Viability Of HaCaT Cells Research Articles

Development and validation of TOF-SIMS and CLSM imaging method for cytotoxicity study of ZnO nanoparticles in HaCaT cells

Zinc oxide nanoparticles (ZnO NPs) exhibit novel physiochemical properties and have found increasing use in sunscreen products and cosmetics. The potential toxicity is of increasing concern due to their close association with human skin. A time-of-flight secondary ion mass spectrometry (TOF-SIMS) and confocal laser scanning microscopy (CLSM) imaging method was developed and validated for rapid and sensitive cytotoxicity study of ZnO NPs using human skin equivalent HaCaT cells as a model system. Assorted material, chemical, and toxicological analysis methods were used to confirm their shape, size, crystalline structure, and aggregation properties as well as dissolution behavior and effect on HaCaT cell viability in the presence of various concentrations of ZnO NPs in aqueous media. Comparative and correlative analyses of aforementioned results with TOF-SIMS and CLSM imaging results exhibit reasonable and acceptable outcome. A marked drop in survival rate was observed with 50μg/ml ZnO NPs. The CLSM images reveal the absorption and localization of ZnO NPs in cytoplasm and nuclei. The TOF-SIMS images demonstrate elevated levels of intracellular ZnO concentration and associated Zn concentration-dependent 40Ca/39K ratio, presumably caused by the dissolution behavior of ZnO NPs. Additional validation by using stable isotope-labeled 68ZnO NPs as tracers under the same experimental conditions yields similar cytotoxicity effect. The imaging results demonstrate spatially-resolved cytotoxicity relationship between intracellular ZnO NPs, 40Ca/39K ratio, phosphocholine fragments, and glutathione fragments. The trend of change in TOF-SIMS spectra and images of ZnO NPs treated HaCaT cells demonstrate the possible mode of actions by ZnO NP involves cell membrane disruption, cytotoxic response, and ROS mediated apoptosis.

Arecoline inhibits epithelial cell viability by upregulating the apoptosis pathway: Implication for oral submucous fibrosis

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as a significant etiological factor for disease manifestation. However, the underlying molecular mechanisms regarding areca nut chewing-induced OSF are only partially understood. Herein, we reported that arecoline markedly induced morphologic change in HaCaT epithelial cells, but had no obvious effect on Hel fibroblast cells. MTS assay revealed that arecoline significantly suppressed HaCaT cell viability. Moreover, flow cytometric analysis indicated that arecoline substantially promoted HaCaT cell, but not Hel cell apoptosis in a dose-dependent manner. Furthermore, arecoline-induced HaCaT cell apoptosis was found to be associated with increased expression and activation of cleaved-Bid, cleaved-PARA and cleaved-caspase-3. Collectively, our results suggest that HaCaT epithelial cells are more sensitive than Hel fibroblast cells to arecoline-induced cytotoxicity, which may be involved in the pathogenesis of OSF.

A lectin from Bothrops leucurus snake venom raises cytosolic calcium levels and promotes B16-F10 melanoma necrotic cell death via mitochondrial permeability transition

BlL, a galactose-binding C-type lectin purified from Bothrops leucurus snake venom, exhibits anticancer activity. The current study was designed to elucidate the cellular mechanisms by which BlL induces melanoma cell death. The viabilities of B16-F10 melanoma cells and HaCaT keratinocytes treated with BlL were evaluated. Necrotic and apoptotic cell death, cytosolic Ca2+ levels, mitochondrial Ca2+ transport and superoxide levels were assessed in B16-F10 melanoma cells exposed to BlL. We found that treatment with BlL caused dose-dependent necrotic cell death in B16-F10 melanoma cells. Conversely, the viability of non-tumorigenic HaCaT cells was not affected by similar doses of BlL. BlL-induced B16-F10 necrosis was preceded by a significant (2-fold) increase in cytosolic calcium concentrations and a significant (3-fold) increase in mitochondrial superoxide generation. It is likely that BlL treatment triggers B16-F10 cell death via mitochondrial permeability transition (MPT) pore opening because the pharmacological MPT inhibitors bongkrekic acid and Debio 025 greatly attenuated BlL-induced cell death. Experiments evaluating mitochondrial Ca2+ transport in permeabilized B16-F10 cells strongly supported the hypothesis that BlL rapidly stimulates cyclosporine A-sensitive Ca2+-induced MPT pore opening. We therefore conclude that BlL causes selective B16-F10 melanoma cell death via dysregulation of cellular Ca2+ homeostasis and Ca2+-induced opening of MPT pore.

HaCaT Cell에서 황기 에탄올 추출물의 Aquaporin-3 발현 효과

Astragalus membranaceus Bunge is used in herbal medicine in Eastern Asian countries including Korea. In this study, we assessed the effects of A. membranaceus extract (AM) on the aquaporin-3 (AQP3) protein expression in HaCaT cells. AM did not affect viability of HaCaT cells. AQP3 expression and cell migration seem to be maximal at <TEX>$100{\mu}g/mL$</TEX> concentration. Epidermal growth factor receptor (EGFR) kinase inhibitor, PD153035, blocked AM-induced AQP3 expression and cell migration. In addition, an 80% ethanol extracts of herbal prescription, SinhyoTakleesan (ST), which is composed of A. membranaceus, Angelicae gigantis, Glycyrrhiza glabra Linne, and Lonicera japonica Flos also induced AQP3 expression at <TEX>$20{\mu}g/mL$</TEX> in HaCaT cells. Collectively, these results suggest that AM induce AQP3 expression via EGFR pathway.

Regulatory effect of serum starvation on autophagy in a human keratinocyte cell line HaCaT.

Objective To evaluate the regulatory effect of serum starvation on autophagy in human HaCaT keratinocytes,and to investigate its molecular mechanism.Methods HaCaT cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10％ fetal calf serum (control group) or in DMEM without fetal calf serum (starvation group),for four hours.Then,cell appearance was observed by light microscopy,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cell viability,transmission electron microscopy to observe ultrastructural changes,monodansylcadaverine (MDC) staining to identify the formation of autophagosomes,and Western blot to measure the expression of microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ,LC3-Ⅰ and autophagy related protein 7 (ATG7).Additionally,the phosphorylation level of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 was semiquantitatively analyzed by Western blot.Results Serum starvation increased the viability of HaCaT cells.Electron microscopy and MDC staining confirmed the formation of autophagosomes in serum-starved HaCaT cells.Compared with the control cells,the serum-starved HaCaT cells showed a significant increase in the transformation of LC3-Ⅰ to LC3-Ⅱ (LC3-Ⅱ/LC3-Ⅰ ratio:3.508 ± 0.415 vs.0.538 ± 0.038,t =13.32,P ＜ 0.01),and expression of ATG7 (ATG7/GAPDH ratio:0.048 ± 0.011 vs.0.021 ± 0.006,t =7.27,P ＜ 0.05).The phosphorylation level of mTOR at ser2448 and ser2481 was significantly lower in the serum-starved HaCaT cells than in the control cells (phosphorylated mTOR (ser2448)/mTOR ratio:0.394 ± 0.048 vs.0.762 ± 0.108,t =7.58,P＜ 0.05; phosphorylated mTOR (ser2481)/mTOR ratio:0.111 ± 0.020 vs.0.263 ± 0.039,t =13.77,P ＜ 0.05).Conclusion Serum starvation can induce autophagy in and increase the viability of HaCaT cells,likely by inhibiting the activation of mTOR. Key words: Keratinocytes; Autophagy; Cells, cultured

Effects of 1-hexadecyl-3-methylimidazolium ionic liquids on the physicochemical characteristics and cytotoxicity of phosphatidylcholine vesicles

This report is the first to study behaviors of nano-scale colloids prepared by long alkyl-chain imidazolium-based ionic liquids (LILs), namely, 1-hexadecyl-3-methylimidazolium hexafluorophosphate (C16MIMPF6) and 1-hexadecyl-3-methylimidazolium bromide (C16MIMBr), as well as phosphatidylcholines (PCs), including hydrogenated soybean phosphatidylcholine (HSPC) and egg phosphatidylcholine (EPC). The effects of LILs on the physicochemical characteristics of PC vesicles are investigated using morphological observations and measurements of particle size, zeta potential, and fluorescence polarization. The experimental data reveal that the incorporation of LILs into PC vesicles shows significant changes in the size, turbidity, zeta potential, storage stability, and membrane fluidity of colloids, which may be correlated with the counter-ions of the LILs, the degree of saturation of the PCs, and the vesicular composition. In addition, the cell viability of HaCaT, A431, and SCC25 cells decreases in a dose-dependent manner and is associated with the composition of the PC vesicles that are incorporated with LIL. The apoptotic characteristic of cells is more significant for the PC vesicles that incorporate a higher molar ratio of LILs at dispersion concentrations of 0.1 and 1mM. This work suggests that cytotoxicity is dependent on the counter-ions of LILs, the composition and concentration of dispersions, and the physicochemical properties of mixed LIL/PC colloids.

Lucidone protects human skin keratinocytes against free radical-induced oxidative damage and inflammation through the up-regulation of HO-1/Nrf2 antioxidant genes and down-regulation of NF-κB signaling pathway

We investigated the protective effects of lucidone, a naturally occurring cyclopentenedione isolated from the fruits of Lindera erythrocarpa Makino, against free-radical and inflammation stimulator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress in human keratinocyte (HaCaT) cells, with the aim of revealing the possible mechanisms underlying the protective efficacy. Lucidone pretreatment (0.5-10 μg/mL) markedly increased HaCaT cell viability and suppressed AAPH-induced reactive oxygen species (ROS) generation, lipid peroxidation, and DNA damage. Notably, the antioxidant potential of lucidone was directly correlated with the increased expression of an antioxidant gene, heme oxygenase 1 (HO-1), which was followed by the augmentation of the nuclear translocation and transcriptional activation of NF-E2-related factor-2 (Nrf2), with or without AAPH. Nrf2 knockdown diminished the protective effects of lucidone. We also observed that lucidone pretreatment inhibited AAPH-induced inflammatory chemokine prostaglandin E₂ (PGE₂) production and the expression of cyclooxygenase-2 (COX-2) in HaCaT cells. Lucidone treatment also significantly inhibited AAPH-induced nuclear factor-κB (NF-κB) activation and suppressing the degradation of inhibitor-κB (I-κB). Furthermore, lucidone significantly diminished AAPH-induced COX-2 expression through the down-regulation of the extracellular signal-regulated kinase (ERK) and p38 MAPK signaling pathways. Therefore, lucidone may possess antioxidant and anti-inflammatory properties and may be useful for the prevention of free radical-induced skin damage.

β-Galactosidase-Catalyzed Synthesis of Galactosyl Chlorphenesin and Its Characterization

We synthesized galactosyl chlorphenesin (CPN-G) using β-gal-containing Escherichia coli (E. coli) cells in which the conversion yield of chlorphenesin (CPN) to CPN-G reached about 64% during 12h. CPN-G was identified and characterized using high-performance liquid chromatography, liquid chromatography-mass spectrometry, Fourier transform-infrared spectrometry, and nuclear magnetic resonance analysis ((1)H and (13)C). We verified that a galactose was covalently bound to a CPN alcohol group during CPN-G synthesis throughout these analyses. In particular, by the hydrolysis of CPN-G using β-gal, it was confirmed that a galactose was bound to CPN. The minimal inhibitory concentration (MIC) results showed that the CPN-G MICs were fairly similar to those of CPN. HACAT cell viability was significantly higher in CPN-G-treated cells than in CPN-treated cells at concentrations of 0.0-20.0mM. Finally, we accomplished the synthesis of less toxic CPN-G, compared with CPN, using β-gal-containing E. coli cells as whole cells without changes in the MICs against microorganisms.

The role of plant metabolism in the mutagenic and cytotoxic effects of four organophosphorus insecticides in Salmonella typhimurium and in human cell lines

This study used a cell/microbe co-incubation assay to evaluate the effect of four organophosphorus insecticides (parathion-methyl, azinphos-methyl, omethoate, and methamidophos) metabolized by coriander (Coriandrum sativum). The reverse mutation of Salmonella typhimurium strains TA98 and TA100 was used as an indicator of genetic damage. Treatments with these insecticides inhibited peroxidase activity in plant cells by between 17% (omethoate) and 98% (azinphos-methyl) and decreased plant protein content by between 36% (omethoate) and 99.6% (azinphos-methyl). Azinphos-methyl was the most toxic when applied directly. In the Ames test, treatments applied directly to strain TA100 killed the bacteria; however, the presence of plant metabolism detoxified the system and permitted the growth of bacteria. In strain TA98, plant metabolites of insecticides were mutagenic. This result suggests that the tested pesticides produce mutations through frameshifting. The same pesticides were applied to human skin (HaCaT) and lung (NL-20) cell lines to evaluate their effects on cell viability. Pesticides applied directly were more cytotoxic than the combination of pesticide plus coriander metabolic fraction. Omethoate and methamidophos did not affect the viability of HaCaT cells, but azinphos-methyl and parathion-methyl at 100 and 1000μgmL−1 significantly decreased viability (p<0.05). The NL-20 cell line was remarkably sensitive to the direct application of insecticides. All of the treatment conditions caused decreases in NL-20 cell viability (e.g., viability decreased to 12.0% after parathion-methyl treatment, to 14.7% after azinphos-methyl treatment, and to 6.9% after omethoate treatment). Similar to the Ames test, all of the insecticides showed decreased toxicity in human cells when they were cultured in the presence of plant metabolism. In conclusion, when the studied organophosphorus insecticides were plant-metabolized, they induced mutations in the bacterial strain TA98. In human cell lines, plant metabolism reduced the cytotoxic properties of the insecticides, and human keratinocytes were more resistant to mortality than bronchial cells.

Curcumin Protects Human Keratinocytes against Inorganic Arsenite-Induced Acute Cytotoxicity through an NRF2-Dependent Mechanism

Human exposure to inorganic arsenic leads to various dermal disorders, including hyperkeratosis and skin cancer. Curcumin is demonstrated to induce remarkable antioxidant activity in a variety of cells and tissues. The present study aimed at identifying curcumin as a potent activator of nuclear factor erythroid 2-related factor 2 (NRF2) and demonstrating its protective effect against inorganic arsenite- (iAs3+-) induced cytotoxicity in human keratinocytes. We found that curcumin led to nuclear accumulation of NRF2 protein and increased the expression of antioxidant response element- (ARE-) regulated genes in HaCaT keratinocytes in concentration- and time-dependent manners. High concentration of curcumin (20 μM) also increased protein expression of long isoforms of NRF1. Treatment with low concentrations of curcumin (2.5 or 5 μM) effectively increased the viability and survival of HaCaT cells against iAs3+-induced cytotoxicity as assessed by the MTT assay and flow cytometry and also attenuated iAs3+-induced expression of cleaved caspase-3 and cleaved PARP protein. Selective knockdown of NRF2 or KEAP1 by lentiviral shRNAs significantly diminished the cytoprotection conferred by curcumin, suggesting that the protection against iAs3+-induced cytotoxicity is dependent on the activation of NRF2. Our results provided a proof of the concept of using curcumin to activate the NRF2 pathway to alleviate arsenic-induced dermal damage.

Hydrogen water intake via tube-feeding for patients with pressure ulcer and its reconstructive effects on normal human skin cells in vitro

BackgroundPressure ulcer (PU) is common in immobile elderly patients, and there are some research works to investigate a preventive and curative method, but not to find sufficient effectiveness. The aim of this study is to clarify the clinical effectiveness on wound healing in patients with PU by hydrogen-dissolved water (HW) intake via tube-feeding (TF). Furthermore, normal human dermal fibroblasts OUMS-36 and normal human epidermis-derived cell line HaCaT keratinocytes were examined in vitro to explore the mechanisms relating to whether hydrogen plays a role in wound-healing at the cellular level.MethodsTwenty-two severely hospitalized elderly Japanese patients with PU were recruited in the present study, and their ages ranged from 71.0 to 101.0 (86.7 ± 8.2) years old, 12 male and 10 female patients, all suffering from eating disorder and bedridden syndrome as the secondary results of various underlying diseases. All patients received routine care treatments for PU in combination with HW intake via TF for 600 mL per day, in place of partial moisture replenishment. On the other hand, HW was prepared with a hydrogen-bubbling apparatus which produces HW with 0.8-1.3 ppm of dissolved hydrogen concentration (DH) and −602 mV to −583 mV of oxidation-reduction potential (ORP), in contrast to reversed osmotic ultra-pure water (RW), as the reference, with DH of < 0.018 ppm and ORP of +184 mV for use in the in vitro experimental research. In in vitro experiments, OUMS-36 fibroblasts and HaCaT keratinocytes were respectively cultured in medium prepared with HW and/or RW. Immunostain was used for detecting type-I collagen reconstruction in OUMS-36 cells. And intracellular reactive oxygen species (ROS) were quantified by NBT assay, and cell viability of HaCaT cells was examined by WST-1 assay, respectively.ResultsTwenty-two patients were retrospectively divided into an effective group (EG, n = 12) and a less effective group (LG, n = 10) according to the outcomes of endpoint evaluation and the healing criteria. PU hospitalized days in EG were significantly shorter than in LG (113.3 days vs. 155.4 days, p < 0.05), and the shortening rate was approximately 28.1%. Either in EG or in LG, the reducing changes (EG: 91.4%; LG: 48.6%) of wound size represented statistically significant difference versus before HW intake (p < 0.05, p < 0.001). The in vitro data demonstrate that intracellular ROS as quantified by NBT assay was diminished by HW, but not by RW, in ultraviolet-A (UVA)-irradiated HaCaT cells. Nuclear condensation and fragmentation had occurred for UVA-irradiated HaCaT cells in RW, but scarcely occurred in HW as demonstrated by Hoechst 33342 staining. Besides, under UVA-irradiation, either the mitochondrial reducing ability of HaCaT cells or the type-I collagen construction in OUMS-36 cells deteriorated in RW-prepared culture medium, but was retained in HW-prepared culture medium as shown by WST-1 assay or immunostain, respectively.ConclusionsHW intake via TF was demonstrated, for severely hospitalized elderly patients with PU, to execute wound size reduction and early recovery, which potently ensue from either type-I collagen construction in dermal fibroblasts or the promoted mitochondrial reducing ability and ROS repression in epidermal keratinocytes as shown by immunostain or NBT and WST-1 assays, respectively.

파라-쿠마린산의 자외선B 차단 효과

Recent studies have uncovered attractive properties of para-coumaric acid (PCA) as a potential skin hywhitening agent. The purpose of the current study was to examine its UVB-shielding effects. Effects of PCA on the viability of HaCaT cells exposed to UVB were assessed in vitro in comparison with other aromatic amino acid metabolites that have similar UV absorption spectra. For in vivo test, PCA cream (1.5 %) and cream base were topically applied to the dorsal skin of SKH-1 hairless mice and the inflammatory responses due to UVB exposure were monitored by changes in skin color (erythema) and thickness (edema). The cream application-UVB exposure regimen was repeated every other day for a total of 12 sessions. When HaCaT cells were irradiated with UVB, there was a dose-dependent decline in cell viability. The cell viability decline due to UVB exposure (10 mJ ) was significantly prevented by 100 PCA, cinnamic acid, urocanic acid, or indole acrylic acid by 39, 27, 39, or 31 %, respectively. Topical application of PCA cream onto the dorsal skin of hairless mice (10 ) attenuated the changes of color parameters, , , values, and thickness of the UVB (150 mJ )-exposed skin by 59, 50, 58, and 53 %, respectively. The current study, together with the previous studies that demonstrated the antimelanogenic effects of PCA, suggested that PCA may prevent not only dyspigmentation but also inflammatory reactions in the UVB-exposed skin.

Fragrance chemicals lyral and lilial decrease viability of HaCat cells’ by increasing free radical production and lowering intracellular ATP level: Protection by antioxidants

We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells.Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death.

Quercetin enhancement of arsenic‐induced apoptosis via stimulating ROS‐dependent p53 protein ubiquitination in human HaCaT keratinocytes

In this study, QUE, but not the structurally related chemical, rutin, enhanced the cytotoxicity of arsenic trioxide (As(+3)) against the viability of normal human HaCaT keratinocytes via induction of apoptosis. QUE enhancement of As(+3)-mediated apoptosis was accompanied by increased intracellular peroxide production according to a DCFH-DA analysis, and DNA ladders induced by QUE/As(+3) were inhibited by adding the antioxidative compound, N-acetyl cysteine (NAC). A loss of the mitochondrial membrane potential by QUE/As(+3) was observed according to DiOC(6) staining in concert with increased Bax protein and cytosolic cytochrome (Cyt) c protein expression in HaCaT cells, which was prevented by the addition of NAC. A decrease in the p53 protein with increased protein ubiquitination was detected in QUE/As(+3)-treated HaCaT cells, and this was prevented by the addition of NAC. The decrease in the p53 protein by QUE/As(+3) was reversed by adding the proteasome inhibitor, MG132. L-Buthionine sulphoximine (BSO) enhanced the cytotoxicity of As(+3) against the viability of HaCaT cells with reduced p53 protein through inducing protein ubiquitination and reactive oxygen species (ROS) production, and disrupting the mitochondrial membrane potential in HaCaT cells. Additionally, QUE and BSO enhanced the cytotoxic effects of monomethylarsonous acid (MMA(+3)) but not other arsenic compounds in accordance with increased p53 protein ubiquitination in HaCaT cells. QUE plus As(+3) stimulation of apoptosis in human HaCaT keratinocytes via activating ROS-dependent p53 protein ubiquitination may offer a rationale for the use of QUE to improve the clinical efficacy of arsenics in treating psoriasis.

여뀌 추출물의 세포 보호 작용과 피부 흡수 증진을 위한 리포좀 제형 연구

이전 연구에서 저자들은 여뀌 추출물의 항산화, 항노화, 항균 활성 및 여뀌 추출물을 함유한 크림의 보습 효과에 대한 결과를 보고한 바 있다. 본 연구에서는 여뀌 추출물과 그 주요 성분인 isoquercitrin의 사람 적혈구와 HaCaT 세포에서의 세포 보호 효과를 측정하였다. 여뀌 추출물의 피부 전달시스템으로 에토좀 및 탄성 리포좀을 제조하여 입자크기, 포집효율, 안정성 및 피부 흡수 증진 능력을 평가하였다. 적혈구 광용혈에서 여뀌 추출물과 isoquercitrin은 <TEX>$5{\mu}g/mL$</TEX>의 농도에서 <TEX>${\alpha}$</TEX>-토코페롤보다도 큰 세포보호효과를 나타내었다. 여뀌 추출물은 HaCaT 세포에 대해 <TEX>$50{\mu}g/mL$</TEX>의 농도에서 독성을 나타내지 않았다, UVB <TEX>$400mJ/cm^2$</TEX>를 HaCaT cell에 조사하였을 때, 여뀌 추출물은 농도 의존적(<TEX>$12.5{\sim}50{\mu}g/mL$</TEX>)으로 자외선으로부터 세포를 보호하였고, <TEX>$25{\mu}g/mL$</TEX>의 농도에서 양성 대조군(세포 생존율, 36 %)에 비하여 큰세포보호 활성(생존율, 90 %)을 나타내었다. 0.04 % 여뀌 추출물을 담지한 에토좀의 입자 크기는 173.0 nm, 포집효율은55.58 %이였다. 에토좀 제조 후 일주일동안 안정한 단분산 형태를 나타내었다. 피부 투과 실험 결과 에토좀은 일반 리포좀이나 에탄올 용액에서보다도 더 큰 피부 투과능을 보여주었다. 0.1 % 여뀌 추출물을 담지한 탄성 리포좀의 최적의 제형은 인지질 대 계면활성제(<TEX>$Tego^{(R)}$</TEX>care 450)의 비율이 95 : 5으로 확인되었다. 0.1 % 여뀌 추출물 함유한 최적의 탄성 리포좀의 입자크기는 176.5 nm, 가변형성은 16.4, 포집효율은 68.8 %이었다. 여뀌 추출물을 담지한 탄성 리포좀은 계면활성제가 포함되지 않은 제형보다 더 큰 피부 투과능을 나타내었다. In our previous studies, the antioxidant, anti-aging, and antibacterial activities of Persicaria hydropipier L. extract, and the moisturizing effect of cream containing P. hydropipier extract were investigated. In this study, the cellular protective effects of P. hydropipier extract and isoquercitrin, main component from P. hydropipier in <TEX>$^1O_2$</TEX>-induced photohemolysis of human erythrocytes and ultraviolet B (UVB)-exposed HaCaT cells were investigated. Liposomes such as ethosome and elastic liposome for enhanced transdermal delivery were prepared. Size, loading efficiency, stability, and cumulative permeated amounts of ethosomes and elastic liposomes were evaluated. P. hydropipier extract and isoquercitrin showed more prominent cellular protective effect than (+)-<TEX>${\alpha}$</TEX>-tocopherol, known as lipid antioxidant at <TEX>$5{\mu}g/mL$</TEX>. P. hydropipier extract didn't show any characteristics of cytotoxicity at <TEX>$50{\mu}g/mL$</TEX>. When HaCaT cells were exposed to a single large dose (<TEX>$400mJ/cm^2$</TEX>) of UVB, the extract protected the cells against UVB radiation in a concentration dependent manner (<TEX>$12.5{\sim}50{\mu}g/mL$</TEX>). Cell viability of HaCaT cells exposed to UVB <TEX>$400mJ/cm^2$</TEX> was increased by treatment with P. hydropipier extract or isoquercitrin from 36 % (cell viability of positve control groups) to 90 % (cell viability of P. hydropipier extract or isoquercitrin- treated groups). The size of 0.04 % P. hydropiper extract loaded ethosomes was 173.0 nm and the loading efficiency was 55.58 %. 0.04 % P. hydropiper extract loaded ethosomes were stable with as monodisperse particles for 1 week. The ethosome exhibited more skin permeability than general liposome and ethanol solution. The optimal ratio of lipid to surfactant (<TEX>$Tego^{(R)}$</TEX> care 450) of 0.1 % P. hydropiper extract loaded elastic liposomes was observed to be 95 : 5. Vesicle size of 0.1 % P. hydropiper extract loaded elastic liposome was 176.5 nm. The deformability index of the elastic liposome was 16.4. The loading efficiency was 68.8 %. The elastic liposome containing P. hydropiper extract showed more skin permeability than liposome without surfactant (<TEX>$Tego^{(R)}$</TEX> care 450).

Differential response of normal human epidermal keratinocytes and HaCaT cells to hydrogen peroxide-induced oxidative stress

Normal human epidermal keratinocytes (NHEKs) and HaCaT cells are the most common models used to study the effects of various factors on skin cells. These cell lines share some common characteristics, but little is known about their differences in handling hydrogen peroxide (H(2) O(2) )-induced oxidative stress. To investigate the differential response of NHEKs and HaCaT cells to H(2) O(2) -induced oxidative stress. We examined differences in NHEKs and HaCaT cells after H(2) O(2) treatment, assessing changes in cell viability; levels of intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and caspase-3/7; percentage of cells arrested in G1 phase; number of senescence-associated β-galactosidase (SA-β-Gal)-positive cells and; expression of senescence-related protein Klotho. The viability of NHEKs and HaCaT cells decreased in a concentration-dependent and time-dependent manner after exposure to H(2) O(2) . The inhibitory effect of 150 μmol/L H(2) O(2) on cell viability was greater in HaCaT cells than in NHEKs (P<0.05). Levels of ROS and caspase-3/7, and the percentage of cells arrested in G1 phase, were higher in HaCaT cells than in NHEKs, whereas intracellular SOD was higher in NHEKs than in HaCaT cells after exposure to 150 μmol/L H(2) O(2) (P < 0.05). SA-β-Gal positive cells increased significantly in NHEKs after treatment with H(2) O(2) (P < 0.05). Klotho was significantly downregulated in both NHEKs and HaCaT cells after H(2) O(2) treatment, but no SA-β-Gal-positive HaCaT cells were seen, even after treatment with H(2) O(2) . Normal human epidermal keratinocytes are more resistant than HaCaT cells to H(2) O(2) -induced oxidative stress. HaCaT cells have senescence phenotypes, but do not express β-Gal.

Ellagic acid protects human keratinocyte (HaCaT) cells against UVA-induced oxidative stress and apoptosis through the upregulation of the HO-1 and Nrf-2 antioxidant genes

UV radiation from the sun is a potent environmental risk factor in the pathogenesis of skin damage. Much of the skin damage caused by ultraviolet A (UVA) irradiation from the sun is associated with oxidative stress. The aim of this study was to investigate the protective role of ellagic acid (25–75μM), a natural antioxidant, against UVA (5–20J/cm2)-induced oxidative stress and apoptosis in human keratinocyte (HaCaT) cells and to reveal the possible mechanisms underlying this protective efficacy. Ellagic acid pre-treatment markedly increased HaCaT cell viability and suppressed UVA-induced ROS generation and MDA formation. Moreover, ellagic acid pre-treatment prevented UVA-induced DNA damage as evaluated by the comet assay. Ellagic acid treatment also significantly inhibited the UVA-induced apoptosis of HaCaT cells, as measured by a reduction of DNA fragmentation, mitochondria dysfunction, ER stress, caspase-3 activation, and Bcl-2/Bax deregulation. Notably, the antioxidant potential of ellagic acid was directly correlated with the increased expression of HO-1 and SOD, which was followed by the downregulation of Keap1 and the augmented nuclear translocation and transcriptional activation of Nrf2 with or without UVA irradiation. Nrf2 knockdown diminished the protective effects of ellagic acid. Therefore, ellagic acid may be useful for the treatment of UVA-induced skin damage.

Human HaCaT cell toxicity associated with oxidative stress on the polished rice grown adjacent to abandoned mines and potential health risk through rice intake

Human HaCaT cell viability, total antioxidants generation, and activities of antioxidant enzymes, i.e. superoxide dismutase (SOD) and catalase (CAT), were estimated on contaminated polished rice grown in abandoned mines. Potential health risk by intake of contaminated polished rice causing cytotoxicity on human HaCaT cell was assessed using the Monte-Carlo simulation. Results showed that the HaCaT cell viability in toxic elements (TEs)-contaminated polished rice was inhibited in a time-dependent manner, ranging from 5–25%. Likewise, time-dependent total antioxidants in contaminated polished rice were significantly decreased to 13–22 mM at 72 h post-incubation than in control demonstrating that the mine-impacted polished rice was associated with oxidative damage and decrement of the total antioxidants. The enzyme activities of scavenging reactive oxygen species, i.e. SOD and CAT were 2–4 folds induced compared to control. Assuming that TEs-contaminated polished rice inducing cytotoxicity on HaCaT cell was ingested by human population, no health risk is expected.

Ex vivo bio-compatibility of honey-alginate fibrous matrix for HaCaT and 3T3 with prime molecular expressions

Honey's inherent compositional diversity, bio-compatibility and time tested therapeutic efficacy, especially in tissue repair as a topical agent, attract researchers towards harnessing its biomaterial potential particularly in developing matrix for tissue engineering applications. Hence, this study fabricates fibrous mat from optimum honey-alginate formulation and alginate solution using wet spinning technology. The physical and morphological properties of the scaffolds are assessed and finally their comparative biological performances are evaluated through in vitro studies on adherence, viability and prime molecular expression of HaCaT and 3T3 cells. The honey-alginate scaffold demonstrates better performance than that of alginate in terms of cellular adherence, viability and proper expression of cell-cell adhesion molecule (E-cadherin) and prime molecules of extra cellular matrix (Collagen I and III) by HaCaT and 3T3 respectively.

Fullerene-C&lt;SUB&gt;60&lt;/SUB&gt; Incorporated in Liposome Exerts Persistent Hydroxyl Radical-Scavenging Activity and Cytoprotection in UVA/B-Irradiated Keratinocytes

The aim of this study is to examine antioxidant activity of fullerene-C60 (C60) incorporated in liposome (LpsmFlln, a diameter of 75.6 nm). LpsmFlln is water-soluble, and composed of hydrogenated lecithin of 89.7%, glycine soja sterol of 10% and C60 of 0.3%. Hydroxyl radicals (*OH), generated from UVA- or UVB-irradiated H2O2, were scavenged by LpsmFlln but not by C60-lacking Lpsm as assessed by ESR, showing that the active principle is C60 as scanty as 1/415 weight versus LpsmFlln; the *OH amount (% of non-additive control) was decreased, LpsmFlln-dose-dependently, and for 0.5% LpsmFlln (C60-eq.:16.7 microM) to 34.1% or 78.3% upon irradiation with UVA (12 J/cm2) or UVB (500 mJ/cm2), respectively, showing the superiority for UVA to UVB in terms of the *OH scavenging of LpsmFlln. Cell viability of human skin keratinocytes HaCaT decreased to 41.1% upon UVA-irradiation at 10 J/cm2, but retained to 60.6% with 0.025% LpsmFlln (C60-eq.: 0.84 microM) together with prevention of cell-morphological degeneration, in contrast to scarce effects of C60-lacking Lpsm. The scavenging activity for Fenton reaction-generated *OH, detected by DMPO/ESR, was 96.2% or 72.2% (% of no-additive control) at 1 min and decreased time-dependently to 24.8% or 28.3% at 12 min with 16.7 microM L-ascorbic acid (Asc) or Trolox, respectively, whereas 0.5% LpsmFlln (C60-eq:16.7 microM, the same concentration as for Asc) diminished *OH by 90.9% at 1 min and 91.5% even at 12 min, demonstrating the superiority of LpsmFlln to Asc or Trolox in terms of persistence of *OH-scavenging ability. Repressive efficacy on beta-carotene discoloration (% of control) for 60 min was in the order, based on the same molar or weight concentration: 1.3%:3.34 microM Asc < 25.0%:0.1% Lpsm < 36.3%:0.1% LpsmFlln (C60-eq.:3.34 microM) < 57.2%:3.34 microM Trolox, indicating the preventive effect of LpsmFlln against beta-carotene oxidation. Thus, LpsmFlln was demonstrated for an antioxidant ability characteristic of long-term persistence, and is attributed to fullerene-C60 but scarcely to Lpsm in all the tests examined, and is expected as the skin-protecting agent against oxidative stress.