Senescence Of Human Skin Fibroblasts Research Articles

Combined dasatinib and quercetin treatment contributes to skin rejuvenation through selective elimination of senescent cells in vitro and in vivo.

The skin's protective functions are compromised over time by both endogenous and exogenous aging. Senescence is well-documented in skin phenotypes, such as wrinkling and sagging, a consequence of the senescence-associated secretory phenotype (SASP) that involves the accumulation of senescent fibroblasts, chronic inflammation, and collagen remodeling. Although therapeutic approaches for eliminating senescent cells from the skin are available, their efficacy remains unclear. Accordingly, we aimed to examine the effects of dasatinib in combination with quercetin (D + Q) on senescent human skin fibroblasts and aging human skin. Senescence was induced in human dermal fibroblasts (HDFs) using approaches such as long-term passaging, ionizing radiation, and doxorubicin treatment. The generated senescent cells were treated with D + Q or vehicle. Additionally, a mouse-human chimera model was generated by subcutaneously transplanting whole-skin grafts of aged individuals onto nude mice. Mouse models were administered D + Q or vehicle by oral gavage for 30 days. Subsequently, skin samples were harvested and stained for senescence-associated beta-galactosidase. Senescence-associated markers were assessed by western blotting, reverse transcription-quantitative PCR and histological analyses. Herein, D + Q selectively eliminated senescent HDFs in all cellular models of induced senescence. Additionally, D + Q-treated aged human skin grafts exhibited increased collagen density and suppression of the SASP compared with control grafts. No adverse events were observed during the study period. Collectively, D + Q could ameliorate skin aging through selective elimination of senescent dermal fibroblasts and suppression of the SASP. Our findings suggest that D + Q could be developed as an effective therapeutic approach for combating skin aging.

A focused natural compound screen reveals senolytic and senostatic effects of Isatis tinctoria

ABSTRACT Natural products and their derivatives historically represent alternatives to conventional synthetic molecules for pharmacotherapy, ranging from cancer chemotherapeutics to cosmetic ingredients that exert anti-aging activities. Cellular senescence is considered a main driver of skin aging, yet natural products that target skin senescence in a specific manner are not thoroughly explored. Here, we performed a focused compound screen to identify natural products that exert anti-senescence effects. We found that Isatis tinctoria, woad extracts, displayed a senolytic effect on senescent human skin fibroblasts. Furthermore, treatment with woad extracts attenuated the expression of pro-inflammatory senescence-associated secretory phenotype (SASP), showing a senostatic activity. Intriguingly, woad extracts displayed only a marginal cytotoxic effect toward senescent human lung fibroblasts. Thus, our results reveal the potential activities of woad extracts for targeting skin senescence and suggest that woad extracts could be an attractive ingredient for cosmetics to prevent skin aging.

Cathepsin F is a potential marker for senescent human skin fibroblasts and keratinocytes associated with skin aging

Cellular senescence is characterized by cell cycle arrest and the senescence-associated secretory phenotype (SASP) and can be triggered by a variety of stimuli, including deoxyribonucleic acid (DNA) damage, oxidative stress, and telomere exhaustion. Cellular senescence is associated with skin aging, and identification of specific markers of senescent cells is essential for development of targeted therapies. Cathepsin F (CTSF) has been implicated in dermatitis and various cancers and participates in cell immortalization through its association with Bcl family proteins. It is a candidate therapeutic target to specifically label and eliminate human skin fibroblasts and keratinocytes immortalized by aging and achieve skin rejuvenation. In this study, we investigated whether CTSF is associated with senescence in human fibroblasts and keratinocytes. In senescence models, created using replicative aging, ionizing radiation exposure, and the anticancer drug doxorubicin, various senescence markers were observed, such as senescence-associated β-galactosidase (SA-β-gal) activity, increased SASP gene expression, and decreased uptake of the proliferation marker BrdU. Furthermore, CTSF expression was elevated at the gene and protein levels. In addition, CTSF-positive cells were abundant in aged human epidermis and in some parts of the dermis. In the population of senescent cells with arrested division, the number of CTSF-positive cells was significantly higher than that in the proliferating cell population. These results suggest that CTSF is a candidate for therapeutic modalities targeting aging fibroblasts and keratinocytes.

509 Ultrasmall prussian blue nanoparticles protect human skin fibroblasts from ultraviolet A stress induced premature senescence

Background: Skin aging is one of the major challenges our society faces, and the generation of reactive oxygen species(ROS)seems to play a major role in age-related skin modifications.Thus, ROS scavengers that can block excessive production of ROS have great therapeutic potential. Ultrasmall prussian blue nanoparticles(USPBNPs),which belong to the iron-based metal-organic frameworks, exhibited an intensive ability of scavenging free radicals as nanozyme and showed great application potential in the treatment of free radical-related diseases. Herein, we propose an efficient treatment strategy in which an artificial nanozyme with multienzyme activity drives photoprotection against ultraviolet A stress induced premature senescence(UVA-SIPS) primarily by scavenging ROS. Methods: In the present study, UVA-SIPS model was established by UVA radiation on human skin fibroblasts .Cell viability was determined using the Cell Counting Kit-8 (CCK-8). Senescent cells were detected by senescence β-galactosidase staining.Fluorescence microscopy and flow cytometry were used to assay the intracellular ROS concentration.Cell cycle was measured by flow cytometry.The expression of cellular γH2A.X,p16,p21 and p53 were also measured. Results: The results revealed that UVA radiation could cause premature senescence in human skin fibroblasts. Interestingly, the pretreatment of USPBNPs significantly reduced senescent cells and attenuated several features of cellular senescence, including morphological and metabolic changes,senescence related DNA damage, cell cycle arrest and more senescence-associated secretory phenotypes (SASPs). Conclusions: This study provides a proof of concept for a novel class of protective nanoagents that might be beneficial for prevention of skin photoaging and other age-related disorders.

Protective and Anti-Aging Effects of 5 Cosmeceutical Peptide Mixtures on Hydrogen Peroxide-Induced Premature Senescence in Human Skin Fibroblasts

Skin aging usually leads to the excessive deterioration of the dermal extracellular matrix, loss of antimicrobial function, loss of skin barrier function, and a series of inflammatory processes. Bioactive peptides have been widely used in cosmetics due to their protective effects on skin and efficient absorption. Combination of different peptides may lead to synergistic or antagonistic effects, so different formulas need to be designed and tested properly. In this study, 5 functional cosmeceutical peptides were tested on their individual and mixed activities to detect a suitable anti-aging and protective formula from our experiments. After the individual activity test, the optimal concentration is 200 μg/mL of carnosine for the superoxide dismutase (SOD) activity, 200 μg/mL of GHK peptide for the hydroxyproline (HYP) content activity, 100 μg/mL of acetyl tetrapeptide-5 for the angiotensin-converting enzyme 1 activity, 400 μg/mL of hexapeptide-11 for the HYP content activity, and 400 μg/mL of acetyl hexapeptide-3 for the catecholamine content activity. According to the optimal concentration of these 5 cosmeceutical peptides, 6 formulations of peptide mixtures were designed and tested for their anti-aging activities and protective effects against hydrogen peroxide-induced premature senescence in human skin fibroblasts. One of the cosmeceutical peptide mixtures (carnosine + acetyl tetrapeptide-5 + hexapeptide-11 + acetyl hexapeptide-3) significantly reduced the intracellular malondialdehyde and hydroxyl free radical contents and increased the HYP and human elastin contents as well as the enzymatic activities of SOD and glutathione peroxidase. Our study suggests that this formula of cosmeceutical peptide mixtures could be a promising agent for use in anti-aging and protective cosmetics.

Regulatory Mechanism of Rosemary Extract (Rosmarinus officinalis) on Human Skin Fibroblasts During Ultraviolet A Photoaging

In this study, human skin fibroblasts human dermis fibroblasts were used as the object, the possible effects and mechanisms of rosemary extract on repeated exposure to Ultraviolet A (UVA) induced senescence of human skin fibroblasts were investigated. During the research, we mainly carried out through two parts. The first part mainly explores the effect of repeated UVA radiation on human skin fibroblasts; the second part mainly discusses the effect of rosemary extract on the induction of human skin formation by repeated UVA radiation. The results showed that repeated UVA irradiation of human skin fibroblasts can induce aging phenomena, such as slow proliferation and an increase in β-galactosidase staining positive rate; repeated exposure of rosemary extract to low-dose UVA can slow down human skin fibroblasts to age. Its mechanism may be related to reducing the total intracellular ROS, the mRNA expression of MMP-1 and MMP-3, and the expression of p53 protein. In short, this study provides a new approach and theoretical basis for the development and application of natural sunscreens.

Reacquisition of a spindle cell shape does not lead to the restoration of a youthful state in senescent human skin fibroblasts.

Senescent fibroblasts are characterized by their inability to proliferate and by a pro-inflammatory and catabolic secretory phenotype, which contributes to age-related pathologies. Furthermore, senescent fibroblasts when cultured under classical conditions in vitro are also characterized by striking morphological changes, i.e. they lose the youthful spindle-like appearance and become enlarged and flattened, while their nuclei from elliptical become oversized and highly lobulated. Knowing the strong relation between cell shape and function, we cultured human senescent fibroblasts on photolithographed Si/poly(vinyl alcohol) (PVA) micro-patterned surfaces in order to restore the classical spindle-like geometry and subsequently to investigate whether the changes in senescent cells' morphology are the cause of their functional alterations. Interestingly, under these conditions senescent cells' nuclei do not revert to the classical elliptical phenotype. Furthermore, enforced spindle-shaped senescent cells retained their deteriorated proliferative ability, and maintained the increased gene expression of the cell cycle inhibitors p16Ink4a and p21Waf1. In addition, Si/PVA-patterned-grown senescent fibroblasts preserved their senescence-associated phenotype, as evidenced by the overexpression of inflammatory and catabolic genes such as IL6, IL8, ICAM1 and MMP1 and MMP9 respectively, which was further manifested by an intense downregulation of fibroblasts' most abundant extracellular matrix component Col1A, compared to their young counterparts. These data indicate that the restoration of the spindle-like shape in senescent human fibroblasts is not able to directly alter major functional traits and restore the youthful phenotype.

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.

Short exposure to cold atmospheric plasma induces senescence in human skin fibroblasts and adipose mesenchymal stromal cells

Cold Atmospheric Plasma (CAP) is a novel promising tool developed in several biomedical applications such as cutaneous wound healing or skin cancer. Nevertheless, in vitro studies are lacking regarding to CAP effects on cellular actors involved in healthy skin healing and regarding to the mechanism of action. In this study, we investigated the effect of a 3 minutes exposure to CAP-Helium on human dermal fibroblasts and Adipose-derived Stromal Cells (ASC) obtained from the same tissue sample. We observed that CAP treatment did not induce cell death but lead to proliferation arrest with an increase in p53/p21 and DNA damages. Interestingly we showed that CAP treated dermal fibroblasts and ASC developed a senescence phenotype with p16 expression, characteristic morphological changes, Senescence-Associated β-galactosidase expression and the secretion of pro-inflammatory cytokines defined as the Senescence-Associated Secretory Phenotype (SASP). Moreover this senescence phenotype is associated with a glycolytic switch and an increase in mitochondria content. Despite this senescence phenotype, cells kept in vitro functional properties like differentiation potential and immunomodulatory effects. To conclude, we demonstrated that two main skin cellular actors are resistant to cell death but develop a senescence phenotype while maintaining some functional characteristics after 3 minutes of CAP-Helium treatment in vitro.

Photoprotection of Cerium Oxide Nanoparticles against UVA radiation-induced Senescence of Human Skin Fibroblasts due to their Antioxidant Properties

Ultraviolet (UV) irradiation, particularly ultraviolet A (UVA), stimulates reactive oxygen species (ROS) production in the epidermis and dermis, which plays a major part in the photoageing of human skin. Several studies have demonstrated that cerium oxide nanoparticles (CeO2 NP) can exhibit an antioxidant effect and free radical scavenging activity. However, the protective role of CeO2 NP in skin photoageing and the underlying mechanisms are unclear. In this study, we investigated the effects of CeO2 NP on UVA-irradiated human skin fibroblasts (HSFs) and explored the potential signalling pathway. CeO2 NP had no apparent cytotoxicity, and could reduce the production of proinflammatory cytokines, intracellular ROS, senescence-associated β-galactosidase activity, and downregulate phosphorylation of c-Jun N-terminal kinases (JNKs) after exposure to UVA radiation. Based on our findings, CeO2 NPs have great potential against UVA radiation-induced photoageing in HSFs via regulating the JNK signal-transduction pathway to inhibit oxidative stress and DNA damage.

Antiaging Effects of Urolithin A on Replicative Senescent Human Skin Fibroblasts.

Although the health benefits attributed to urolithin A, such as anticancer, anti-inflammatory, and antioxidant effects, are based on numerous, diverse studies carried out in vitro, the biological effects of urolith A are still not entirely understood. In this study, we explored the biological effects of urolithin A using senescent human skin fibroblasts (HSFs) to determine whether urolithin A has any antiaging potential. Our results showed that urolithin A significantly increased type I collagen expression and reduced matrix metalloproteinase 1 (MMP-1) expression. Urolithin A also reduced intracellular reactive oxygen species, which may be partially due to activation of the Nrf2-mediated antioxidative response. These results indicate that urolithin A is a promising antiaging agent. Meanwhile, we noticed that the 50 μM urolithin A could cause changes in cell morphology and inhibition in cell proliferation, which were due to cell cycle arrest in G2/M phase. However, SA-β-gal (senescence-associated β-galactosidase) staining and γH2AX immunofluorescence staining showed cellular senescence status of HSFs did not change. Results of DAPI (4'6-diamidino-2-phenylindole) staining (no significant change) increased BCL2 gene expression and mitochondrial membrane potential (no significant change) after urolithin A treatment showed that the cells did not undergo apoptosis. These results provided further insights into the molecular mechanism of urolithin A. In conclusion, urolithin A showed a strong potential of antiaging.

Blocking negative effects of senescence in human skin fibroblasts with a plant extract

There is increasing evidence that senescent cells are a driving force behind many age-related pathologies and that their selective elimination increases the life- and healthspan of mice. Senescent cells negatively affect their surrounding tissue by losing their cell specific functionality and by secreting a pro-tumorigenic and pro-inflammatory mixture of growth hormones, chemokines, cytokines and proteases, termed the senescence-associated secretory phenotype (SASP). Here we identified an extract from the plant Solidago virgaurea subsp. alpestris, which exhibited weak senolytic activity, delayed the acquisition of a senescent phenotype and induced a papillary phenotype with improved functionality in human dermal fibroblasts. When administered to stress-induced premature senescent fibroblasts, this extract changed their global mRNA expression profile and particularly reduced the expression of various SASP components, thereby ameliorating the negative influence on nearby cells. Thus, the investigated plant extract represents a promising possibility to block age-related loss of tissue functionality.

Research on the senescence of human skin fibroblasts induced by ultraviolet b and its mechanism

Objective: To discuss the effect of ultraviolet B (UVB) on inducing the senescence of human skin fibroblasts and investigate its mechanism. Methods: The tissue explants method was used to isolate and culture the primary human skin fibroblasts, and after the observation of changes in cell morphology and β-galactosidase (β-gal) cell senescence staining via irradiation with different doses of UVB, the single irradiation dose of UVB in the induction of senescence of skin fibroblasts in subsequent experiment was determined. The cells and supernatant were collected at different time points (12, 24, 48 and 72 h) after irradiation with established doses of UVB. Western blotting was performed to detect the expression of marker P16 protein in induced senescent cells, and the expression of type I collagen, type III collagen, matrix metalloproteinase (MMP)-1 and MMP-3 of senescent cells was detected via ELISA. Results: According to Western blotting analysis, the expression of P16 protein in induced senescent skin fibroblasts was increased with the prolongation of irradiation time of UVB. ELISA showed that the expression of type I and III collagen was significantly reduced with the prolongation of irradiation time of UVB, while the expression of MMP1 and MMP3 was significantly increased. Conclusion: UVB irradiation can induce the senescence of human skin fibroblasts, whose mechanism may be related to the decrease of collagen synthesis and the increase of collagen degradation.

693 Blocking negative effects of senescence in human skin fibroblasts with a plant extract

693 Blocking negative effects of senescence in human skin fibroblasts with a plant extract

Diffuse colonies of human skin fibroblasts in relation to cellular senescence and proliferation.

Development of personalized skin treatment in medicine and skin care may benefit from simple and accurate evaluation of the fraction of senescent skin fibroblasts that lost their proliferative capacity. We examined whether enriched analysis of colonies formed by primary human skin fibroblasts, a simple and widely available cellular assay, could reveal correlations with the fraction of senescent cells in heterogenic cell population. We measured fractions of senescence associated β-galactosidase (SA-βgal) positive cells in either mass cultures or colonies of various morphological types (dense, mixed and diffuse) formed by skin fibroblasts from 10 human donors. Although the donors were chosen to be within the same age group (33-54 years), the colony forming efficiency of their fibroblasts (ECO-f) and the percentage of dense, mixed and diffuse colonies varied greatly among the donors. We showed, for the first time, that the SA-βgal positive fraction was the largest in diffuse colonies, confirming that they originated from cells with the least proliferative capacity. The percentage of diffuse colonies was also found to correlate with the SA-βgal positive cells in mass culture. Using Ki67 as a cell proliferation marker, we further demonstrated a strong inverse correlation (r=−0.85, p=0.02) between the percentage of diffuse colonies and the fraction of Ki67+ cells. Moreover, a significant inverse correlation (r=−0.94, p=0.0001) between the percentage of diffuse colonies and ECO-f was found. Our data indicate that quantification of a fraction of diffuse colonies may provide a simple and useful method to evaluate the extent of cellular senescence in human skin fibroblasts.

MiR-377 induces senescence in human skin fibroblasts by targeting DNA methyltransferase 1

Skin aging is a complicated physiological process and epigenetic feature, including microRNA-mediated regulation and DNA methylation, have been shown to contribute to this process. DNA methylation is regulated by DNA methyltransferase, of which DNA methyltransferase 1 (DNMT1) is the most abundantly known. But evidence supporting its role in skin aging remains scarce, and no report regards its specifical upstream-regulating molecules in the process of skin aging so far. Here, we found that DNMT1 expression was markedly higher in young human skin fibroblasts (HSFs) than that in passage-aged HSFs, and DNMT1 knockdown significantly induced the senescence phenotype in young HSFs. We predicted the upstream miRNAs which could regulate DNMT1 with miRNA databases and found miR-377 had high homology with a sequence in the 3′-UTR of human DNMT1 mRNA. We confirmed that miR-377 was a potential regulator of DNMT1 by luciferase reporter assays. miR-377 expression in passage-aged HSFs was markedly higher than that in the young HSFs. miR-377 overexpression promoted senescence in young HSFs, and inhibition of miR-377 reduced senescence in passage-aged HSFs. Moreover, these functions were mediated by targeting DNMT1. Microfluidic PCR and next-generation bisulfite sequencing of 24 senescent-associated genes’ promoters revealed alterations of the promoter methylation levels of FoxD3,p53, and UTF1 in HSFs treated with miR-377 mimics or inhibitors. We also verified that the miR-377-mediated changes in p53 expression could be reversed by regulation of DNMT1 in HSFs. Similarly, there was a negative correlation between miR-377 and DNMT1 expression in young and photoaged HSFs, HSFs, or skin tissues from UV-unexposed areas of different aged donors. Our results highlight a novel role for miR-377-DNMT1-p53 axis in HSF senescence. These findings shed new light on the mechanisms of skin aging and identify future opportunities for its therapeutic prevention.

599 Melatonin delays UV-induced premature senescence in human skin fibroblasts

Melatonin (N-acetyl-5-methoxytryptamine), an indolamine produced in pinealis gland, is the main human molecule involved in the circadian rhythms regulation, reaching maximum concentration levels during the night and under darkness. Melatonin is widely distributed to all body tissues, and in addition to its circadian effects, it has several other biological effects such as antioxidant effect, anti-inflammatory effect and protection to nuclear and mitochondrial DNA. Due to these properties, we hypothesized that melatonin would have the potential to reduce or to delay the appearance of molecular markers related to the premature cell aging. Results of flow cytometry and western blotting experiments showed that melatonin is able to reduce senescence-associated β-galactosidase, p16, p21 and IL-8 expression in human fibroblasts submitted to an UV-induced premature senescence protocol. Inducing suitable levels of topical nocturnal melatonin may be a promisor strategy for anti-aging cosmetics, especially for people exposed to excessive artificial light during the night.

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.

Inhibitory effect of chlorogenic acid on senescence of human skin fibroblasts

Objective To evaluate the inhibitory effect of chlorogenic acid on senescence of human skin fibroblasts (HSFs). Methods Fibroblasts isolated from human foreskin were treated with 1 mmol/L glyoxal in vitro to develop a model for cellular senescence. In order to select effective concentrations of chlorogenic acid, some HSFs were treated with 1 mmol/L glyoxal alone or in combination with chlorogenic acid at different concentrations (5, 10, 20, 40, 80 μmol/L) for 3 days, with those receiving no treatment serving as the blank control group. Then, methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of HSFs. Some HSFs were divided into 5 groups to be cultured alone (blank control group) , or treated with 1 mmol/L glyoxal (glyoxal group) or the combination of 1 mmol/L glyoxal and chlorogenic acid at effective concentrations of 10, 20 and 40 μmol/L (glyoxal+ chlorogenic acid groups). Senescence associated β-galactosidase (SA-β-gal) staining and real-time fluorescence-based quantitative PCR were conducted to determine the percentage of senescent cells and expression level of p16INK4a mRNA respectively. Statistical analysis was carried out by one-way analysis of variance followed by the least significant difference (LSD) -t test. Results Compared with the blank control group, the glyoxal group showed significantly decreased cellular proliferative activity of HSFs (55.65% ± 2.00% vs. 100% ± 6.90%, P 0.05). Therefore, 10- 40 μmol/L was selected as the effective concentrations of chlorogenic acid. The glyoxal group showed significant increases in the percentage of senescent (SA-β-gal-positive) cells (35.65% ± 2.24% vs. 13.00% ± 2.22%, P< 0.01) and expression level of p16INK4a mRNA (2-ΔΔCt: 1.00 ± 0.06 vs.0.26 ± 0.05, P< 0.01) compared with the blank control group, while the glyoxal+ 10-, 20-, 40-μmol/L chlorogenic acid groups showed significantly decreased percentage of senescent cells (31.50% ± 2.13%, 22.31% ± 3.11% and 19.32% ± 3.01% respectively) and expression level of p16INK4a mRNA (2-ΔΔCt: 0.88 ± 0.08, 0.73 ± 0.06 and 0.68 ± 0.04 respectively) compared with the glyoxal group (all P< 0.05). Additionally, the percentage of senescent cells decreased with the increase in chlorogenic acid concentrations in the glyoxal+ chlorogenic acid groups. Conclusion Chlorogenic acid can protect HSFs from glyoxal-induced senescence. Key words: Skin aging; Fibroblasts; Glyoxal; Chlorogenic acid; Cell aging; Cell proliferation; p16INK4a mRNA

Basal level of autophagy is increased in aging human skin fibroblasts in vitro, but not in old skin.

Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubule-associated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5-fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP.