Fucoidan from Undaria pinnatifida Ameliorates Epidermal Barrier Disruption via Keratinocyte Differentiation and CaSR Level Regulation

Abstract

The epidermal barrier acts as a line of defense against external agents as well as helps to maintain body homeostasis. The calcium concentration gradient across the epidermal barrier is closely related to the proliferation and differentiation of keratinocytes (KCs), and the regulation of these two processes is the key to the repair of epidermal barrier disruption. In the present study, we found that fucoidan from Undaria pinnatifida (UPF) could promote the repair of epidermal barrier disruption in mice. The mechanistic study demonstrated that UPF could promote HaCaT cell differentiation under low calcium condition by up-regulating the expression of calcium-sensing receptor (CaSR), which could then lead to the activation of the Catenin/PLCγ1 pathway. Further, UPF could increase the expression of CaSR through activate the ERK and p38 pathway. These findings reveal the molecular mechanism of UPF in the repair of the epidermal barrier and provide a basis for the development of UPF into an agent for the repair of epidermal barrier repair.

Keywords: CaSR; ERK; UPF; epidermal barrier; p38.

Figure 1

Molecular weight and monosaccharide composition of UPF. (A) The molecular weight and molecular mass distributions of UPF were determined by GPC-MALLS consisting of a refractive index detector Waters 2414 (RI) and a Wyatt DAWN EOS MALLS detector (B) The UPF were dissolved in ammonia, mixed with PMP, and neutralized with 200 µL of formic acid. The derivatization chromatomap was collected by UPLC/Q-TOF-MS. (C) Derivatization chromatomap of standard monosaccharides (Man, Rib, Rha, GluUA, GalUA, Glc, Gal, Xyl, Ara, Fuc).

Figure 2

The effect of UPF on the recovery of epidermal barrier. Epidermal barrier disruption of ICR mice was induced by tape stripping on their shaved back skin until the TEWL reached 40 mg/cm²/hour. UPF hydrogel was administrated topically on the dorsal skin. (A) The photos were taken every 12 h after disruption. (B) The values of TEWL were measured at 0 h, 12 h, 24 h, 48 h, 72 h, 84 h. Data are presented as means ± SEM obtained from three independent experiments, * p < 0.05 and ** p < 0.01 versus the vehicle control. (C) The back skin were harvested at 84 h and the skin sections were prepared and stained with hematoxylin–eosin. (D) The back skin were harvested at 84 h and the skin sections were prepared and stained with immunohistochemistry.

Figure 3

The effect of UPF on proliferation, differentiation and sustained Ca²⁺ concentration in HaCaT cells. HaCaT cells were treated with or without indicated concentrations (10, 20, 50 μg/mL of UPF. (A) The involucrin and filaggrin mRNA levels in HaCaT cells were assayed after 72 h treatment by qPCR. (B) The involucrin and filaggrin proteins levels in HaCaT cells were assayed after 72 h treatment by Western blot. (C) The involucrin and filaggrin mRNA levels in NHEK cells were assayed after 72 h treatment by qPCR. (D) The involucrin and filaggrin proteins levels in NHEK cells were assayed after 72 h treatment by Western blot. (E) The proliferation of HaCaT cells were assayed after 24 h, 48 h or 72 h treatment by BrdU assay. (F) HaCaT cells were treated with or without indicated concentrations (10, 20, 50 µg/mL) of UPF for 24 h, loaded with Fura-4 AM, and the fluorescence was recorded using a confocal microscope. Data are presented as means ± SEM obtained from three independent experiments, * p < 0.05 and ** p < 0.01 versus Control.

Figure 4

The effect of UPF on the expression of CaSR and CaSR mediated signaling pathway. (A) HaCaT cells were treated with or without indicated concentrations (10, 20, 50 µg/mL) of UPF, the CaSR mRNA levels in HaCaT cells were assayed at 12 h, 24 h, and 48 h by qPCR. (B) HaCaT cells were treated with or without indicated concentrations (10, 20, 50 µg/mL) of UPF, the CaSR protein levels in HaCaT cells were assayed at 12 h, 24 h, and 48 h by Western blot. (C) CaSR mediated signaling pathway. (D) HaCaT cells were treated with or without indicated concentrations (10, 20, 50 µg/mL) of UPF, the levels of PLC-γ1, p120-catenin, phospho-PLCγ1 and phospho- p120-catenin in cytoplasm and β-catenin in the nucleus were detected by Western blot. (E) HaCaT cells were exposed to indicate concentrations of UPF with or without NPS-2143 (CaSR inhibitor, 150 nM), the protein levels of PLC-γ1, p120-catenin, phospho-PLCγ1, phospho-p120-catenin, the involucrin and filaggrin were assayed by Western blot. (F) HaCaT cells were exposed to indicate concentrations of UPF with or without NPS-2143 (CaSR inhibitor) for 72 h, the involucrin and filaggrin mRNA levels were assayed by qPCR.

Figure 5

UPF increases the expression of CaSR via the ERK and p38 signaling pathway. (A) HaCaT cells were treated with or without indicated concentrations (10, 20, 50 μg/mL) of UPF, the levels of ERK, p38, phospho-ERK and phospho-p38 in cells were detected by Western blotting. (B) HaCaT cells were exposed to indicated concentrations of UPF with or without LY3214996 (ERK inhibitor, 100 nM) and SB203580 (p38 inhibitor, 20 µM) for 12 h, the levels of PLC-γ1, p120-catenin, phospho-PLCγ1 and phospho-p120-catenin in cells were detected by Western blotting. (C) The relative expression levels of phosphorylation of PLCγ1 and p120-catenin were detected by gray analysis. (D) HaCaT cells were exposed to indicated concentrations of UPF with or without LY3214996 and SB203580 for 72 h, the CaSR mRNA levels in HaCaT cells were assayed by qPCR. (E) HaCaT cells were exposed to indicated concentrations of UPF with or without LY3214996 and SB203580 for 72 h, the involucrin and filaggrin mRNA levels in HaCaT cells were assayed by qPCR.