Propolis suppresses atopic dermatitis through targeting the MKK4 pathway

Abstract

Propolis is a natural resinous substance made by bees through mixing various plant sources. Propolis has been widely recognized as a functional food due to its diverse range of beneficial bioactivities. However, the therapeutic effects of consuming propolis against atopic dermatitis (AD) remain largely unknown. The current study aimed to investigate the potential efficacy of propolis against AD and explore the active compound as well as the direct molecular target. In HaCaT keratinocytes, propolis inhibited TNF-α-induced interleukin (IL)-6 and IL-8 secretion. It also led to a reduction in chemokines such as monocyte chemoattractant protein-1 (MCP-1) and macrophage-derived chemokine (MDC), while restoring the levels of barrier proteins, filaggrin and involucrin. Propolis exhibited similar effects in AD-like human skin, leading to the suppression of AD markers and the restoration of barrier proteins. In DNCB-induced mice, oral administration of propolis attenuated AD symptoms, improved barrier function, and reduced scratching frequency and transepidermal water loss (TEWL). In addition, propolis reversed the mRNA levels of AD-related markers in mouse dorsal skin. These effects were attributed to caffeic acid phenethyl ester (CAPE), the active compound identified by comparing major components of propolis. Mechanistic studies revealed that CAPE as well as propolis could directly and selectively target MKK4. Collectively, these findings demonstrate that propolis may be used as a functional food agent for the treatment of AD.

Keywords: MKK4; atopic dermatitis; caffeic acid phenethyl ester; propolis; skin barrier.

FIGURE 1

Effects of propolis on the level of cytokines, chemokines, and barrier proteins in HaCaT keratinocytes. Production level of cytokines (A) IL‐6 and (B) IL‐8, and chemokines (C) MCP‐1 and (D) MDC in culture media was determined using ELISA (n = 3). The expression of skin barrier proteins, (E) FLG and (F) IVL was determined by qRT‐PCR. Data represent the mean ± standard deviation (SD). ^# p < 0.05; ^### p < 0.001 versus control. *p < 0.05; **p < 0.01; ***p < 0.001 versus induced group. FLG, filaggrin; IVL, involucrin; MCP‐1, monocyte chemoattractant protein‐1; MDC, macrophage‐derived chemokine; qRT‐PCR, quantitative reverse transcription polymerase chain reaction.

FIGURE 2

Effects of propolis on the levels of AD‐related biomarkers and barrier proteins in human skin tissues. (A) Experimental process and timeline of an ex vivo human skin tissue model (created with BioRender.com). (B) The expression of AD‐related markers in tissues was determined using western blotting. GAPDH served as the loading control. (C–E) The culture media of skin tissues at days 2, 6, 10, and 14 were collected, and the secretion levels of cytokines (C) IL‐6 and (D) IL‐8, and chemokine (E) MCP‐1 were measured by ELISA (n = 3). Data represent the mean ± standard deviation (SD). ^### p < 0.001 versus control group of each day. **p < 0.01; ***p < 0.001 versus induced group of each day. (F) Expression of filaggrin was detected using immunofluorescence staining (scale bar: 100 μm). Representative images of filaggrin (green), bright field (gray), and merged images were presented. AD, atopic dermatitis; ELISA, enzyme‐linked immunosorbent assay; TSLP, thymic stromal lymphopoietin; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; MCP‐1, monocyte chemoattractant protein‐1.

FIGURE 3

Effects of propolis on atopic dermatitis (AD) symptoms, barrier dysfunction, and pruritus in 2,4‐dinitrochlorobenzene (DNCB)‐induced mice. (A) Experimental timeline of AD mouse model (created with BioRender.com). (B) Representative photographs of mice of each group. (C–E) During the experimental period, the effect of propolis on (C) body weight, (D) transepidermal water loss (TEWL), and (E) scratching frequency of mice was determined once a week (n = 7). (F) The ear thickness of all mice was measured at the 35th day before sacrificing the mice. Cetirizine served as a positive control (reference drug). Data represent the mean ± standard deviation (SD). ^### p < 0.001 versus vehicle group. *p < 0.05; ***p < 0.001 versus AD‐induced group.

FIGURE 4

Effects of propolis on histopathological symptoms and atopic dermatitis (AD)‐related biomarkers in the skin of mice. (A) Representative images of mice dorsal skin tissues stained with hematoxylin and eosin (H&E) (scale bar: 200 μm). (B–G) Propolis was treated to mice at 40 and 120 mg/kg B.W. and cetirizine was treated at 5 mg/kg B.W. The mRNA expressions of Th2 cytokines, (B) IL‐4 and (C) IL‐13, barrier proteins, (D) filaggrin (FLG) and (E) involucrin (IVL), and itch‐inducing cytokines, (F) IL‐25 and (G) IL‐33, were examined by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). Data represent the mean ± standard error of the mean (SEM). ^# p < 0.05; ^## p < 0.01; ^### p < 0.001 versus Naïve group, analyzed by t‐test. *p < 0.05; **p < 0.01; ***p < 0.001 versus AD group, analyzed by t‐test. (H) The expression of itch‐inducing cytokine (TSLP) and barrier protein (loricrin) was examined using western blotting. β‐Actin served as the loading control.

FIGURE 5

Effect of propolis on MAPK, NF‐κB, Akt, and MAP2K signaling pathways. Effects of propolis on TNF‐α‐induced (A) MAPK pathway, (B) NF‐κB pathway, and (C) Akt phosphorylation were determined by western blotting in HaCaT keratinocytes. The levels of phosphorylated and basal forms of indicated proteins were examined. (D) The levels of phosphorylated and basal forms of MKK3/6 and MKK4 were examined by western blotting. Vinculin served as the loading control. (E) Kinase activities were determined after propolis treatment. Results are expressed as percentage inhibition of the kinase activity compared to the dimethyl sulfoxide (DMSO) control. (F) The MKK4‐p38 signaling pathway is regulated by propolis (created with BioRender.com). MAPK, mitogen‐activated protein kinase; NF‐κB, nuclear factor kappa B; Akt, protein kinase B; MAP2K, mitogen‐activated protein kinase kinase.

FIGURE 6

Effects of the constituents in propolis extract on the level of atopic dermatitis (AD)‐related biomarkers in HaCaT keratinocytes. (A) Cells were treated with the indicated 10 compounds (2.5 and 10 μM) for 1 h and then TNF‐α (10 ng/mL) was added and incubated for 24 h. The concentration of monocyte chemoattractant protein‐1 (MCP‐1) and IL‐8 in culture supernatants was measured by enzyme‐linked immunosorbent assay (ELISA) (n = 3), and all values are indicated by the percent of the induced group.

FIGURE 7

Effects of caffeic acid phenethyl ester on the activities of p38 and MAP2K signaling pathway. (A) The molecular structure of caffeic acid phenethyl ester (CAPE). (B) Kinase activities were determined after CAPE treatment (10 μM). Results are expressed as percentage inhibition of the kinase activity measured in the dimethyl sulfoxide (DMSO) control wells. (C) Effects of CAPE on regulating the phosphorylation of p38 and MKKs were determined by western blotting in HaCaT keratinocytes. Vinculin served as the loading control. (D–E) Docking prediction of CAPE to the MKK4's adenosine triphosphate (ATP) binding site. (D) The binding conformation among CAPE (represented in cyan), Adenylyl imidodiphosphate (AMP‐PNP) (represented in purple), and the active site of MKK4 was depicted. (E) The chemical binding residues and interactions were shown in a 2D diagram.