Extract of Wheatgrass and Aronia Mixture Ameliorates Atopic Dermatitis-Related Symptoms by Suppressing Inflammatory Response and Oxidative Stress In Vitro and In Vivo

Abstract

Atopic dermatitis is regulated by the production of pro-inflammatory cytokines and chemokines via the nuclear factor kappa B or mitogen-activated protein kinase signaling pathways, as well as, the release of oxidative stress-related factors via the NF-E2 p45-related factor 2 signaling pathway. Both wheatgrass (Triticum aestivum L., TA) and aronia (Aronia melanocarpa, AR) are known for their anti-inflammatory and antioxidant properties, however, the anti-inflammatory and antioxidant effects of TA and AR (TAAR) mixture extract have not been elucidated in an atopic dermatitis model. In this study, we assessed the inhibitory effects and underlying molecular mechanism of TAAR extract against lipopolysaccharide-induced inflammation and tumor necrosis factor-α/interferon-γ-induced inflammation and oxidative stress in vitro. We also investigated the alleviating effect of TAAR extract on DNCB-induced atopic dermatitis-like skin lesions in mice in vivo. We found that TAAR extract treatment inhibited inflammatory mediators in both RAW 264.7 cells and HaCaT cells, and increased the expression of oxidative stress defense enzymes in HaCaT cells. Furthermore, treatment of the DNCB-induced mouse model with TAAR extract ameliorated the overall symptoms of atopic dermatitis. Therefore, TAAR extract as a novel natural therapeutic agent may be used for the treatment of atopic dermatitis.

Keywords: Aronia melanocarpa; Triticum aestivum L.; atopic dermatitis; inflammation; oxidative stress.

Figure 1

HPLC analysis of the TA and AR (TAAR) mixture extract. HPLC chromatograms of the (A) standard compound and (B) TAAR extract sample.

Figure 2

Effects of TAAR extract on the viability of and inflammatory response in LPS-induced RAW 264.7 cells. (A) Cytotoxicity of TAAR extract. mRNA levels of pro-inflammatory cytokines (B) IL-1β and (C) IL-6 in LPS-induced RAW 264.7 cells. Protein levels of (D) iNOS and COX-2 and the mRNA levels of (E) iNOS and (F) COX-2 in LPS-induced RAW 264.7 cells. Expression levels of (G) NO and (H) PGE₂ in LPS-induced RAW 264.7 cells. All values are presented as the mean ± SEM of three experiments. Data were analyzed by Tukey’s post hoc test. ^### p < 0.001 vs. no treatment group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. LPS treatment only group.

Figure 3

Effects of TAAR extract on the viability of and the inflammatory response in TNF-α/IFN-γ-induced HaCaT cells. (A) Cytotoxicity of TAAR extract. mRNA expression of (B–F) cytokines IL-1β, IL-6, TNF-α, IL-5, and TSLP and (G–L) chemokines CCL2, CCL5, CCL17, CCL22, CXCL8, CXCL10. All values are presented as the mean ± SEM of three experiments. Data were analyzed by Tukey’s post hoc test. ^### p <0.001 vs. no treatment group; *** p < 0.001 vs. TNF-α/IFN-γ treatment only group.

Figure 4

Effects of TAAR extract on the NFκB and MAPK signaling pathways in TNF-α/IFN-γ-induced HaCaT cells. (A) Protein levels of cytosol IκB and nucleus NFκB. (B) Bar graph showing the relative density of the Western blot band pIκB/β-actin and NFκB/lamin B. (C) Protein expression levels of phosphorylated and total MAPKs (p38, ERK, and JNK). (D) Bar graph showing the relative density of the Western blot band pp38/p38, pERK/ERK, and pJNK/JNK. All values are presented as the mean ± SEM of three experiments. Data were analyzed by Tukey’s post hoc test. ^### p < 0.001 vs. no treatment group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. TNF-α/IFN-γ treatment only group.

Figure 5

Effects of TAAR extract on the activation of the Nrf2/HO-1/NQO1 signaling pathway in TNF-α/IFN-γ-induced HaCaT cells. (A) Protein levels of Nrf2, HO-1, and NQO1. (B) Bar graph showing the relative density of the Western blot band Nrf2/β-actin, HO-1/β-actin, and NQO1/β-actin. All values are presented as the mean ± SEM of three experiments. Data were analyzed by Tukey’s post hoc test. ^### p < 0.001 vs. no treatment group; *** p < 0.001 vs. TNF-α/IFN-γ treatment only group.

Figure 6

Effect of TAAR extract on DNCB-induced AD-like clinical symptoms in mice. (A) Representative photographic images of AD-like dorsal skin lesions from mice from each group. (B) Atopic dermatitis score and (C) skin thickness. (D) Representative photographs of mouse spleen and (E) spleen weight of mice from each group. (F) Moisture content of dorsal skin. (G) Total serum IgE and (H) serum IL-4 levels. All values are presented as the mean ± SEM of three experiments. Data were analyzed by Tukey’s post hoc test. ^### p < 0.001 vs. normal control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. DNCB-treated only group.

Figure 7

Effects of TAAR extract on histological features in the dorsal tissue of DNCB-induced mice. (A) Representative images of H&E and TB staining of the mice dorsal skin tissue (scale bar: 200 µm). Mast cells are indicated by red arrows. (B) Epidermal thickness, (C) dermal thickness, and (D) number of infiltrated mast cells. All values are presented as the mean ± SEM of three experiments. Data were analyzed by Tukey’s post hoc test. ^### p < 0.001 vs. normal control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. DNCB-treated only group.

Figure 8

Effects of TAAR extract on the mRNA levels of DNCB-induced pro-inflammatory cytokines and chemokines in the dorsal skin tissue of DNCB-induced mice. mRNA expression of (A–D) cytokines IL-1β, IL-6, TNF-α, and IL-5 and (E–I) chemokines CCL17, CCL22, CXCL8, CXCL10, CXCL11. All values are presented as the mean ± SEM of three experiments. Data were analyzed by Tukey’s post hoc test. ^### p < 0.001 vs. normal control group; ** p < 0.01, and *** p < 0.001 vs. DNCB-treated only group.

Figure 9

Effects of TAAR extract on the levels of oxidative stress-related factors in the dorsal skin tissue of DNCB-induced mice. (A–C) mRNA levels of Nrf2, HO-1, and NQO1 in the dorsal skin tissue of DNCB-induced mice. (D) Protein expression of Nrf2, HO-1, and NQO1 in dorsal skin tissue of DNCB-induced mice. (100× magnification). All values are presented as the mean ± SEM of three experiments. Data were analyzed by Tukey’s post hoc test. ^### p < 0.001 vs. normal control group; ** p < 0.01, and *** p < 0.001 vs. DNCB-treated only group.

Figure 10

A pathomechanism summary of the anti-inflammatory and antioxidant effects of the TAAR extract.