Cinnamomum camphora Leaves Alleviate Allergic Skin Inflammatory Responses In Vitro and In Vivo

Abstract

In this study, we investigated the therapeutic potential of Cinnamomum camphora leaves on allergic skin inflammation such as atopic dermatitis. We evaluated the effects of C. camphora leaves on human adult low-calcium high-temperature keratinocytes and atopic dermatitis mice. C. camphora leaves inhibited Macrophage-derived chemokine (an inflammatory chemokine) production in interferon-γ (10 ng/mL) stimulated Human adult low-calcium high-temperature keratinocytes in a dose dependent manner. C. camphora leaves suppressed the phosphorylation of janus kinase signal transducer and activator of transcription 1. C. camphora leaves also suppressed the phosphorylation of extracellular signal-regulated kinase 1/2, a central signaling molecule in the inflammation process. These results suggest that C. camphora leaves exhibits anti-inflammatory effect via the phosphorylation of signal transducer and activator of transcription 1 and extracellular signal-regulated kinase 1/2. To study the advanced effects of C. camphora leaves on atopic dermatitis, we induced experimental atopic dermatitis in mice by applying 2,4-dinitrochlorobenzene. The group treated with C. camphora leaves (100 mg/kg) showed remarkable improvement of atopic dermatitis symptoms: reduced serum immunoglobulin E levels, smaller lymph nodes with reduced thickness and length, decreased ear edema, and reduced levels of inflammatory cell infiltration in the ears. Interestingly, the effects of C. camphora leaves on atopic dermatitis symptoms were stronger than those of hydrocort cream, a positive control. Taken together, C. camphora leaves showed alleviating effects on the inflammatory chemokine production in vitro and atopic dermatitis symptoms in vivo. These results suggest that C. camphora leaves help in the treatment of allergic inflammation such as atopic dermatitis.

Keywords: 2,4-Dinitrochlorobenzene; Atopic dermatitis; Cinnamomum camphora; Immunoglobulin E; Inflammation; Signal transducer and activator of transcription 1.

Fig. 1

Effect of CCex on the MDC production in the IFN-γ-stimulated HaCaT keratinocytes. (A) Cells (1.5 x 10⁵ cells/mL) were pre-incubated for 18 hr, and then treated with IFN-γ (10 ng/mL) in the presence or absence of CCex for 24 hr (12.5–100 μg/mL). Cell viability was determined by the WST assay. (B) Cells (1.5 × 10⁵ cells/mL) were pre-incubated for 18 hr and then treated with CCex (12.5–100 μg/mL) in the presence of IFN-γ (10 ng/mL) for 24 hr. The amounts of MDC were measured from the culture supernatants by ELISA. EGCG was used as a positive control. Data are the mean ± SD of three independent experiments. **p < 0.01 and ***p< 0.001 vs. CCex-untreated cells in the presence of IFN-γ.

Fig. 2

Effect of CCex on the phosphorylation of STAT1 in IFN-γ-stimulated HaCaT keratinocytes. (A) Cells (5.0 × 10⁵ cells/mL) were pretreated with CCex (12.5–100 μg/mL) for 120min and stimulated with IFN-γ (10 ng/mL) for 120 min. The levels of phosphorylated STAT1 (Tyr701, Ser727) were assessed by Western blotting from whole cell lysates. (B, C) Data represent the density ration of STAT 1 (Tyr701) and STAT1 (Ser727) phosphorylation in IFN-γ-stimulated HaCaT keratinocytes. EGCG was used as a positive control.

Fig. 3

Effect of CCex on the phosphorylation of ERK1/2 in IFN-γ-stimulated HaCaT keratinocyte. (A) Cells (5.0 × 10⁵ cells/mL) were pretreated with CCex (12.5–100 μg/mL) for 120 min and stimulated with IFN-γ (10 ng/mL) for 5min. The levels of phosphorylated ERK1/2 were assessed by Western blotting from whole cell lysates. (B) Data represent the density ratio of ERK1/2 phosphorylation in IFN-γ-stimulated HaCaT keratinocytes. PD98059 (ERK1/2 inhibitor) was used as a positive control.

Fig. 4

CCex decreases serum IgE level. (A) Mice were sensitized by applying 1% DNCB or vehicle on their abdomen as the first sensitization (day-7). On day 0, mice were challenged again by applying 0.3% DNCB to the ears on every other day for up to 30 days. Starting on day 12, the mice were treated with hydrocort cream and CCex (10 and 100 mg/kg) on their ears every other day. The mice were sacrificed on day 31. (B) After sacrifice, the IgE in mouse serum was measured by ELISA. Values represent the mean ± SD. ***p< 0.001 compared to mice stimulated with DNCB alone (induction group).

Fig. 5

CCex alleviates the development of experimental AD. (A) Photos of the ears. (B) Ear thickness at indicated days. (C) Paraffin-embeded sections of ear tissue stained with hematoxylin and eosin. (D) Photos of the lymph nodes (LNs). Values represent the mean ± SD. ***p< 0.001 compared to mice stimulated with DNCB alone (induction group).