Pretreatment Of HaCaT Cells Research Articles

Photoprotective activities of Lignosus rhinocerus in UV-irradiated human keratinocytes

Ethnopharmacological relevanceLignosus rhinocerus, also known as Tiger Milk Mushroom has been used traditionally to treat a variety of human conditions, including asthma, diabetes, respiratory disease, skin allergy, and food poisoning. The reported activities of Lignosus rhinocerus extracts include anti-inflammatory, anti-oxidant, anti-asthmatic, anti-microbial, anti-cancer, neuroprotection, and immune modulation effects. However, its effect on human skin is not well documented, including human skin exposed to ultraviolet light (UV). Exposure to UV can trigger various cellular responses, including inflammation, oxidative stress, DNA damage, cell death, and cellular aging. Aim of the studyThe study aims to investigate the effects of methanolic extract prepared from cultured Lignosus rhinocerus (herein referred to as TM02 and its methanol extract as TM02-ME) on UV-irradiated human keratinocytes. Materials and methodsPowdered stock of TM02 was dissolved and sequentially extracted with different solvents to prepare the extracts and the methanol extract was subsequently characterized based on its bio-activities on HaCaT human keratinocytes. The keratinocytes were pre-treated with the methanol extract followed by UV-irradiation. Cellular responses of the HaCaT cells such as cell viability, DNA damage, as well as gene and protein expressions that were responsive to the treatments, were characterized by using bio-assays, including reverse-transcription based PCR, Western blot, cell viability, and mitochondrial Cytochrome C release assays. ResultsTM02-ME protected HaCaT cells from UV-induced DNA damage and cell death in a dose-dependent manner. Pre-treatment of HaCaT cells with TM02-ME led to a 39% reduction of cyclobutane pyrimidine dimers (CPD) and up-regulated the gene expression of REV1 and SPINK5 in UVB-irradiated HaCaT cells when compared to the control. In addition, TM-02-ME treated HaCaT cells increased the expression of BCL-XL and BCL-2 proteins which coincided with the down-regulation of mitochondrial Cyt. C release in the UV-B irradiated HaCaT cells. The results were further supported by data that showed the stable clones of HaCaT cells stably expressed BCL-XL were resistant to UVB-induced cell death. Conclusions__The results showed that TM02-ME confers photoprotective activities to UVB-irradiated HaCaT cells, leading to a reduction in DNA damage and cell death as well as up-regulated the expression of REV1 and SPINK5 which are involved in DNA repair and skin barrier function, respectively. The up-regulation of pro-survival members of the BCL-2 family by TM02-ME confers protection against UVB-induced cell death.

Novel Antioxidant Peptides from Crassostrea Hongkongensis Improve Photo-Oxidation in UV-Induced HaCaT Cells.

Enzymatic hydrolysates from Oysters (OAH) display multiple biological activities. Previously, a 3~5 KDa oyster ultrafiltration component (OUP) showed a high property of preventing skin oxidation. Subsequently, we identified specific peptides with such activity. OUP was fractionated stepwise by Sephadex-G25 and RP-HPLC, and active fractions were screened using UV-irradiated HaCaT cells. The most active fractions (OP5-3) were analyzed by LC-MS/MS and a total of 17 peptides were identified. Results from mass spectrometry showed that OP5-3 consisted of peptides with a molecular weight range of 841.51–1786.92 Da. Six of these peptides were synthesized for validating the activity of resisting skin oxidation in the same cell model. All six peptides showed varying degrees of antioxidant activity, while pretreatment of HaCaT cells with AIVAEVNEAAK alleviated UV cytotoxicity, inhibited metalloproteinase 1 (MMP-1) expression, and showed the highest activity to resist UV-induced skin photo-oxidation among these peptides. In addition, results from molecular docking analysis of MMP-1 with AIVAEVNEAAK showed that AIVAEVNEAAK suppresses its enzymatic activity by directly interacting with MMP-1 and thus exhibit anti-photoaging activity.

Abstract P030: Chemoprevention by the mutant p53 reactivator SLMP53-2 on ultraviolet radiation-induced skin cancer

Abstract The growing incidence of skin cancer (SC) is a global public health concern. Preventive approaches mostly embrace the sunscreen use against ultraviolet radiation (UVR), the main SC etiologic factor. Yet, the increasing SC incident have suggested that additional preventive agents could complement the sunscreen, helping to reverse this trend. Mutant p53 (mutp53) represents a promising target in SC prevention, since its occurrence is a key early event in UVR-induced SC. Thus, the inhibition of mutp53 formation by mutp53 reactivators, which are able to restore the wild-type-like function to p53, would be a valuable strategy for SC prevention. Recently, our group identified the tryptophanol-derived oxazoloisoindolinone SLMP53-2 as a new mutp53 reactivator. Herein, we aimed to study the potential of SLMP53-2 as a chemopreventive drug against UVR-induced SC. For that, assays with normal human keratinocyte HaCaT cells were performed. Cells were pre-treated with SLMP53-2 for 24h, followed by exposure to distinct UVB doses and further analysis. For in vivo studies, FVB/N mice were topically pre-treated for 1h prior to UVB radiation (180 mJ/cm2) and the skin was collected 24h after for immunohistochemistry/immunofluorescence analysis. Pre-treatment of HaCaT cells with SLMP53-2 enhanced cell survival in response to UVB with promotion of cell cycle arrest in G0/G1 phase, upregulation of p21 protein levels, reduction of UVB-induced apoptosis, and inhibition of JNK activity. SLMP53-2 also protected cells from UVB-induced ROS generation and from its consequent lipid peroxidation and protein carbonylation. It enhanced DNA repair through upregulation of the protein and mRNA levels of XPC (xeroderma pigmentosum complementation group C) and DDB-2 (DNA damaged binding protein 2) from the NER (nucleotide excision repair) pathway. In fact, SLMP53-2 depleted UVB-induced DNA damage, as evidenced by a reduction of DNA in comet tails, γH2AX staining and cyclobutane pyrimidine dimers (CPDs) levels. SLMP53-2 also showed promising activity in suppressing UVB-induced inflammation, decreasing COX-2, IL-6 and TNF-α protein levels and reducing NF-kB nuclear translocation and DNA binding ability. Moreover, SLMP53-2 enhanced the expression of key players in keratinocytes differentiation, namely NOTCH1 and keratin 1. Consistently, pre-treatment with SLMP53-2 reduced mutp53 protein levels by potentially restoring its wild-type p53 form, which may underlie the increased resistance of HaCaT cells to UVB harmful effects. Importantly, in vivo studies supported that SLMP53-2 pre-treatment of UVB-irradiated mice promoted the survival of epidermal cells, reducing the DNA damage by enhancement of DNA repair and downregulating mutp53 expression levels. Further, it reduced the expression of inflammatory-related proteins and promoted cell differentiation. Collectively, these results support a promising chemopreventive activity for SLMP53-2 against UVB-induced skin damage. We thank FCT/MCTES for the projects UIDB/50006/2020, UIDP/04423/2020 and fellowship SFRH/BD/128673/2017 (J.B. Loureiro). Citation Format: Joana B. Loureiro, Rita Ribeiro, Nair Nazareth, Tiago Ferreira, Adelina Quaresma, Valentina Barcherini, Laura Marabini, Paula A. Oliveira, Maria M. M. Santos, Lucília Saraiva. Chemoprevention by the mutant p53 reactivator SLMP53-2 on ultraviolet radiation-induced skin cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P030.

Bucillamine Inhibits UVB-Induced MAPK Activation and Apoptosis in Human HaCaT Keratinocytes and SKH-1 Hairless Mouse Skin.

Ultraviolet B (UVB) radiation is known as a culprit in skin carcinogenesis. We have previously reported that bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine), a cysteine derivative with antioxidant and anti-inflammatory capacity, protects against UVB-induced p53 activation and inflammatory responses in mouse skin. Since MAPK signaling pathways regulate p53 expression and activation, here we determined bucillamine effect on UVB-mediated MAPK activation in vitro using human skin keratinocyte cell line HaCaT and in vivo using SKH-1 hairless mouse skin. A single low dose of UVB (30mJcm-2 ) resulted in increased JNK/MAPK phosphorylation and caspase-3 cleavage in HaCaT cells. However, JNK activation and casaspe-3 cleavage were inhibited by pretreatment of HaCaT cells with physiological doses of bucillamine (25 and 100µm). Consistent with these results, bucillamine pretreatment in mice (20mgkg-1 ) inhibited JNK/MAPK and ERK/MAPK activation in skin epidermal cells at 6-12 and 24h, respectively, after UVB exposure. Moreover, bucillamine attenuated UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in mouse skin. These findings demonstrate that bucillamine inhibits UVB-induced MAPK signaling, cell proliferation and apoptosis. Together with our previous report, we provide evidence that bucillamine has a photoprotective effect against UV exposure.

Ethanol extract of Gynura bicolor (GB) protects against UVB-induced photodamage of skin by inhibiting P53-mediated Bcl-2/BAX/Caspase-3 apoptosis pathway.

To investigate the protective effect of ethanol extract of Gynura bicolor (GB) against UVB-induced photodamage of skin and the possible mechanisms. DPPH (1,1-diphenyl-2-pico radical) test was used to detect the antioxidant capacity of ethanol extract of Gynura bicolor (GB). The protective effects of GB against UVB irritation were detected both in Hacat cells and photodamage rat models. UVB irradiation could inhibit viability and induce apoptosis of Hacat cells in a dose-dependent manner. The pretreatment of Hacat cells by GB could obviously reverse the effects in a dose-dependent manner. The mRNA and protein expressions of p53, Bax, caspase-3 were increased, while anti-apoptotic protein Bcl-2 was decreased and this effect could be reversed by GB pretreatment in a dose-dependent manner. In vivo, the application of GB could alleviate the skin damage of SD rats and improve the superficial inflammation of the dermis as well as inhibit the expressions of P53 and Caspase-3 induced by UVB irradiation. Ethanol extract of Gynura bicolor could protect the photodamage of human Hacat keratinocytes and SD rats against UVB irradiation by inhibiting P53-mediated Bcl-2/ BAX/Casaspe-3 apoptosis pathway.

Effects of tacrolimus in treating vitiligo on the secretion of local chemokine

Objective To analyze the abnormalities of local chemokines in patients with vitiligo and to explore the effect of tacrolimus on the secretion of chemokines in keratinocytes. Methods Blister fluids of 50 patients with vitiligo were collected, including lesion areas and normal areas. Luminex was used to analyze the concentration of local chemokines in patients with vitiligo to determine whether the chemokines were closely related to vitiligo. The effect of tacrolimus on chemokine secretion of was analyzed by Western blot in HaCaT cells. Results By Luminex analysis of blister fluid, it was found that CXCL9 and CXCL10 were significantly higher in the leukoplakia of vitiligo, and there was a significant difference, compared with the blister fluid in the normal site (P<0.01). IFN-γ significantly stimulated the keratinocyte cell line HaCat to express CXCL9 and CXCL10. After pretreatment of HaCaT cells with 20 mg tacrolimus, the expression of CXCL9 and CXCL10 was significantly decreased, compared with the blank control (P<0.01). Conclusions The leukoplakia chemokines CXCL9 and CXCL10 are highly expressed in vitiligo patients. The tacrolimus can significantly reduce the expression of CXCL9 and CXCL10 in keratinocytes under stress, and it therefore plays a therapeutic role in vitiligo. Key words: Vitiligo; Tacrolimus; Chemokine CXCL9; Chemokine CXCL10; HaCaT cells

피부각질세포에서 梔子柏皮湯의 아토피 피부염 개선효과

Chijabaegpi-tang (CHG) is an oriental herbal medicine that has been used for its various pharmacological effects, which include anti-inflammatory, anti-oxidant and immunoregulation activities. In the present study, we investigated which skin inflammations are involved in the TNF-α/IFNγ-induced HaCaT cells. We investigated the suppressive effect of CHG on TNF-α/IFNγ-induced HaCaT cell production of the following chemokines: macrophage-derived chemokine (MDC)/CCL22; regulated on activation, normal T-cell expressed and secreted (RANTES)/CCL5; and interleukin-8 (IL-8); thymus and activation-regulated chemokine (TARC)/CCL17. The pre-treatment of HaCaT cells with CHG suppressed TNF-α/IFNγ-induced nuclear transcription factor kappa-B (NF-κB). In addition, CHG inhibited TNF-α/IFNγ-induced phosphorylation of ERK and p38. TNF-α/IFNγ suppressed the expression of skin barrier proteins, including filaggrin (FLG), Involucrin (IVL) and loricrin (LOR). By contrast, CHG restored the expression of FLG, IVL and LOR. Taken together, our findings suggest that CHG could be a therapeutic agent for prevention of skin disease, including atopic dermatitis.

Protective roles of NRF2 signaling pathway in cobalt chloride-induced hypoxic cytotoxicity in human HaCaT keratinocytes

Hypoxia is a key pathological process involved in many cutaneous diseases. Nuclear factor E2-related factor 2 (NRF2) is a central regulator of antioxidant response element (ARE)-dependent transcription and plays a pivotal role in the cellular adaptive response to oxidative stress. Kelch-like ECH-associated protein 1 (KEAP1) is a cullin-3-adapter protein that represses the activity of NRF2 by mediating its ubiquitination and degradation. In the present study, we examined the role of NRF2 signaling pathway in the cytotoxicity induced by cobalt chloride(CoCl2), a hypoxia-mimicking agent, in human keratinocyte HaCaT cells with stable knockdown of NRF2 (NRF2-KD) and KEAP1 (KEAP1-KD). Acute CoCl2 exposure markedly increased the levels of intracellular reactive oxygen species (ROS), and resulted in hypoxic damage and cytotoxicity of HaCaT cells. Stable knockdown of NRF2 dramatically reduced the expression of many antioxidant enzymes and sensitized the cells to acute CoCl2-induced oxidative stress and cytotoxicity. In contrast, KEAP1-KD cells observably enhanced the activity of NRF2 and ARE-regulated genes and led to a significant resistance to CoCl2-induced cellular damage. In addition, pretreatment of HaCaT cells with tert-butylhydroquinone, a well-known NRF2 activator, protected HaCaT cells from CoCl2-induced cellular injury in a NRF2-dependent fashion. Likewise, physical hypoxia-induced cytotoxicity could be significantly ameliorated through NRF2 signaling pathway in HaCaT cells. Together, our results suggest that NRF2 signaling pathway is involved in antioxidant response triggered by CoCl2-induced oxidative stress and could protect human keratinocytes against acute CoCl2 -induced hypoxic cytotoxicity.

546 Kaempferol and Isorhamnetin isolated from the Inula britannica prevents heat-induced MMP-1 expression by inhibiting MAPKs and AP-1 signaling in HaCaT cells

The skin aging process can be divided into intrinsic aging and extrinsic aging like photo-aging. However, recent studies have shown that heat also can cause skin aging, called thermal skin aging, by fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). In this study, we investigated the influence of Kaempferol and Isorhamnetin isolated from the Inula Britannica on heat-exposed HaCaT cells (immortalized human keratinocytes) and the relationship among MMP-1 expression, c-Jun and MAP kinases. Kaempferol and Isorhamnetin showed very good reactive oxygen species (ROS) activity in DPPH assay and increased the viability of heat-exposed HaCaT cells. Especially, Pre-treatment of HaCat cells with Kaempferol and Isorhamnetin declined MMP-1 level, which is over expressed by heat. We studied the influence of heat on c-Jun, MAP Kinases, and AP-1 by Western blotting and enzymic activity assay with and without Kaempferol and Isorhamnetin treatment. They suppressed c-Jun and the phosphorylation of MAP Kinases, upstream modulators of AP-1 after heat exposure condition. These results indicate that Kaempferol and Isorhamnetin can inhibit heat-induced MMP-1 expression by interfering with MAP Kanases and AP-1 signaling. Our studies demonstrate that Kaempferol and Isorhamnetin isolated from the Inula Britannica may be potentially useful in the prevention and treatment of thermal skin aging.

Protective effects of TES trioleate, an inhibitor of phospholipase A2, on reactive oxygen species and UVA-induced cell damage

2-[Tris(oleoyloxymethyl)methylamino]-1-ethane sulfonic acid (TES trioleate) is an inhibitor of phospholipase A2 (PLA2), which hydrolyzes cell membrane phospholipids to produce arachidonic acid (AA) and lysophospholipids (LysoPLs). Here, we investigated the protective effects of TES trioleate on cell damage caused by ultraviolet A (UVA) light and reactive oxygen species (ROS). Pre-incubation with 250–1000μM TES trioleate resulted in concentration-dependent protection from UVA-induced damage in HaCaT cells. Additionally, 25–1000μM TES trioleate provided protection against H2O2 in a concentration-dependent manner. In human erythrocytes treated with 1O2, 10–100μM TES trioleate showed concentration-dependent protective effects, similar to but stronger than the controls, 4-BPB and lipophilic antioxidant (+)-α-tocopherol at 100μM. TES trioleate did not have detectable radical scavenging activity. Moreover, compared with (+)-α-tocopherol and rutin, TES trioleate showed low ROS scavenging activity. Thus, although TES trioleate showed cell protective effects against UVA, H2O2, and 1O2-induced damages, these effects were not caused by the scavenging ability of the radical or ROS. Finally, pretreatment of HaCaT cells and human erythrocytes with l-α-lysophosphatidylcholine produced by PLA2 promoted increased cell damage at low concentrations. Thus, the protective effects of TES trioleate on cellular damage by UVA and ROS may be associated with inhibition of PLA2-dependent cell damage rather than ROS scavenging.

UVB Generates Microvesicle Particle Release in Part Due to Platelet-activating Factor Signaling.

The lipid mediator platelet-activating factor (PAF) and oxidized glycerophosphocholine PAF agonists produced by ultraviolet B (UVB) have been demonstrated to play a pivotal role in UVB-mediated processes, from acute inflammation to delayed systemic immunosuppression. Recent studies have provided evidence that microvesicle particles (MVPs) are released from cells in response to various signals including stressors. Importantly, these small membrane fragments can interact with various cell types by delivering bioactive molecules. The present studies were designed to test if UVB radiation can generate MVP release from epithelial cells, and the potential role of PAF receptor (PAF-R) signaling in this process. We demonstrate that UVB irradiation of the human keratinocyte-derived cell line HaCaT resulted in the release of MVPs. Similarly, treatment of HaCaT cells with the PAF-R agonist carbamoyl PAF also generated equivalent amounts of MVP release. Of note, pretreatment of HaCaT cells with antioxidants blocked MVP release from UVB but not PAF-R agonist N-methyl carbamyl PAF (CPAF). Importantly, UVB irradiation of the PAF-R-negative human epithelial cell line KB and KB transduced with functional PAF-Rs resulted in MVP release only in PAF-R-positive cells. These studies demonstrate that UVB can generate MVPs in vitro and that PAF-R signaling appears important in this process.

Ginsenoside Rg5:Rk1 attenuates TNF-α/IFN-γ-induced production of thymus- and activation-regulated chemokine (TARC/CCL17) and LPS-induced NO production via downregulation of NF-κB/p38 MAPK/STAT1 signaling in human keratinocytes and macrophages

Atopic dermatitis (AD) is a chronic skin disease that affects millions of people worldwide. Keratinocytes and macrophages are two cells types that play a pivotal role in the development of AD. These cells produced different chemokines and cytokines, especially thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), as well as nitric oxide (NO) through inducible nitric oxide synthase (iNOS) and COX2 in response to stimulation by TNF-α/IFN-γ and lipopolysaccharide (LPS) respectively. These mediators are thought to be crucial regulators of the pathogenesis of AD. Although several natural compounds to treat AD have been studied, the effect of Rg5:Rk1 from Panax ginseng (P. ginseng) on AD has not yet been investigated. In this study, we evaluated the inhibitory effect of Rg5:Rk1 on TNF-α/IFN-γ stimulated keratinocytes (HaCaT cells) and LPS-stimulated macrophages (RAW 264.7 cells). Enzyme-linked immunosorbent assay (ELISA) data showed that pretreatment of HaCaT cells with Rg5:Rk1 significantly reduced the TNF-α/IFN-γ-induced increase in TARC/CCL17 expression in a dose-dependent manner. In addition, Rg5:Rk1 decreased LPS-mediated nitric oxide (NO) and reactive oxygen species (ROS) production in RAW 264.7 cells. A considerable reduction in messenger RNA (mRNA) expression of the aforementioned AD mediators was also observed. Pretreatment with Rg5:Rk1 attenuated the TNF-α/IFN-γ-induced phosphorylation of p38 MAPK, STAT1, and NF-κB/IKKβ in HaCaT cells. Together, these findings suggest that ginsenoside Rg5:Rk1 may have a potential anti-AD effect by suppressing NF-κB/p38 MAPK/STAT1 signaling.

Size is an essential parameter in governing the UVB-protective efficacy of silver nanoparticles in human keratinocytes.

BackgroundUltraviolet (UV) radiation from sun, particularly its UVB component (290–320 nm), is considered the major etiological cause of skin cancer that impacts over 2 million lives in the United States alone. Recently, we reported that polydisperse colloidal suspension of silver nanoparticles (AgNPs) protected the human keratinocytes (HaCaT) against UVB-induced damage, thus indicating their potential for prevention of skin carcinogenesis. Here we sought out to investigate if size controlled the chemopreventive efficacy of AgNPs against UVB-induced DNA damage and apoptosis.MethodsPercent cell viability was examined by WST-1 assay after treating the cells with various doses (1–10 μg/mL) of AgNPs of different sizes (10, 20, 40, 60 and 100 nm) for 12 and 24 h. For protection studies, cells were treated with AgNPs of different sizes at a uniform concentration of 1 μg/mL. After 3 h, cells were irradiated with UVB (40 mJ/cm2) and dot-blot analysis was performed to detect cyclobutane pyrimidine dimers (CPDs) as an indication of DNA damage. Apoptosis was analyzed by flow cytometry after staining the cells with 7-Amino-Actinomycin (7-AAD) and PE Annexin V. Immunoblot analysis was accomplished by processing the cells for protein extraction and Western blotting using specific antibodies against various proteins.ResultsThe data show that the pretreatment of HaCaT cells with AgNPs in the size range of 10–40 nm were effective in protecting the skin cells from UVB radiation-induced DNA damage as validated by reduced amounts of CPDs, whereas no protection was observed with AgNPs of larger sizes (60 and 100 nm). Similarly, only smaller size AgNPs (10–40 nm) were effective in protecting the skin cells from UV radiation-induced apoptosis. At the molecular level, UVB –irradiation of HaCaT cells led to marked increase in expression of pro-apoptotic protein (Bax) and decrease in anti-apoptotic proteins (Bcl-2 and Bcl-xL), while it remained largely unaffected in skin cells pretreated with smaller size AgNPs (10–40 nm).ConclusionsAltogether, these findings suggest that size is a critical determinant of the UVB-protective efficacy of AgNPs in human keratinocytes.

Spinasterol‐glucose inhibits IFN‐γ/TNF‐α‐induced expression of TARC/CCL17 via blocking NF‐κB and STAT1 activation in human HaCaT keratinocytes

TARC/CCL17 is a Th2‐type chemokine and is implicated in the development of Th2‐skewed allergic diseases. TARC/CCL17 expression is induced by pro‐inflammatory cytokines such as IFN‐γ and TNF‐α. A phytosterol, 3‐O‐β‐D‐glucopyanosylspinasterol (spinasterol‐Glc), was isolated from Stewartia koreana leaves, and was previously shown to inhibit the production of NO and pro‐inflammatory cytokines in macrophage cells. In the present study, we investigated the effect of Spinasterol‐Glc on production of TARC/CCL17 induced by IFN‐γ and TNF‐α in HaCaT keratinocytes. Pretreatment of HaCaT cells with spinasterol‐Glc inhibited the mRNA and protein expression of TARC/CCL17 induced by IFN‐γ/TNF‐α in a dose‐dependent manner. Spinasterol‐ Glc suppressed IFN‐γ/TNF‐α‐induced activation of NF‐κB and STAT1. Spinastrol‐Glc also inhibited phosphorylation of IκB‐α and IKKα/β as well as nuclear translocation of NF‐κB. We demonstrated that spinasterol‐Glc reduced IFN‐γ/TNF‐α‐induced transcription of the NF‐κB‐ and the GAS‐ minimal promoters. In addition, spinasterl‐Glc suppressed the DNA binding of NF‐κB and STAT1 to its cognate consensus site. These results suggest that spinasterol‐Glc has effective inhibitory effects on production of TARC/CCL17 via inhibition of NF‐κB as well as STAT activation, and may be used as a potential anti‐inflammatory agent for the prevention and treatment of skin inflammatory diseases.

Cross-regulations among NRFs and KEAP1 and effects of their silencing on arsenic-induced antioxidant response and cytotoxicity in human keratinocytes.

Background: Nuclear factor E2-related factors (NRFs), including NRF2 and NRF1, play critical roles in mediating the cellular adaptive response to oxidative stress. Human exposure to inorganic arsenic, a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer.Objective: We investigated the cross-regulations among NRF2, NRF1, and KEAP1, a cullin-3–adapter protein that allows NRF2 to be ubiquinated and degraded by the proteasome complex, in arsenic-induced antioxidant responses.Results: In human keratinocyte HaCaT cells, selective knockdown (KD) of NRF2 by lentiviral short hairpin RNAs (shRNAs) significantly reduced the expression of many antioxidant enzymes and sensitized the cells to acute cytotoxicity of inorganic arsenite (iAs3+). In contrast, silencing KEAP1 led to a dramatic resistance to iAs3+-induced apoptosis. Pretreatment of HaCaT cells with NRF2 activators, such as tert-butylhydroquinone, protects the cells against acute iAs3+ toxicity in an NRF2-dependent fashion. Consistent with the negative regulatory role of KEAP1 in NRF2 activation, KEAP1-KD cells exhibited enhanced transcriptional activity of NRF2 under nonstressed conditions. However, deficiency in KEAP1 did not facilitate induction of NRF2-target genes by iAs3+. In addition, NRF2 silencing reduced the expression of KEAP1 at transcription and protein levels but increased the protein expression of NRF1 under the iAs3+-exposed condition. In contrast, silencing KEAP1 augmented protein accumulation of NRF2 under basal and iAs3+-exposed conditions, whereas the iAs3+-induced protein accumulation of NRF1 was attenuated in KEAP1-KD cells.Conclusions: Our studies suggest that NRF2, KEAP1, and NRF1 are coordinately involved in the regulation of the cellular adaptive response to iAs3+-induced oxidative stress.

Saponins from the roots of Platycodon grandiflorum suppress ultraviolet A-induced matrix metalloproteinase-1 expression via MAPKs and NF-κB/AP-1-dependent signaling in HaCaT cells

Saponins from the roots of Platycodon grandiflorum (CKS) have been shown to exhibit many pharmacological activities, including anti-cancer and anti-inflammatory activities and antioxidant effects. However, anti-skin photoaging effects of CKS have not yet been reported. In this study, we investigated the protective effects of CKS against UVA damage on immortalized human keratinocytes (HaCaT). We then explored the inhibitory effects of CKS on UVA-induced MMP-1 and investigated the molecular mechanism underlying those effects. CKS increased the cell viability and inhibited reactive oxygen species (ROS) production in HaCaT cells exposed to UVA irradiation. Pre-treatment of HaCaT cells with CKS inhibited UVA-induced production of MMP-1 and MMP-9. In addition, CKS decreased UVA-induced expression of the inflammatory cytokines IL-1β and IL-6. Western blot analysis further revealed that CKS markedly suppressed the enhancement of collagen degradation in UVA-exposed HaCaT cells. CKS also suppressed UVA-induced activation of NF-κB or c-Jun and c-Fos, and the phosphorylation of MAPKs, which are upstream modulators of NF-κB and AP-1.

Hydrogen Sulfide Protects against Chemical Hypoxia-Induced Cytotoxicity and Inflammation in HaCaT Cells through Inhibition of ROS/NF-κB/COX-2 Pathway

Hydrogen sulfide (H2S) has been shown to protect against oxidative stress injury and inflammation in various hypoxia-induced insult models. However, it remains unknown whether H2S protects human skin keratinocytes (HaCaT cells) against chemical hypoxia-induced damage. In the current study, HaCaT cells were treated with cobalt chloride (CoCl2), a well known hypoxia mimetic agent, to establish a chemical hypoxia-induced cell injury model. Our findings showed that pretreatment of HaCaT cells with NaHS (a donor of H2S) for 30 min before exposure to CoCl2 for 24 h significantly attenuated CoCl2-induced injuries and inflammatory responses, evidenced by increases in cell viability and GSH level and decreases in ROS generation and secretions of IL-1β, IL-6 and IL-8. In addition, pretreatment with NaHS markedly reduced CoCl2-induced COX-2 overexpression and PGE2 secretion as well as intranuclear NF-κB p65 subunit accumulation (the central step of NF-κB activation). Similar to the protective effect of H2S, both NS-398 (a selective COX-2 inhibitor) and PDTC (a selective NF-κB inhibitor) depressed not only CoCl2-induced cytotoxicity, but also the secretions of IL-1β, IL-6 and IL-8. Importantly, PDTC obviously attenuated overexpression of COX-2 induced by CoCl2. Notably, NAC, a ROS scavenger, conferred a similar protective effect of H2S against CoCl2-induced insults and inflammatory responses. Taken together, the findings of the present study have demonstrated for the first time that H2S protects HaCaT cells against CoCl2-induced injuries and inflammatory responses through inhibition of ROS-activated NF-κB/COX-2 pathway.

The flavonoids apigenin and luteolin suppress ultraviolet A-induced matrix metalloproteinase-1 expression via MAPKs and AP-1-dependent signaling in HaCaT cells

Ultraviolet (UV) irradiation causes major changes in skin connective tissues as a result of the degradation of collagen, a major structural component of the extracellular matrix. This process is likely mediated by matrix metalloproteinases (MMPs). Such changes in collagenous skin tissues have been suggested to be causes of cutaneous aging and skin cancer. We investigated the protective effects of apigenin and luteolin on immortalized human keratinocytes (HaCaT) against UVA damage. We then explored the inhibitory effects of apigenin and luteolin on UVA-induced MMP-1 and investigated the molecular mechanism underlying those effects. HaCaT cells were treated with apigenin and luteolin for the indicated times followed by irradiation with UVA. Those effects were assessed by semi-quantitative PCR, Western blotting and enzymic activity assays. These two compounds, at concentrations of 1-5μM, increased the viability of, and inhibited ROS production in HaCaT cells exposed to UVA irradiation. Pre-treatment of HaCaT cells with apigenin and luteolin also inhibited UVA-induced production of the collagenases MMP-1. They also suppressed UVA-induced expression of c-Jun and c-Fos and the phosphorylation of three MAP kinases, upstream modulators of AP-1. Furthermore, the same two flavonoids decreased the UVA-induced influx of Ca(2+) into HaCaT cells and the phosphorylation of Ca(2+)/calmodulin-dependent kinases (CaMKs). The results indicate that apigenin and luteolin inhibited UVA-induced collagenolytic MMP-1 production by interfering with Ca(2+)-dependent MAPKs and AP-1 signaling. They may thus be potentially useful in the prevention and treatment of skin photoaging.

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta- O-galloyl-β- d-glucose via blockade of NF-κB and STAT1 activation in the HaCaT cells

Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta- O-galloyl-β- d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-α/IFN-γ induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-α/IFN-γ-induced NF-κB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-γ. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-α and/or IFN-γ-induced activation of NF-κB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.

Inhibition of UVB‐mediated Oxidative Stress and Markers of Photoaging in Immortalized HaCaT Keratinocytes by Pomegranate Polyphenol Extract POMx

In recent years there has been an increase in use of botanicals with antioxidant properties as skin photoprotective agents. Pomegranate (Punica granatum L.) fruit possesses strong antioxidant and antiinflammatory properties. Recently, we have shown that pomegranate-derived products rich in anthocyanidins and ellagitannins inhibit UVB-mediated activation of nuclear factor kappa B and modulate UVA-mediated cell proliferation pathways in normal human epidermal keratinocytes. In this study, we evaluated the effect of polyphenol-rich pomegranate fruit extract (POMx) on UVB-induced oxidative stress and photoaging in human immortalized HaCaT keratinocytes. Our data show that pretreatment of HaCaT cells with POMx (10-40 microg mL(-1)) inhibited UVB (15-30 mJ cm(-2))-mediated (1) decrease in cell viability, (2) decrease in intracellular glutathione content and (3) increase in lipid peroxidation. Employing immunoblot analysis we found that pretreatment of HaCaT cells with POMx inhibited UVB-induced (1) upregulation of MMP-1, -2, -7 and -9, (2) decrease in TIMP-1, (3) phosphorylation of MAPKs and (iv) phosphorylation of c-jun, whereas no effect was observed on UVB-induced c-fos protein levels. These results suggest that POMx protects HaCaT cells against UVB-induced oxidative stress and markers of photoaging and could be a useful supplement in skin care products.