Z‐Ligustilide inhibits benzo(a)pyrene‐induced CYP1A1 upregulation in cultured human keratinocytes via ROS‐dependent Nrf2 activation

Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is an environmental contaminant that can induce cytochrome P4501A1 (CYP1A1) upregulation via aryl hydrocarbon receptor (AhR) activation and provoke inflammation. Here, we investigated the effect of Z‐Ligustilide, an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on BaP‐induced CYP1A1 upregulation in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z‐Ligustilide significantly inhibited BaP‐induced CYP1A1 upregulation in NHEKs. Treatment of NHEKs with Z‐Ligustilide induced Nuclear factor‐E2‐related factor 2 (Nrf2) nuclear translocation and expression of the Nrf2‐regulated genes for haeme oxygenase‐1 (HO‐1) and NAD(P)H:quinine oxidoreductase‐1 (NQO1). AhR silencing, SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor) and LY294002 (a PI3K inhibitor) did not suppress Z‐Ligustilide‐induced Nrf2 activation. Moreover, treatment of NHEKs with Z‐Ligustilide increased reactive oxygen species (ROS) and L‐N‐acetylcysteine (L‐NAC, an antioxidant) attenuated Z‐ligustilide‐induced Nrf2 nuclear translocation and HO‐1 expression. L‐NAC or knock‐down of Nrf2 significantly attenuated the inhibitory effects of Z‐Ligustilide on BaP‐induced CYP1A1 upregulation in NHEKs. Taken together, these findings suggest that Z‐Ligustilide can suppress BaP‐induced CYP1A1 upregulation through ROS‐dependent Nrf2 pathway activation and may be beneficial in preventing or treating BaP‐induced skin damage.     

Glycyrrhizic acid prevents ultraviolet‐B‐induced photodamage: a role for mitogen‐activated protein kinases,nuclear factor kappa B and mitochondrial apoptotic pathway

Glycyrrhizic acid (GA), a natural triterpene, has received attention as an agent that has protective effects against chronic diseases including ultraviolet UV‐B‐induced skin photodamage. However, the mechanism of its protective effect remains elusive. Here, we used an immortalized human keratinocyte cell line (HaCaT) and a small animal model (BALB/c mice), to investigate the protective effects of GA against UV‐B‐induced oxidative damage, and additionally, delineated the molecular mechanisms involved in the UV‐B‐mediated inflammatory and apoptotic response. In the HaCaT cells, GA inhibited the UV‐B‐mediated increase in intracellular reactive oxygen species (ROS) and down‐regulated the release of pro‐inflammatory cytokines interleukin (IL)‐1α, ‐1β and ‐6, tumor necrosis factor (TNF)‐α and prostaglandin E2 (PGE2). GA inhibited UV‐B‐mediated activation of p38 and JNK MAP kinases, COX‐2 expression and nuclear translocation of NF‐κB. Furthermore, GA inhibited UV‐B‐mediated apoptosis by attenuating translocation of Bax from the cytosol to mitochondria, thus preserving mitochondrial integrity. GA‐treated HaCaT cells also exhibited elevated antiapoptotic Bcl‐2 protein, concomitant with reduced caspase‐3 cleavage and decreased PARP‐1 protein. In BALB/c mice, topical application of GA on dorsal skin exposed to UV‐B irradiation protected against epidermal hyperplasia, lymphocyte infiltration and expression of several inflammatory proteins, p38, JNK, COX‐2, NF‐κB and ICAM‐1. Based on the above findings, we conclude that GA protects against UV‐B‐mediated photodamage by inhibiting the signalling cascades triggered by oxidative stress, including MAPK/NF‐κB activation, as well as apoptosis. Thus, GA has strong potential to be used as a therapeutic/cosmeceutical agent against photodamage.     

NF‐κB is involved in inhibition of lipoxin A4 on dermal inflammation and hyperplasia induced by mezerein

Please cite this paper as: NF‐κB is involved in inhibition of lipoxin A₄ on dermal inflammation and hyperplasia induced by mezerein. Experimental Dermatology 2010; 19 : e286–e288. Abstract: The mechanisms by which lipoxin A₄ (LXA₄) inhibit skin inflammation remain unclear. In the present studies, the ear inflammatory model was induced by topical application of mezerein. Treatment of the mouse ear with LXA₄ exhibited the inhibitory effects on oedema, neutrophil infiltration, vascular permeability, expressions of interleukin (IL)‐1, IL‐6 and IL‐8 mRNA, DNA‐binding activity of nuclear factor‐κB (NF‐κB), and on dermal hyperplasia. NF‐κB reporter activities and nuclear translocations of NF‐κB p65 in cultured keratinocytes stimulated by mezerein were inhibited by pretreatment of the cells with LXA₄. LXA₄ reduced degradation, but not phosphorylation of IκBα in cultured keratinocytes stimulated by mezerein, suggesting that LXA₄‐attenuated IκBα degradation may restore the mezerein‐blocked inhibitory effects of IκB on nuclear translocation and DNA‐binding activity of NF‐κB. Our results demonstrated that LXA₄ displays the anti‐inflammatory and anti‐proliferative role on ear inflammatory model induced by mezerein and these effects were related with downregulation of DNA‐binding activity of NF‐κB.     

Modulation of HMGB1 translocation and RAGE/NFκB cascade by quercetin treatment mitigates atopic dermatitis in NC/Nga transgenic mice

Quercetin, glycosylated form of flavonoid compound, has potent antioxidant and anti‐inflammatory properties. In this study, we have investigated the effects of quercetin on skin lesion, high‐mobility group box (HMGB)1 cascade signalling and inflammation in atopic dermatitis (AD) mouse model. AD‐like lesion was induced by the application of house dust mite extract to the dorsal skin of NC/Nga transgenic mouse. After AD induction, quercetin (50 mg/kg, p.o) was administered daily for 2 weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for HMGB1, receptor for advanced glycation end products (RAGE), toll‐like receptor (TLR)4, nuclear factor (NF)κB, nuclear factor erythroid‐2‐related factor (Nrf)2, kelch‐like ECH‐associated protein (Keap)1, extracellular signal‐regulated kinase (ERK)1/2, cyclooxygenase (COX)2, tumor necrosis factor (TNF)α, interleukin (IL)‐1β, IL‐2Rα and other inflammatory markers in the skin of AD mice. In addition, serum levels of T helper (Th) cytokines (interferon (IFN)γ, IL‐4) were measured by enzyme‐linked immunosorbent assay. Quercetin treatment attenuated the development of AD‐like skin lesions. Histological analysis showed that quercetin inhibited hyperkeratosis, parakeratosis, acanthosis, mast cells and infiltration of inflammatory cells. Furthermore, quercetin treatment downregulated cytoplasmic HMGB1, RAGE, nuclear p‐NFκB, p‐ERK1/2, COX2, TNFα, IL‐1β, IL‐2Rα, IFNγ and IL‐4 and upregulated nuclear Nrf2. Our data demonstrated that the HMGB1/RAGE/NFκB signalling might play an important role in skin inflammation, and quercetin treatment could be a promising agent for AD by modulating the HMGB1/RAGE/NFκB signalling and induction of Nrf2 protein.     

Curcumin inhibits UVB‐induced matrix metalloproteinase‐1/3 expression by suppressing the MAPK‐p38/JNK pathways in human dermal fibroblasts

Curcumin (diferuloylmethane) is a polyphenol derived from turmeric (Curcuma longa), which is commonly used as a spice. Recent studies have shown that curcumin has a wide range of pharmacological activities, including anticarcinogenic, antioxidant, anti‐inflammatory and antiangiogenic activities. However, the antiphotoageing effects of curcumin have yet to be characterized. In this study, we investigated the inhibitory effects of curcumin on matrix metalloproteinase (MMP)‐1 and MMP‐3 expression in human dermal fibroblast cells. Western blot analysis revealed that curcumin inhibited ultraviolet (UV) B‐induced MMP‐1 and MMP‐3 expression. Furthermore, curcumin significantly blocked UVB‐induced reactive oxygen species generation in fibroblasts. Curcumin treatment significantly blocked the UVB‐induced activation of nuclear factor (NF)‐κB and activator protein (AP)‐1. Additionally, curcumin strongly repressed the UVB‐induced phosphorylation of p38 and c‐Jun N‐terminal kinase. Curcumin prevented UVB‐induced MMP expression through mitogen‐activated protein kinase/NF‐κB inhibition and AP‐1 activation. In conclusion, curcumin may be useful for preventing and treating skin photoageing.     

Licochalcone A activates Nrf2 in vitro and contributes to licorice extract‐induced lowered cutaneous oxidative stress in vivo

The retrochalcone licochalcone A (LicA) has previously been shown to possess antimicrobial and anti‐inflammatory properties. In this study, we focused on pathways responsible for the antioxidative properties of LicA. In vitro, LicA protected from oxidative stress mediated by reactive oxygen species (ROS) by activating the expression of cytoprotective phase II enzymes. LicA induced nuclear translocation of NF‐E2‐related factor 2 (Nrf2) in primary human fibroblasts and elevated the expression of the cytoprotective and anti‐inflammatory enzymes heme oxygenase 1 and glutamate–cysteine ligase modifier subunit. LicA‐treated cells displayed a higher ratio of reduced to oxidized glutathione and decreased concentrations of ROS in UVA‐irradiated human dermal fibroblasts, as well as in activated neutrophils. In vivo, ultraweak photon emission analysis of skin treated with LicA‐rich licorice extract revealed a significantly lowered UVA‐induced luminescence, indicative for a decrease in oxidative processes. We conclude from these data that topical application of licorice extract is a promising approach to induce Nrf2‐dependent cytoprotection in human skin.     

Antitumor activity of sulforaphane in mice model of skin cancer via blocking sulfatase‐2

Although there are many treatment options for skin cancer, the chemotherapeutic agents for skin cancer are linked with many adverse effects as well as the development of multidrug resistance. Sulforaphane is an isothiocyanate, which is found in cruciferous vegetables. Consumption of sulforaphane‐rich diet has been linked to inhibition of UV‐exposed skin carcinogenesis. Therefore, the goal of this study was to determine the ability of sulforaphane to reduce skin cancer in mice through inhibition of sulfatase‐2 enzyme. Epicutaneous application of 7,12‐dimethylbenz (a) anthracene was performed on the shaved dorsal skin of mice followed by croton oil. Sulforaphane (9 μmol/mouse/day) was administered to mice orally. Skin was removed from the dorsal area for assessment of sulfatase‐2, glypican‐3, heparan sulphate proteoglycans (HSPGs), nuclear factor (NF)κB, nuclear factor E2‐related factor 2 (Nrf2), tumor necrosis factor (TNF)‐α, IL‐1β and caspase‐3. In addition, skin sections were stained with haematoxylin/eosin, Mallory and cytokeratin immunostaining. We found that, sulforaphane blocked sulfatase‐2 activity, leading to significant elevation in HSPGs as well as significant reduction in glypican‐3. In addition, sulforaphane significantly activated Nrf2 and reduced both the gene and protein expression of NFκB, TNF‐α, IL‐1β and caspase‐3. In parallel, stained sections obtained from skin cancer mice treated with sulforaphane showed significant reduction in hyperkeratosis, acanthosis and epithelial dysplasia. The collective results indicate that sulforaphane suppresses skin cancer via blocking sulfatase‐2 with subsequent elevation in HSPGs and reduction in glypican‐3. Moreover, sulforaphane attenuated skin cancer‐induced activation of inflammatory and apoptotic pathways.     

P38 and JNK signal pathways are involved in the regulation of phlorizin against UVB‐induced skin damage

Phlorizin is well known to inhibit sodium/glucose cotransporters in the kidney and intestine for the treatment of diabetes, obesity and stress hyperglycaemia. However, the effects of phlorizin against ultraviolet B (UVB) irradiation and its molecular mechanism are still unknown. We examined the effects of phlorizin on skin keratinocyte apoptosis, reactive oxygen species (ROS) production, pro‐inflammatory responses after UVB irradiation and the changes of some signal molecules by in vitro and in vivo assay. We observed that phlorizin pretreatments inhibited HaCaT cell apoptosis and overproduction of ROS induced by UVB. Phlorizin also decreased the expression of UVB‐induced pro‐inflammatory cytokines, such as interleukin‐1 beta (IL‐1β), interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) at the mRNA level. Topical application of phlorizin on UVB‐exposed skin of nude mice prevented the formation of scaly skin and erythema, inhibited the increase of epidermal thickness and reduced acute inflammation infiltration in skin. Additionally, PCR, Western blot and immunohistochemical data showed that phlorizin reversed the overexpression of cyclooxygenase‐2 (Cox‐2) induced by UVB irradiation both in vitro and in vivo. The activation of p38 and JNK mitogen‐activated protein kinases (MAPK) after UVB irradiation was also inhibited by phlorizin. These findings suggest that phlorizin is effective in protecting skin against UVB‐induced skin damage by decreasing ROS overproduction, Cox‐2 expression and the subsequent excessive inflammation reactions. It seemed that p38 and JNK MAPK signal pathways are involved in the regulation of the protective function of phlorizin.     

Dihydroavenanthramide D prevents UV‐irradiated generation of reactive oxygen species and expression of matrix metalloproteinase‐1 and ‐3 in human dermal fibroblasts

Ultraviolet B (UVB) radiation induces photoageing by upregulating the expression of matrix metalloproteinases (MMPs) in human skin cells. Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component in oats. Although anti‐inflammatory, anti‐atherosclerotic and antioxidant effects have been reported, the antiphotoageing effects of DHAvD are yet to be understood. In this study, we investigated the inhibitory effects of DHAvD on UVB‐induced production of reactive oxygen species (ROS) and expression of MMPs, and its molecular mechanism in UVB‐irradiated human dermal fibroblasts. Western blot and real‐time PCR analyses revealed that DHAvD inhibited UVB‐induced MMP‐1 and MMP‐3 expression. It also significantly blocked UVB‐induced ROS generation in fibroblasts. Additionally, DHAvD attenuated UVB‐induced phosphorylation of MAPKs, activation of NF‐κB and AP‐1. DHAvD regulates UVB‐irradiated MMP expression by inhibiting ROS‐mediated MAPK/NF‐κB and AP‐1 activation. DHAvD may be a useful candidate for preventing UV light‐induced skin photoageing.     

Baicalein inhibits matrix metalloproteinase 1 expression via activation of TRPV1‐Ca‐ERK pathway in ultraviolet B–irradiated human dermal fibroblasts

Increased matrix metalloproteinase 1 (MMP‐1) expression is a feature of photo‐aged skin. We investigated the effects of baicalein and sulphoraphane on ultraviolet B (UVB) irradiation–induced MMP‐1 expression and apoptosis using human dermal fibroblasts. UVB irradiation not only increased MMP‐1 expression, but also caused apoptosis. Both baicalein and sulphoraphane protected cells from UVB irradiation–induced apoptosis, but only baicalein inhibited MMP‐1 expression. UVB irradiation activated 12‐lipoxygenase, and its product, 12‐hydroxyeicosatetraenoic acid, activated TRPV1 channels. The resulting UVB irradiation–induced Ca²⁺ increase was blocked by the 12‐lipoxygenase inhibitor baicalein and the TRPV1 blocker capsazepine, but not by the Nrf2 inducer sulphoraphane. UVB irradiation also increased ROS generation and decreased Nrf2 protein levels. UVB irradiation–induced MMP‐1 expression was blocked by the Ca²⁺ chelator BAPTA, by capsazepine and by TRPV1 silencing. However, induction was unaffected by the antioxidant N‐acetylcysteine. ERK phosphorylation and JNK phosphorylation were induced by UVB irradiation, but only ERK phosphorylation was Ca²⁺ sensitive. Increased MMP‐1 expression was blocked by PD98059, but not by SP600125. Thus, increased MMP‐1 expression is mediated by increased cytosolic Ca²⁺ and ERK phosphorylation. UVB irradiation–induced ROS generation is also Ca²⁺ sensitive, and UVB irradiation–induced apoptosis is caused by increased ROS. Thus, baicalein, by blocking the UVB irradiation–induced cytosolic Ca²⁺ increase, protects cells from UVB irradiation–induced MMP‐1 expression and apoptosis. In contrast, sulphoraphane, by decreasing cellular ROS, protects cells from only UVB‐induced apoptosis. Thus, targeting 12‐lipoxygenase may provide a therapeutic approach to improving the health of photo‐aged human skin.     

Increased serum HMGB1 levels in patients with Henoch–Schönlein purpura

High‐mobility group box‐1 (HMGB1) has been implicated as a pro‐inflammatory cytokine in the pathogenesis of various inflammatory and autoimmune diseases. However, information about HMGB1 in Henoch–Schönlein purpura (HSP) is still unclear. Herein, we investigated the role of HMGB1 in patients with HSP and the pro‐inflammatory effects of HMGB1 on human dermal microvascular endothelial cell line (HMEC‐1). Serum HMGB1 levels in patients with HSP together with patients with allergic vasculitis (AV) and urticarial vasculitis (UV) were detected by enzyme‐linked immunosorbent assay (ELISA). HMEC‐1 cells were treated with HMGB1 at concentrations ranging from 4 ng/ml to 100 ng/ml. Serum HMGB1 levels were significantly increased in patients with HSP, AV and UV, when compared with those in control group. Moreover, abundant cytoplasmic expression of HMGB1 was observed in endothelial cells in lesional skin of HSP patients. Using membrane cytokine antibody array, we indicate that HMGB1 markedly induced TNF‐α and IL‐6 release in cultured supernatant. Furthermore, by real‐time quantitative PCR and ELISA, the effects of HMGB1 on these cytokines production in HMEC‐1 cells were established. Finally, Western blot data revealed that HMGB1 can induce phosphorylation of inhibitor of κB‐α (IκBα) and the nuclear translocation of nuclear factor‐κB (NF‐κB) p65 in HMEC‐1 cells. In conclusion, this study provides first observations on the association of HMGB1 with HSP. We suggest that HMGB1 may be an important mediator of endothelial inflammation through the induction of TNF‐α and IL‐6 production and may play a crucial role in the pathogenesis of HSP.     

Loss of INK4a/Arf gene enhances ultraviolet radiation‐induced cutaneous tumor development

The CDKN2A locus encodes for tumor suppressor genes p16^INK4a and p14^Arf which are frequently inactivated in human skin tumors. The purpose of this study was to determine the relationship between loss of INK4a/Arf activity and inflammation in the development of ultraviolet (UV) radiation‐induced skin tumors. Panels of INK4a/Arf^‐/? mice and wild‐type (WT) mice were treated with a single dose of UVB (200 mJ/cm²). For long‐term studies, these mice were irradiated with UVB (200 mJ/cm²) three times weekly for 30 weeks. At the end of the experiment, tissues were harvested from mice and assayed for inflammatory biomarkers and cytokines. A single dose of UVB resulted in a significant increase in reactive oxygen species (ROS) and 8‐dihydroxyguanosine (8‐oxo‐dG) lesions in INK4a/Arf^?/? mice compared to WT mice. When subjected to chronic UVB, we found that 100% of INK4a/Arf^?/‐ mice had tumors, whereas there were no tumors in WT controls after 24 weeks of UVB exposure. The increase in tumor development correlated with a significant increase in nuclear factor (NF)‐κB, cyclooxygenase‐2 (COX‐2), prostaglandin E₂ (PGE₂) and its receptors both in UVB‐exposed skin and in the tumors. A significant increase was seen in inflammatory cytokines in skin samples of INK4a/Arf^‐/‐ mice following treatment with chronic UVB radiation. Furthermore, significantly more CD11b⁺Gr1⁺ myeloid cells were present in UVB‐exposed INK4a/Arf^‐/‐ mice compared to WT mice. Our data indicate that by targeting UVB‐induced inflammation, it may be possible to prevent UVB‐induced skin tumors in individuals that carry CDKN2A mutation.     

Melanocyte‐protective effect of afzelin is mediated by the Nrf2‐ARE signalling pathway via GSK‐3β inactivation

Vitiligo is an acquired condition characterized by depigmented, cutaneous lesions that result from the death of pigment‐producing cells, melanocytes. The occurrence of oxidative stress has been proposed as a pathogenetic mechanism for melanocyte degeneration in vitiligo. Therefore, in this study, we investigated the cytoprotective effects of afzelin against oxidative stress and its mechanism of action in human epidermal melanocytes. We found that afzelin significantly inhibited hydrogen peroxide‐induced cell death, cellular reactive oxygen species production and lipid peroxidation in melanocytes. In an antioxidant response element (ARE)‐luciferase reporter assay, afzelin increased ARE‐luciferase reporter activity in a concentration‐dependent manner. Consistently, the expression of antioxidant genes, including NF‐E2‐related factor 2 (Nrf2), haem oxygenase‐1 (HO‐1) and catalase, was enhanced by afzelin treatment. Nuclear translocation of Nrf2 also increased in response to afzelin treatment. In addition, the phosphorylation of glycogen synthase kinase‐3β (GSK‐3β) was induced by afzelin treatment. The enhancement of HO‐1 gene expression by afzelin treatment was reduced by Nrf2‐siRNA expression. Furthermore, we found that the expression of Nrf2‐siRNA significantly attenuated the cytoprotective effect of afzelin against hydrogen peroxide. These data suggest that the cytoprotective effects of afzelin against hydrogen peroxide may be mediated by Nrf2‐ARE signalling via GSK‐3β inactivation. Our data suggest the novel use of afzelin for the prevention of oxidative stress‐induced damage in melanocytes and its potential as a therapeutic agent for vitiligo.     

Intercellular pathway through hyaluronic acid in UVB‐induced inflammation

Ultraviolet B (UVB) radiation induces inflammation in the skin specifically at the site of exposure. We unexpectedly found that UVB‐induced inflammation was not induced in gp91phox‐depleted mice. To test whether gp91phox is directly involved in UVB‐induced inflammation, neutrophil‐ and hyaluronic acid–depleted mice were also irradiated and examined for their response. Hyaluronic acid–depleted mice showed strongly inhibited UVB‐induced inflammation, but the neutrophil‐depleted mice did not exhibit any suppressed UVB‐induced inflammation. To elucidate the pathway by which UVB irradiation induced inflammation, we examined the expression of nucleotide‐binding domain, leucine‐rich‐containing family, pyrin domain‐containing‐3 (NLRP3) and caspase‐1 in the mouse skin. An increase in the expression of NLRP3 and caspase‐1 was seen following the UVB irradiation of C57BL mice; however, the UVB‐irradiated gp91phox‐knockout (gp91phox^?/?) mice did not have this increase in expression. Furthermore, the plasma IL‐1β level increased after the UVB irradiation in C57BL mice, but there was no change in the gp91phox^?/? mice. These results clearly indicate that nicotinamide adenine dinucleotide phosphate oxidase is activated by gp91phox, which is expressed on the surface in response to the increased expression of hyaluronic acid induced by UVB irradiation, and as result, the generation of reactive oxygen species (ROS) increases. This ROS activate NLRP3, and NLRP3 leads to the production of caspase‐1, which subsequently increases IL‐1β, thereby finally inducing inflammation. It is thought that this system may play an important role in the damage and ageing of skin, and further studies are necessary to confirm these finding.