Portulaca oleracea L. extracts alleviate 2,4-dinitrochlorobenzene-induced atopic dermatitis in mice

Abstract

Atopic dermatitis (AD) is a common chronic allergic skin disease characterized clinically by severe skin lesions and pruritus. Portulaca oleracea L. (PO) is a resourceful plant with homologous properties in medicine and food. In this study, we used two different methods to extract PO, and compared the therapeutic effects of PO aqueous extract (POAE) and PO ultrasound-assisted ethanol extract (POEE) on 2,4-dinitrochlorobenzene (DNCB)-induced AD mice. The results showed that in POAE and POEE, the extraction rates of polysaccharides were 16.95% and 9.85%, while the extraction rates of total flavonoids were 3.15% and 3.25%, respectively. Compared with AD mice, clinical symptoms such as erythema, edema, dryness and ulceration in the back and left ear were alleviated, and pruritus behavior was reduced after POAE and POEE treatments. The thickness of the skin epidermis was thinned, the density of skin nerve fibers labeled with protein gene product 9.5 (PGP9.5) was decreased, and mast cell infiltration was reduced. There was a decrease in blood lymphocytes, eosinophils and basophils, a significant decrease in spleen index and a noticeable decrease in serum immunoglobulin E (Ig E). POEE significantly reduced the concentration of the skin pruritic factor interleukin (Il)-31. POAE and POEE reduced the concentration of skin histamine (His), down-regulated mRNA expression levels of interferon-γ (Ifnγ), tumor necrosis factor-α (Tnf-α), thymic stromal lymphopoietin (Tslp) and Il-4, with an increase of Filaggrin (Flg) and Loricrin (Lor) in skin lesions. These results suggested that POAE and POEE may inhibit atopic response and alleviate the clinical symptoms of AD by inhibiting the expression of immune cells, inflammatory mediators and cytokines. PO may be a potential effective drug for AD-like diseases.

Keywords: 2,4-dinitrochlorobenzene (DNCB); Portulaca oleracea L.; anti-inflammatory; anti-pruritic; atopic dermatitis; immunomodulation.

FIGURE 1

Polysaccharide and total flavonoid extraction rates of POAE and POEE in this study. (A) Polysaccharide and total flavonoid extraction rates of POAE. (B) Polysaccharide and total flavonoid extraction rates of POEE. Absorbance was measured at least 3 times and calculated based on the standard products (Glucose and Rutin). The data were expressed as mean.

FIGURE 2

POAE and POEE significantly alleviate DNCB-induced AD clinical symptoms in mice. (A) Animal experiments. (B) Representative dorsal skin photographs of each group of mice. (C) SCORAD scores of each group of mice. (D) Pruritus scores of mice in each group. The data were expressed as mean ± SD (n = 5 per group). ^###P < 0.001, vs. control groups; *P < 0.05, **P < 0.01, ***P < 0.001, vs. model (DNCB) groups.

FIGURE 3

POAE and POEE reduced the density of nerve fibers in the dorsal skin. (A) Representative photographs of H&E staining of the dorsal skin of each group of mice (Scale bar: 200 μm). The black arrow indicates the epidermal layer of the skin. (B) Thickness of the epidermal layer of the dorsal skin of each group of mice (n = 3 per group). (C) Immunofluorescence staining of representative dorsal skin nerve fibers from each group of mice (Scale bar: 50 μm). The nuclei showed blue fluorescence after DAPI staining, and PGP9.5 showed red fluorescence under the labeling of fluorescent secondary antibody. (D) The ratio of PGP9.5/DAPI fluorescence area in each group of mice (n = 4 per group). The data were expressed as mean ± SD. ^###P < 0.001, vs. control groups; **P < 0.01, ***P < 0.001, vs. DNCB (model) groups.

FIGURE 4

POAE and POEE reduced the number of immune cells and abnormal increase in serum Ig E in AD mice. (A) Spleen indices of mice in each group (n = 5 per group). (B–F) The number of lymphocytes, eosinophils, basophils, monocytes and neutrophils in each group of mice (n = 5 per group). (G) Pictures of representative dorsal skin TB staining of each group. Red circles mark some of the mast cells (Scale bar: 200 μm). (H) The number of mast cells in the dermis of each group (n = 3 per group). (I) Serum Ig E concentration of mice in each group (n = 3 per group). The data were expressed as mean ± SD. ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001, vs. control groups; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. model (DNCB) groups.

FIGURE 5

POAE and POEE inhibit skin lesions and pruritus-associated cytokines in AD mice. (A) Skin His concentrations in each group of mice. (B) Skin Il-31 concentrations in each group of mice. (C–H) Relative mRNA expression of Ifn-γ, Tnf-α, Tslp, Il-4, Flg, and Lor in each group of mice. The data were expressed as mean ± SD (n = 3 per group). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, vs. control groups; *P < 0.05, **P < 0.01, ***P < 0.001, vs. model (DNCB) groups.