Oral Administration of p-Hydroxycinnamic Acid Attenuates Atopic Dermatitis by Downregulating Th1 and Th2 Cytokine Production and Keratinocyte Activation
Fig 8
p-hydroxycinammic acid reduces the production of proinflammatory cytokines by activated HaCaT keratinocytes by inhibiting NF-κB and AP-1 activity.
(A) Human keratinocyte HaCaT cells (1 × 106 /well) were pre-treated with p-hydroxycinammic acid (HCA) at different concentrations (10–100 μM) for 30 min and stimulated with tumor necrosis factor (TNF)α (10 ng/ml) and interferon (IFN)γ (10 ng/ml). After 3 h of incubation, the cells were harvested, total RNA was isolated, and the expression of genes encoding inflammatory cytokines (TNFα, IL-1β, IL-6, and TSLP) was measured by conventional RT-PCR. (B) HaCaT cells were pretreated with HCA (50 μM) for 30 min and then activated with TNFα and IFNγ. At different time points (0, 3, and 6 h), the cells were harvested, total RNAs were isolated, and the expression of genes encoding inflammatory cytokines (TNFα, IL-1β, IL-6, and TSLP) was measured by conventional RT-PCR. (C) HaCaT cells (2×106) were transfected with either the pGL3-NF-κB (C) or AP-1 (D) construct in combination with pRL-TK and stabilized for 24 h. The cells were further incubated for 1 h in the absence or presence of HCA (50 μM) and then stimulated for 16 h with TNFα (10 ng/ml) and IFNγ (10 ng/ml). The luciferase activities were measured by a luminometer. The data from three independent treatments are expressed as the mean ±SD. *P<0.05, versus mock (A) or versus treatment without HCA (B–D). (D) Microphotographs of sections of the left ear 28 days after the start of AD induction. The serial sections were immunostained for either keratin 5 (KRT5) or p65 plus DAPI. Lower inserts (left panels) show the costaining for p65 (green) and DAPI (blue) in the squares. At least three randomly chosen sites (squares) in the KRT5 regions were analyzed for nuclear p65 positive cells on the right panel. The data are expressed as mean ± SD (n = 3/group).