UVB-irradiated HaCaT Keratinocytes Research Articles

Antiphotoaging Effect of AGEs Blocker™ in UVB-Irradiated Cells and Skh:HR-1 Hairless Mice.

Chronic exposure to ultraviolet (UV) radiation is a major cause of photoaging. It involves extrinsic aging, wrinkle formation, and skin dehydration, and leads to excessive production of active oxygen that adversely affects the skin. Here, we investigated the antiphotoaging effect of AGEs BlockerTM (AB), which comprises Korean mint aerial part and fig and goji berry fruits. Compared to its individual components, AB was more potent at increasing the expression of collagen and hyaluronic acid and decreasing MMP-1 expression in UVB-irradiated Hs68 fibroblasts and HaCaT keratinocytes. In Skh:HR-1 hairless mice exposed to 60 mJ/cm2 UVB for 12 weeks, oral administration of 20 or 200 mg/kg/day AB restored skin moisture by improving UVB-induced erythema, skin moisture, and transepidermal water loss, and alleviated photoaging by improving UVB-induced elasticity and wrinkles. Moreover, AB upregulated the mRNA levels of hyaluronic acid synthase and collagen-related Col1a1, Col3a1, and Col4a1 genes, increasing hyaluronic acid and collagen expression, respectively. AB inhibited UVB-induced MAPK and AP-1 (c-fos) activation, resulting in significantly downregulated expression of MMP-1 and -9, which are responsible for collagen degradation. AB also stimulated the expression and activity of antioxidative enzymes and reduced lipid peroxidation. Thus, AB is a potential preventive and therapeutic agent for photoaging.

Ginsenoside Rk1 Prevents UVB Irradiation-Mediated Oxidative Stress, Inflammatory Response, and Collagen Degradation via the PI3K/AKT/NF-κB Pathway In Vitro and In Vivo.

Long-term exposure to ultraviolet (UV) irradiation, especially UVB, can trigger destructive intracellular effects, including various types of DNA damage, oxidative stress, and inflammatory responses, leading to accelerated skin aging. Ginsenoside Rk1, a rare ginsenoside pertaining to panaxadiol saponins, has been certified to possess underlying anti-inflammatory effects. Nevertheless, the efficiency of Rk1 against the photoaging of human skin and the latent molecular mechanisms are still unclear. Here, UVB-irradiated HaCaT keratinocytes were used as an in vitro model, and UVB-irradiated BALB/c nude mouse dorsal skin was established as an in vivo model to explore the mechanism by which Rk1 protects skin. Consequently, we found that Rk1 administration significantly attenuated oxidative stress by suppressing reactive oxygen species (ROS) overproduction and strengthening the activities of antioxidant enzymes. The UVB-induced inflammatory response was alleviated by Rk1 application via regulation of the secretion of various proinflammatory cytokines. Additionally, western blot assays illustrated that Rk1 intervention inhibited collagen degradation by reducing the expression of matrix metalloproteinases. Further studies revealed that Rk1 could suppress the PI3K/AKT/NF-κB signaling pathways in vitro and in vivo. Molecular docking results indicated that Rk1 might effectively bind to the active pockets of PI3K, AKT, and NF-κB. The PI3K activator 740 Y-P clearly reversed the effects of Rk1 on oxidative stress, the inflammatory response, and collagen degradation in UVB-irradiated HaCaT cells. Moreover, histological and Masson staining verified that the administration of Rk1 to BALB/c nude mice remarkably ameliorated UVB-induced skin roughness, epidermal thickening, collagen fiber arrangement disorder, and wrinkles. Overall, the evidence provided in this study suggested that Rk1 could be applied for the development of effective natural antiphotoaging agents for skin health.

Protective effects of Quercus acuta Thunb. fruit extract against UVB-induced photoaging through ERK/AP-1 signaling modulation in human keratinocytes

BackgroundQuercus acuta Thunb. (Fagaceae) or Japanese evergreen oak is cultivated as an ornamental plant in South Korea, China, Japan, and Taiwan and used in traditional medicine. The acorn or fruit of Quercus acuta Thunb. (QAF) is the main ingredient of acorn jelly, a traditional food in Korea. Its leaf was recently shown to have potent xanthine oxidase inhibitory and anti-hyperuricemic activities; however, there have been no studies on the biological activity of QAF extracts. Solar ultraviolet light triggers photoaging of the skin, which increases the production of reactive oxygen species (ROS) and expression of matrix metalloproteinase (MMPs), and destroys collagen fibers, consequently inducing wrinkle formation. The aim of this study was to investigate the effect of water extracts of QAF against UVB-induced skin photoaging and to elucidate the underlying molecular mechanisms in human keratinocytes (HaCaT).MethodsIn this study, we used HPLC to identify the major active components of QAF water extracts. Anti-photoaging effects of QAF extracts were evaluated by analyzing ROS procollagen type I in UVB-irradiated HaCaT keratinocytes. Antiradical activity was determined using 2,2-diphenyl-1-picrylhydrazyl and 2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) assays. The expression of MMP-1 was tested by western blotting and ELISA kits. QAF effects on phosphorylation of the MAPK (p38, JNK, and ERK) pathway and transcription factor AP-1, which enhances the expression of MMPs, were analyzed by western blots.ResultsWe identified two major active components in QAF water extracts, gallotannic acid and ellagic acid. The QAF aqueous extracts recovered UVB-induced cell toxicity and reduced oxidative stress by inhibiting intracellular ROS generation in HaCaT cells. QAF rescued UVB-induced collagen degradation by suppressing MMP-1 expression. The anti-photoaging activities of QAF were associated with the inhibition of UVB-induced phosphorylation of extracellular signal-regulated kinase (ERK) and activator protein 1 (AP-1). Our findings indicated that QAF prevents UVB-induced skin damage due to collagen degradation and MMP-1 activation via inactivation of the ERK/AP-1 signaling pathway. Overall, this study strongly suggests that QAF exerts anti-skin-aging effects and is a potential natural biomaterial that inhibits UVB-induced photoaging.ConclusionThese results show that QAF water extract effectively prevents skin photoaging by enhancing collagen deposition and inhibiting MMP-1 via the ERK/AP-1 signaling pathway.

Topical Artocarpus communis Nanoparticles Improved the Water Solubility and Skin Permeation of Raw A. communis Extract, Improving Its Photoprotective Effect.

Antioxidants from plant extracts are often used as additives in skincare products to prevent skin problems induced by environmental pollutants. Artocarpus communis methanol extract (ACM) has many biological effects, such as antioxidant, anti-inflammatory, wound healing, and photoprotective effects; however, the poor water solubility of raw ACM has limited its applications in medicine and cosmetics. Topical antioxidant nanoparticles are one of the drug-delivery systems for overcoming the poor water solubility of antioxidants for increasing their skin penetration. The present study demonstrated that ACM-loaded hydroxypropyl-β-cyclodextrin and polyvinylpyrrolidone K30 nanoparticles (AHP) were successfully prepared and could effectively increase the skin penetration of ACM through changing the physicochemical characteristics of raw ACM, including reducing the particle size, increasing the surface area, and inducing amorphous transformation. Our results also revealed that AHP had significantly better antioxidant activity than raw ACM for preventing photocytotoxicity because the AHP formulation increased the cellular uptake of the ACM in UVB-irradiated HaCaT keratinocytes. In conclusion, our results suggest that AHP may be used as a good topical antioxidant nanoparticle for delivering ACM into deep layers of the skin for preventing UVB-induced skin problems.

Effect of Fish Bone Bioactive Peptides on Oxidative, Inflammatory and Pigmentation Processes Triggered by UVB Irradiation in Skin Cells.

In the present study, we evaluated for the first time the photoprotective effect of fish bone bioactive peptides (FBBP) preparation isolated from silver carp (Hypophthalmichthys molitrix) discarded tissue using in vitro experimental models of skin cells exposed to ultraviolet B (UVB) irradiation and stressing agents. FBBP preparation was obtained by papain treatment of minced bones and centrifugal ultrafiltration, and the molecular weight (MW) distribution was characterized by size exclusion and reversed-phase high performance liquid chromatography (RP-HPLC). In vitro assessment of the effect of FBBP pretreatment in UVB-irradiated L929 fibroblasts and HaCaT keratinocytes revealed their cytoprotective activity. Their capacity to efficiently reduce reactive oxygen species (ROS) production and lipid peroxidation varied in a dose-dependent manner, and it was greater in fibroblasts. A decrease of proinflammatory cytokines secretion, in particular of tumor necrosis factor alpha (TNF-α), was found after FBBP pretreatment of THP-1-derived inflamed macrophages. Melanin production and tyrosinase activity investigated in UVB-irradiated Mel-Juso cells were lowered in direct relation to FBBP concentrations. FBBP fractions with high radical scavenging activity were separated by ion exchange chromatography, and two collagenic sequences were identified. All these results offer new scientific data on aquaculture fish bone-derived peptides confirming their ability to control the antioxidant, anti-inflammatory and pigmentation processes developed during UV irradiation of skin cells and recommend their use as valuable natural ingredients of photoprotective cosmeceutical products.

Lentinula edodes extract inhibits matrix metalloproteinase expression and increases type I procollagen expression via the p38 MAPK/c-Fos signaling pathway in ultraviolet A and B-irradiated HaCaT keratinocytes

Objective: To determine the effect of Lentinula edodes extract on ultraviolet (UV) A and UVB-induced changes in matrix metalloproteinase (MMP) and type I procollagen expression using human immortalized HaCaT keratinocytes. Methods: Lentinula edodes ethanol extract (LEE) was obtained by extraction with 80% ethanol for 4 h at 80 °C. Effect of LEE on UV-induced alteration on the expression and production of MMPs and type I procollagen in keratinocytes was investigated using ELISA, RT-PCR, and Western blotting assay. To determine the underlying mechanism of LEE-mediated effects, mitogen-activated protein kinase (MAPK) and activator protein 1 signaling pathways were analysed by Western blotting assay. Results: LEE significantly inhibited the expression of MMP-1 and MMP-9 and increased the expression of type I procollagen in UVA and UVB-irradiated HaCaT keratinocytes. The phosphorylation levels of p38 were significantly inhibited by LEE whereas it did not affect c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation. Suppression of p38 phosphorylation was also accompanied by downregulation of UVA and UVB-induced increase in c-Fos. Conclusions: LEE effectively inhibits the expression of MMP-1 and MMP-9 and increases type I procollagen production through the p38 MAPK/c-Fos signaling pathway in UVA and UVB-irradiated HaCaT keratinocytes. This findings suggest that Lentinula edodes may be developed as a cosmetic material to suppress UV exposuremediated skin aging.

Regulation of Anti-Oxidative, Anti-Inflammatory, and Anti-Apoptotic Activity of Advanced Cooling Composition (ACC) in UVB-Irradiated Human HaCaT Keratinocytes.

We recently demonstrated that advanced cooling composition (ACC) has effective ingredients that exhibit anti-inflammatory effects in RAW 264.7 cells stimulated with lipopolysaccharide (LPS) and exhibit strong antimicrobial effects on Pseudomonas aeruginosa, Staphylococcus aureus, MRSA (methicillin-resistant Staphylococcus aureus), Candida albicans, and Streptococcus mutans. To further investigate whether ACC has beneficial effects in ultraviolet B (UVB)-irradiated human keratinocytes (HaCaT cells), HaCaT cells were pretreated with ACC prior to UVB irradiation. Our data showed that ACC, which is effective at 100 µg/mL, is nontoxic and has an antioxidative effect against UVB-induced intracellular reactive oxygen species (ROS) in HaCaT cells. In addition, ACC exerts cytoprotective effects against UVB-induced cytotoxicity in HaCaT cells by inhibiting abnormal inflammation and apoptosis through the regulation of mitogen-activated protein kinase (MAPK) signals, such as jun-amino-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). Therefore, these results indicate that ACC is a potentially beneficial raw material that possesses antioxidative, anti-inflammatory, and antiapoptotic effects against UVB-induced keratinocytes and may have applications in skin health.

ATR Kinase Activity Limits Mutagenesis and Promotes the Clonogenic Survival of Quiescent Human Keratinocytes Exposed to UVB Radiation

The ATR protein kinase has well-described roles in maintaining genomic integrity during the DNA synthesis phase of the cell cycle. However, ATR function in cells that are not actively replicating DNA remains largely unexplored. Using HaCaT and telomerase-immortalized human keratinocytes maintained in a confluent, nonreplicating state in vitro, ATR was found to be robustly activated in response to UVB radiation in a manner dependent on the nucleotide excision repair factor and DNA translocase XPB. Inhibition of ATR kinase activity under these conditions negatively impacted acute cell survival and cytotoxicity and severely inhibited the ability of UVB-irradiated HaCaT keratinocytes to proliferate upon stimulation with growth factors. Furthermore, ATR kinase inhibition in quiescent HaCaT keratinocytes potentiated UVB mutagenesis at the hypoxanthine phosphoribosyltransferase locus. Though ATR inhibition did not impact the rate of removal of cyclobutane pyrimidine dimers from genomic DNA, elevated levels of PCNA mono-ubiquitination and chromatin-associated PCNA and RPA indicate that excision gap-filling synthesis was altered in the absence of ATR signaling. These results indicate that the ATR kinase plays important roles in preventing mutagenesis and in promoting the proliferative potential of quiescent keratinocytes exposed to UVB radiation.

Fucoxanthin and Rosmarinic Acid Combination Has Anti-Inflammatory Effects through Regulation of NLRP3 Inflammasome in UVB-Exposed HaCaT Keratinocytes.

Excessive exposure to ultraviolet (UV) radiation is the main risk factor to develop skin pathologies or cancer because it encourages oxidative condition and skin inflammation. In this sense, strategies for its prevention are currently being evaluated. Natural products such as carotenoids or polyphenols, which are abundant in the marine environment, have been used in the prevention of oxidative stress due to their demonstrated antioxidant activities. Nevertheless, the anti-inflammatory activity and its implication in photo-prevention have not been extensively studied. Thus, we aimed to evaluate the combination of fucoxanthin (FX) and rosmarinic acid (RA) on cell viability, apoptosis induction, inflammasome regulation, and anti-oxidative response activation in UVB-irradiated HaCaT keratinocytes. We demonstrated for the first time that the combination of FX and RA (5 µM RA plus 5 μM FX, designated as M2) improved antioxidant and anti-inflammatory profiles in comparison to compounds assayed individually, by reducing UVB-induced apoptosis and the consequent ROS production. Furthermore, the M2 combination modulated the inflammatory response through down-regulation of inflammasome components such as NLRP3, ASC, and Caspase-1, and the interleukin (IL)-1β production. In addition, Nrf2 and HO-1 antioxidant genes expression increased in UVB-exposed HaCaT cells pre-treated with M2. These results suggest that this combination of natural products exerts photo-protective effects by down-regulating NRLP3-inflammasome and increasing Nrf2 signalling pathway.

1162 BMAL1 and CLOCK proteins in regulating the sensitivity of human keratinocytes to UVB-induced apoptosis

A diverse array of biological processes is under circadian controls. It has been reported that UV-induced apoptosis and DNA damage responses in mouse skin are time-of-day dependent and regulated by core clock proteins, including BMAL1, CLOCK, PERs, and CRYs. The aim of this study is to investigate the circadian controls of UV responses in human keratinocytes, using the siRNA-mediated loss-of-function approach. We found that low-dose (5 mJ/cm2) of UVB triggered an oscillation of BMAL1 and CLOCK mRNA expression in the immortal HaCat human keratinocyte cell line, as well as primary human keratinocytes. In HaCat cells, depletion of BMAL1 or CLOCK reduced the percentage of UVB-induced apoptotic cells from 43% (in controls) to 29% (p < 0.001) and 31% (P < 0.05), respectively. Moreover, knockdown of either clock gene strongly blocked UVB-induced expression of tumor necrosis factor α (TNFα) and phosphorylation of NF-κB. These results suggest that BMAL1-CLOCK proteins may promote cell death by regulating the TNFα-NF-κB mediated extrinsic apoptotic pathway in UVB-irradiated HaCat keratinocytes. Given the critical impact of UVB on photo-aging and photo-carcinogenesis, mechanistic elucidation and understanding of circadian controls on UVB effects in human skin will be beneficial for prevention and treatment of skin-related diseases including cancers.

The effects of antioxidant and anti-aging treatment of UVB-irradiated human HaCaT keratinocytes with ethanol extracts of colored rice varieties

The effects of antioxidant and anti-aging treatment of UVB-irradiated human HaCaT keratinocytes with ethanol extracts of colored rice varieties

Youngiasides A and C Isolated from Youngia denticulatum Inhibit UVB-Induced MMP Expression and Promote Type I Procollagen Production via Repression of MAPK/AP-1/NF-κB and Activation of AMPK/Nrf2 in HaCaT Cells and Human Dermal Fibroblasts.

This study investigated the effects of youngiaside A (YA), youngiaside C (YC), and Youngia denticulatum extract (YDE) on extrinsic aging and assessed its molecular mechanisms in UVB-irradiated HaCaT keratinocytes and human dermal fibroblasts (HDFs). The results showed that YA, YC, and YDE decreased matrix metalloproteinase (MMP) expression and production in HaCaT cell and HDFs and increased collagen expression and production in HDFs. In addition, YA, YC, and YDE significantly increased antioxidant enzyme expression, thereby down-regulating UVB-induced reactive oxygen species (ROS) production and ROS-induced mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) signaling in HaCaT cells. Furthermore, YA, YC, and YDE reduced phosphorylation of IκBα and IKKα/β, blocked nuclear factor-κB (NF-κB) p65 nuclear translocation, and strongly suppressed pro-inflammatory mediators. Finally, YA, YC, and YDE augmented UVB-induced adenosine monophosphate activated protein kinase (AMPK) phosphorylation and YA and YC did not inhibit MMP-1 production in AMPK inhibitor or nuclear factor-erythroid 2-related factor-2 (Nrf2) siRNA-treated HaCaT cells. The results suggest that these compounds could be potential therapeutic agents for prevention and treatment of skin photoaging.

The Role of p21 in Apoptosis, Proliferation, Cell Cycle Arrest, and Antioxidant Activity in UVB-Irradiated Human HaCaT Keratinocytes.

BackgroundSkin cancer is the most common cancer in the United States, and ultraviolet B (UVB) radiation-induced DNA damage is the major environmental factor underlying skin cancer development. p21, a p53-inducible protein, plays a key role in the cellular response to UVB-induced DNA damage.Material/MethodsThrough p21 silencing and overexpression, we investigated the role of p21 in apoptosis, proliferation, cell cycle arrest, and oxidative stress in UVB-irradiated HaCaT keratinocytes.ResultsWe found that UVB exposure induced significant p21 downregulation (p<0.05) and was associated with significantly increased apoptosis, significantly decreased proliferation, and significantly increased G2 phase arrest (p<0.05) in UVB-irradiated HaCaT keratinocytes. p21 silencing significantly promoted apoptosis, significantly inhibited G2 phase arrest, and significantly inhibited proliferation (p<0.05), but after UVB irradiation, p21 silencing demonstrated a less significant pro-apoptotic effect and a more significant inhibition of G2 phase arrest (p<0.05), which was reflected in significantly higher proliferative activity (p<0.05). p21 overexpression acted in an anti-apoptotic manner in the absence of UVB-induced DNA damage but acted in a pro-apoptotic manner in the presence of UVB-induced DNA damage, displaying an “antagonistic duality” similar to other growth-promoting oncoproteins. p53 expression mirrored p21 expression, suggesting a regulatory feedback mechanism between p21 and p53 expression. p21 overexpression significantly downregulated glutathione peroxidase and superoxide dismutase antioxidant activity (p<0.05) while significantly upregulating hydrogen peroxide and malondialdehyde content (p<0.05), suggesting a role in decreasing antioxidant defense capabilities in UVB-irradiated HaCaT keratinocytes.ConclusionsThese findings reveal that p21 may play a key role in HaCaT keratinocytes’ response to UVB exposure.

Antioxidant, cell-protective, and anti-melanogenic activities of leaf extracts from wild bitter melon (Momordica charantia Linn. var. abbreviata Ser.) cultivars.

BackgroundSeveral wild bitter melon (WBM; Momordica charantia Linn. var. abbreviata Ser.) cultivars were developed in Taiwan. However, little information is available regarding biological function of WBM leaf. Therefore, the objectives of this study were to investigate the nutrient content, antioxidant, cell protection and anti-melanogenic properties of wild bitter melon leaf.ResultsMethanolic leaf extracts were prepared from a variety and two cultivars of WBM. All extracts exerted potent nitric oxide and hydroxyl radical scavenging capacities. Furthermore, all extracts effectively reduce the production of reactive oxygen species and prevent cell death in UVB-irradiated HaCaT keratinocytes. The cell protective effect of leaf extract was also investigated by the prevention of HaCaT cells from sodium nitroprusside or menadione-induced toxicity, and significant cyto-protective activities were observed for all of them. Additionally, all extracts significantly suppressed tyrosinase activity and melanin levels in B16-F10 melanocytes.ConclusionsWBM leaf extract showed significant antioxidant, cyto-protective and anti-melanogenic activities. These findings suggested that WBM leaves may be beneficial for preventing the photo-oxidative damage and melanogenesis of skin.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-014-0078-y) contains supplementary material, which is available to authorized users.

Ficus deltoidea Extract Protects HaCaT Keratinocytes from UVB Irradiation-Induced Inflammation

Ficus deltoidea from the Moraceae family is a popular medicinal herb in Malaysia. It possesses strong antioxidant and anti-inflammatory properties. In the present study, the anti-inflammatory effects of F deltoidea extract on UVB-irradiated HaCaT Keratinocytes were investigated HaCaT Keratinocytes were UVB-iradiated (12,5 mj/cm ) and were treated with 0.05,0.08 or 0.1% of F. deltoidea extract. Cell viability following UVB irradiation was sigfi cantly higher in the groups treated with the F. deltoidea extract at doses of 0.05,0.0B or 0.1% than in control group with UVB irradiation only. TNF-o, lL-1a, IL-6. and cydooxygenase (COX-2) play pnmary roles In the inflammation process upon UV eradiation and are known to be stimulated by UVB irradiatwn. Treatment with the F. deltoidea extract dramatically inhibited the UV-induced TNF-o. Il-1a. IL-6, and COX-2 expression. These results suggest that the f. deltoidea extract may exert a protective effect against UVB-induced skin inflammation.

Protective effect of indole-3-pyruvate against ultraviolet b-induced damage to cultured HaCaT keratinocytes and the skin of hairless mice.

Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB) radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr), 4-hydroxyphenylpyruvate (HPPyr), and indole-3-pyruvate (IPyr)) against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm2) and maintained with or without test compounds (1–25 mM). In addition, the dorsal skin of hairless mice (HR-1) was treated with test compounds (100 µmol) and exposed to UVB light (1 J/cm2) for two times. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1β (IL-1β) and interleukin 6 (IL-6). IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2) expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1β and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.

잎새버섯 균사체를 이용한 감초추출발효물의 플라보노이드 생성과 항염 활성 연구

리퀴리티게닌과 이소리퀴리티게닌은 감초의 주요 플라보노이드 성분이다. 이들 플라보노이드는 수용성 감초 추출물과 <TEX>${\beta}$</TEX>-glucosidase를 생성하는 잎새버섯 HB0071 균사체 발효배양을 통하여 생산하였다. 감초추출물 내 리퀴리티게닌과 이소리퀴리티게닌은 잎새버섯 발효배양 동안 현저히 증가하였다. 이 균주의 <TEX>${\beta}$</TEX>-glucosidase의 활성은 배양 96시간을 기준으로 최고 91.5 mU/mL로 확인되었으며, 감초추출발효물로부터 생성된 리퀴리티게닌과 이소리퀴리티게닌의 함량은 HPLC 분석을 통하여 최대 <TEX>$568.5{\mu}g/mL$</TEX>과 <TEX>$89.6{\mu}g/mL$</TEX>로 확인되었다. 본 연구에서는 감초추출물의 잎새버섯 발효 전 후의 시료가 처리된 각질형성세포를 이용하여 자외선 UVB에 조사로 발현된 염증유발인자(COX-2)와 사이토카인(IL-<TEX>$1{\beta}$</TEX>, IL-6) 모두 감초추출발효물(FLEx)에서 농도의존적으로 발현이 억제되는 것을 확인하였다. 결론적으로 리퀴리티게닌과 이소리퀴리티게닌의 함량이 증가된 감초추출발효물은 자외선으로부터 손상된 피부 염증반응을 완화시켜줄 것으로 사료된다. Liquiritigenin and isoliquiritigenin are the major flavonoids present in licorice. These flavonoid compounds were prepared by submerged culture of Grifola frondosa (G. frondosa) HB0071 mycelia producing <TEX>${\beta}$</TEX>-glucosidase in the aqueous extract of licorice. The contents of liquiritigenin and isoliquiritigenin were increased during the fermentation. This fungus produced a high <TEX>${\beta}$</TEX>-glucosidase (activity of 91.5 mU/mL), thereby achieving high amounts of liquiritigenin and isoliquiritigenin (<TEX>$568.5{\mu}g/mL$</TEX> and <TEX>$89.6{\mu}g/mL$</TEX>), respectively at 96 h. A reversed- phase high-performance liquid chromatography method was established for simultaneous determination of liquiritigenin and isoliquiritigenin in fermented licorice extract (FLEx). The anti-inflammatory activities were investigated by licorice extract (LEx) before and after fermentation with G. frondosa HB0071. The treatment of UVB-irradiated HaCaT keratinocytes with FLEx resulted in a dose-dependent decrease in the expression level of cyclooxygenase-2 (COX-2) mRNA. Furthermore, FLEx dose-dependently decreased mRNA of the pro-inflammatory cytokines of IL-<TEX>$1{\beta}$</TEX> and IL-6 in UVB-irradiated HaCaT cells. These results suggest that FLEx may mitigate the effects of skin inflammation by reducing UVB-induced adverse skin reactions.

Caffeine Promotes Ultraviolet B-induced Apoptosis in Human Keratinocytes without Complete DNA Repair

In response to ultraviolet B damage, keratinocytes undergo apoptosis to eliminate damaged cells, thereby preventing tumorigenic transformation. Caffeine, the most widely consumed psychoactive substance, produces complex pharmacological actions; it has been shown to be chemopreventive in non-melamona skin cancer in mice through increasing apoptosis. Here we have investigated the molecular and cellular mechanisms in the pro-apoptotic effect of caffeine on UVB-irradiated human HaCaT keratinocytes. Pretreatment with caffeine increased UVB-induced apoptosis in HaCaT cells. Caffeine blocked UVB-induced Chk1 phosphorylation. In addition, similar to the effect of the PI3K inhibitor LY294002, caffeine also inhibited phosphorylation of AKT and up-regulation of COX-2, two critical oncogenic pathways in skin tumorigenesis. However, phosphorylation of EGFR or ERK was unaffected. Inhibiting ATR pathways by siRNA targeting ATR had little effect on UVB-induced apoptosis or AKT activation, indicating that the inhibitory effect of caffeine on apoptosis and the AKT pathway does not require the ATR pathway. Inhibiting AKT by caffeine blocked UVB-induced COX-2 up-regulation. Expression of constitutively active AKT that was not inhibited by caffeine was found to protect cells from caffeine-promoted apoptosis post-UVB irradiation, indicating that AKT is an essential inhibitory target for caffeine to promote apoptosis. Caffeine specifically sensitized cells with unrepaired DNA damage to UVB-induced apoptosis. These findings indicate that in HaCaT keratinocytes, inhibiting the AKT/COX-2 pathways through an ATR-independent pathway is a critical molecular mechanism by which caffeine promotes UVB-induced apoptosis of unrepaired keratinocytes for elimination.

Modification of gene expression by melatonin in UVB-irradiated HaCaT keratinocyte cell lines using a cDNA microarray

Excessive ultraviolet B (UVB) irradiation causes apoptotic cell death or induction of tumors in skin. Melatonin is a promising antioxidant and direct radical scavenger. Recently, it was reported that melatonin increases the survival of ultraviolet-B (UVB)-irradiated HaCaT keratinocyte cell lines. However, the precise molecular mechanisms underlying protective effect of melatonin on UVB damage are largely unknown. In this study, to gain more insight into the molecular mechanisms involved in melatonin-induced cell survival on UVB-irradiated HaCaT keratinocytes, we performed cDNA microarray analysis. HaCaT keratinocytes were incubated without or with melatonin at 100 nm for 30 min prior to UVB irradiation at 100 mJ/cm(2), and total RNA was isolated. Our data showed that the expression of apoptosis regulator genes (apoptosis related protein-3, apoptotic chromatin condensation inducer in the nucleus), cancer related genes (tumor suppressor deleted in oral cancer-related 1), cell cycle regulator (cyclin-dependent kinase 2 interacting protein), enzymes (glutathione peroxidase 1, ubiquitin-conjugating enzyme E2M), and signal transducer genes [fibroblast growth factor (acidic) intracellular binding protein, transforming growth factor beta-stimulated protein TSC-22] were decreased by melatonin treatment in the UVB-irradiated HaCaT keratinocyte cell lines, compared to that of UVB-irradiated HaCaT cells without melatonin. Thus, findings of the present study demonstrate that melatonin modulates the expression of apoptosis related genes in UVB-irradiated HaCaT cells, resulting in increasing cell survival, thereby suggesting that melatonin may be used as a promising sunscreen substance to reduce cell death of keratinocytes after excessive UVB irradiation.

E‐cadherin and its downstream catenins are proteolytically cleaved in human HaCaT keratinocytes exposed to UVB

It has been reported that ultraviolet B (UVB) irradiation causes the loss of E-cadherin of melanocytes, leading them to escape from neighboring keratinocytes during melanoma development. However, little has been paid on its effect on E-cadherin of keratinocytes. In the present study we therefore focus on whether UVB affects expression of E-cadherin-catenin complex in human HaCaT keratinocytes. We found that E-cadherin, beta-, and gamma-catenin but not alpha-catenin were proteolytically cleaved in UVB-irradiated HaCaT keratinocytes. The effect was only observed in keratinocyte undergoing apoptosis. Cleavage of beta- and gamma-catenin was fully abolished by caspase-3 and caspase-8 inhibitors, whereas cleavage of E-cadherin was inhibited by neither caspase nor metalloproteinase inhibitors. Functional analysis showed that the cleavage resulted in the disruption of the physical association between E-cadherin and catenins, indicating that E-cadherin signaling was compromised in UVB-irradiated HaCaT keratinocytes. Because E-cadherin in keratinocytes plays important roles in mediating cell-cell adhesion in epidermis of skin, the loss of E-cadherin and signaling components in keratinocytes may lead to the disruption of skin integrity after UVB exposure.