Live-cell imaging Unveils stimulus-specific dynamics of Nrf2 activation in UV-exposed melanoma cells: Implications for antioxidant compound screening.

The transcription factor Nuclear factor e2-related factor 2 (Nrf2) is pivotal in orchestrating cellular antioxidant defense mechanisms, particularly in skin cells exposed to ultraviolet (UV) radiation and electrophilic phytochemicals. To comprehensively investigate Nrf2's role in maintaining cellular redox equilibrium following UV-induced stress, we engineered a novel Nrf2 fusion-based reporter system for real-time, live-cell quantification of Nrf2 activity in human melanoma cells. Utilizing live quantitative imaging, we dissected the kinetic profiles of Nrf2 activation in response to an array of stimuli, including UVA and UVB radiation, as well as a broad spectrum of phytochemicals including ferulic acid, gallic acid, hispidulin, p-coumaric acid, quercetin, resveratrol, tannic acid, and vanillic acid as well as well-known Nrf2 inducers, tert-butylhydroquinone (tBHQ) and sulforaphane (SFN). Intriguingly, we observed distinct dynamical patterns of Nrf2 activity contingent on the specific stimuli applied. Sustained activation of Nrf2 was empirically correlated with the increased antioxidant response element (ARE) activity. Our findings demonstrate the nuanced impact of different phenolic compounds on Nrf2 activity and the utility of our Nrf2-CTΔ16-YFP reporter in characterizing the dynamics of Nrf2 translocation in response to diverse stimuli. In summary, our innovative reporter system not only revealed compounds capable of modulating UVA-induced Nrf2 activity but also showcased its utility as a robust tool for future antioxidant compound screening efforts.

A pilot study comparing the efficacy of autologous cultured fibroblast injections with hyaluronic acid fillers for treating nasolabial folds.

Autologous cultured fibroblast injections for soft tissue augmentation are a potential alternative to other filler materials. No studies have compared autologous fibroblast injections and hyaluronic acid (HA) fillers for treating nasolabial folds (NLFs). To compare the efficacies and safeties of autologous cultured fibroblast injections and HA fillers for treating NLFs. This prospective, evaluator-blinded, pilot study enrolled 60 Thai female adult patients diagnosed with moderate to severe NLFs. They were randomized to receive either 3 treatments of autologous fibroblasts at 2-week intervals or 1 treatment with HA fillers. The primary outcome was the clinical improvement of the NLFs graded by 2 blinded dermatologists immediately after injection and at 1-, 3-, 6-, and 12-month follow-ups. Objective measurement of the NLF volume was evaluated. Patient self-assessment scores, pain scores, and adverse reactions were recorded. Of the 60 patients, 55 (91.7%) completed the study protocol. The NLF volumes improved significantly in the autologous fibroblast group at all follow-ups relative to baseline (P = 0.000, 0.004, 0.000, 0.000, and 0.003). The patients in the autologous fibroblast group rated more noticeable NLF improvements than those in the HA filler group (3-month follow-up, 58.41% vs. 54.67%; 6-month follow-up, 52.50% vs. 46%; 12-month follow-up, 44.55% vs. 31.33%). No serious adverse reactions were recorded. Autologous fibroblast injections are safe and effective for treating NLFs. These injections also promise sustained growth of living cells, possibly leading to a greater persistence than shown by other fillers.

Role of Phytochemicals in Skin Photoprotection via Regulation of Nrf2.

Ethnopharmacological studies have become increasingly valuable in the development of botanical products and their bioactive phytochemicals as novel and effective preventive and therapeutic strategies for various diseases including skin photoaging and photodamage-related skin problems including abnormal pigmentation and inflammation. Exploring the roles of phytochemicals in mitigating ultraviolet radiation (UVR)-induced skin damage is thus of importance to offer insights into medicinal and ethnopharmacological potential for development of novel and effective photoprotective agents. UVR plays a role in the skin premature aging (or photoaging) or impaired skin integrity and function through triggering various biological responses of skin cells including apoptosis, oxidative stress, DNA damage and inflammation. In addition, melanin produced by epidermal melanocytes play a protective role against UVR-induced skin damage and therefore hyperpigmentation mediated by UV irradiation could reflect a sign of defensive response of the skin to stress. However, alteration in melanin synthesis may be implicated in skin damage, particularly in individuals with fair skin. Oxidative stress induced by UVR contributes to the process of skin aging and inflammation through the activation of related signaling pathways such as the mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1), the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), the nuclear factor kappa B (NF-κB) and the signal transducer and activator of transcription (STAT) in epidermal keratinocytes and dermal fibroblasts. ROS formation induced by UVR also plays a role in regulation of melanogenesis in melanocytes via modulating MAPK, PI3K/Akt and the melanocortin 1 receptor (MC1R)-microphthalmia-associated transcription factor (MITF) signaling cascades. Additionally, nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated antioxidant defenses can affect the major signaling pathways involved in regulation of photoaging, inflammation associated with skin barrier dysfunction and melanogenesis. This review thus highlights the roles of phytochemicals potentially acting as Nrf2 inducers in improving photoaging, inflammation and hyperpigmentation via regulation of cellular homeostasis involved in skin integrity and function. Taken together, understanding the role of phytochemicals targeting Nrf2 in photoprotection could provide an insight into potential development of natural products as a promising strategy to delay skin photoaging and improve skin conditions.

Mitochondria-Targeted Hydrogen Sulfide Delivery Molecules Protect Against UVA-Induced Photoaging in Human Dermal Fibroblasts, and in Mouse Skin In Vivo.

Aims: Oxidative stress and mitochondrial dysfunction play a role in the process of skin photoaging via activation of matrix metalloproteases (MMPs) and the subsequent degradation of collagen. The activation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor controlling antioxidant and cytoprotective defense systems, might offer a pharmacological approach to prevent skin photoaging. We therefore investigated a pharmacological approach to prevent skin photoaging, and also investigated a protective effect of the novel mitochondria-targeted hydrogen sulfide (H2S) delivery molecules AP39 and AP123, and nontargeted control molecules, on ultraviolet A light (UVA)-induced photoaging in normal human dermal fibroblasts (NHDFs) in vitro and the skin of BALB/c mice in vivo. Results: In NHDFs, AP39 and AP123 (50-200 nM) but not nontargeted controls suppressed UVA (8 J/cm2)-mediated cytotoxicity and induction of MMP-1 activity, preserved cellular bioenergetics, and increased the expression of collagen and nuclear levels of Nrf2. In in vivo experiments, topical application of AP39 or AP123 (0.3-1 µM/cm2; but not nontargeted control molecules) to mouse skin before UVA (60 J/cm2) irradiation prevented skin thickening, MMP induction, collagen loss of oxidative stress markers 8-hydroxy-2'-deoxyguanosine (8-OHdG), increased Nrf2-dependent signaling, as well as increased manganese superoxide dismutase levels and levels of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α). Innovation and Conclusion: Targeting H2S delivery to mitochondria may represent a novel approach for the prevention and treatment of skin photoaging, as well as being useful tools for determining the role of mitochondrial H2S in skin disorders and aging. Antioxid. Redox Signal. 36, 1268-1288.

The Protective Effect of Polyherbal Formulation, Harak Formula, on UVA-Induced Photoaging of Human Dermal Fibroblasts and Mouse Skin via Promoting Nrf2-Regulated Antioxidant Defense.

Polyherbal formulation combining multiple herbs is suggested to achieve enhanced therapeutic effects and reduce toxicity. Harak herbal formula (HRF) extracts were proposed to regulate skin responses to UVR through their ability to suppress UVA-induced matrix metalloproteinase-1 (MMP-1) and pigmentation via promoting antioxidant defenses in in vitro models. Therefore, natural products targeting Nrf2 (nuclear factor erythroid 2-related factor 2)-regulated antioxidant response might represent promising anti-photoaging candidates. Hesperetin (HSP) was suggested as a putative bioactive compound of the HRF, as previously shown by its chemical profiling using the liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In this study, we explored the anti-photoaging effects of HRF extracts and HSP on normal human dermal fibroblasts (NHDFs) and mouse skin exposed to UVA irradiation. Pretreatment of NHDFs with HRF extracts and HSP protected against UVA (8 J/cm2)-mediated cytotoxicity and reactive oxygen species (ROS) formation. The HRF and HSP pretreatment also attenuated the UVA-induced MMP-1 activity and collagen depletion concomitant with an upregulation of Nrf2 activity and its downstream genes (GST and NQO-1). Moreover, our findings provided the in vivo relevance to the in vitro anti-photoaging effects of HRF as topical application of the extracts (10, 30 and 100 mg/cm2) and HSP (0.3, 1, and 3 mg/cm2) 1 h before UVA exposure 3 times per week for 2 weeks (a total dose of 60 J/cm2) mitigated MMP-1 upregulation, collagen loss in correlation with enhanced Nrf2 nuclear accumulation and its target protein GST and NQO-1 as well as reduced 8-hydroxy-2'-deoxyguanosine (8-OHdG) in irradiated mouse skin. Thus, our findings revealed that HRF extracts and HSP attenuated UVA-induced photoaging via upregulating Nrf2, together with their abilities to reduce ROS formation and oxidative damage. Our study concluded that the HRF and its bioactive ingredient HSP may represent potential candidates for preventing UVA-induced photoaging via restoration of redox balance.

Hydroxylumisterols, Photoproducts of Pre-Vitamin D3, Protect Human Keratinocytes against UVB-Induced Damage.

Lumisterol (L3) is a stereoisomer of 7-dehydrocholesterol and is produced through the photochemical transformation of 7-dehydrocholesteol induced by high doses of UVB. L3 is enzymatically hydroxylated by CYP11A1, producing 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3. Hydroxylumisterols function as reverse agonists of the retinoic acid-related orphan receptors α and γ (RORα/γ) and can interact with the non-genomic binding site of the vitamin D receptor (VDR). These intracellular receptors are mediators of photoprotection and anti-inflammatory activity. In this study, we show that L3-hydroxyderivatives significantly increase the expression of VDR at the mRNA and protein levels in keratinocytes, both non-irradiated and after UVB irradiation. L3-hydroxyderivatives also altered mRNA and protein levels for RORα/γ in non-irradiated cells, while the expression was significantly decreased in UVB-irradiated cells. In UVB-irradiated keratinocytes, L3-hydroxyderivatives inhibited nuclear translocation of NFκB p65 by enhancing levels of IκBα in the cytosol. This anti-inflammatory activity mediated by L3-hydroxyderivatives through suppression of NFκB signaling resulted in the inhibition of the expression of UVB-induced inflammatory cytokines, including IL-17, IFN-γ, and TNF-α. The L3-hydroxyderivatives promoted differentiation of UVB-irradiated keratinocytes as determined from upregulation of the expression at the mRNA of involucrin (IVL), filaggrine (FLG), and keratin 14 (KRT14), downregulation of transglutaminase 1 (TGM1), keratins including KRT1, and KRT10, and stimulation of ILV expression at the protein level. We conclude that CYP11A1-derived hydroxylumisterols are promising photoprotective agents capable of suppressing UVB-induced inflammatory responses and restoring epidermal function through targeting the VDR and RORs.

CYP11A1-derived vitamin D3 products protect against UVB-induced inflammation and promote keratinocytes differentiation.

UVB radiation mediates inflammatory responses causing skin damage and defects in epidermal differentiation. 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) interacts with the vitamin D3 receptor (VDR) to regulate inflammatory responses. Additionally, 1,25(OH)2D3/VDR signaling represents a potential therapeutic target in the treatment of skin disorders associated with inflammation and poor differentiation of keratinocytes. Since the protective effect of 1,25(OH)2D3 against UVB-induced skin damage and inflammation is recognized, CYP11A1-derived vitamin D3-hydroxyderivatives including 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3 and 1,20,23(OH)3D3 were tested for their anti-inflammatory and skin protection properties in UVB-irradiated human epidermal keratinocytes (HEKn). HEKn were treated with secosteroids for 24 h pre- and post-UVB (50 mJ/cm2) irradiation. Secosteroids modulated the expression of the inflammatory response genes (IL-17, NF-κB p65, and IκB-α), reducing nuclear-NF-κB-p65 activity and increasing cytosolic-IκB-α expression as well as that of pro-inflammatory mediators, IL-17, TNF-α, and IFN-γ. They stimulated the expression of involucrin (IVL) and cytokeratin 10 (CK10), the major markers of epidermal differentiation, in UVB-irradiated cells. We conclude that CYP11A1-derived hydroxyderivatives inhibit UVB-induced epidermal inflammatory responses through activation of IκB-α expression and suppression of NF-kB-p65 activity and its downstream signaling cytokines, TNF-α, and IFN-γ, as well as by inhibiting IL-17 production and activating epidermal differentiation.

Photoprotective Properties of Vitamin D and Lumisterol Hydroxyderivatives.

We have previously described new pathways of vitamin D3 activation by CYP11A1 to produce a variety of metabolites including 20(OH)D3 and 20,23(OH)2D3. These can be further hydroxylated by CYP27B1 to produce their C1α-hydroxyderivatives. CYP11A1 similarly initiates the metabolism of lumisterol (L3) through sequential hydroxylation of the side chain to produce 20(OH)L3, 22(OH)L3, 20,22(OH)2L3 and 24(OH)L3. CYP11A1 also acts on 7-dehydrocholesterol (7DHC) producing 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone (7DHP) which can be converted to the D3 and L3 configurations following exposure to UVB. These CYP11A1-derived compounds are produced in vivo and are biologically active displaying anti-proliferative, anti-inflammatory, anti-cancer and pro-differentiation properties. Since the protective role of the classical form of vitamin D3 (1,25(OH)2D3) against UVB-induced damage is recognized, we recently tested whether novel CYP11A1-derived D3- and L3-hydroxyderivatives protect against UVB-induced damage in epidermal human keratinocytes and melanocytes. We found that along with 1,25(OH)2D3, CYP11A1-derived D3-hydroxyderivatives and L3 and its hydroxyderivatives exert photoprotective effects. These included induction of intracellular free radical scavenging and attenuation and repair of DNA damage. The protection of human keratinocytes against DNA damage included the activation of the NRF2-regulated antioxidant response, p53-phosphorylation and its translocation to the nucleus, and DNA repair induction. These data indicate that novel derivatives of vitamin D3 and lumisterol are promising photoprotective agents. However, detailed mechanisms of action, and the involvement of specific nuclear receptors, other vitamin D binding proteins or mitochondria, remain to be established.

The effect of botulinum toxin type A in different dilution on the contraction of fibroblast-In vitro study.

BACKGROUND: Botulinum toxin type A (BoNT-A) may directly remodel dermal tissues or induce a loss of normal morphology and cytoplasmic retraction and spread. Intradermal injection was claimed to produce a dermo-lifting effect, including midface lifting by using low concentration with variable dilution. OBJECTIVE: To understand how intradermal BoNT-A achieves tissue lifting, we examined different type of BoNT-A and their effects on dermal fibroblast contraction. METHODS: Normal human dermal fibroblasts were treated with onabotulinumtoxin (ONA), abobotulinumtoxin (ABO), prabotulinumtoxinA (PRABO), incobotulinumtoxinA (INCO), and letibotulinumtoxin A (LETI) in dilutions used in real-world practice. Fifty fibroblasts per dilution were photographed and measured the length to demonstrate their contraction every 2 hours from baseline (0 hours) to 12 hours post-treatment. RESULTS: ONA did not significantly decrease fibroblast lengths, at any timepoint or dilution. At 1:7 dilution ratios, ABO decreased fibroblast lengths after 2 hours and significantly after 10-12 hours. At 1:7, 1:8, 1:9, and 1:10 dilution, PRABO decreased length, and most rapidly at 1:7 and 1:8. At 1:6, 1:8, 1:9, and 1:10 dilution, INCO decreased lengths almost immediately. At 1:6 dilution, INCO decreased lengths almost immediately. At 1:7 dilution, INCO decreased lengths after 2-4 hours, while at 1:8, 1:9, and 1:10 dilution, INCO decreased lenghts nearly imediately. LETI decreased lengths at all dilutions except 1:9, with near-immediate effects at 1:6, 1:7, 1:8, and 1:10. At 1:4 dilution, LETI decreased lengths from 1 hour. CONCLUSIONS: Different commercial preparations of BoNT-A toxins cause different fibroblast contractions in vitro. Product selection and dilution used may affect the clinical outcome of intradermal injection of BoNT-A for face lifting.

Effect of Sucrier Banana Peel Extracts on Inhibition of Melanogenesis through the ERK Signaling Pathway.

Hyperpigmentation is a type of pigmentary disorder induced by overexpression of melanin content activated severe esthetic problems as melasma, freckle, ephelides, lentigo and other forms on human skin. Several whitening agents have restricted use because of their side effects or stability such as kojic acid, ascorbic acid and hydroquinone can act as cytotoxic substance which associated to dermatitis and skin cancer. To find for the safe substance, this study aimed to find for the ability of several components in Sucrier banana peel (SBP) extracts to inhibit melanogenesis process through p38 signaling pathway in B16F10 mouse melanoma cells. Tyrosinase activity and the cellular melanin content were dose dependent manner decreasing after SBP treatment. Furthermore, SBP decreased the expression of melanogenesis relate protein as microphthalmia-associated transcription factor (MITF) and tyrosinase protein after 24 hours incubation with α-melanocyte stimulating hormones (MSH) stimulating. The findings demonstrated that SBP contained an effective agent for hyperpigmentation inhibitor through p38 signaling pathways without any effect to ERK pathway, and subsequent down-regulate MITF expression and tyrosinase enzyme family production.

Protective effects of novel derivatives of vitamin D3 and lumisterol against UVB-induced damage in human keratinocytes involve activation of Nrf2 and p53 defense mechanisms.

We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50-200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.

Ethanol enhances endothelial ionic currents and nitric oxide release via intermediate-conductance calcium-activated potassium channel.

AIMS: Ethanol is known to induce NO release and coronary vasorelaxation. Evidence suggests that K+ channels, especially a Ca2+-activated K+ channel (KCa), may regulate endothelial NO production. We aimed to investigate the ethanol effect on K+ currents in human coronary artery endothelial cells (HCAECs), identify the K+ channel type/subtype and signaling pathway involved, and demonstrate the relevance to ethanol-induced NO release. MAIN METHODS: Ionic currents of cultured HCAECs were studied using whole-cell patch clamp technique. NO production were measured using the fluorescent probe, 2,3-diaminonaphthalene. KEY FINDINGS: We found that ethanol significantly potentiated HCAEC current (maximal increase to 155.68 ±â€¯18.93%, 20 mM ethanol, +80 mV; mean ±â€¯SEM, n = 9). Ethanol-induced current was significantly inhibited by blockers of IKCa or SKCa (intermediate- or small-conductance KCa), but not by blocking other K+ channels. When other known HCAEC channels were inhibited except IKCa, 20 mM ethanol significantly increased IKCa current to 198 ±â€¯25.11% (n = 6), but it could not enhance SKCa current that was similarly isolated. Moreover, ethanol-induced NO release was prevented by blocking IKCa channel, adenosine A2A receptor (A2AR), Gs protein, or protein kinase A (PKA). SIGNIFICANCE: This study was the first to demonstrate that acute ethanol exposure could activate endothelial IKCa channel, via A2AR-Gs-PKA signaling, leading to increased whole-cell current and NO release, which could be an important mechanism underlying ethanol-induced NO release and vasodilation.

Cyclin D1 depletion interferes with oxidative balance and promotes cancer cell senescence.

Expression of cyclin D1 (CCND1) is required for cancer cell survival and proliferation. This is presumably due to the role of cyclin D1 in inactivation of the RB tumor suppressor. Here, we investigated the pro-survival function of cyclin D1 in a number of cancer cell lines. We found that cyclin D1 depletion facilitated cellular senescence in several cancer cell lines. Senescence triggered by cyclin D1 depletion was more extensive than that caused by the prolonged CDK4 inhibition. Intriguingly, the senescence caused by cyclin D1 depletion was independent of RB status of the cancer cell. We identified a build-up of intracellular reactive oxygen species in the cancer cells that underwent senescence upon depletion of cyclin D1 but not in those cells where CDK4 was inhibited. The higher ROS levels were responsible for the cell senescence, which was instigated by the p38-JNK-FOXO3a-p27 pathway. Therefore, expression of cyclin D1 prevents cancer cells from undergoing senescence, at least partially, by keeping the level of intracellular oxidative stress at a tolerable sub-lethal level. Depletion of cyclin D1 promotes the RB-independent pro-senescence pathway and the cancer cells then succumb to the endogenous oxidative stress levels.This article has an associated First Person interview with the first author of the paper.

Melatonin and its derivatives counteract the ultraviolet B radiation-induced damage in human and porcine skin ex vivo.

Melatonin and its derivatives (N1 -acetyl-N2 -formyl-5-methoxykynurenine [AFMK] and N-acetyl serotonin [NAS]) have broad-spectrum protective effects against photocarcinogenesis, including both direct and indirect antioxidative actions, regulation of apoptosis and DNA damage repair; these data were primarily derived from in vitro models. This study evaluates possible beneficial effects of melatonin and its active derivatives against ultraviolet B (UVB)-induced harm to human and porcine skin ex vivo and to cultured HaCaT cells. The topical application of melatonin, AFMK, or NAS protected epidermal cells against UVB-induced 8-OHdG formation and apoptosis with a further increase in p53ser15 expression, especially after application of melatonin or AFMK but not after NAS use. The photoprotective action was observed in pre- and post-UVB treatment in both human and porcine models. Melatonin along with its derivatives upregulated also the expression of antioxidative enzymes after UVB radiation of HaCaT cells. The exogenous application of melatonin or its derivatives represents a potent and promising tool for preventing UVB-induced oxidative stress and DNA damage. This protection results in improved genomic, cellular, and tissue integrity against UVB-induced carcinogenesis, especially when applied prior to UV exposure. In addition, our ex vivo experiments provide fundamental justification for further testing the clinical utility of melatonin and metabolites as protectors again UVB in human subjects. Our ex vivo data constitute the bridge between vitro to vivo translation and thus justifies the pursue for further clinical utility of melatonin in maintaining skin homeostasis.

Thai herbal antipyretic 22 formula (APF22) inhibits UVA-mediated melanogenesis through activation of Nrf2-regulated antioxidant defense.

Thai herbal antipyretic 22 formula (APF22), a polyherbal formula, has been traditionally used to treat dermatologic problems including hyperpigmentation. Exposure of the skin to ultraviolet A (UVA) causes abnormal melanin production induced by photooxidative stress. This study thus aimed to investigate the protective effects of APF22 extracts and phenolic compounds, ferulic acid (FA), and gallic acid (GA; used as positive control and reference compounds), on melanogenesis through modulation of nuclear factor E2-related factor 2 (Nrf2) signaling and antioxidant defenses in mouse melanoma (B16F10) cells exposed to UVA. Our results revealed that the APF22 extracts, FA, and GA reduced melanin synthesis as well as activity and protein levels of tyrosinase in UVA-irradiated B16F10 cells. Moreover, APF22 extracts and both FA and GA were able to activate Nrf2-antioxidant response element signaling and promote antioxidant defenses including glutathione, catalase, glutathione peroxidase, and the glutathione-S-transferase at both mRNA and enzyme activity levels in irradiated cells. In conclusion, APF22 extracts suppressed UVA-mediated melanogenesis in B16F10 cells possibly via redox mechanisms involving activation of Nrf2 signaling and upregulation of antioxidant defenses. Moreover, pharmacological action of the APF22 extracts may be attributed to the phenolic compounds, FA, and GA, probably serving as the APF22's active compounds.

Nrf2 in keratinocytes modulates UVB-induced DNA damage and apoptosis in melanocytes through MAPK signaling.

Responses of melanocytes (MC) to ultraviolet (UV) irradiation can be influenced by their neighbouring keratinocytes (KC). We investigated the role of Nrf2 in regulating paracrine effects of KC on UVB-induced MC responses through phosphorylation of MAPKs in association with oxidative stress in primary human MC cocultured with primary human KC using a transwell co-culture system and small-interfering RNA-mediated silencing of Nrf2 (siNrf2). The mechanisms by which Nrf2 modulated paracrine factors including α-melanocyte-stimulating hormone (α-MSH) and paracrine effects of KC on UVB-mediated apoptosis were also assessed. Our findings showed that co-culture of MC with siNrf2-transfected KC enhanced UVB-mediated cyclobutane pyrimidine dimer (CPD) formation, apoptosis and oxidant formation, together with phosphorylation of ERK, JNK and p38 in MC. Treatment of MC with conditioned medium (CM) from Nrf2-depleted KC also increased UVB-mediated MC damage, suggesting that KC modulated UVB-mediated MC responses via paracrine effects. Additionally, depletion of Nrf2 in KC suppressed UVB-induced α-MSH levels as early as 30min post-irradiation, although pretreatment with N-acetylcysteine (NAC) elevated its levels in CM from siNrf2-transfected KC. Furthermore, NAC reversed the effect of CM from Nrf2-depleted KC on UVB-induced apoptosis and inflammatory response in MC. Our study demonstrates for the first time that KC provided a rescue effect on UVB-mediated MC damage, although depletion of Nrf2 in KC reversed its protective effects on MC in a paracrine fashion in association with elevation of ROS levels and activation of MAPK pathways in MC. Nrf2 may indirectly regulate the paracrine effects of KC probably by affecting levels of the paracrine factor α-MSH via a ROS-dependent mechanism.

IL-1ß-induced modulation of gene expression profile in human dermal fibroblasts: the effects of Thai herbal Sahatsatara formula, piperine and gallic acid possessing antioxidant properties.

BACKGROUND: Pain is the main symptom of most musculoskeletal disorders and can be caused by inflammation in association with oxidative stress. Thai herbal Sahatsatara formula (STF), a polyherbal formula, has been traditionally used for relieving muscle pain and limb numbness. This study aimed to investigate biologically active compounds of STF and its pharmacological effects related to antioxidant and anti-inflammatory activities. METHODS: The identification of possibly active compounds of STF was performed by high performance liquid chromatography (HPLC). Moreover, this study also assessed the free radical scavenging activities of STF and its components using DPPH radical scavenging assay and their inhibitory effects on IL-1ß-induced intracellular reactive oxygen species (ROS) formation in primary human dermal fibroblasts (NHDFs) using DCFDA-flow cytometry analysis. Modulation of human gene expression by STF and its active compounds was investigated by microarray analyzed through Gene Ontology (GO) classification and pathway enrichment analysis. RESULTS: HPLC analysis has revealed the presence of gallic acid (GA) and piperine (PP) as the major compounds in STF extracts. Our finding discovered that STF and its active compounds (GA and PP) yielded free radical scavenging activities and abilities to inhibit IL-1ß-induced cellular ROS formation in NHDFs. Furthermore, microarray analysis demonstrated that a total of 84 genes (54 upregulated and 30 downregulated) were significantly affected by IL-1ß involved in inflammatory cytokines, chemokines, transcription factors, cell adhesion molecules and other immunomodulators participating in NF-κB signaling. The significantly upregulated genes in IL-1ß-treated in NHDFs participate in interleukin and cholecystokinin (CCRK) signaling pathways. The GO analysis of the target genes showed that all test compounds including indomethacin, STF and its active compounds, can downregulate the genes involved in NF-кB signaling pathway in IL-1ß-treated NHDFs compared to the cells treated with IL-1ß alone. CONCLUSIONS: STF and its active compounds possessing antioxidant actions can modulate the effects of IL-1ß-mediated alteration of gene expression profiles associated with inflammatory signaling in NHDFs.

Activation of Nrf2 Reduces UVA-Mediated MMP-1 Upregulation via MAPK/AP-1 Signaling Cascades: The Photoprotective Effects of Sulforaphane and Hispidulin.

UVA irradiation plays a role in premature aging of the skin through triggering oxidative stress-associated stimulation of matrix metalloproteinase-1 (MMP-1) responsible for collagen degradation, a hallmark of photoaged skin. Compounds that can activate nuclear factor E2-related factor 2 (Nrf2), a transcription factor regulating antioxidant gene expression, should therefore serve as effective antiphotoaging agents. We investigated whether genetic silencing of Nrf2 could relieve UVA-mediated MMP-1 upregulation via activation of mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) signaling using human keratinocyte cell line (HaCaT). Antiphotoaging effects of hispidulin (HPD) and sulforaphane (SFN) were assessed on their abilities to activate Nrf2 in controlling MMP-1 and collagen expressions in association with phosphorylation of MAPKs (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38), c-Jun, and c-Fos, using the skin of BALB/c mice subjected to repetitive UVA irradiation. Our findings suggested that depletion of Nrf2 promoted both mRNA expression and activity of MMP-1 in the UVA-irradiated HaCaT cells. Treatment of Nrf2 knocked-down HaCaT cells with MAPK inhibitors significantly suppressed UVA-induced MMP-1 and AP-1 activities. Moreover, pretreatment of the mouse skin with HPD and SFN, which could activate Nrf2, provided protective effects against UVA-mediated MMP-1 induction and collagen depletion in correlation with the decreased levels of phosphorylated MAPKs, c-Jun, and c-Fos in the mouse skin. In conclusion, Nrf2 could influence UVA-mediated MMP-1 upregulation through the MAPK/AP-1 signaling cascades. HPD and SFN may therefore represent promising antiphotoaging candidates.