Gromwell (Lithospermum erythrorhizon) supplementation enhances epidermal levels of ceramides, glucosylceramides, β-glucocerebrosidase, and acidic sphingomyelinase in NC/Nga mice

Abstract

We have previously reported that dietary gromwell (Lithospermum erythrorhizon; LE) prevents the development of atopic dermatitis (AD) with increased epidermal levels of total ceramide (Cer), the major lipid maintaining epidermal barrier. In this study, we investigated whether the increased level of total Cer induced by dietary LE would be related to the altered metabolism of glucosylceramide (GlcCer) and sphingomyelin (SM), two major precursor lipids in Cer generation. NC/Nga mice, an animal model of AD, were fed a control diet (group CA: atopic control) or a diet with 70% ethanol LE extracts (1% in diet; group LE) for 10 weeks. Individual species of Cer, GlcCer, and SM were analyzed by high-performance thin layer chromatography. In the epidermis of group CA, total Cer (including Cer2 and Cer5-7) and total GlcCer (including GlcCer-B/C/D) were significantly reduced; these levels in group LE were increased to levels similar to the normal control group of BALB/c mice (group C). In addition, protein expressions and activities of β-glucocerebrosidase (β-GlcCer'ase) and acidic sphingomyelinase (aSMase), enzymes for GlcCer or SM hydrolysis, respectively, were increased in group LE. However, alterations of Cer1, Cer3/4, GlcCer-A, and all SM species (including SM1-3) were not significant among groups C, CA, and LE. Dietary gromwell increases GlcCer-B/C/D, and further enhances the generation of Cer2 and Cer5-7 with high protein expressions and activities of β-GlcCer'ase and aSMase.

FIG. 1.

Epidermal levels of ceramide (Cer), glucosylceramide (GlcCer), and sphingomyelin (SM) in Balb/c mice fed a control diet (group C) and NC/Nga mice fed either a control diet (group CA) or Lithospermum erythrorhizon (LE) diet (group LE) for 10 weeks. Total lipids were fractionated into total Cer, total GlcCer, and total SM, of which individual species were further separated into (A) Cer1–7, (B) GlcCer-A/B/C/D, and (C) SM1–3 by high-performance thin layer chromatography. Values are mean±SEM (n=10). The data were evaluated for statistical significance by one-way ANOVA and the Duncan's multiple comparison test. ^abcDifferent letters indicate significant differences at P<.05.

FIG. 2.

Activities of β-glucocerebrosidase (β-GlcCer'ase) and acidic sphingomyelinase (aSMase) in the epidermis of Balb/c mice fed a control diet (group C) and NC/Nga mice fed either a control diet (group CA) or LE diet (group GW) for 10 weeks. (A) β-GlcCer'ase activity was determined in epidermal homogenates using fluorescent 4-methylumbelliferyl-β-D-glucopyranoside as a substrate and expressed as nmole/μg protein. (B) aSMase activity was determined in epidermal homogenates with using [¹⁴C] sphingomyelin as a substrate and expressed as cpm/μg protein. Values are mean±SEM (n=10). The data were evaluated for statistical significance by one-way ANOVA and the Duncan's multiple comparison test. ^abcDifferent letters indicate significant differences at P<.05.

FIG. 3.

Protein expressions of β-glucocerebrosidase (β-GlcCer'ase) and aSMase in the epidermis of BALB/c mice fed a control diet (group C) and NC/Nga mice fed either a control diet (group CA) or LE diet (group LE) for 10 weeks. (A) Representative expressions of β-GlcCer'ase and aSMase in the epidermis of groups. (B) The signal intensities from multiple experiments of A were quantified and integrated areas were normalized, first to the corresponding value of the keratin 5 loading control. Values are mean±SEM (n=10). The data were evaluated for statistical significance by one-way ANOVA and the Duncan's multiple comparison test. ^abcDifferent letters indicate significant differences at P<.05.