OBJECTIVE: To investigate the effectiveness and preliminary mechanisms of icariin (ICA) in enhancing the reparative effects of adipose-derived stem cells (ADSCs) on skin radiation damagies in rats. METHODS: Twelve SPF-grade Sprague Dawley rats [body weight (220±10) g] were subjected to a single dose of 10 Gy X-ray irradiation on a 1.5 cm×1.5 cm area of their dorsal skin, with a dose rate of 200 cGy/min to make skin radiation damage model. After successful modelling, the rats were randomly divided into 4 groups ( n=3), and on day 2, the corresponding cells were injected subcutaneously into the irradiated wounds: group A received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL), group B received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL)+1 μmol/L ICA (0.1 mL), group C received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a hypoxia-inducible factor 2α (HIF-2α) inhibitor+1 μmol/L ICA (0.1 mL), and group D received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a Notch1 inhibitor+1 μmol/L ICA (0.1 mL). All treatments were administered as single doses. The skin injury in the irradiated areas of the rats was observed continuously from day 1 to day 7 after modelling. On day 28, the rats were sacrificed, and skin tissues from the irradiated areas were harvested for histological examination (HE staining and Masson staining) to assess the repair status and for quantitative collagen content detection. Immunohistochemical staining was performed to detect CD31 expression, while Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to measure the protein and mRNA relative expression levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), fibroblast growth factor 2 (FGF-2), interleukin 10 (IL-10), transforming growth factor β (TGF-β), HIF-2α, and Notch1, 2, and 3. RESULTS: All groups exhibited skin ulcers and redness after irradiation. On day 3, exudation of tissue fluid was observed in all groups. On day 7, group B showed significantly smaller skin injury areas compared to the other 3 groups. On day 28, histological examination revealed that the epidermis was thickened and the dermal fibers were slightly disordered with occasional inflammatory cell aggregation in group A. In group B, the epidermis appeared more normal, the dermal fibers were more orderly, and there was an increase in new blood vessels without significant inflammatory cell aggregation. In contrast, groups C and D showed significantly increased epidermal thickness, disordered and disrupted dermal fibers. Group B had higher collagen fiber content than the other 3 groups, and group D had lower content than group A, with significant differences ( P<0.05). Immunohistochemical staining showed that group B had significantly higher CD31 expression than the other 3 groups, while groups C and D had lower expression than group A, with significant differences ( P<0.05). Western blot and qRT-PCR results indicated that group B had significantly higher relative expression levels of VEGF, PDGF-BB, FGF-2, IL-10, TGF-β, HIF-2α, and Notch1, 2, and 3 proteins and mRNAs compared to the other 3 groups ( P<0.05). CONCLUSION ICA may enhance the reparative effects of ADSCs on rat skin radiation damage by promoting angiogenesis and reducing inflammatory responses through the HIF-2α-VEGF-Notch signaling pathway.
Animals ; Rats, Sprague-Dawley ; Skin/pathology* ; Rats ; Vascular Endothelial Growth Factor A/genetics* ; Basic Helix-Loop-Helix Transcription Factors/genetics* ; Signal Transduction ; Flavonoids/pharmacology* ; Adipose Tissue/cytology* ; Stem Cells/cytology* ; Receptors, Notch/metabolism* ; Radiation Injuries, Experimental/metabolism* ; Wound Healing/drug effects* ; Male

Objective To investigate the effect of liquid-liquid phase separation(LLPS)of YTH domain family protein 2(YTHDF2)on the sodium arsenite-induced malignant transformation of skin cells,providing a new intervention target for the prevention and control of sodium arsenite-induced carcinogenesis.Methods The HaCaT cell model of malignant transformation was constructed by continuous treatment with 1 μmol/L sodium arsenite for 22 weeks,including cells with normal YTHDF2 LLPS(YTHDF2-wt)and cells with inhibited YTHDF2 LLPS(YTHDF2-mut).Confocal microscopy was employed to observe and characterize the LLPS droplets formed by YTHDF2 during sodium arsenite-induced malignant transformation of skin cells.Cell proliferation,scratch healing,and colony formation assays were performed to detect malignant phenotypes.Western blotting,quantitative reverse transcription PCR,and immunofluorescence experiments were conducted to examine the effects of YTHDF2 LLPS on the mRNA and protein levels of phosphatase and tensin homolog deleted on chromosome ten(PTEN)during sodium arsenite-induced malignant transformation of skin cells.Results After 4 weeks of sodium arsenite treatment,LLPS droplets of YTHDF2 appeared in YTHDF2-wt cells,and the number of droplets gradually increased as the treatment time was prolonged(F=35.252,P<0.001),while no phase-separated droplets were observed in YTHDF2-mut cells.Compared with YTHDF2-mut cells,YTHDF2-wt cells showed enhanced proliferation at the time points of 48 h(t=3.654,P=0.006)and 72 h(t=5.458,P<0.001)after 22 weeks of sodium arsenite treatment.The scratch healing rate of YTHDF2-wt cells was increased at the 8th(t=12.137,P<0.001)and 22th(t=4.484,P=0.011)weeks of sodium arsenite treatment.The number of colonies formed by YTHDF2-wt cells was higher at the 4th(t=3.365,P=0.027),8th(t=5.580,P=0.005),and 22th(t=3.328,P=0.029)weeks of sodium arsenite treatment.Compared with YTHDF2-mut cells,YTHDF2-wt cells showed down-regulated protein(t=-3.119,P=0.036)and mRNA(t=4.051,P=0.015) levels of PTEN after 22 weeks of sodium arsenite treatment.Immunofluorescence results showed that after 4 weeks of sodium arsenite treatment,YTHDF2 LLPS droplets in YTHDF2-wt cells were localized to stress granules,translation-related membrane-less organelles.Conclusions During sodium arsenite-induced malignant transformation of skin cells,YTHDF2 undergoes LLPS and localizes to stress granules,translation-related membrane-less organelles.YTHDF2 LLPS participates in sodium arsenite-induced malignant transformation of skin cells by down-regulating the mRNA level of the key tumor suppressor PTEN.
Arsenites/toxicity* ; Sodium Compounds/toxicity* ; Humans ; Cell Transformation, Neoplastic/drug effects* ; PTEN Phosphohydrolase/metabolism* ; Cell Proliferation ; Skin/cytology* ; RNA-Binding Proteins ; Skin Neoplasms/chemically induced* ; Cell Line

OBJECTIVESTo investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.

METHODSMSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), β-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.

RESULTSMSP (7.81 μg/mL) down-regulated TGF-β1 and up-regulated HGF and β-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-β1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).

CONCLUSIONSThe MSP flower extract may have hair growth-promotion activities.

Animals ; Antioxidants ; pharmacology ; Cell Count ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flowers ; chemistry ; Hair Follicle ; cytology ; drug effects ; growth & development ; Hepatocyte Growth Factor ; metabolism ; Humans ; Mast Cells ; cytology ; Mice, Inbred C57BL ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Poaceae ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Skin ; metabolism ; Stem Cell Factor ; metabolism ; Stress, Psychological ; pathology ; Substance P ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; beta Catenin ; metabolism

BACKGROUNDCathepsin L (CatL) is a cysteine protease with strong matrix degradation activity that contributes to photoaging. Mannose phosphate-independent sorting pathways mediate ultraviolet A (UVA)-induced alternate trafficking of CatL. Little is known about signaling pathways involved in the regulation of UVA-induced CatL expression and activity. This study aims to investigate whether a single UVA irradiation affects CatL expression and activity and whether mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway is involved in the regulation of UVA-induced CatL expression and activity in human dermal fibroblasts (HDFs).

METHODSPrimary HDFs were exposed to UVA. Cell proliferation was determined by a cell counting kit. UVA-induced CatL production and activity were studied with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and fluorimetric assay in cell lysates collected on three consecutive days after irradiation. Time courses of UVA-activated JNK and p38MAPK signaling were examined by Western blotting. Effects of MAPK inhibitors and knockdown of Jun and Fos on UVA-induced CatL expression and activity were investigated by RT-PCR, Western blotting, and fluorimetric assay. Data were analyzed by one-way analysis of variance.

RESULTSUVA significantly increased CatL gene expression, protein abundance, and enzymatic activity for three consecutive days after irradiation (F = 83.11, 56.14, and 71.19, respectively; all P < 0.05). Further investigation demonstrated phosphorylation of JNK and p38MAPK activated by UVA. Importantly, inactivation of JNK pathway significantly decreased UVA-induced CatL expression and activity, which were not affected by p38MAPK inhibition. Moreover, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatL expression and activity.

CONCLUSIONSUVA enhances CatL production and activity in HDFs, probably by activating JNK and downstreaming AP-1. These findings provide a new possible molecular approach for antiphotoaging therapy.

Anthracenes ; pharmacology ; Cathepsin L ; metabolism ; Cells, Cultured ; Child ; Child, Preschool ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Fibroblasts ; cytology ; drug effects ; metabolism ; radiation effects ; Humans ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; drug effects ; radiation effects ; Oncogene Proteins v-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Pyridines ; pharmacology ; Skin ; cytology ; Ultraviolet Rays

BACKGROUNDShikonin is a major active chemical component extracted from Lithospermi Radix, an effective traditional herb in various types of wound healing. Shikonin can accelerate granulomatous tissue formation by the rat cotton pellet method and induce neovascularization in granulomatous tissue. The purpose of the study was to investigate its mechanism of action in human skin cells.

METHODSMTS assay was used to measure cell growth. The collagen type I (COL1 ) mRNA expression and procollagen type I C-peptide (PIP) production were detected by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Immunofluorescence and western blot analyses were carried out to investigate nuclear factor-κB (NF-κB) signaling pathway. Cell-based proteasome activity assay was used to determine proteasome activity.

RESULTSIn this study, we found that 10 μmol/L shikonin stimulated the growth of normal human keratinocytes and 1 μmol/L shikonin promoted growth of human dermal fibroblasts. However, shikonin did not directly induce COL1 mRNA expression and PIP production in dermal fibroblasts in vitro. In addition, 1 μmol/L shikonin inhibited translocation of NF-κB p65 from cytoplasm to nucleus induced by tumor necrosis factor-α stimulation in dermal fibroblasts. Furthermore, shikonin inhibited chymotrypsin-like activity of proteasome and was associated with accumulation of phosphorylated inhibitor κB-α in dermal fibroblasts.

CONCLUSIONSThese results suggested that shikonin may promote wound healing via its cell growth promoting activity and suppress skin inflammation via inhibitory activity on proteasome. Thus, shikonin may be a potential therapeutic reagent both in wound healing and inflammatory skin diseases.

Cell Proliferation ; drug effects ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; drug effects ; Humans ; Keratinocytes ; drug effects ; NF-kappa B ; metabolism ; Naphthoquinones ; pharmacology ; Polymerase Chain Reaction ; Proteasome Endopeptidase Complex ; drug effects ; Skin ; cytology

PURPOSE: The purpose of this study was to evaluate the effects of essential oil on oxidative stress, immunity, and skin condition in atopic dermatitis (AD) induced mice. METHODS: This study was a 3x3 factorial design. Factors were oil type (Lavender, Thyme, and 2:1 mixture of lavender and thyme oil [blending oil]) and treatment period (0 day, 7 days, and 21 days). The samples were 45 mice with AD and randomly assigned to nine groups of five mice per group. The dependent variables such as superoxide radical, IgE, degranulated mast cells, and epidermal thickness were measured. Data were collected from February to April in 2014. Descriptive statistics, One-way ANOVA, Two-way ANOVA, and Tukey's HSD test were performed using the SPSS WIN 20.0 program. RESULTS: Dependent variables were not statistically significantly different by the three oil types (p >.05). Essential oils such as lavender, thyme, and blending oil were all effective in reducing AD symptoms and especially 2:1 blending oil were most effective. There were statistically significant differences by the three treatment periods in all dependent variables (p <.001). There were statistically significant interactions between oil types and treatment periods in all dependent variables (p <.01). For decreasing superoxide radical, degranulated mast cells, and epidermal thickness, 2:1 mixed oil should be applied for at least 21 days. Otherwise to reduce IgE, 2:1 mixed oil should be used for at least 7 days. CONCLUSION: These findings provide bases for developing effective interventions for AD patients to manage their AD symptoms.
Animals ; Dermatitis, Atopic/chemically induced/*drug therapy/pathology ; Disease Models, Animal ; *Immunity/drug effects ; Immunoglobulin E/blood ; Lavandula/*chemistry/metabolism ; Mast Cells/cytology/metabolism ; Mice ; Oils, Volatile/chemistry/pharmacology/therapeutic use ; *Oxidative Stress/drug effects ; Picryl Chloride/toxicity ; Plant Oils/chemistry/pharmacology/*therapeutic use ; Singlet Oxygen/metabolism ; Skin/drug effects/pathology ; Thymus Plant/*chemistry/metabolism

FTY720 is a synthetic compound derived from the metabolites of Isaria sinclairii. Its unique chemical structure and mechanism appear to be distinctive from other known immunosuppressors. In the present study, the effect of FTY720 on immunosuppression and toxicity to heart was evaluated by detection of lymphocytes count, heart rate in rats, the survival time of the allografts of skin slice in mice and binding to S1P1 and S1P3 receptors by confocal. The results showed that FTY720 could induce lymphopenia, reduce the heart rates in rats and prolong the survival time of the allografts of skin slice in mice. The assay results on confocal showed that FTY720 can bind with S1P1 and S1P3 on surface of CHO-S1P1 and CHO-S1P3 cells. FTY720 could be developed for wide application for organ transplantation and self-immunity diseases.
Animals ; Cricetinae ; Cricetulus ; Female ; Fingolimod Hydrochloride ; Graft Survival ; drug effects ; Heart Rate ; drug effects ; Immunosuppressive Agents ; pharmacology ; Lymphocyte Count ; Lymphocytes ; cytology ; drug effects ; Lymphopenia ; chemically induced ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Microscopy, Confocal ; Propylene Glycols ; pharmacology ; Protein Binding ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Lysosphingolipid ; agonists ; metabolism ; Skin Transplantation ; Sphingosine ; analogs & derivatives ; pharmacology ; Transplantation, Homologous

The link of hedgehog (Hh) signaling activation to human cancer and synthesis of a variety of Hh signaling inhibitors raise great expectation that inhibiting Hh signaling may be effective in human cancer treatment. Cyclopamine (Cyc), an alkaloid from the Veratrum plant, is a specific natural product inhibitor of the Hh pathway that acts by targeting smoothened (SMO) protein. However, its poor solubility, acid sensitivity, and weak potency relative to other Hh antagonists prevent the clinical development of Cyc as a therapeutic agent. Here, we report properties of cyclopamine tartrate salt (CycT) and its activities in Hh signaling-mediated cancer in vitro and in vivo. Unlike Cyc, CycT is water soluble (5-10 mg/mL). The median lethal dose (LD50) of CycT was 62.5 mg/kg body weight compared to 43.5 mg/kg for Cyc, and the plasma half-life (T1/2) of CycT was not significantly different from that of Cyc. We showed that CycT had a higher inhibitory activity for Hh signaling-dependent motor neuron differentiation than did Cyc (IC50 = 50 nmol/L for CycT vs. 300 nmol/L for Cyc). We also tested the antitumor effectiveness of these Hh inhibitors using two mouse models of basal cell carcinomas (K14cre:Ptch1(neo/neo) and K14cre:SmoM2(YFP)). After topical application of CycT or Cyc daily for 21 days, we found that all CycT-treated mice had tumor shrinkage and decreased expression of Hh target genes. Taken together, we found that CycT is an effective inhibitor of Hh signaling-mediated carcinogenesis.
Animals ; Carcinoma, Basal Cell ; pathology ; Cell Differentiation ; drug effects ; Embryonic Stem Cells ; cytology ; Hedgehog Proteins ; antagonists & inhibitors ; metabolism ; Mice ; Motor Neurons ; cytology ; Plants, Medicinal ; chemistry ; Receptors, G-Protein-Coupled ; antagonists & inhibitors ; metabolism ; Signal Transduction ; drug effects ; Skin Neoplasms ; pathology ; Smoothened Receptor ; Solubility ; Tartrates ; blood ; pharmacology ; Tumor Burden ; drug effects ; Veratrum ; chemistry ; Veratrum Alkaloids ; blood ; isolation & purification ; pharmacology

OBJECTIVETo evaluate the effects of epicutaneous application of anticoagulant warfarin, by examining the presence of tissue injury and immune/inflammatory activity in exposed skin.

METHODSRats were exposed to warfarin by applying 10 μg of warfarin-sodium to 10-12 cm(2) skin (range 0.8-1 μg per 1 cm(2)) for 3 consecutive days. Tissue injury was evaluated by lipid peroxidation, histomorphological changes and signs of reparative activity in skin. T cell infiltration and selected aspects of epidermal cell activity were examined as indicators of immune/inflammatory skin response to warfarin application.

RESULTSRepeated warfarin application exerted no effect on skin metabolic viability, but resulted in tissue injury (increased malondialdehyde, MDA, production, evident histo-morphological changes in epidermis and dermis depicting cell injury and death). Increased numbers of proliferating cell nuclear antigen (PCNA(+)) cells indicated reparative processes in injured skin. Infiltration of CD3(+) cells (T lymphocytes) along with the increased production of tumor necrosis factor-a (TNF-a) by epidermal cells from warfarin-treated skin and their co-stimulatory effect in an in vitro T-cell activation assay demonstrated immunomodulatory effects of epicutaneous warfarin.

CONCLUSIONPresented data have documented tissue damage associated with immune/inflammatory activity in skin exposed to warfarin. Observed effects are relevant to immunotoxic potential of this anticoagulant in settings of external exposure.

Animals ; CD3 Complex ; genetics ; metabolism ; Dermatitis, Contact ; pathology ; Epidermis ; cytology ; Gene Expression Regulation ; physiology ; Inflammation ; metabolism ; Lipid Peroxidation ; Male ; Proliferating Cell Nuclear Antigen ; genetics ; metabolism ; Rats ; Rodenticides ; pharmacology ; Skin ; cytology ; drug effects ; metabolism ; T-Lymphocytes ; physiology ; Warfarin ; pharmacology

OBJECTIVETo study the effect of binding activities of the NH(2) terminus of E1A to the proteins regulating cell growth on ras-induced cell senescence and explore the mechanism of E1A-mediated escape from ras-induced senescence by E1A in human fibroblast.

METHODSIn primary human fibroblasts, the proteins regulating cell growth in association with E1A NH(2) terminus, including the Rb family proteins, p300/CBP, and p400, were inactivated or interfered. The effect of alterations in the binding activities of these proteins on cell senescence bypass mediated by E1A was evaluated by cell growth curve.

RESULTSThe Inactivation of Rb family proteins alone was not sufficient to rescue ras-induced cell senescence, whereas inactivation of both the Rb proteins and p300/CBP blocked ras-induced senescence of human fibroblasts.

CONCLUSIONRb and p300/CBP binding activities are both required for E1A to bypass ras-induced senescence in human fibroblasts.

Adenovirus E1A Proteins ; pharmacology ; Cellular Senescence ; drug effects ; Fibroblasts ; cytology ; Humans ; Primary Cell Culture ; Retinoblastoma Protein ; metabolism ; Skin ; cytology ; p300-CBP Transcription Factors ; metabolism ; ras Proteins ; antagonists & inhibitors ; pharmacology