Therapeutic Effects of Fermented Flax Seed Oil on NC/Nga Mice with Atopic Dermatitis-Like Skin Lesions

Abstract

Background. Atopic Dermatitis (AD) is one of the most common chronic inflammatory skin diseases. Objective. This experiment aimed to study the effects of Fermented Flax Seed Oil (FFSO) on symptoms such as redness, eczema, and pruritus induced by AD. Materials and Methods. AD-induced NC/Nga mice were used to observe the immunological and therapeutic effects of FFSO on skin in vivo. Raw 264.7 cells were used to investigate the effects of FFSO in cells. Fc receptor expression and concentration of beta-hexosaminidase were measured. Nitric oxide assay, Western blotting, real-time PCR, image analysis, and statistical analysis were performed in vitro. Results. In the immunohistochemical results, p-ERK 1/2 expression decreased, fibrogenesis strongly increased, and distribution reduction is observed. Distribution of IL-4-positive cells in the corium near the basal portion of the epithelium in the AT group was reduced. FFSO treatment reduced the number of cells showing NF-κB p65 and iNOS expression. The level of LXR in the AT group was higher than that in the AE group, and elevation of PKC expression was significantly reduced by FFSO treatment. Conclusion. FFSO could alleviate symptoms of AD such as epithelial damage, redness, swelling, and pruritus.

Figure 1

The mitigative effect of FFSO treatment for AD. (a) The skin damage as eczema was mitigated in AT. (b) The damage of intercellular space of stratum corneum, hyperplasia, and infiltration of lymphocytes were increased in AE but decreased in AT. (H&E satin, Bar size, 100 μm). The p-ERK 1/2 positive reaction in the AT was decreased in stratum granulosum. (p-ERK 1/2 immunohistochemistry, Bar size, 100 μm). The decrease of edema was shown in AT as compared with AE. (Masson's trichrome stain, Bar size, 100 μm). The distribution of capillary was decreased in AT as compared with AE (Phloxine-tartrazine stain, Bar size, 100 μm). Abbreviations. EP: epidermis; DE: dermis; M/T: Masson's trichrome stain; P/T: Phloxine-tartrazine stain; Ctrl, induced AD and given water; AE, induced AD and given water; AT, induced AD with FFSO treatment.

Figure 2

Regulation of mast cell activation. (a1) FFS Oil, FFSO; Ratio of Fc receptor to GAPDH expression in cells was determined by real-time PCR. Real-time PCR product-stimulated macrophages were analyzed as described in Section 2. Data were expressed as the mean ± SD of duplicate experiments in three different wells. (a2) Cells were treated with the indicated concentration of FFSO. Degranulation was assessed by β-hexosaminidase release into the supernatant. β-hexosaminidase released into the medium is presented as the mean ± SE. (b) Numbers of degranulated mast cell (white arrow) in the dermal papillae increased in the AE group but decreased in the AT group (Luna's method; Bar size, 50 µm). Fc ε receptor-positive cells (arrow indicates dark brown) in the HTT remarkably decreased (substance-P immunohistochemistry; Bar size, 50 µm). Mast cells secreting substance-P, MMP-9, and 5-HT (arrow indicates dark brown) in the AT group remarkably decreased (immunohistochemistry; Bar size, 50 µm). MMP-9: matrix metalloproteinases-9, 5-HT: serotonin. Other abbreviations are the same as in Figure 1. ^∗P < 0.05 compared with AE; ^∗∗P < 0.01 compared with AE; ^##P < 0.01 compared with Ctrl.

Figure 3

Downregulation of inflammation. (a1) Cells were pretreated with different concentrations of FFSO for 30 minutes and then stimulated with LPS (1 microg/mL) for 12 hours. Ratio of TNF-α to GAPDH expression in cells was determined by real-time PCR, as described in Section 2. (a2) Cells were pretreated with the indicated concentrations of FFSO for 2 hours and then stimulated with LPS (1 microg/mL) for 1 hour, after which nuclear extracts were prepared. Detection of NF-κB-binding activities was performed as described in Section 2. This experiment was repeated two times with similar results. (a3) Ratio of COX-2 to GAPDH expression on cells same as (a1). (a4) Cells were pretreated with different concentrations of FFSO for 30 minutes and then stimulated with LPS (1 microg/mL) for another 24 hours. Expression of iNOS and COX-2 proteins was detected by Western blotting using specific anti-iNOS and anti-COX-2 antibodies. β-Actin protein was used as an internal control. Expression of TNF-α, NF-kB p65, iNOS, and COX-2 (arrow indicates dark brown) was induced by D. farinae in the AE group. These positive reactions in the AT group were remarkably decreased compared with those in the AE group (immunohistochemistry; Bar size, 50 μm). Abbreviations are the same as in Figure 1. ^∗P < 0.05 compared with AE; ^∗∗P < 0.01 compared with AE; ^##P < 0.01 compared with Ctrl.

Figure 4

Regulation of Th2 differentiation. (a) Ratio of IL-6 to GAPDH expression in cells was determined by real-time PCR. Real-time PCR product-stimulated macrophages were analyzed as described in Section 2. Data were expressed as the mean ± SD of two separate experiments. (b) Expression of IL-4 and IL-6 (arrow indicates dark brown) in cells decreased in the AT group compared with the AE group (IL-4 and IL-6 immunohistochemistry; Bar size, 50 µm). EP: epidermis, DE: dermis. STAT6 and STAT3 expression (arrow indicates dark brown) in cells decreased in the AT group compared with the AE group (STAT6 and STAT3 immunohistochemistry; Bar size, 50 µm). Abbreviations are the same as in Figure 1. ^∗∗P < 0.01 compared with AE; ^##P < 0.01 compared with Ctrl.

Figure 5

Maintenance of lipid barrier in epidermis. LXR expression (arrow indicates dark brown) remarkably decreased in the AE group but was maintained in the AT group (LXR immunohistochemistry; Bar size, 50 µm). The lipid barrier (arrow indicates bright blue) in the intercellular space from the stratum granulosum to stratum corneum was absent in the AE group but present in the AT group (Sudan black B; Bar size, 50 µm). PKC expression (arrow indicates dark brown) in the AE group remarkably decreased (PKC immunohistochemistry; Bar size, 50 µm). LXR: liver X receptor, PKC: Protein kinase C; other abbreviations are the same as in Figure 1.