Gyogamdan, a Traditional Medicine Prescription, Ameliorated Dermal Inflammation and Hyperactive Behavior in an Atopic Dermatitis Mouse Model Exposed to Psychological Stress

Abstract

Psychological stress (PS) plays a significant role as an aggravating factor in atopic dermatitis (AD). The traditional medicine prescription, Gyogamdan, has been used to treat chest discomfort and mood disorders caused by PS. This study investigated the effects of an ethanolic extract of Gyogamdan (GGDE) on stress-associated AD models and the underlying mechanisms. 2,4-Dinitrochlorobenzene- (DNCB-) treated BALB/c mice were exposed to social isolation (SI) stress. The effects of orally administered GGDE (100 or 500 mg/kg) were evaluated by ELISA, western blotting, and an open field test (OFT). SI stress exaggerated the skin inflammation and induced locomotor hyperactivity in the AD mouse model. GGDE reduced the levels of IgE, TNF-α, IL-13, eotaxin, and VEGF and mast cell/eosinophil infiltration and prevented the decreases in the levels of involucrin and loricrin in the skin. GGDE also suppressed the SI-induced increases in corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and corticosterone (CORT) in socially isolated AD mice. Furthermore, GGDE reduced traveling distances and mean speed significantly in the OFT. The in vitro experiments were performed using HaCaT, HMC-1, PC12, and BV2 cells. In the TNF-α/IFN-γ- (TI-) stimulated HaCaT cells, GGDE decreased the thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) production significantly by inhibiting p-STAT1 and NF-κB signaling. GGDE also reduced VEGF production in HMC-1 cells stimulated with CRH/substance P (SP) by inhibiting p-ERK signaling pathway. GGDE increased the cell viability significantly and suppressed apoptosis in CORT-stimulated PC12 cells. Moreover, GGDE suppressed the LPS-induced production of NO, TNF-α, IL-1β, and IL-6 in BV2 cells. These results suggest that GGDE might be useful in patients with AD, which is exacerbated by PS.

Figure 1

Effects of GGDE on AD mice exposed to PS. (a) Schematic diagram of the animal experiment. (b) Representative images of skin lesions. Clinical severity scores (c) and body weights (d) were measured. Results are presented as means ± SDs (n = 5 per experiment). +P < 0.05, vs. the CON group; ^#P < 0.05, vs. the AD group; ^∗P < 0.05, vs. the SI-AD group.

Figure 2

Effects of GGDE on the inflammatory response in AD mice exposed to PS. (a) The results of histological staining in the dorsal skin. Scale bar: 250 μm. (b) Skin tissue levels of inflammatory mediators and serum IgE levels were measured. The results are presented as means ± SDs (n = 5 per experiment). (c) Effects of GGDE on the skin barrier protein expression in AD mice exposed to PS. Western blot of loricrin and involucrin in dorsal skin lesions. Results are presented as the means ± SDs (n = 3 per experiment). +P < 0.05, vs. the CON group; #P < 0.05, vs. the AD group; ^∗P < 0.05, vs. the SI-AD group.

Figure 3

Effects of GGDE on hypothalamic-pituitary-adrenal (HPA) axis and behavioral changes in AD mice exposed to PS. (a) CRH, ACTH, and CORT levels in serum. (b) Behavior in the open field test. Representative open field test track sheets. Total distances (cm) traveled, distances traveled in the peripheral zone and central zone, and mean speeds (cm/s) in the 10-minute test. The results are presented as the means ± SDs (n = 5 per experiment). +P < 0.05, vs. the CON group; #P < 0.05, vs. the AD group; ^∗P < 0.05, vs. the SI-AD group.

Figure 4

Effects of GGDE on inflammatory mediator release in HaCaT and HMC-1 cells. (a) The HaCaT cell viabilities were assessed using XTT assays. (b) Effects of GGDE on TI (TNF-α and IFN-γ)-induced TARC and MDC productions. (c) Western blot of p-STAT1, STAT1, p-IκB-α, IκB-α, β-actin, NF-κB p65, and lamin B in HaCaT cells. (d) HMC-1 cell viabilities were assessed using XTT assays. (e) Effects of GGDE on CRH/SP-induced VEGF production. (f) Western blot of p-ERK, ERK, p-p38, p38, p-JNK, JNK, and β-actin in HMC-1 cells. Dexamethasone (DEX) was used as a positive control. The results are presented as means ± SDs (n = 3 per experiment). #P < 0.05, vs. CON; ^∗P < 0.05, vs. T/I-treated cells; ∗P < 0.05, vs. CRH/SP-treated cells.

Figure 5

Effects of GGDE on CORT-induced neurotoxicity in PC12 cells. (a) Effects of CORT on the viability of PC12 cells. (b) Effects of GGDE on cell viability in CORT-stimulated PC12 cells. (c) Effects of GGDE on apoptosis rate in CORT-stimulated PC12 cells. The percentage of apoptotic cells was measured using Muse Annexin V and Dead Cell kit. Results are presented as means ± SDs (n = 3 per experiment). #P < 0.05, vs. CON; ^∗P < 0.05, vs. CORT-treated cells.

Figure 6

Effects of GGDE on LPS-induced neuroinflammation in BV2 cells. (a) Effects of GGDE on the viability of BV2 cells. (b) Effects of GGDE on NO production in LPS-stimulated BV2 cells. (c) Effects of GGDE on the production of TNF-α, IL-1β, and IL-6 in LPS-stimulated BV2 cells. Results are presented as means ± SDs (n = 3 per experiment). #P < 0.05, vs. CON; ^∗P < 0.05, vs. LPS-treated cells.

Figure 7

(a) Composition and chemical components in GGDE. (b) HPLC chromatograms of commercial standards for detecting nootkatone and α-cyperone, ergosterol, dehydrotrametenolic acid, pachymic acid, and tumulosic acid. (c) HPLC chromatograms of GGDE for the detection of nootkatone and α-cyperone.