Boesenbergia rotunda extract accelerates human keratinocyte proliferation through activating ERK1/2 and PI3K/Akt kinases.

Boesenbergia rotunda (BR) has long been used as tradition medicine. For its pharmacological effects on wound healing, previous studies in an animal model provided convincing results that the ethanolic extract from the rhizome of this plant can stimulate wound healing. However, the mechanism about how this plant promotes wound healing at the molecular level has not been elucidated. As a step towards the development of wound healing agents, our current study utilized a human keratinocyte cell line (HaCaT) as an in vitro model to define the potential molecular mechanisms of BR extract in enhancing wound-healing. Our HPLC results showed that BR extract contained kaempferol as one of its potential compounds. The extract strongly promoted wound healing of HaCaT cell monolayer. This effect was eventually defined to be regulated through the ability of BR extract to induce cell proliferation. At the signaling level, we discovered that BR extract rapidly activated ERK1/2 and Akt phosphorylation upon the addition of the extract. Additionally, our experiments where specific inhibitors of MEK (U0126) and PI3K (LY294002) were utilized verified that BR enhanced cell proliferation and wound healing through stimulating the MAPK and PI3K/Akt signal transduction pathways. Moreover, direct inhibition of keratinocyte DNA synthesis by mitomycin C (MMC) could completely block the proliferative effects of BR extract. Nevertheless, data from Transwell migration assay revealed that BR extract did not promote keratinocyte migration. Altogether, we provided more evidence that BR possesses its wound healing-promoting action through the activation of proliferation and survival pathways, and our study suggests that BR is an interesting candidate to be developed as a wound healing-promoting agent.

Selective Janus Kinase 1 Inhibition Is a Promising Therapeutic Approach for Lupus Erythematosus Skin Lesions.

Background: Cutaneous lupus erythematosus (CLE) is an interferon (IFN) -driven autoimmune skin disease characterized by an extensive cytotoxic lesional inflammation with activation of different innate immune pathways. Aim of our study was to investigate the specific role of Janus kinase 1 (JAK1) activation in this disease and the potential benefit of selective JAK1 inhibitors as targeted therapy in a preclinical CLE model. Methods: Lesional skin of patients with different CLE subtypes and healthy controls (N = 31) were investigated on JAK1 activation and expression of IFN-associated mediators via immunohistochemistry and gene expression analyses. The functional role of JAK1 and efficacy of inhibition was evaluated in vitro using cultured keratinocytes stimulated with endogenous nucleic acids. Results were confirmed in vivo using an established lupus-prone mouse model. Results: Proinflammatory immune pathways, including JAK/STAT signaling, are significantly upregulated within inflamed CLE skin. Here, lesional keratinocytes and dermal immune cells strongly express activated phospho-JAK1. Selective pharmacological JAK1 inhibition significantly reduces the expression of typical proinflammatory mediators such as CXCL chemokines, BLyS, TRAIL, and AIM2 in CLE in vitro models and also improves skin lesions in lupus-prone TREX1-/- -mice markedly. Conclusion: IFN-associated JAK/STAT activation plays a crucial role in the pathophysiology of CLE. Selective inhibition of JAK1 leads to a decrease of cytokine expression, reduced immune activation, and decline of keratinocyte cell death. Topical treatment with a JAK1-specific inhibitor significantly improves CLE-like skin lesions in a lupus-prone TREX1-/- -mouse model and appears to be a promising therapeutic approach for CLE patients.

Phospholipase Cγ1 is required for normal irritant contact dermatitis responses and sebaceous gland homeostasis.

Differentiation and proliferation of keratinocyte are controlled by various signalling pathways. The epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Inhibition of EGFR signalling disturbs keratinocyte proliferation, differentiation and migration. Previous studies have revealed that one of the EGFR downstream signalling molecules, phospholipase Cγ1 (PLCγ1), regulates differentiation, proliferation and migration of keratinocytes in in vitro cell culture system. However, the role of PLCγ1 in the regulation of keratinocyte functions in animal epidermis remains unexplored. In this study, we generated keratinocyte-specific PLCγ1 knockout (KO) mice (PLCγ1 cKO mice). Contrary to our expectations, loss of PLCγ1 did not affect differentiation, proliferation and migration of interfollicular keratinocytes. We further examined the role of PLCγ1 in irritant contact dermatitis (ICD), in which epidermal cells play a pivotal role. Upon irritant stimulation, PLCγ1 cKO mice showed exaggerated ICD responses. Further study revealed that epidermal loss of PLCγ1 induced sebaceous gland hyperplasia, indicating that PLCγ1 regulates homeostasis of one of the epidermal appendages. Taken together, our results indicate that, although PLCγ1 is dispensable in interfollicular keratinocyte for normal differentiation, proliferation and migration, it is required for normal ICD responses. Our results also indicate that PLCγ1 regulates homeostasis of sebaceous glands.

Horse Oil Mitigates Oxidative Damage to Human HaCaT Keratinocytes Caused by Ultraviolet B Irradiation.

Horse oil products have been used in skin care for a long time in traditional medicine, but the biological effects of horse oil on the skin remain unclear. This study was conducted to evaluate the protective effect of horse oil on ultraviolet B (UVB)-induced oxidative stress in human HaCaT keratinocytes. Horse oil significantly reduced UVB-induced intracellular reactive oxygen species and intracellular oxidative damage to lipids, proteins, and DNA. Horse oil absorbed light in the UVB range of the electromagnetic spectrum and suppressed the generation of cyclobutane pyrimidine dimers, a photoproduct of UVB irradiation. Western blotting showed that horse oil increased the UVB-induced Bcl-2/Bax ratio, inhibited mitochondria-mediated apoptosis and matrix metalloproteinase expression, and altered mitogen-activated protein kinase signaling-related proteins. These effects were conferred by increased phosphorylation of extracellular signal-regulated kinase 1/2 and decreased phosphorylation of p38 and c-Jun N-terminal kinase 1/2. Additionally, horse oil reduced UVB-induced binding of activator protein 1 to the matrix metalloproteinase-1 promoter site. These results indicate that horse oil protects human HaCaT keratinocytes from UVB-induced oxidative stress by absorbing UVB radiation and removing reactive oxygen species, thereby protecting cells from structural damage and preventing cell death and aging. In conclusion, horse oil is a potential skin protectant against skin damage involving oxidative stress.

Moringa oleifera stem extract protect skin keratinocytes against oxidative stress injury by enhancement of antioxidant defense systems and activation of PPARα.

Oxidative stress is an important cause of skin injury induced by UVB radiation. Moringa oleifera also known as horseradish tree or drumstick tree, have multiple nutraceutical or pharmacological functions. However, whether Moringa oleifera protects skin against oxidative stress injury remains unknown. To investigate the effects of the ethanol extract of Moringa oleifera stem (MSE) on skin oxidative stress injury and its molecular mechanism, we first determined the effect of MSE on epidermal oxidative stress injury induced by H2O2 in keratinocytes (HaCaT cells) and by UVB-radiation in mice. Then we investigated the effect of MSE on the enhancement of antioxidant system and activation of PPARα in vitro and in vivo. Furthermore, the flavonoids compositions in MSE were assayed by high-performance liquid chromatography (HPLC), and then molecular docking study was used to assess the major component in MSE to activate PPARα. Our results indicate that MSE (100-400 µg/mL) protected the epidemic cell against oxidative stress injury in vitro and topical treatment with MSE cream (6%) inhibit UVB-induced oxidative stress injury in the epidermis of the mouse skin. PPARα activation is involved in the protective effect of MSE. HPLC assay and molecular docking study indicated that rutin might be the main component in MSE to activate PPARα. These results confirm that MSE exerts the protective effect on oxidative stress induced skin keratinocytes injury. Moreover, the protective effect of MSE is mediated by enhancement of antioxidant defense systems and activation of PPARα in skin keratinocytes.

Brazilin and Caesalpinia sappan L. extract protect epidermal keratinocytes from oxidative stress by inducing the expression of GPX7.

Caesalpinia sappan L., belonging to the family Leguminosae, is a medicinal plant that is distributed in Southeast Asia. The dried heartwood of this plant is used as a traditional ingredient of food, red dyes, and folk medicines in the treatment of diarrhea, dysentery, tuberculosis, skin infections, and inflammation. Brazilin is the major active compound, which has exhibited various pharmacological effects, including anti-platelet activity, anti-hepatotoxicity, induction of immunological tolerance, and anti-inflammatory and antioxidant activities. The present study aimed to evaluate the antioxidant activity and expression of antioxidant enzymes of C. sappan L. extract and its major compound, brazilin, in human epidermal keratinocytes exposed to UVA irradiation. Our results indicated that C. sappan L. extract reduced UVA-induced H2O2 production via GPX7 activation. Moreover, brazilin exhibited antioxidant effects that were similar to those of C. sappan L. via glutathione peroxidase 7 (GPX7), suggesting that C. sappan L. extract and its natural compound represent potential treatments for oxidative stress-induced photoaging of skin.

Eupafolin ameliorates COX-2 expression and PGE2 production in particulate pollutants-exposed human keratinocytes through ROS/MAPKs pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Eupafolin is a major bioactive compound derived from the methanolic extract of the medicinal herb Phyla nodiflora, which has been used in traditional Chinese medicine to treat various inflammatory diseases. Recently, particulate air pollutants have been shown to induce inflammation of the skin. In this study, we seek to determine whether eupafolin can inhibit the production of inflammatory mediators in a human skin keratinocyte cell line exposed to particulate air pollutants (particulate matter, PM), and determine the molecular mechanisms involved. MATERIALS AND METHODS: Human keratinocyte HaCaT cells were treated with PM in the presence or absence of eupafolin. Cyclooxygenase-2 (COX-2) protein and gene expression levels were determined by Western blotting, RT-PCR and luciferase activity assay. Prostaglandin E2 (PGE2) production was evaluated by the enzyme immunoassay method. Generation of intracellular reactive oxygen species (ROS) was measured by the dichlorofluorescin (DCFH) oxidation assay, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was determined by a chemiluminescence assay. For in vivo studies, COX-2 expression in the skin of BALB/c nude mice was analyzed by immunohistochemistry. RESULTS: Eupafolin inhibited PM-induced COX-2 protein and gene expression and PGE2 production in HaCaT cells. In addition, eupafolin suppressed PM-induced intracellular ROS generation, NADPH oxidase activity, MAPK (ERK, JNK and p38) activation and NK-κB activation. In vivo studies showed that topical treatment with eupafolin inhibited COX-2 expression in the epidermal keratinocytes of PM-treated mice. CONCLUSIONS: Eupafolin exerts anti-inflammatory and antioxidant effects on skin keratinocytes exposed to particulate air pollutants, and may have potential use in the treatment or prevention of air pollutant-induced inflammatory skin diseases in the future.

Cynaropicrin attenuates UVB-induced oxidative stress via the AhR-Nrf2-Nqo1 pathway.

Due to its antioxidant and anti-inflammatory activities, artichoke (Cynara scolymus) has been used as folk medicine to treat various diseases. Cynaropicrin (Cyn), a sesquiterpene lactone, is the major bioactive phytochemical in the artichoke; however, its pharmacological mechanism remains unknown. Because some phytochemicals exert their antioxidant activity by activating aryl hydrocarbon receptor (AhR), leading to subsequent induction of the antioxidant pathway including nuclear factor E2-related factor 2 (Nrf2) and NAD(P)H: quinone oxidoreductase 1 (Nqo1), we investigated whether Cyn also activates the AhR-Nrf2-Nqo1 pathway. Cyn indeed induced the activation (nuclear translocation) of AhR, leading to nuclear translocation of Nrf2 and dose-dependent upregulation of Nrf2 and Nqo1 mRNAs in human keratinocytes. The Cyn-induced AhR-Nrf2-Nqo1 activation was AhR- and Nrf2-dependent, as demonstrated by the observation that it was absent in keratinocytes transfected by siRNA against either AhR or Nrf2. In accordance with these findings, Cyn actively inhibited generation of reactive oxygen species from keratinocytes irradiated with ultraviolet B (UVB) in a Nrf2-dependent manner. Cyn also inhibited the production of proinflammatory cytokines such as interleukin 6 and tumor necrosis factor-α from UVB-treated keratinocytes. Our findings demonstrate that Cyn is a potent activator of the AhR-Nrf2-Nqo1 pathway, and could therefore be applied to prevention of UVB-induced photo aging.

CDK4-mediated MnSOD activation and mitochondrial homeostasis in radioadaptive protection.

Mammalian cells are able to sense environmental oxidative and genotoxic conditions such as the environmental low-dose ionizing radiation (LDIR) present naturally on the earth's surface. The stressed cells then can induce a so-called radioadaptive response with an enhanced cellular homeostasis and repair capacity against subsequent similar genotoxic conditions such as a high dose radiation. Manganese superoxide dismutase (MnSOD), a primary mitochondrial antioxidant in mammals, has long been known to play a crucial role in radioadaptive protection by detoxifying O2(•-) generated by mitochondrial oxidative phosphorylation. In contrast to the well-studied mechanisms of SOD2 gene regulation, the mechanisms underlying posttranslational regulation of MnSOD for radioprotection remain to be defined. Herein, we demonstrate that cyclin D1/cyclin-dependent kinase 4 (CDK4) serves as the messenger to deliver the stress signal to mitochondria to boost mitochondrial homeostasis in human skin keratinocytes under LDIR-adaptive radioprotection. Cyclin D1/CDK4 relocates to mitochondria at the same time as MnSOD enzymatic activation peaks without significant changes in total MnSOD protein level. The mitochondrial-localized CDK4 directly phosphorylates MnSOD at serine-106 (S106), causing enhanced MnSOD enzymatic activity and mitochondrial respiration. Expression of mitochondria-targeted dominant negative CDK4 or the MnSOD-S106 mutant reverses LDIR-induced mitochondrial enhancement and adaptive protection. The CDK4-mediated MnSOD activation and mitochondrial metabolism boost are also detected in skin tissues of mice receiving in vivo whole-body LDIR. These results demonstrate a unique CDK4-mediated mitochondrial communication that allows cells to sense environmental genotoxic stress and boost mitochondrial homeostasis by enhancing phosphorylation and activation of MnSOD.

Antibacterial and antigelatinolytic effects of Satureja hortensis L. essential oil on epithelial cells exposed to Fusobacterium nucleatum.

The present report examined the effects of essential oils (EOs) from Satureja hortensis L. and Salvia fruticosa M. on the viability and outer membrane permeability of the periodontopathogen Fusobacterium nucleatum, a key bacteria in oral biofilms, as well as the inhibition of matrix metalloproteinase (MMP-2 and MMP-9) activities in epithelial cells exposed to such bacteria. Membrane permeability was tested by measuring the N-phenyl-1-naphthylamine uptake and bacterial viability by using the commercially available Live/Dead BacLight kit. In addition, gelatin zymography was performed to analyze the inhibition of F. nucleatum-induced MMP-2 and MMP-9 activities in HaCaT cells. We showed that 5, 10, and 25 µL/mL of Sat. hortensis L. EO decreased the ratio of live/dead bacteria and increased the outer membrane permeability in a range of time from 0 to 5 min. Treatments with 10 and 25 µL/mL of Sal. fruticosa M. also increased the membrane permeability and 5, 10, and 25 µL/mL of both EOs inhibited MMP-2 and MMP-9 activities in keratinocytes induced after exposure of 24 h to F. nucleatum. We conclude that antibacterial and antigelatinolytic activities of Sat. hortensis L. EO have potential for the treatment of periodontal inflammation.

Illicium verum extract suppresses IFN-γ-induced ICAM-1 expression via blockade of JAK/STAT pathway in HaCaT human keratinocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Interferon-gamma (IFN-γ) signaling in keratinocytes plays an important role in mediating inflammatory conditions such as psoriasis and contact dermatitis. Illicium verum Hook. f. has been used in traditional medicine for treating skin inflammation, rheumatism, asthma, and bronchitis in Asia. AIM OF THE STUDY: To investigate the anti-inflammatory effects and regulatory mechanisms of Illicium verum extract (IVE) in the human keratinocyte cell line HaCaT. MATERIALS AND METHODS: In this study, we examined the effect of IVE on IFN-γ-induced ICAM-1 expression in HaCaT cells. The levels of IFN-γ receptor α (IFN-γRα), phosphorylated Janus kinase 2 (pJak2), phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and suppressor of cytokine signaling 1 (SOCS1) were analyzed by western blot. Expression of intercellular adhesion molecule-1 (ICAM-1) on the HaCaT cells was determined by reverse transcription-polymerase chain reaction, western blot, and cell-surface enzyme-linked immunosorbent assay. The effects of IVE and its constituents on the adherence of T lymphocytes to IFN-γ-treated HaCaT cells were also investigated. RESULTS: IVE significantly inhibited IFN-γ-induced mRNA and protein expression of ICAM-1. IVE inhibited IFN-γ-induced IFN-γRα, pJak2 and pSTAT1 expression in HaCaT cells. The expression of SOCS1 was up-regulated by treatment of IVE. In addition, IVE and its constituents (p-anisaldehyde and trans-anethole) effectively suppressed IFN-γ-induced adherence of Jurkat T cells to HaCaT cells and ICAM-1 expression on the cell surface. CONCLUSIONS: These results indicate that the anti-inflammatory effects of IVE may contribute to therapeutic efficacy in IFN-γ-dependent inflammatory skin diseases by modulating the IFN-γ signal pathway.

Carnosic acid, a phenolic diterpene from rosemary, prevents UV-induced expression of matrix metalloproteinases in human skin fibroblasts and keratinocytes.

Exposure of the skin to ultraviolet (UV) irradiation induces photoageing through the up-regulation of matrix metalloproteinases (MMPs) and subsequent breakdown of extracellular matrices. Reactive oxygen species (ROS) and epidermal growth factor receptor (EGFR) activation play central roles in UV-induced MMP expression through initiating extracellular signal-regulated kinase (ERK)-mediated AP-1 signalling. We aimed to explore the effects of carnosic acid (CA), a phenolic diterpene from rosemary, on UV-induced MMP expression in human skin cells. Molecular mechanism underlying the effects of CA was also examined in the aspect of MMP expression, ERK/AP-1 pathway, ROS generation and EGFR activation. Human dermal fibroblast cell line (Hs68), primary normal human dermal fibroblasts (HDFs) and normal human epidermal keratinocytes (HEKs) were employed, and antiphotoageing effects of CA were assessed by Western blotting, quantitative real-time PCR and enzyme assays. CA significantly inhibited UVA- and UVB-induced expression of MMP-1, MMP-3 and MMP-9 in a concentration-dependent manner in Hs68 cells. UVB-induced ERK activation and the formation of transcription factor, AP-1, were significantly suppressed by CA. Among the upstream events of MMP expression, UVB-induced ROS generation was attenuated by CA, while EGFR activation was not affected. Confirming the antiphotoageing effects of CA through the suppression of UV-induced ROS generation, UVB-enhanced GADD45 expression, a marker for oxidative DNA damages was significantly reduced by CA. Inhibitory effects of CA on UVB-induced MMP expression could be also seen in HDFs and HEKs. Collectively, our study demonstrates that CA inhibits the UV-enhanced MMPs in human skin cells through the inhibition of ROS and the suppression of ERK/AP-1 activation.

Zinc inhibits apoptosis and maintains NEP downregulation, induced by ropivacaine, in HaCaT cells.

Zinc (Zn), a cell-protective metal against various toxic compounds, is the key agent for neutral endopeptidase (NEP) functional structure. NEP is a zinc metalloenzyme which degrades endogenous opioids and is expressed in human keratinocytes (HaCaT). Ropivacaine, a widely used opiate local anaesthetic, exerts cell toxic and apoptotic effects against HaCaT cells. The aim of the present study is to investigate whether zinc modulates the effects of ropivacaine on proliferation, viability, apoptosis and NEP expression in HaCaT cells. To investigate the role of ropivacaine in NEP function, HaCaT cells overexpressing NEP were generated via cell transfection with plasmids carrying NEP cDNA. Ropivacaine's anti-proliferative effect was tested by Neubauer's chamber cell counting, and induction of cell death was demonstrated by trypan blue exclusion assay. Apoptosis due to ropivacaine was tested via DNA fragmentation and poly-ADP-ribose-polymerase (PARP) cleavage. NEP and PARP expression was performed by western blot analysis. Results showed that zinc (15 µΜ) inhibited proliferation and cell death induction by ropivacaine (0.5, 1 and 2 mM) (p < 0.05) as well as apoptosis induced by the drug (0.5 and 1 mM) in HaCaT cells. Ropivacaine (1.0, 2.0 and 5.0 mM) downregulated NEP expression in the presence of zinc (15 µΜ) while NEP overexpression enhanced ropivacaine's apoptotic effect. In conclusion, the abilities of zinc to inhibit the toxic and apoptotic effects of ropivacaine, to maintain NEP downregulation induced by the drug and, consequently, to enhance its anaesthetic result suggest that zinc may have a significant role in pain management and tissue protection.

Bioinformatical and in vitro approaches to essential oil-induced matrix metalloproteinase inhibition.

CONTEXT: Essential oils carry diverse antimicrobial and anti-enzymatic properties. OBJECTIVE: Matrix metalloproteinase (MMP) inhibition characteristics of Salvia fruticosa Miller (Labiatae), Myrtus communis Linnaeus (Myrtaceae), Juniperus communis Linnaeus (Cupressaceae), and Lavandula stoechas Linnaeus (Labiatae) essential oils were evaluated. MATERIALS AND METHODS: Chemical compositions of the essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS). Bioinformatical database analysis was performed by STRING 9.0 and STITCH 2.0 databases, and ViaComplex software. Antibacterial activity of essential oils against periodontopathogens was tested by the disc diffusion assay and the agar dilution method. Cellular proliferation and cytotoxicity were determined by commercial kits. MMP-2 and MMP-9 activities were measured by zymography. RESULTS: Bioinformatical database analyses, under a score of 0.4 (medium) and a prior correction of 0.0, gave rise to a model of protein (MMPs and tissue inhibitors of metalloproteinases) vs. chemical (essential oil components) interaction network; where MMPs and essential oil components interconnected through interaction with hydroxyl radicals, molecular oxygen, and hydrogen peroxide. Components from L. stoechas potentially displayed a higher grade of interaction with MMP-2 and -9. Although antibacterial and growth inhibitory effects of essential oils on the tested periodontopathogens were limited, all of them inhibited MMP-2 in vitro at concentrations of 1 and 5 µL/mL. Moreover, same concentrations of M. communis and L. stoechas also inhibited MMP-9. MMP-inhibiting concentrations of essential oils were not cytotoxic against keratinocytes. DISCUSSION AND CONCLUSION: We propose essential oils of being useful therapeutic agents as MMP inhibitors through a mechanism possibly based on their antioxidant potential.

Nanomolar melatonin enhances nNOS expression and controls HaCaT-cells bioenergetics.

A novel role of melatonin was unveiled, using immortalized human keratinocyte cells (HaCaT) as a model system. Within a time window compatible with its circadian rhythm, melatonin at nanomolar concentration raised both the expression level of the neuronal nitric oxide synthase mRNA and the nitric oxide oxidation products, nitrite and nitrate. On the same time scale, a depression of the mitochondrial membrane potential was detected together with a decrease of the oxidative phosphorylation efficiency, compensated by glycolysis as testified by an increased production of lactate. The melatonin concentration, ∼ nmolar, inducing the bioenergetic effects and their time dependence, both suggest that the observed nitric oxide-induced mitochondrial changes might play a role in the metabolic pathways characterizing the circadian melatonin chemistry.

Antioxidative and anti-inflammatory effects of phenolic compounds from the roots of Ulmus macrocarpa.

The roots of Ulmus macrocarpa Hance (Ulmaceae) have been used as an oriental traditional medicine for the treatment of inflammation, ulcers, cancers, and parasites. Activity guided isolation from the roots of U. macrocarpa yielded three flavonoids [catechin 7-O-ß-D-apiofuranoside (1), (+)-catechin (2), taxifolin 6-C-glucopyranoside (3)], and one coumarin [fraxin (4)]. To investigate the antioxidative and anti-inflammatory effects of these compounds, DPPH radical scavenging activity and inhibitory activity against nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells were examined and the expression of inducible NO synthase (iNOS) and cyclooxidase-2 (COX-2) were measured by RT-PCR and Western Blotting in HaCaT cells. Compounds 1, 2, and 3 showed moderate antioxidative activities compared with L-ascorbic acid as a positive control. NO production was reduced and the expressions of iNOS and COX-2 and their mRNA were inhibited by the addition of samples (1-4). These results suggest that the phenolic compounds from the roots of U. macrocarpa might be developed as antioxidant and anti-inflammatory agents.

Celastrol-induced apoptosis in human HaCaT keratinocytes involves the inhibition of NF-κB activity.

Psoriasis is a chronic inflammatory skin disease affecting 1-3% of the world's population. Traditional Chinese medicines have been extensively used for treating psoriasis with promising clinical results. Celastrol, a triterpenoid isolated from a Chinese herb Celastrus orbiculatus caulis, has been known to have diverse pharmacological effects such as anti-inflammatory, anti-cancer and antioxidant activities. The present study aimed at evaluating the anti-proliferative action of celastrol on cultured HaCaT cells and elucidating the mechanisms of action involved. Celastrol was shown to inhibit HaCaT cells growth with an IC50 value of 1.1 µM as measured by MTT assay. The ability of celastrol to induce apoptosis was studied by flow cytometric and western blot analyses. Celastrol was found to be capable of inducing apoptosis in HaCaT cells as characterized by phosphatidyl-serine (PS) externalization, depolarization of mitochondrial membrane potential and activation of caspase-3. The apoptosis induced by celastrol could be suppressed by Z-IETD-FMK and Z-LEHD-FMK, the respective caspase-8 and caspase-9 inhibitor. In addition, western blot analysis revealed a significant augmentation in the protein expression of Bax and attenuation in Bcl-2, suggesting that the celastrol-induced apoptosis acts through both death receptor and mitochondrial pathways. Moreover, western blot analysis on the expression of Rel/NF-κB demonstrated that the celastrol-mediated apoptosis on HaCaT cells was associated with the inhibition of the NF-κB pathway. Taken together, the present project has for the first time identified celastrol as a naturally occurring compound with potent apoptogenic action on cultured human keratinocytes, rendering it a promising candidate for further development into an anti-psoriatic agent.

(2S)-2'-methoxykurarinone from Sophora flavescens suppresses cutaneous T cell-attracting chemokine/CCL27 expression induced by interleukin-ß/tumor necrosis factor-α via heme oxygenase-1 in human keratinocytes.

One of the CC chemokines, cutaneous T cell-attracting chemokine (CTACK/CCL27), is a skin-specific CC chemokine that is produced constitutively by keratinocytes and is highly up-regulated in inflammatory skin conditions such as atopic dermatitis and contact dermatitis. (2S)-2'-Methoxykurarinone (MOK) from Sophora flavescens has been demonstrated to have antioxidant effects. Heme oxygenase (HO)-1 has recently emerged as an important cytoprotective enzyme against oxidative stress and inflammatory responses in many cell types. This study aimed to define whether and how MOK regulates skin specific CTACK/CCL27 chemokine production in human HaCaT keratinocytes. The level of CTACK/CCL27 and HO-1 expression was measured by reverse transcription-polymerase chain reaction, and signaling was evaluated by western blot analysis. CTACK/CCL27 production was determined by enzyme-linked immunosorbent assay. Pretreatment with MOK suppressed tumor necrosis factor-α (TNF-α)- and interleukin (IL)-1ß-induced CTACK/CCL27 production in human HaCaT keratinocytes. MOK inhibited TNF-α- and IL-1ß-induced nuclear factor κB (NF-κB) activation. Interestingly, pretreatment with MOK significantly suppressed TNF-α- and IL-1ß-induced CTACK/CCL27 production through the induction of HO-1. This suppression was completely abolished by HO-1 small interfering RNA. Furthermore, carbon monoxide, but not other end products of HO-1 activity, also suppressed TNF-α- and IL-1ß-induced CTACK/CCL27 production. These results demonstrate that MOK attenuates TNF-α- and IL-1ß-induced production of CTACK/CCL27 in human HaCaT keratinocytes by inhibiting NF-κB activation and induction of HO-1.

Phospholipid scramblase 1 is secreted by a lipid raft-dependent pathway and interacts with the extracellular matrix protein 1 in the dermal epidermal junction zone of human skin.

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.

Date seed oil inhibits hydrogen peroxide-induced oxidative stress in human epidermal keratinocytes.

BACKGROUND: Oxidative stress has been implicated in various skin diseases through the generation of reactive oxygen species and the depletion of endogenous antioxidant systems. The administration of antioxidants is reportedly helpful, notably to enhance the healing process. To protect the skin against oxidative damages, we have studied the effect of new oil: "date seed oil" (DSO). This oil, may serve as a potential source of natural antioxidants such as phenols and tocopherols. METHODS: Here, we report the protective effect of DSO against hydrogen peroxide (H(2)O(2))-induced oxidative stress in terms of lipid peroxidation, depletion of endogenous antioxidant defense enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) using normal human epidermal keratinocytes (NHEK). RESULTS: In the investigated model system, DSO has significant chemoprotective effect, by inhibition of damage caused by H(2)O(2) compared with cells without such addition endowing with a radical scavenging ability. Treatment of NHEK with DSO inhibited H(2)O(2)-induced lipid peroxidation. In addition, this oil inhibited H(2)O(2)-induced depletion of antioxidant defense components, such as SOD, CAT and GPx. CONCLUSIONS: Our findings demonstrate that DSO is an efficient extract that is able to prevent keratinocytes oxidative damage induced by H(2)O(2) exposure and may thus be a potential promising candidate, as a chemopreventive agent, in the development of keratinocytes-related pathologies.