anti-photoageing algal compounds using an in-vitro model of photoageing

Stress-induced premature senescence (SIPS) has been proposed as an in-vitro model for testing the long-term effects of stressful events and to find molecules/natural extracts that protect against such stress. Premature senescence of human skin diploid fibroblasts (HDFs) can be induced by repeated subcytotoxic exposure to UVB, with the appearance of so-called biomarkers of senescence such as growth arrest, senescence-associated beta-galactosidase activity, senescence-associated gene over-expression and the common 4977-bp mitochondrial DNA deletion. This model of UVB-induced premature senescence has been acknowledged as a robust in-vitro model in photoageing research. In this study, the potential anti-photoageing effects of a series of algal extracts were tested. The appearance of the biomarkers of UVB-induced premature senescence of HDFs was studied with or without algal extracts. One algal extract was shown to be particularly protective against UVB-induced SIPS. The results obtained here reinforce the notion that UVB-induced premature senescence of HDFs can be used to screen potential anti-photoageing compounds.