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Dietary cerebroside from sea cucumber (Stichopus japonicus): absorption and effects on skin barrier and cecal short-chain fatty acids Jingjing Duan, Marina Ishida, Kazuhiko Aida, Tsuyoshi Tsuduki, jin Zhang, Yuki Manabe, Takashi Hirata, and Tatsuya Sugawara J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02564 • Publication Date (Web): 02 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 2016
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Journal of Agricultural and Food Chemistry
Dietary cerebroside from sea cucumber (Stichopus japonicus): absorption and effects on skin barrier and cecal short-chain fatty acids
Jingjing Duan1,2*, Marina Ishida1, Kazuhiko Aida3, Tsuyoshi Tsuduki4, Jin Zhang2, Yuki Manabe1, Takashi Hirata1,5 and Tatsuya Sugawara1*,
1
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan 2
Present address of J. Duan: Department of Cardiology, Boston Children's Hospital,
Boston, MA 02115, USA 3
Central Laboratory, Nippon Flour Mill Company Ltd., Kanagawa 243-0041, Japan
4
Laboratory of Food and Biomolecular Science, Graduate School of Life Science and
Agriculture, Tohoku University, Sendai 981-8555, Japan 5
Present address of T. Hirata: Shijonawate Gakuen University, Daito, Osaka 574-0011,
Japan
*Corresponding author: Tatsuya Sugawara (Tel: +81-75-753-6212; Fax: +81-75-753-6212; E-mail:
[email protected]) Jingjing Duan (Tel: +1-617-919-4654; Fax: +1-617-731-0787; E-mail:
[email protected])
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Abstract
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Sphingolipids from marine source attract more attentions recently because of their
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distinctiveness on structures and expected functions. In this study, the content and
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component of cerebroside from sea cucumber Stichopus japonicus was analyzed. The
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absorption of cerebroside from S. japonicus was investigated with an in vivo lipid
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absorption assay. Our result revealed that S. japonicus is a rich source of cerebroside
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that contained considerable amounts of odd carbon chain sphingoid bases. The
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cumulative recovery of d17:1 and d19:2 consisting cerebrosides were 0.31±0.16% and
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0.32±0.10%, respectively for 24 h after administration. To the best of our knowledge,
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this is the first work that shows sphingolipids from marine source could be absorbed
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in vivo and incorporated into ceramides. In addition, dietary supplementation with sea
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cucumber cerebroside to hairless mouse improved the skin barrier function and
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increased short-chain fatty acids in cecal contents, that shown beneficial effects on
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host.
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Keywords: cerebroside; dietary sphingolipids; sea cucumber Stichopus japonicus;
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sphingoid base
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Introduction
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It has been estimated that the consumption of sphingolipids in selected foods
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has been estimated over 100 g per year by Vesper et al. in 1999.1 Most of the dietary
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studies to date have been conducted with sphingolipids from milk, soy and other
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“terrestrial” foods; however, aquatic organisms are also rich sources.2-4 In recent years,
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researchers found several atypical structures of sphingolipids from squid, octopus, sea
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cucumbers, and many other marine organisms.3,
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invertebrates have atypical types of chain length,unsaturation and hydroxylation in
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sphingoid bases such as 2-amino-4,8,10-octatriene-1,3-diol (d18:3) , 2-amino-9-
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methyl-4,8,10- octatriene-1,3-diol (d19:3) and 2-amino-1,3-dihydroxy-4-heptadecene
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(d17:1).3, 5-9 With the shift of dietary habits of modern humans in recent years, and the
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development of the sphingolipid analysis platform “sphingolipidomics”,10-11 we
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believe that humans consume more sphingolipids than currently known and it is thus
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necessary to re-survey the sphingolipids in diet.
5-7
Sphingolipids of marine
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Most of the cellular sphingolipids are usually synthesized de novo, however,
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biosynthesis of sphingolipids can be affected by many dietary factors, including the
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availability of the precursors and their regulators in metabolic pathways.7,
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Sphingolipids that are taken up from exogenous sources can be recycled and/or
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degraded and participate in lipid raft formation.13 Therefore, exogenous (dietary)
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sphingolipids and modulators of sphingolipid metabolism affect raft related signaling
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pathways,
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including glucosylceramide (GlcCer) and galactosylceramide (GalCer), as one of the
which
might
explain
their
biological
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Cerebroside,
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most significant components of sphingolipids, has emerged as an important ingredient
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in our diet. Several bioactivities of dietary cerebrosides, such as their
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anti-inflammatory,14 improving the barrier function of the skin,15-17 cancer-protective
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effects on the intestine,18-19 and preventing melanin formation20 have been reported.
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Cerebrosides from sea cucumbers have unique sphingolipid molecules3, 5 known to
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have anti-tumor8, 21, anti-adipogenic22, and against oxidative damage23 activities, and
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can induce apoptosis in human hepatoma HepG2 cells through p-AKT and DR5.24 It
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has been found that cerebroside from different species of sea cucumbers consists of
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both GlcCer and GalCer,25-29 whereas there is no report to date that cerebroside from
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the sea cucumber S. japonicus contains any GalCer. S. japonicus is used in fresh
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(whole body) or dried (body wall only) as an important food and traditional medicine
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in Asian countries. 30,31 Its body wall or viscera soft capsules have been consumed as
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a dietary supplement recently.32 Du et al. found that liposomes prepared from sea
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cucumber could be transport and uptake in small intestinal epithelial cell models,33
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that would be beneficial to understanding the fate of dietary sea cucumber
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sphingolipids. However, in vivo absorption of cerebroside from sea cucumber and its
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function as a dietary component are still not well understood.
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In this study, we evaluated the amount of cerebroside from edible sea cucumber
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S. japonicus and investigated its absorption in the rat intestine by a lipid absorption
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assay. The effects of dietary cerebroside from sea cucumber on the skin barrier and
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cecal short-chain fatty acids were investigated with a hairless murine model.
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Materials and Methods 4
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Analysis of cerebroside from sea cucumber Stichopus japonicus
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Dried sea cucumber (Stichopus japonicus) was purchased from a local
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fisherman in Nagasaki, Japan. Fresh sea cucumber (S. japonicus) was kindly donated
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by Isonoya Co. Ltd. (Maizuru, Japan). After washing with saline, fresh sea cucumbers
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were necropsied to acquire different body parts, which were then lyophilized and
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milled. Total lipids were extracted by Folch’s method and saponified with 0.4 M KOH
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in methanol at 38 °C for 2 h to remove glycerolipids. The alkali-stable lipid fractions
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were applied to high-performance liquid chromatography with evaporative light
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scattering detector (HPLC- ELSD) with a TSKgel CN-80Ts ( 250 × 4.6 i.d. mm, 5 µm)
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(TOSOH, Tokyo, Japan) and maintained at 40 °C. The mobile phase consisted of
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hexane-2-propanol (99:1, v/v) and chloroform-methanol (60:40, v/v) with flow rate
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1.0 mL/min. The amounts of cerebroside were calculated with our previous published
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quantitative analysis method.4 The cerebroside fractions from body wall and viscera
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of fresh sea cucumber were collected after HPLC and applied for further structural
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analysis.
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A Shimadzu high performance liquid chromatography-ion trap-time of flight
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mass spectrometer (LCMS-IT-TOF) equipped with an electrospray ionization
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interface (Shimadzu, Kyoto, Japan) was used for Liquid chromatography-mass
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spectrometry (LC-MS) analyses. A TSK gel ODS-100Z column (2.0 × 50 mm, 3 µm,
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Tosoh, Tokyo, Japan) was eluted with methanol/water (95:5, v/v) containing 5 mM
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ammonium acetate at a flow rate of 0.2 mL/min. The MS was operated under
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previously reported conditions.3, 34 For the structural analysis of cerebroside from S. 5
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japonicus, [M+H]+ and [M+H-18]+ (loss of water) in the positive scan mode were
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used for MS/MS analysis as precursor ions to obtain the product ions. The
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characteristic signals of sphingoid base moieties were observed as the product ions
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using the auto MS/MS detection mode. Pairs of the structurally specific product ions
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of sphingoid bases and their precursor ions were used and calculated for identification
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of cerebroside molecules (for example, m/z 700.7/264.4, 728.7/264.4 and 810.9/264.4,
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for d18:1/C16:0, d18:1/C18:0 and d18:1/C24:1 cerebroside, respectively) .34-35 For
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analysis of sphingolipids in rat lymph, the MS was operated under the same
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conditions as we reported previously.36
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Analysis of the sphingoid base fraction prepared from sea cucumber cerebroside
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The cerebroside powder from dried sea cucumber (S. japonicus) was kindly
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prepared by Nippon Flour Mills Co. Ltd. (Atsugi, Japan), and its purity was 96% as
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determined by HPLC.37 The powder was hydrolyzed with 1 M HCl in methanol at
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70 °C for 18 h to release free sphingoid bases, then o-phthalaldehyde (OPA)
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derivatives of the free sphingoid bases were quantified with reverse-phase HPLC
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system consisted of an LC-10AD pump and an RF-10AXL fluorescence detector
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(Shimadzu). Sphingoid bases were separated with acetonitrile/water (80:20, v/v) on a
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TSK gel ODS-80Ts column (Tosoh), 4.6 × 250 mm that isocratic analyses were
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performed at 1.0 mL/min. The OPA derivatives were detected with excitation
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wavelength and emission wavelength of 334 nm and 440 nm, respectively. 38
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Evaluation of intestinal absorption of sea cucumber cerebroside
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Sea cucumber cerebroside absorption was studied via a lipid absorption assay. 6
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This study was conducted in conformity with the policies and procedures detailed in
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the Animal Experiment Guidelines of Tohoku University. Surgeries and maintenance
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of rats and all other procedures were the same as in our previous study
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male Sprague-Dawley rats (9 weeks old, n=5 for each experimental and control
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groups) were obtained from Japan SLC (Hamamatsu, Japan) and were housed at 23 ±
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1 °C with a 12 h light/dark cycle. After acclimatization, the rats were anesthetized and
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a cannula (SV35, Dural Plastics) was inserted into their left thoracic channel for
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collecting lymphatic fluid, and a catheter (SP-55, Dural Plastics) was inserted into
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their stomach. After surgery, each rat was placed in a restraining cage in a warm
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recovery room. A physiological solution containing 139 mmol/L glucose and 85
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mmol/L NaCl was infused continuously overnight at a rate of 3 mL/h through the
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stomach cannula, and the same solution was also provided for drinking. The next
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morning, after collection of lymph for 2 h as a blank control, the rats were infused
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with 3 mL emulsions prepared by ultrasonication (containing 200 mg triolein, 50 mg
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fatty acid-free albumin, 200 mg sodium taurocholate, and with or without 5 mg
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cerebroside powder from dried sea cucumber as described above) as a single bolus
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through the stomach catheter. After those emulsions, infusion of the glucose/NaCl
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solution was continued and subsequently lymph samples were collected in
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EDTA-containing tubes and stored at -30 °C until analysis. Lipids were extracted and
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saponified, the alkali-stable fraction was subjected to HPLC analysis for
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quantification of free sphingoid bases (free sphingoid base fraction-“Free”). A portion
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of the alkali-stable fraction of lymph extract was degraded with aqueous methanolic 1 7
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. Briefly,
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M HCl at 70 °C for 18 h. Free sphingoid bases liberated from the complex
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sphingolipids were then subjected to HPLC for quantification analysis of total
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sphingolipids (total sphingoid base fraction-“Total”). The OPA derivatives of the free
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forms of sphingoid bases were analyzed as described above. The method for
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quantifying the recovery of sea cucumber sphingoid bases in this study was based on
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our previously study 36.
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Effect of dietary cerebroside on the barrier function of skin in a murine model
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Four-week-old female hairless mice (Hos: HR-1) were purchased from Japan
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SLC Inc. (Shizuoka, Japan). Animals were kept at 25 °C with a 12 h light/dark cycle
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and allowed free access to food and water. All experiments were performed according
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to the guidelines of Kyoto University for the use and care of laboratory animals. After
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acclimatization, mice were randomly allocated to 2 groups (n=6) with feeding
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standard AIN-93G (control group) or sea cucumber cerebroside supplemented (SC
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group) diets. The cerebroside supplemented diet was made with sea cucumber
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cerebroside powder (0.1% of total weight) that mixed uniformly into 0.1% soybean
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oil-reduced AIN-93G growth purified diet. After 2 weeks, the dorsal skin of mice was
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tape-stripped to remove the stratum corneum by using 25 mm × 40 mm adhesive
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cellophane tape (Nichiban, Tokyo, Japan) until the trans epidermal water
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loss (TEWL) reached
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room temperature and 40% relative humidity, every 2 days before skin damage and
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every 6 h after tape stripping until it recovered to the normal level. Mice were
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sacrificed by cervical dislocation under isoflurane anesthesia. The cecum of each
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g/h/m2.15
TEWL
was
measured
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mouse was immediately excised, and cecal content samples were quick frozen in
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-80 °C until use.
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Determination of short-chain fatty acids in cecal contents
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Short-chain fatty acids in cecal contents of hairless mouse were determined by
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gas chromatography with slight modifications.39 Briefly, 100 µL diethyl ether and 5
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µL 35% HCl were added to 100 µL 15% cecal content homogenates containing 0.95
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mM 2-ethylbutyric acid as an internal standard. After centrifugation at 1500×g at 4 °C
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for 20 min, the diethyl ether layer (upper layer) was collected. The ether extract
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sample (1 µL) was injected into a gas chromatograph (GC-14B, Shimadzu Co., Kyoto,
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Japan) equipped with a DB-FFAP capillary column (15 m × 0.530 mm × 0.5 µm,
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Agilent Technologies, CA, USA). Injector and detector temperatures were 145 °C and
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175 °C, respectively. The initial oven temperature was 80 °C for 1 min, subsequently
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increased at a rate of 10 °C/min, and then held at 130 °C for 1 min.
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Statistical analysis
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Data are reported as mean ± S.E. Statistical analyses were performed using
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Student’s t-test to identify levels of significance between the control and the other
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groups (p