Fig 1.
TCA contributes to the inhibitory effect of BPLE on UVB-induced MMP-1 expression in HaCaT cells.
A and C, Protein expression was analyzed by Western blotting (MMP-1), Zymography (MMP-2), and ELISA (MMP-1 contents). Cells were treated with BPLE/PLE (A) or TCA/DAA (C) at the indicated concentration for 1 h before being exposed to 0.01 J/cm2 of UVB, and media was harvested 48 h later. B and D, Cell viability of HaCaT cells in the presence or absence of BPLE, PLE (B) or TCA, DAA (D). Cell viability was measured using MTT assay. Each experiment was performed in triplicate. The data are presented as the mean ±S.D. of MMP-1 protein content and cell viability. Means with different letters (a-c) within a graph were significantly different from each other at p < 0.05.
Fig 2.
BPLE and TCA inhibit UVB-induced MMP-1 mRNA expression and AP-1 transactivation in HaCaT cells.
A and B, Cells were treated with BPLE and TCA at the indicated concentrations for 1 h before being exposed to 0.01 J/cm2 of UVB and mRNA was collected 12 h later. MMP-1 mRNA expression was analyzed as qRT-PCR. C and D, BPLE and TCA attenuated UVB-induced AP-1 transactivation in HaCaT cells. Cells were treated with BPLE and TCA at the indicated concentration for 1 h before being exposed to 0.01 J/cm2 of UVB and cells were lysed 12 h later. AP-1 transactivation was measured using Luciferase assay. All data are presented as the mean ±S.D. determined from three independent experiments. Means with different letters (a-c) within a graph were significantly different from each other at p < 0.05.
Fig 3.
BPLE and TCA inhibit UVB-induced binding affinity of AP-1 residues in HaCaT cells.
A, B, and C, DNA binding activity of phospho-c-jun (A), c-Fos (B) and Fra-1 (C). Cells were treated with BPLE and TCA at the indicated concentrations for 1 h before being exposed to 0.01 J/cm2 of UVB and nuclear extract was collected 4 h later. DNA binding activity was measured by TransAM AP-1 DNA-binding ELISA kit. Means with different letters (a-d) within a graph were significantly different from each other at p < 0.05. D, Phosphorylated and total protein levels of c-Jun were measured by Western blot assay. Relative band intensity is expressed as fold of control and indicated on top of each band.
Fig 4.
BPLE and TCA inhibit UVB-induced Akt phosphorylation but not Mitogen-activated kinase (MAPK) signaling pathway in HaCaT cells.
A-D, Cells were treated with BPLE (A, C) and TCA (B, D) at the indicated concentration for 1 h before exposed to 0.01 J/cm2 of UVB and collected after 30 min. Phosphorylated and total protein levels were analyzed by Western blot assay. Relative band intensity is expressed as fold of control and indicated on top of each band.
Fig 5.
BPLE and TCA inhibit Phosphoinositide 3-kinase (PI3K) activity.
A, B and C, PI3K activity was measured by HTRF described in the Materials and Methods. PI3K isoforms, p110α/p85α (A), p110β/p85α (B), and p110δ/p85α (C) were used for measuring activity. Means with different letters (a-d) within a graph were significantly different from each other at p < 0.05.
Fig 6.
BPLE and TCA decrease collagen degradation and MMP-1 expression in a human skin equivalent model A, A schematic diagram of the 3D human skin cell culture system.
The experimental procedure was described in the Materials and Methods. B and C, BPLE and TCA inhibit UVB-induced MMP-1 protein expression levels and collagen degradation. The serial sections, from the human skin equivalent, were mounted onto silane-coated slides and subjected to Masson’s trichrome staining (B and D) or to immunohistochemical staining using anti-MMP-1 antibody (C and E). MMP-1 appears brown. Relative density was measured using the ImageJ program. Means with different letters (a-d) within a graph were significantly different from each other at p < 0.05.