ETAS®50 Attenuates Ultraviolet-B-Induced Interleukin-6 Expression by Suppressing Akt Phosphorylation in Normal Human Dermal Fibroblasts.

We recently reported that ETAS 50, a standardized extract from the Asparagus officinalis stem, exerted anti-inflammatory effects on ultraviolet-B- (UV-B-) irradiated normal human dermal fibroblasts (NHDFs) by inhibiting nuclear factor-κB p65 nuclear import and the resulting interleukin-1ß (IL-1ß) expression. To further elucidate the antiphotoaging potency of ETAS 50, we examined the anti-inflammatory effects on UV-B-irradiated NHDFs by focusing on the stress-activated mitogen-activated protein kinase (MAPK) and Akt signaling pathways. NHDFs were treated with 1 mg/mL of ETAS 50 or dextrin (vehicle control) after UV-B irradiation (20 mJ/cm2) for different time periods. Phosphorylation levels of c-Jun N-terminal kinase (JNK), p38 MAPK, and Akt were analyzed by western blotting. IL-6 mRNA levels were analyzed by real-time polymerase chain reaction. UV-B-irradiated NHDFs showed increased phosphorylation levels of JNK, p38 MAPK, and Akt, as well as increased mRNA levels of IL-6. ETAS 50 treatment after UV-B irradiation suppressed the increased phosphorylation levels of Akt without affecting those of JNK and p38 MAPK. ETAS 50 as well as Akt inhibitor Perifosine repressed UV-B irradiation-induced IL-6 mRNA expression. These results suggest that ETAS 50 treatment represses UV-B irradiation-induced IL-6 expression by suppressing Akt phosphorylation. The present findings demonstrate the potential of ETAS 50 to prevent photoaging by attenuating UV-B irradiation-induced proinflammatory responses in skin fibroblasts.

A standardized extract of Asparagus officinalis stem prevents reduction in heat shock protein 70 expression in ultraviolet-B-irradiated normal human dermal fibroblasts: an in vitro study.

BACKGROUND: Heat shock protein 70 (HSP70) exhibits protective effects against ultraviolet (UV)-induced premature skin aging. A standardized extract of Asparagus officinalis stem (EAS) is produced as a novel and unique functional food that induces HSP70 cellular expression. To elucidate the anti-photoaging potencies of EAS, we examined its effects on HSP70 expression levels in UV-B-irradiated normal human dermal fibroblasts (NHDFs). METHODS: NHDFs were treated with 1 mg/mL of EAS or dextrin (vehicle control) prior to UV-B irradiation (20 mJ/cm2). After culturing NHDFs for different time periods, HSP70 mRNA and protein levels were analyzed using real-time polymerase chain reaction and western blotting, respectively. RESULTS: UV-B-irradiated NHDFs showed reduced HSP70 mRNA levels after 1-6 h of culture, which were recovered after 24 h of culture. Treatment with EAS alone for 24 h increased HSP70 mRNA levels in the NHDFs, but the increase was not reflected in its protein levels. On the other hand, pretreatment with EAS abolished the UV-B irradiation-induced reduction in HSP70 expression at both mRNA and protein levels. These results suggest that EAS is capable to preserve HSP70 quantity in UV-B-irradiated NHDFs. CONCLUSIONS: EAS exhibits anti-photoaging potencies by preventing the reduction in HSP70 expression in UV-irradiated dermal fibroblasts.

Anti-Inflammatory Effect of ETAS®50 by Inhibiting Nuclear Factor-κB p65 Nuclear Import in Ultraviolet-B-Irradiated Normal Human Dermal Fibroblasts.

Ultraviolet (UV) irradiation induces proinflammatory responses in skin cells, including dermal fibroblasts, accelerating premature skin aging (photoaging). ETAS 50, a standardized extract from the Asparagus officinalis stem, is a novel and unique functional food that suppresses proinflammatory responses of hydrogen peroxide-stimulated skin fibroblasts and interleukin- (IL-) 1ß-stimulated hepatocytes. To elucidate its antiphotoaging potencies, we examined whether ETAS 50 treatment after UV-B irradiation attenuates proinflammatory responses of normal human dermal fibroblasts (NHDFs). UV-B-irradiated NHDFs showed reduced levels of the cytosolic inhibitor of nuclear factor-κB α (IκBα) protein and increased levels of nuclear p65 protein. The nuclear factor-κB nuclear translocation inhibitor JSH-23 abolished UV-B irradiation-induced IL-1ß mRNA expression, indicating that p65 regulates transcriptional induction. ETAS 50 also markedly suppressed UV-B irradiation-induced increases in IL-1ß mRNA levels. Immunofluorescence analysis revealed that ETAS 50 retained p65 in the cytosol after UV-B irradiation. Western blotting also showed that ETAS 50 suppressed the UV-B irradiation-induced increases in nuclear p65 protein. Moreover, ETAS 50 clearly suppressed UV-B irradiation-induced distribution of importin-α protein levels in the nucleus without recovering cytosolic IκBα protein levels. These results suggest that ETAS 50 exerts anti-inflammatory effects on UV-B-irradiated NHDFs by suppressing the nuclear import machinery of p65. Therefore, ETAS 50 may prevent photoaging by suppressing UV irradiation-induced proinflammatory responses of dermal fibroblasts.

Enzyme-Treated Asparagus Extract (ETAS) Facilitates the Turnover of UV-B-Irradiated Keratinocytes.

Enzyme-treated asparagus extract (ETAS) is prepared from the lower, residual parts of asparagus, and some functionalities, such as anti-oxidative and neuroprotective activities, have been suggested. The purpose of the present study was to investigate the effects of ETAS on photoaging in the epidermal layer of the skin using cultured keratinocytes. Normal human epidermal keratinocytes were irradiated or left unirradiated with UV-B (10 mJ/cm2) and incubated with ETAS (0.5 or 2 mg/mL) or vehicle. After 3 or 13 h, molecular examinations were performed, and after 24 or 48 h, cell viabilities were determined by a CCK-8 assay. ETAS addition may induce keratinocyte migration and proliferation as well as apoptosis under molecular examination. These results suggest that ETAS might accelerate turnover of keratinocytes.

Rutin supplementation in the diet has protective effects against toxicant-induced hippocampal injury by suppression of microglial activation and pro-inflammatory cytokines: protective effect of rutin against toxicant-induced hippocampal injury.

AIMS: Rutin is one of the flavonoids that has many beneficial effects on the health. Previously, we showed that rutin has a protective effect on trimethyltin (TMT)-induced memory dysfunction in rats. The aim of this study was to investigate the protective effects of rutin on TMT-induced hippocampal injury and the time course profiles of these effects in rats. METHODS: Four-week-old male Sprague-Dawley (SD) rats were fed chow with or without rutin (0.75%) during the experimental period and were administered with a single dose of TMT (8.5 mg/kg b.w., p.o.) or vehicle at 6 weeks of age. The rats were sacrificed 5, 10, or 20 days after the TMT administration and then histological and molecular examinations of the hippocampus were performed. RESULTS: Rutin supplementation suppressed the TMT-induced decrease in the number of hippocampal pyramidal neurons 20 days after TMT administration. The TMT-induced up-regulation of the mRNA expression levels of reactive microglia marker and pro-inflammatory cytokines were reversed by rutin supplementation 10 or 20 days after the TMT administration. CONCLUSIONS: These results suggested that the neuroprotective effect of rutin on TMT-induced spatial memory impairment could be attributable to its inhibitory effect against microglial activation and its role in synapse formation via neurotrophic factors in the hippocampus.

Protective effect of rutin against spatial memory impairment induced by trimethyltin in rats.

Rutin is a flavonoid with various biological activities that are beneficial to human health. Trimethyltin is a toxic organotin compound, and rats injected with trimethyltin serve as a useful in vivo model for studying spatial memory impairment and neurodegeneration in the hippocampus. The protective effect of rutin against the trimethyltin-induced spatial memory impairment and hippocampal neuron damage in rats was examined. Peroral administration of a single dose of trimethyltin (8.5 mg/kg) induced spatial memory loss and the extensive loss of CA3 pyramidal neurons in hippocampi, as indicated by the results of a Morris water maze task and histologic examination, respectively. Prolonged supplementation of rutin significantly reversed the trimethyltin-induced spatial memory impairment and the damage to pyramidal neurons in the hippocampal CA3b region, indicating an antioxidative effect of rutin. These results suggest that rutin in the diet may provide a protective effect against spatial memory impairment accompanied by hippocampal pyramidal neuron loss.

[Protective effects of buckwheat hull extract against experimental hippocampus injury induced by trimethyltin in rats].

OBJECTIVES: The main objective of this study is to clarify the protective effects of buckwheat hull extract (BWHE) against toxicant-induced spatial memory impairment and hippocampal neuron injury in rats. METHODS: Male Sprague-Dawley (Jsl: SD) rats were fed chow containing 0.75% (w/w) BWHE during the experimental period. Two weeks after the start of the experiment, trimethyltin (TMT) (8 mg/kg bw) was administered orally to 6-week-old rats. After another two weeks, the rats were subjected to the Morris water maze task, which was used to determine spatial memory impairment. On the day after the Morris water maze task was performed, the right hemi-hippocampi were removed from the right half of the brain and weighed. Coronal sections of the left half of the brain were cut into 16-mum sections using a cryostat, and the number of neurons in each hippocampal region was evaluated by counting the surviving neurons using a light microscope. RESULTS: The impairment of spatial memory and the decrease in the hippocampal weight were observed after the TMT administration. Prolonged supplementation of BWHE seemed to reverse these TMT-induced toxic effects, and also improved the spatial memory of rats. CONCLUSIONS: The present results suggest that the BWHE supplementation of foods enhanced the spatial memory of rats and may have protective effects against hippocampal neurodegeneration accompanied by spatial memory impairment.

Retinoic acid inhibits uterotrophic activity of bisphenol A in adult ovariectomized rats.

Bisphenol A (BPA) is used in the production of polycarbonate and epoxy resins. The weak estrogenic activity of BPA has been confirmed by both in vitro and in vivo assays. Retinal acetate has been reported to inhibit the adverse effects of BPA on male mice reproduction. All-trans-retinoic acid (ATRA) is a potent natural derivative of vitamin A and is reported to inhibit the estrogen-induced proliferation of human breast carcinoma cells. In this study, we investigated the possible inhibitory effects of ATRA on the estrogenic activity of BPA by a standard in vivo uterotrophic assay. Proliferated and apoptotic uterine cells were identified by 5-bromo-2'deoxyuridine (BrdU) incorporation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. We observed that ATRA supplementation significantly inhibits a BPA-induced uterine weight increase in adult ovariectomized rats. However, there were no significant differences in the increases in the numbers of BrdU-positive cells and TUNEL-positive cells between the BPA and BPA+ATRA groups. These results show that ATRA may have an inhibitory effect on the estrogenic activity of BPA in an in vivo assay.

Antiestrogenic activity of vitamin A in in vivo uterotrophic assay.

All-trans-retinoic acid (ATRA), the primary active metabolite of vitamin A, was examined for its antiestrogenic activity in rats using an in vivo uterotrophic assay. All rats were ovariectomized 2 weeks prior to receiving 5 mg/kg/day ATRA or 0.3 micro g/kg/day ethynyl estradiol (EE) subcutaneously once a day for 3 consecutive days. Rats were sacrificed 1, 3, 6, 12 or 24 h after the last treatment. EE increased uterine weight and the coinjection of ATRA with EE significantly suppressed this effect 3 and 24 h after treatment. mRNA expression was examined during this 24-h period and the mRNA expression levels of estrogen receptor alpha (ER alpha), retinoic acid receptor beta (RAR beta), retinoid X receptor gamma (RXR gamma) and cellular retinol-binding protein I (CRBP I) were found to have significantly increased in the ATRA+EE group compared with those in the EE group. This is the first report on the antiestrogenic activity of ATRA determined using an in vivo adult rat uterotrophic assay. The up-regulation of RAR or RXR mRNA expression level was probably responsible for the antiestrogenic activity of ATRA.

RNA constitution and estrogen-responsive gene expression in the ovariectomized rat uterus.

Because reproductive tracts in ovariectomized rodents, which are commonly used in endocrinological studies, exhibit drastic changes in response to exogenous estrogens, quantitative evaluation of gene expression requires extra caution. We found that the whole mRNA content of total RNA in the uterus of the ovariectomized rat was dose-dependently reduced by treatment with 17alpha-ethynylestradiol (EE) for 3 consecutive days. Because of the decline in the ratio of mRNA/total RNA, real-time reverse transcriptase-polymerase chain reaction analysis seemingly showed that the relative ratios of the levels of stable RNAs, rRNA18S, rRNA5S, and tRNA-Gly, to whole mRNA were increased by EE. These results indicate that applying a fixed concentration of total RNA and stable RNA as an internal control to uterine mRNA quantification should be reconsidered. On the other hand, the beta-actin gene showed the most stable expression among the housekeeping genes examined. Using beta-actin mRNA as an internal control, we observed that tissue inhibitor of metalloproteinase-3 (TIMP3) mRNA was dose-dependently reduced 24 h after treatment with EE or bisphenol A for 3 days. Continued investigation of TIMP3 with an appropriate internal control may be helpful in elucidating the mechanisms involved in uterine activation.

Uterotrophic effects of benzophenone derivatives and a p-hydroxybenzoate used in ultraviolet screens.

Ultraviolet (UV) sunscreen products are popular because of concerns about UV radiation and skin cancer. Unfortunately, some of these products contain agents with estrogenic activity. We used an ovariectomized rat uterotrophic assay to measure the estrogenic activities of 2,4-dihydroxybenzophenone (2,4-DHBP), 2,2',4,4'-tetrahydroxybenzophenone (2,2',4,4'-THBP), and 4-hydroxybenzoic acid isobutyl ester (isobutyl-paraben), which are agents in UV sunscreens, and ethynyl estradiol (EE) and bisphenol A (BPA), which are positive controls. All chemicals increased rat uterine weights. The 10% effective doses (ED10, mg/kg/day) of EE, BPA, 2,4-DHBP, 2,2',4,4'-THBP, and isobutyl-paraben, as determined by Hill equation analysis, where 5E-5, 41.1, 544.6, 33.0, and 230.9, respectively, and their relative potencies against EE were about 1/800,000, 1/10,000,000, 1/600,000, and 1/4,000,000, respectively. Our findings indicated that UV screens contain weak estrogenic compounds.

Synthesis and estrogenic activity of bisphenol a mono- and di-beta-D-glucopyranosides, plant metabolites of bisphenol A.

The syntheses and characterization of bisphenol A mono- and di-beta-D-glucopyranosides were undertaken to confirm that these compounds are major plant metabolities of bisphenol A (BPA) and to allow an assessment of their estrogenicity. Synthesis involved the glucosidation of unprotected BPA with glucose penta-acetate with phosphorus oxychloride as catalyst. The estrogenic activity of BPA and its mono- and di-beta-D-glucopyranosides were measured with an enzyme-linked immunosorbent assay (ELISA)-based estrogen receptor competitive binding assay and with a yeast two-hybrid assay adapted to a chemiluminescent reporter gene (for beta-galactosidase). Both methods showed that the estrogenicity of BPA was eliminated by formation of the di-glucoside, but whereas the ELISA-based method indicated that reduced activity remained in the monoglucoside, the yeast two-hybrid method showed the monoglucoside to be inactive. Presumably these results reflect the more complex interactions of test compound and cellular components required to demonstrate estrogenicity in the yeast two-hybrid assay. As these processes parallel those in mammalian cells, the yeast two-hybrid method is likely to be the more realistic assay. The uptake and metabolism of BPA by plants offers the possibility of phytoremediation of contaminated water, but also provides an additional route for the compound to enter the human food chain.

Higher concentrations of interferon-gamma enhances uptake and transport of dietary antigens by human intestinal cells: a study using cultured Caco-2 cells.

Besides functioning as a mucosal barrier and transporting nutrients, intestinal epithelial cells (IECs) also serve as antigen-presenting cells (APCs). Modification of protein antigens by proteolysis is one of the principal steps in antigen presentation to Th cells. We used a Caco-2 intestinal epithelial cell line to investigate the transepithelial transport of the dietary antigen, ovalbumin (OVA). We also examined the effects of the proinflammatory cytokine interferon-gamma (IFN-gamma) on the antigen transport process in Caco-2 cell layers. Caco-2 cell layers transferred both intact and degraded OVA from the mucosal to the serosal side. IFN-gamma stimulated OVA transport and most of the transported OVA in such cells were degraded. We also examined OVA uptake by Caco-2 cells using immunohistochemical means. Caco-2 cells incorporated OVA in a time-dependent manner and IFN-gamma significantly enhanced antigen internalization. Flow cytometry also demonstrated that IFN-gamma elevated the internalization of FITC-OVA. We also determined the effects of low and high concentrations of IFN-gamma on mucosal permeability and internalization of FITC-OVA. Although both 10 and 50 ng/mL IFN-gamma stimulated mucosal permeability to the same extent, more FITC-OVA was internalized by Caco-2 cells incubated with 50 than with 10 ng/mL IFN-gamma. These results suggest that the effects of IFN-gamma on mucosal permeability and the internalization of antigens by intestinal epithelial cells are brought about by different mechanisms. Therefore, higher concentrations of IFN-gamma stimulate the uptake, processing, and transport of dietary antigens by IECs.

Improvement of a sensitive enzyme-linked immunosorbent assay for screening estrogen receptor binding activity.

A competitive enzyme-linked immunosorbent assay (ELISA) with estrogen receptor (alpha) and a fluorescence depolarization method with Full-Range Beacon were examined as estrogen receptor binding assays to prescreen endocrine-disrupting chemicals (EDCs). In this study, because it is difficult to measure the receptor binding ability of sparingly water-soluble chemicals using these methods, the competitive enzyme immunoassay was further modified for improved sensitivity by changing the operational parameters, such as receptor concentration, ligand concentration, and the reaction temperature. The method was applied to 10 test chemicals, including alkylphenols and bisphenol A (BPA). The diethylstilbestrol (DES) relative binding affinity (RBA) of ELISA kit was set equal to 1 (RBA = IC50/IC50 of DES). The RBAs of BPA, 4-nonylphenol (p-NP), and 4-t-octylphenol (p-t-OP) are 5386, 8619. and 8121 before using the improved competitive enzyme immunoassay and 883, 699, and 2832 using improved it respectively. Mixtures of BPA, p-NP, and p-t-OP gave results that the estrogen binding affinities of these chemicals are additive or slightly more than additive.