Anti-Inflammatory Effects of the LK5 Herbal Complex on LPS- and IL-4/IL-13-Stimulated HaCaT Cells and a DNCB-Induced Animal Model of Atopic Dermatitis in BALB/c Mice

Abstract

Atopic dermatitis (AD) is a chronic inflammatory skin disease influenced by a complex interplay of genetic and environmental factors. The activation of the JAK-STAT pathway increases the expression of inflammatory cytokines such as IL-4 and IL-13, further deteriorating AD. Therefore, for the treatment of AD, the JAK-STAT pathway is emerging as a significant target, alongside inflammatory cytokines. This study investigates the potential therapeutic effects of a novel herbal complex, LK5, composed of Scutellaria baicalensis, Liriope platyphylla, Sophora flavescens, Dictammus dasycarpus, and Phellodendron schneider, known for their anti-inflammatory and immune-modulating properties. We examined the anti-inflammatory and anti-AD effects of the LK5 herbal complex in HaCaT cells stimulated by LPS and IL-4/IL-13, as well as in a mouse model of AD induced by DNCB. In HaCaT cells stimulated with LPS or IL-4/IL-13, the LK5 herbal complex demonstrated anti-inflammatory effects by inhibiting the expression of inflammatory cytokines including TNF-α, IL-6, and IL-1β, and downregulating the phosphorylation of STAT proteins. In a murine AD-like model induced by DNCB, administration of the LK5 herbal complex significantly ameliorated clinical symptoms, including dermatitis, ear thickness, and TEWL. Histological analysis revealed a reduction in epidermal thickness and mast cell infiltration. The LK5 herbal complex also inhibited pruritus induced by compound 48/80. Furthermore, the LK5 herbal complex treatment significantly decreased the levels of inflammatory cytokines such as TSLP, IL-6, and IgE in plasma and ear tissue of AD-induced mice. These findings suggest that the LK5 herbal complex may modulate the immune response and alleviate AD symptoms by inhibiting STAT pathways.

Keywords: LK5 herbal complex; anti-inflammation; atopic dermatitis; itching; signal transducers and activators of transcription.

Figure 1

Time schedule for the DNCB-induced AD model. The mice were sensitized with 1% DNCB twice. Then, the mice were treated with the LK5 herbal complex (12.5, 25, 50 mg/kg and dexamethasone 1 mg/kg) for 10 days.

Figure 2

HPLC chromatograms of (A) the standard compounds and (B) the LK5 herbal complex. The indicator substances are oxymatrine, chlorogenic acid, baicalin, palmatine, and obacunone, which were analyzed sequentially via HPLC. Oxymatrine was separated sequentially on a YMC J’sphere ODS-H80 column (4.6 × 250 mm, 4 μm) at 13 min, with chlorogenic acid at 15 min 20 s, baicalin at 23 min, palmatine at 24 min 20 s, and obacunone at 36 min. The detection of the five standard compounds was confirmed by comparison with the standards of each compound.

Figure 3

Anti-inflammatory effects of the LK5 herbal complex in LPS-stimulated HaCaT cells. (A) Cell viability. (B) NO production. (C–F) iNOS, IL-1β, TNF-α, and IL-6 mRNA expressions via RT-qPCR. (G–I) IL-1β, TNF-α, and IL-6 production via ELISA. The data were determined after LPS stimulation for 24 h. Statistical analysis was performed using one-way ANOVA. The data represent the means ± S.E.M. ^# p < 0.05, ^### p < 0.001 vs. the Con group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the 0 (LPS-stimulated) group. Con, control; LK5, LK5 herbal complex.

Figure 4

Effects of the LK5 herbal complex on LPS-stimulated STAT signaling in HaCaT cells. HaCaT cells were pretreated with the LK5 herbal complex for 1 h, and then exposed to 1 μg/mL LPS for 3 h. The treated cells were assessed for activation of (A) STAT1, (B) STAT3, and (C) STAT6 expressions using Western blot analysis. Statistical analysis was performed using one-way ANOVA. The data represent the means ± S.E.M. ^# p < 0.05, ^### p < 0.001 vs. the Con group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the 0 (LPS-stimulated) group. Con, control; LK5, LK5 herbal complex.

Figure 5

Effects of the LK5 herbal complex on IL-4/IL-13-stimulated STAT signaling and inflammatory cytokines in HaCaT cells. HaCaT cells were pretreated with the LK5 herbal complex for 1 h, and then exposed to IL-4/IL-13 (30 ng/mL) for 45 min. The treated cells were assessed for (A,B) STAT3, and (A,C) STAT6 expression using Western blot analysis. HaCaT cells were pretreated with the LK5 herbal complex for 1 h, and then exposed to IL-4/IL-13 (30 ng/mL) for 24 h. The LK5 herbal complex-treated cells were evaluated for AD-related inflammatory cytokines such as (D) IL-4, (E) IL-13, (F) IL-25, and (G) IL-33 mRNA expressions via RT-qPCR. Statistical analysis was performed using one-way ANOVA. The data represent the means ± S.E.M. ^# p < 0.05, ^### p < 0.001 vs. the Con group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the 0 (IL-4/IL-13-stimulated) group. Con, control; LK5, LK5 herbal complex.

Figure 6

Effects of the LK5 herbal complex on clinical symptoms in DNCB-induced skin lesions. (A) Images of the dorsal skin were taken on day 10, and are shown as representative images for group. Every 2 days, the clinical symptoms, including (B) body weight, (C) ear thickness (µm), (D) dermatitis score, and (E) TEWL, were assessed (n = 8). Statistical analysis was performed using two-way ANOVA. The data represent the means ± S.E.M. ^### p < 0.001 vs. the control group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the DNCB-only group. Con, control; Dexa, dexamethasone; LK5, LK5 herbal complex.

Figure 7

Effects of the LK5 herbal complex on histological changes in DNCB-induced skin lesions and itching in compound 48/80-induced mice. (A) Dorsal skin images stained with H&E and TB were captured at 200× magnification (scale bar: 200 μm). Analysis was conducted on (B) epidermal thickness (black line), and (C) mast cell infiltration (red arrows) was assessed (n = 4). (D) Changes in pruritus with the LK5 herbal complex administration following compound 48/80 treatment. Statistical analysis was performed using two-way ANOVA. The data represent the means ± S.E.M. ^### p < 0.001 vs. the control group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the DNCB- and compound 48/80-only group. Con, control; Dexa, dexamethasone; Terf, terfenadine, LK5; LK5 herbal complex.

Figure 8

Effect of the LK5 herbal complex on the levels of IgE and inflammatory cytokines, including IL-4, IL-13, and TSLP, in AD-like lesioned mice. The plasma protein levels of (A) IgE, (B) IL-6, and (C) TSLP were determined using an ELISA kit (n = 6). The mRNA expressions of (D) IL-4 (E) IL-13, and (F) TSLP in DNCB-induced ear tissue were evaluated via RT-qPCR. Statistical analysis was performed using one-way ANOVA. The data represent the means ± S.E.M. ^# p < 0.05, ^### p < 0.001 vs. the control group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the DNCB-only group. Con, control; Dexa, dexamethasone.