Table 1.
Dermatitis score of auricular [23].
Table 2.
List of primers used in this study.
Fig 1.
Treatment with (S)-(-)-blebbistatin O-benzoate improves mite antigen-induced AD-like dermatitis in NC/Nga mice.
(A) The experimental scheme for induction of house dust mite-induced AD-like skin inflammation and treatment (n = 8 in each group). (B) Dermatitis score and (C) Ear thickness were measured on Days 15, 18, 22, 25, 29, 32, and 35 of the experimental period in the right ear. (S)-(-)-blebbistatin O-benzoate showed a significant reduction in dermatitis score compared to the vehicle. (D) Weight changes of mice were measured once a week during the study period. (E) On Day 35, serum was collected and IgE levels were measured by enzyme-linked immunosorbent assay (ELISA). (S)-(-)-blebbistatin O-benzoate showed a significant decrease compared to the vehicle. Data represent mean ± SE of all experiments, *p < 0.05 vs. vehicle.
Fig 2.
(S)-(-)-blebbistatin O-benzoate improves skin barrier function in AD-like lesions of NC/Nga mice.
Histological observation of auricular tissues on Day 35. (A) Sections recovered from vehicle-and (S)-(-)-blebbistatin O-benzoate-treated mice were stained with hematoxylin and eosin. Scale bar = 100 μm. (B) Immunostaining of filaggrin (FLG). Scale bar = 30 μm. FLG expression in the epidermis was enhanced by (S)-(-)-blebbistatin O-benzoate treatment compared to the vehicle treatment. The data are representative of each group. (C) Epidermal thickness of each tissue was measured based on H&E staining images and evaluated quantitatively (n = 8 per group). (S)-(-)-blebbistatin O-benzoate treatment led to a significant reduction in epidermal thickening. Data represent mean ± SE, *p < 0.05 vs. vehicle.
Fig 3.
(S)-(-)-blebbistatin O-benzoate reduces mast cell infiltration in AD-like lesions of NC/Nga mice.
(A) Toluidine blue staining of auricular tissue of vehicle and (S)-(-)-blebbistatin O-benzoate on Day 35. Scale bar = 50 μm. (B) The number of mast cells stained with toluidine blue was measured and quantitatively evaluated in six areas from each tissue (n = 8 per group). (S)-(-)-blebbistatin O-benzoate treatment significantly decreased mast cell numbers. Data represent mean ± SE, *p < 0.05 vs. vehicle.
Fig 4.
(S)-(-)-blebbistatin O-benzoate upregulates expression of skin barrier function genes in keratinocytes.
(A) Viability of keratinocytes exposed to (S)-(-)-blebbistatin O-benzoate for 48 h. (B) Morphology of keratinocytes differentiation incubated Ca2+, (S)-(-)-blebbistatin O-benzoate, or normal medium for 48 h. (C) Gene expression of epidermal barrier-related molecules FLG, loricrin (LOR), involucrin (INV), and (D) ceramide production pathway-related genes SMPD1, SPTLC2, and GBA in keratinocytes incubated with (S)-(-)-blebbistatin O-benzoate for 72 h. Data represent mean ± SE (n = 3/group) and are average values of three independent experiments, *p < 0.05 and **p < 0.01 vs. PBS control.
Fig 5.
(S)-(-)-blebbistatin O-benzoate suppressed type 2 alarmin cytokine gene in keratinocytes stimulated by mite feces-derived antigen.
mRNA expression levels of TSLP, IL-33, IL-1α, and IL-1β were measured in keratinocytes with stimulation with mite feces-derived antigen, IL-4, and TNF-α for 24 h. Treatment of cells with (S)-(-)-blebbistatin O-benzoate simultaneously with the above stimulation significantly suppressed gene expression of TSLP, IL-33, and IL-1α. Data represent mean ± SE (n = 3/group) and are average values of three independent experiments, *p < 0.05 and **p < 0.01 vs. HDM stimulation control. HDM, house dust mite feces-derived antigen.