Phlorizin, an Active Ingredient of Eleutherococcus senticosus, Increases Proliferative Potential of Keratinocytes with Inhibition of MiR135b and Increased Expression of Type IV Collagen.

E. senticosus extract (ESE), known as antioxidant, has diverse pharmacologic effects. It is also used as an antiaging agent for the skin and phlorizin (PZ) is identified as a main ingredient. In this study, the effects of PZ on epidermal stem cells were investigated. Cultured normal human keratinocytes and skin equivalents are used to test whether PZ affects proliferative potential of keratinocytes and how it regulates these effects. Skin equivalents (SEs) were treated with ESE and the results showed that the epidermis became slightly thickened on addition of 0.002% ESE. The staining intensity of p63 as well as proliferating cell nuclear antigen (PCNA) is increased, and integrin α6 was upregulated. Analysis of ESE confirmed that PZ is the main ingredient. When SEs were treated with PZ, similar findings were observed. In particular, the expression of integrin α6, integrin ß1, and type IV collagen was increased. Levels of mRNA for type IV collagen were increased and levels of miR135b were downregulated. All these findings suggested that PZ can affect the proliferative potential of epidermal cells in part by microenvironment changes via miR135b downregulation and following increased expression of type IV collagen.

Phloridzin isolated from Acanthopanax senticosus promotes proliferation of α6 integrin (CD 49f) and ß1 integrin (CD29) enriched for a primary keratinocyte population through the ERK-mediated mTOR pathway.

We investigated the proliferative effect of a Acanthopanax senticosus extract (ASE) on human CD49f(+)/CD29(+) keratinocytes and isolated phloridzin from A. senticosus as an active compound. In addition, the possible mechanisms of action were examined. We found that the ASE and phloridzin-promoted proliferation of CD49f(+)/CD29(+) cells using MTT and Click-iT™ EdU flow cytometry assays. In addition, phosphorylation of the p44/42 MAPK (ERK), mTOR, p70 S6 kinase (p70S6K), S6 ribosomal protein (S6RP), eukaryotic initiation factor 4B (eIF4B), and eIF4E was stepwise induced in CD49f(+)/CD29(+) cells. Furthermore, the ASE and phloridzin significantly induced the production of vascular endothelial growth factor and interleukin-6 in CD49f(+)/CD29(+) cells. Similarly, ASE and phloridzin-induced phosphorylation of the mTOR/p70S6K/S6RP/eIF4B/eIF4E pathway was blocked in response to pretreatment with PD98059, a specific ERK inhibitor. Taken together, these results indicate that ASE and phloridzin-induced proliferation of CD49f(+)/CD29(+) cells under serum-free conditions was mediated by the ERK-dependent mTOR pathway.

Human keratinocyte caspase-14 expression is altered in human epidermal 3D models by dexamethasone and by natural products used in cosmetics.

Caspase-14 is a cysteinyl-aspartate-specific proteinase that is specifically expressed in epidermal keratinocytes. Dysregulation of caspase-14 expression is implicated in impaired skin barrier formation. To elucidate the regulation of caspase-14 in differentiated keratinocytes, we characterized the expression of caspase-14 in normal human epidermal keratinocytes (NHEKs) and two types of three-dimensional (3D) human epidermis culture models, EPI-200 and EPI-201, via RT-PCR and immunoblot analyses. Caspase-14 expression was absent in subconfluent NHEKs, but was present in confluent NHEKs as well as those induced to differentiate by calcium. Caspase-14 expression levels in the 3D epidermis models were almost equal to that in the Ca(2+)-treated differentiated NHEKs. Despite the presence of caspase-14 expression in these models, caspase-14 activity was found only in the mature 3D skin model, EPI-200. This was confirmed by detection of a 17 kDa cleaved fragment of caspase-14 present only in the EPI-200 model. Since glucocorticoid (GC) receptor is required for skin barrier competence, we investigated whether the GC dexamethasone (Dex) and various natural components of common skin moisturizers affect caspase-14 expression in keratinocytes. Dex decreased caspase-14 expression in undifferentiated, but not differentiated, NHEKs. Conversely, Dex increased caspase-14 expression in both 3D skin models, although it did not alter caspase protease activity. Similar to treatment with Dex, treatment of the premature 3D skin mode, EPI-201 with a Galactomyces ferment filtrate markedly increased expression of caspase-14. Further, these results suggest that the effect of Dex, or lack thereof, on caspase-14 expression is dependent on the stage of keratinocyte differentiation.

Noninvasive biosensor for cathepsin L in the stratum corneum.

PURPOSE: The objective is to propose an on-site testing biosensor of cathepsin L (CatL) activity in the stratum corneum, which can be used for the evaluation of skin conditions noninvasively and easily. METHODS: The biosensor comprises of a disposable test strip and a desktop-sized reader (260 × 150 × 290 mm(3), 1.9 kg), incorporating a charge-coupled device image sensor (CCD) unit to measure the reflectance of the test strip. A novel immuno-chromatographic test strip was proposed for CatL analysis in the stratum corneum. In order to realize the test strip, a colloidal gold technique was selected as the molecular recognition method for the CatL. A human skin sample was collected noninvasively by adhesive tape stripping. RESULTS: Based on optimal assay conditions, the sensitivity of the biosensor was evaluated. It required 10 min from a sample dropping to appear the test line on the test strip. The optical density was proportion to the CatL. Bioanalytical validation indicated that, within the biosensor's detection limit (172.2 µU/mL), its accuracy (R(2) = 0.94), and precision (CV = 15%) approach more elaborate laboratory-based analyzers. In addition, the truncated sampling-reporting cycle (<15 min) allows speedy reporting of CatL levels. CONCLUSION: It was indicated that this noninvasive and easy-to use biosensor might be a novel tool for the semi-quantitative analysis of CatL in the stratum corneum.

Age- and sun exposure-dependent differences in 8-hydroxy-2'-deoxyguanosine and N-(carboxymethyl)lysine in human epidermis.

Aging and exposure to sunlight are two major factors in the deterioration of skin function. In this study, thirty-six fixed human skin samples from sun-exposed and unexposed areas from young and old individuals were used to evaluate the localization of oxidative stress according to levels and distribution of 8-hydroxy-2'-deoxyguanosine and N(ε)-(carboxymethyl)lysine in samples using immunohistochemistry. In the epidermis of the young, negligible amounts of 8-hydroxy-2'-deoxyguanosine and N(ε)-(carboxymethyl)lysine were detected in unexposed areas, whereas nuclear 8-hydroxy-2'-deoxyguanosine and cytoplasmic N(ε)-(carboxymethyl)lysine were increased in the lower epidermis in sun-exposed areas. In contrast, the aged presented prominent nuclear 8-hydroxy-2'-deoxyguanosine and nuclear N(ε)-(carboxymethyl)lysine in the epidermis of unexposed areas, concomitant with dermal increase in N(ε)-(carboxymethyl)lysine. However, the immunostaining of 8-hydroxy-2'-deoxyguanosine and N(ε)-(carboxymethyl)lysine revealed a decrease in the epidermis of sun-exposed areas in the aged. These results suggest an age-dependent difference in the adaptation and protective mechanisms of the epidermis against sunlight-associated oxidative stress, thus necessitating distinct standards for evaluation in each age group. Further investigation is warranted to elucidate underlying molecular mechanisms.

Sulfation of estradiol in human epidermal keratinocyte.

Epidermis is one of the well-known estrogen target tissues. Information regarding estrogen metabolism in epidermis is still very limited compared to that of estrogen action. In the breast cancer tissue, 17ß-estradiol (E(2)) is inactivated by sulfation and the expression level of estrogen sulfotransferase (SULT1E1) is inversely correlated with its malignancy. However, there is little datum about inactivation of estradiol in skin. In order to detect and measure E(2) and its metabolites simultaneously, we established an assay method with radio HPLC. A majority of [(3)H] labeled E(2) was converted to E(2) sulfate in normal human epidermal keratinocyte (NHEK) cells. The estimated activity of sulfotransferase toward E(2) at 20 nM was 0.11±0.01 (pmol/min/mg protein). Significant induction of estrogen sulfotransferase activity was observed in calcium-differentiated NHEK cells (0.58±0.07 (pmol/min/mg protein)). The gene expression of SULT1E1 was fifteen-fold higher in differentiated keratinocyte than in proliferating keratinocyte, whereas that of steroid sulfatase was reduced. These results suggest that E(2) inactivation is primarily mediated by SULT1E1 in keratinocyte and E(2) action is likely suppressed in epidermal differentiation.

Comparison of cathepsin L activity in cheek and forearm stratum corneum in young female adults.

PURPOSE: The objective is to determine the usefulness of cathepsin L (Cat L) activity derived from the stratum corneum as an index of skin conditions by comparing the cheek and the forearm, in 20 female young adults. METHODS: The sebum quantity, hydration index, melanin index and pH were measured with a sensor system consisting of a sebumeter, a corneometer, a mexameter and a pH meter. The stratum corneum was collected using tape stripping. Cat L and total protein (TP) were analyzed and the Cat L/TP ratio was calculated. RESULTS: Our results indicated that (i) chronic sun exposure causes an increase in Cat L activity; and (ii) in order to cancel out the influence of the collecting technique, it is very important to measure the Cat L/TP ratio. CONCLUSION: It is considered that the Cat L/TP ratio may be a useful index of the skin conditions in humans.

Evaluation of anti-wrinkle effects of a novel cosmetic containing niacinamide.

Niacinamide is known to have effectiveness on sallowness, wrinkling, red blotchiness and hyperpigmented spots in aging skin. In this study, we have evaluated the anti-wrinkle effects of a new cosmetic containing niacinamide. A randomized, placebo-controlled, split face study was performed in 30 healthy Japanese females who had wrinkles in the eye areas. The tested cosmetic containing 4% niacinamide was applied on wrinkles of one side for 8 weeks, and a control cosmetic without niacinamide on another site. Anti-wrinkle effects were evaluated with two methods: (i) doctors' observation and photographs based on the guideline of the Japan Cosmetic Industry Association; and (ii) average roughness of skin surface (Ra value) using skin replica. This cosmetic showed marked and moderate improvement in 64% of the subjects with a significant difference as compared with the control site (P < 0.001). Wrinkle grades in the tested area significantly reduced more than pre-application (P < 0.001) and the control (P < 0.001). Reduction in Ra value on the tested area was more than pre-application (P < 0.01) and the control site (P < 0.05) with significant differences. Only one subject stopped the study with minimal irritation. These results indicated that the tested lotion was well tolerated and may be an optional preparation for the treatment of wrinkles in the eye areas.

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model.

Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, beta-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2'-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with beta-carotene and SOD effectively suppressed CL intensity, indicating the generation of 1O2 and O2 .- in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.

Real-time chemiluminescent imaging and detection of reactive oxygen species generated in the UVB-exposed human skin equivalent model.

The objective of this study is to image and detect reactive oxygen species (ROS) generated in the UVB-exposed three-dimensional human skin equivalent model (HSEM), EpiDermtrade mark 200, because the alternative system needs to be urgently established as a replacement for the skin of experimental animals. Evidence that the ROS generation is enhanced in the skin of live animals after the UVB exposure was already obtained, by using the real-time chemiluminescent (RT-CL) method consisting of a sensitive CL probe (CLA) and an ultra-low light imaging apparatus. In this study, CL emission due to the reaction of CLA with endogenously generated ROS increased significantly in the UVB-treated HSEM compared with that in the intact HSEM, the maximum level being observed at a dose of 27mJ/cm(2). ROS under UVB exposure was identified to be ()O2- and (1)O(2) as observed by suppressive effects of SOD and beta-carotene topically applied on sample surface before the UVB exposure. The results for UVB-induced ROS generation in HSEM were consistent with those observed in the skin of live animals. HSEM combined with the RT-CL method was shown to be useful system not only to predict UVB-induced ROS-related skin responses in human but also to find protective agents against UVB-stimulated oxidative stress in place of animals and ex vivo human skin.