Effect of Neferine on DNCB-Induced Atopic Dermatitis in HaCaT Cells and BALB/c Mice

Abstract

Atopic dermatitis (AD) is a chronic and persistent inflammatory skin disease characterized by eczematous lesions and itching, and it has become a serious health problem. However, the common clinical treatments provide limited relief and are accompanied by adverse effects. Therefore, there is a need to develop novel and effective therapies to treat AD. Neferine is a small molecule compound isolated from the green embryo of the mature seeds of lotus (Nelumbo nucifera). It has a bisbenzylisoquinoline alkaloid structure. Relevant studies have shown that neferine has many pharmacological and biological activities, including anti-inflammatory, anti-thrombotic, and anti-diabetic activities. However, there are very few studies on neferine in the skin, especially the related effects on inflammatory skin diseases. In this study, we proved that it has the potential to be used in the treatment of atopic dermatitis. Through in vitro studies, we found that neferine inhibited the expression of cytokines and chemokines in TNF-α/IFN-γ-stimulated human keratinocyte (HaCaT) cells, and it reduced the phosphorylation of MAPK and the NF-κB signaling pathway. Through in vivo experiments, we used 2,4-dinitrochlorobenzene (DNCB) to induce atopic dermatitis-like skin inflammation in a mouse model. Our results show that neferine significantly decreased the skin barrier damage, scratching responses, and epidermal hyperplasia induced by DNCB. It significantly decreased transepidermal water loss (TEWL), erythema, blood flow, and ear thickness and increased surface skin hydration. Moreover, it also inhibited the expression of cytokines and the activation of signaling pathways. These results indicate that neferine has good potential as an alternative medicine for the treatment of atopic dermatitis or other skin-related inflammatory diseases.

Keywords: MAPK; NF-κB; atopic dermatitis; keratinocytes; natural product; neferine.

Figure 1

Cell viability of HaCaT cells after pretreatment with different concentrations of neferine (1, 3, 10, and 30 μM). HaCaT cells were treated with different concentrations of neferine at 37 °C for 24 h in 5% CO₂. Values represent the mean ± SEM from at least three experiments.

Figure 2

The effect of neferine on the mRNA expression levels of cytokines in TNF-α/IFN-γ-stimulated HaCaT cells. HaCaT cells were pretreated with different concentrations of neferine (1, 3, and 10 μM) for 20 min, and then the cells were treated with TNF-α/IFN-γ (10 ng/mL) for 1 h or 24 h (for TSLP). Total RNA was isolated, and the mRNA expression level of (A) IL-1β, (B) IL-6, (C) IL-8, and (D) TSLP was determined using qPCR. Values represent the mean ± SEM from three independent experiments. ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ treatment condition.

Figure 3

The effect of neferine on the mRNA expression levels of chemokines in TNF-α/IFN-γ-stimulated HaCaT cells. HaCaT cells were pretreated with different concentrations of neferine (1, 3, and 10 μM) for 20 min, and then the cells were treated with TNF-α/IFN-γ (10 ng/mL) for 24 h. The mRNA expression level of (A) TARC (CCL17), (B) MDC (CCL22), and (C) RANTES was determined using qPCR. Values represent the mean ± SEM from three independent experiments. ^# p < 0.05, ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ treatment condition.

Figure 3

The effect of neferine on the mRNA expression levels of chemokines in TNF-α/IFN-γ-stimulated HaCaT cells. HaCaT cells were pretreated with different concentrations of neferine (1, 3, and 10 μM) for 20 min, and then the cells were treated with TNF-α/IFN-γ (10 ng/mL) for 24 h. The mRNA expression level of (A) TARC (CCL17), (B) MDC (CCL22), and (C) RANTES was determined using qPCR. Values represent the mean ± SEM from three independent experiments. ^# p < 0.05, ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ treatment condition.

Figure 4

Neferine reduced TNF-α/IFN-γ-induced JNK (A), p38 (B), and ERK (C) activation in human keratinocyte (HaCaT) cells. Human keratinocyte (HaCaT) cells were pretreated with different concentrations of neferine (1, 3, and 10 μM) for 20 min, and then the cells were treated with TNF-α/IFN-γ (10 ng/mL) for 30 min. Western blots were analyzed quantitatively. Values represent the mean ± SEM from three independent experiments. ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ treatment condition.

Figure 5

Neferine inhibited TNF-α/IFN-γ-induced IκB (A) and NF-κB (B) activation in HaCaT cells. Cells were pretreated with different concentrations of neferine (1, 3, and 10 μM) for 20 min and then were treated with TNF-α/IFN-γ (10 ng/mL) for 30 min. Western blots were analyzed quantitatively. Values represent the mean ± SEM from three independent experiments. ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ treatment condition.

Figure 6

Neferine ameliorated skin inflammation in DNCB-induced BALB/c mice. (A) Histopathological variation due to DNCB induction was evaluated using hematoxylin–eosin staining (scale bar, 50 μm). (B). Quantitative analysis of epidermal thickness. Values represent the mean ± SEM from at least six independent experiments. ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the DNCB-induced group.

Figure 7

Change in physiological parameters of DNCB-induced BALB/c mouse skin after treatment with neferine. Analysis of the effect of the change in trans-epidermal water loss (TEWL), erythema, blood flow, hydration, and ear thickness in an atopic-dermatitis-like phenotype in BALB/c mice. Values represent the mean ± SEM from at least six independent experiments. ^# p < 0.05; ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the DNCB-induced group.

Figure 7

Change in physiological parameters of DNCB-induced BALB/c mouse skin after treatment with neferine. Analysis of the effect of the change in trans-epidermal water loss (TEWL), erythema, blood flow, hydration, and ear thickness in an atopic-dermatitis-like phenotype in BALB/c mice. Values represent the mean ± SEM from at least six independent experiments. ^# p < 0.05; ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the DNCB-induced group.

Figure 8

(A) Analysis of the effect of the change in the number of scratches in BALB/c mice with an atopic-dermatitis-like phenotype. (B) Effect of neferine on the weight of the spleen. Values represent the mean ± SEM from at least six independent experiments. ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the DNCB-induced group.

Figure 9

Effect of neferine on p38 (A), ERK (B), and IκB (C) activation in BALB/c mice with an atopic-dermatitis-like phenotype. Western blots were analyzed quantitatively. Values represent the mean ± SEM from at least six independent experiments. ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the DNCB-induced group.

Figure 10

Effect of neferine on cytokine mRNA expression in BALB/c mice with an atopic-dermatitis-like phenotype. Total RNA was isolated and an mRNA analysis was performed via real-time RT-PCR. (A) IL-1β, (B) IL-6, (C) TNF-α, and (D) TSLP. Values represent the mean ± SEM from at least six independent experiments. ^# p < 0.05; ^## p < 0.01 compared with the no-treatment condition; * p < 0.05 and ** p < 0.01 compared with the DNCB-induced AD group.