Regulatory effects of Ishige okamurae extract and Diphlorethohydroxycarmalol on skin barrier function

Abstract

Ethnopharmacological relevance: The pharmacological potential of marine organisms remains largely unexplored. Ishige Okamurae, commonly known as Pae, is extensively distributed over Asia. Its antioxidant, antibacterial, antiobesity, and anti-inflammatory properties are also being investigated.

Aim of the study: In most cases of atopic dermatitis, the stratum corneum, the outermost layer of the epidermis, is damaged, causing symptoms such as dryness and hyperproliferation of the epidermis. In particular, the disruption of cell junctions leads to damage of the skin barrier, exacerbating the disease and becoming a target for therapeutic development. Our study aims to investigate of Ishige okamurae extract (IOE) and a major compound derived from it, called Diphlorethohydroxycarmalol (DPHC), can help strengthen the skin barrier in animals with atopic dermatitis induced by 2,4-dinitrochlorobenzene (DNCB).

Materials and methods: In keratinocyte cell lines, HaCaT cells, the cytotoxicity of IOE and DPHC was assessed by MTT analysis. The gene expression of skin barrier factors and tight junctions were determined by real-time PCR in tumor necrosis factor-α/interferon-γ-stimulated HaCaT cells. In addition, JAK/STAT signaling pathway was performed to evaluating the mechanism of drugs by Western blot. Next, we studied the effects of IOE and DPHC on the skin of animals with DNCB-induced atopic dermatitis. We measured the expression of genes related of the skin barrier and tight junctions in their ear tissue.

Results: As a result, IOE and DPHC confirmed that the expression of skin barrier proteins (thymic stromal lymphopoietin, filaggrin, loricrin, and involucrin) was improved in the DNCB-induced atopic dermatitis model and HaCaT cells. In addition, the expression of tight junction-related proteins (claudin, occludin, and tight junction protein-1) were improved.

Conclusion: IOE and DPHC ameliorated the atopic dermatitis lesions through alleviating the pro-inflammatory responses and tight junction protein destruction. Our results suggest that IOE and DPHC could be promising candidates for enhancing skin barrier function.

Keywords: 2,4-Dinitrochlorobenzene; Ceramide; Diphlorethohydroxycarmalol; Ishige okamurae; Skin barrier; Tight junction.

Fig. 1

Illustrates the impact of IOE on the expression levels of skin barrier genes in HaCaT cells stimulated with TNF-α/IFN-γ. Skin barrier protein levels were assessed through real-time quantitative PCR. Statistical analysis indicated a significant difference (∗p < 0.05, ∗∗p < 0.01) when compared to the TNF-α/IFN-γ-stimulated group.

Fig. 2

Displays the influence of IOE on the gene expression levels of tight junction and stratum corneum proteins in TNF-α/IFN-γ stimulated HaCaT cells. The quantification of skin barrier proteins was conducted using real-time quantitative PCR. Statistical analysis indicated a significant difference (∗p < 0.05, ∗∗p < 0.01) when compared to the TNF-α/IFN-γ-stimulated group.

Fig. 3

Demonstrates the impact of IOE on intracellular signal transduction in TNF-α/IFN-γ stimulated HaCaT cells. The cells were pre-treated with 3 or 30 mg/mL IOE for 1 h before being stimulated with TNF-α/IFN-γ for 30 min. Total protein was extracted using Thermo Fisher total protein extraction reagent.

Fig. 4

Showcases the effect of IOE on skin lesions and IgE levels in a DNCB-induced atopic dermatitis mouse model. (A) The image depicts skin lesions on the ears of mice with DNCB-induced atopic dermatitis 24 h after the final DNCB treatment. Ear thickness was measured using a dial thickness gauge 24 h post-DNCB induction (B), while serum IgE levels were quantified using ELISA (C). Statistical analysis indicated a significant difference (∗p < 0.05, ∗∗p < 0.01) when compared to the DNCB-treated group.

Fig. 5

Examines the impact of IOE on skin barrier gene expression and production in a DNCB-induced atopic dermatitis mouse model. Total RNA was extracted from ear tissues, and the levels of skin barrier proteins such as TSLP, FLG, LOR, and IVL were assessed via real-time PCR. Statistical analysis indicated a significant difference (∗p < 0.05, ∗∗p < 0.01) when compared to the TNF-α/IFN-γ-stimulated group.

Fig. 6

Illustrates the impact of DPHC on the expression levels of skin barrier genes in HaCaT cells stimulated with TNF-α/IFN-γ. The quantification of skin barrier proteins was conducted through real-time PCR. Statistical analysis indicated a significant difference (∗p < 0.05, ∗∗p < 0.01) when compared to the TNF-α/IFN-γ-stimulated group.

Fig. 7

Illustrates the impact of DPHC on the tight junction and SPT gene expression levels in TNF-α/IFN-γ-stimulated HaCaT cells. The quantification of skin barrier proteins was conducted through real-time PCR. Statistical analysis indicated a significant difference (∗p < 0.05, ∗∗p < 0.01) when compared to the TNF-α/IFN-γ-stimulated group.

Fig. 8

Shows the effect of DPHC on intracellular signal transduction in TNF-α/IFN-γ-stimulated HaCaT cells. Prior to being stimulated with TNF-α/IFN-γ for 30 min, HaCaT cells were pre-exposed to either 10 or 60 mg/mL of DPHC for a duration of 1 h. Subsequently, total protein was isolated using the Thermo Fisher total protein extraction reagent. Statistical analysis indicated a significant difference (∗p < 0.05, ∗∗p < 0.01) when compared to the TNF-α/IFN-γ-stimulated group.

Fig. 9

Shows the effect of DPHC on skin lesions and IgE levels in a DNCB-induced atopic dermatitis mouse model. (A) Displays skin lesions on the ears of mice afflicted with DNCB-induced atopic dermatitis, with the image depicting the condition of the ear skin 24 h post the final DNCB treatment. (B) Demonstrates the measurement of ear thickness utilizing a dial thickness gauge 24 h following the initiation of DNCB. (C) Showcases the quantification of serum IgE levels through ELISA. Statistical analysis indicated a significant difference (∗p < 0.05, ∗∗p < 0.01) when compared to the DNCB-treated group.

Fig. 10

Illustrates the impact of DPHC on skin barrier gene expression and production in a DNCB-induced atopic dermatitis mouse model. RNA was isolated from ear tissues, and the quantities of skin barrier proteins including TSLP, FLG, LOR, and IVL were assessed through real-time PCR. Statistical analysis indicated a significant difference (∗p < 0.05, ∗∗p < 0.01) when compared to the DNCB-treated group.