Ozonated Sunflower Oil (OSO) Alleviates Inflammatory Responses in Oxazolone-Induced Atopic Dermatitis (AD)-Like Mice and LPS-Treated RAW 264.7 Cells

Abstract

Ozone, a highly reactive oxidant molecule, is widely used as a complementary therapy for various skin diseases, including wound healing, pressure ulcers, diabetic foot, and infections. However, there is limited research on the effectiveness of ozone for atopic dermatitis (AD). Ozonated sunflower oil (OSO) is an active ingredient obtained from partially ozonated sunflower oil (SO). OSO markedly reduced the LPS-induced increase in IL-1β and nitric oxide (NO) levels in RAW 264.7 mouse macrophage cells. Oxazolone (OXZ) was applied to hairless mice to induce AD-like skin symptoms and immune response. OSO significantly alleviated the OXZ-induced increases in the number of infiltrating mast cells, epidermal thickness, AD symptoms, thymic stromal lymphopoietin (TSLP), and filaggrin, as well as the serum levels of NO, IgE, IL-1β, and TNF-α. Furthermore, OSO inhibited the IL-4/STAT3/MAPK pathway and the expression of NF-κB. Our results suggest that OSO treatment could relieve AD-mediated skin damage through its anti-inflammatory and antioxidant activities. Therefore, it can be used as a therapeutic agent against AD-related skin diseases.

Keywords: Atopic dermatitis (AD); Ozonated sunflower oil (OSO); RAW 264.7; TSLP; filaggrin; inflammation.

Fig. 1. Anti-inflammatory and antioxidant effects of OSO on LPS-stimulated RAW 264.7 cells.

(A) DPPH radical scavenging activity of OSO at various concentrations. (B-C) Viability of RAW 264.7 cells was measured using a WST-8 assay. The cells were treated with OSO for 24 h (B) or co-treated with OSO and LPS for 24 h (C). (D-E) NO assay and ELISA were performed to determine NO (D) and IL-1β (E) levels in the culture supernatant after treatment with OSO in the presence of LPS for 24 h. The results are expressed as the mean ± SEM (n = 3). *, p < 0.05; **, p < 0.01 compared with DPPH-only. ^$$, p < 0.01 compared with DPPH+SO (A). **, p < 0.01 compared with control (B). **, p < 0.01 compared with LPS-only (C). **, p < 0.01 compared with LPS-only. ^$$, p < 0.01 compared with LPS + SO. ^##, p < 0.01 compared with the non-treated samples (D–E).

Fig. 2. Topical administration of OSO improves AD symptoms.

(A) Experimental procedure. (B) Representative photographs of mouse dorsal skin. (C) Histological examination of dorsal skin lesion epidermal thickness using H&E staining. Scale bar, 100 μm. (D) Number of infiltrated mast cells as determined using TB staining. Scale bar, 100 μm. (E) Dermatitis score was analyzed by summing the observed signs in the clinical trials, such as excoriation, scaling, edema, and erythema, with scores of 0 (almost clear), 1 (mild), 2 (moderate), and 3 (severe) for each sign. (F) Histological examination of dorsal skin lesion epidermal thickness was measured using H&E staining. Scale bar, 100 μm. (G) Number of infiltrated mast cells was measured using TB staining. Scale bar, 100 μm. (H) ELISA was performed to determine IgE levels in the serum. (I–J) At the end of the experiment, the mice were euthanized, and spleen weight (I) and inguinal lymph node length (J) were measured. The results are expressed as the mean ± SEM (n = 3–4 per group). *, p < 0.05; **, p < 0.01 compared with the OXZ-only group. $, p < 0.05; ^$$, p < 0.01 compared with the OXZ + SO group. ^##, p < 0.01 compared with the normal group (E-J).

Fig. 3. OSO treatment restores the skin barrier function in an AD-like hairless mouse model.

A-B. At the end of the experiment, TEWL (A) and hydration (B) levels in the AD-like mouse skin lesions were measured. C–G. AD skin lesion filaggrin (Scale bar, 100 μm) (C), TSLP (Scale bar, 50 μm) (D), and IL-4 (Scale bar, 100 μm) (E) expression levels were analyzed using DAB staining. Analysis of epidermis filaggrin (F) and TSLP (G) DAB staining intensity was performed using Image (J). (H) Quantification of the protein ratios p-STAT3 (Tyr⁷⁰⁵)/STAT3 and p-ERK(Thr²⁰²/Tyr²⁰⁴)/ERK in AD-like mouse skin lesion. The results are expressed as the mean ± SEM (n = 3–4 per group). *, p < 0.05; **, p < 0.01 compared with the OXZ-only group. ^$, p < 0.05; ^$$, p < 0.01 compared with the OXZ + SO group. ^##, p < 0.01 compared with the normal group (A, B, F, G, and H).

Fig. 4. OSO exerts anti-inflammatory and antioxidant effects in AD-like hairless mice.

(A) The effect of OSO on serum NO levels was determined using NO assay. (B) The iNOS/GAPDH ratio in skin tissue. (C-D) IL-1β (C) and TNF-α (D) levels in the serum were determined using ELISA. (E) Quantification of the protein ratios p-JNK (Thr¹⁸³/Tyr¹⁸⁵)/JNK and p-p38 (Thr¹⁸⁰/Tyr¹⁸²)/p38. (F–G) NF-κB (F) and NRF2 (G) on an AD skin lesion (Scale bar, 100 μm) were confirmed using DAB staining. The results are expressed as the mean ± SEM (n = 3–4/group). *, p < 0.05; **, p < 0.01 compared with the OXZ-only group. ^$, p < 0.05; ^$$, p < 0.01 compared with the OXZ + SO group. ^##, p < 0.01 compared with the normal group (A–E).