Effects of trichostatin A on migration ability of TGF-β1-stimulated A549 cells were measured using cell migration assay (A) and transwell invasion assay (B).

<p>Values are expressed as the mean ± SEM of independent experiments. *P < 0.05 vs. control. †P < 0.05 vs. TGF-β1 alone. Scale bar = 50 μm.</p

Cytotoxicity of histamine determined by MTT assay.

<p>MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide, *P < 0.05 vs. control.</p

Trichostatin A Inhibits Epithelial Mesenchymal Transition Induced by TGF-β1 in Airway Epithelium - Fig 7

<p>Effects of trichostatin A on expression of E-cadherin, vimentin, fibronectin, α-smooth muscle actin, snail, and slug proteins in TGF-β1-stimulated primary nasal epithelial cells were determined by immunofluorescent staining (A). Effects of trichostatin A on expression of E-cadherin, vimentin, fibronectin, and α-smooth muscle actin protein in TGF-β1-stimulated inferior turbinate tissue were determined by western blotting (B). Representative of independent experiments. Scale bar = 50 μm.</p

Effects of trichostatin A on expression of snail and slug mRNA and protein in TGF-β1-stimulated A549 cells were determined by RT-PCR (A) and western blotting (B) (Representative of independent experiments).

<p>Values are expressed as the mean ± standard error of the mean (SEM) of independent experiments. *P < 0.05 vs. control. †P < 0.05 vs. TGF-β1 alone. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.</p

Effect of baicalin on IL-1β-stimulated collagen contraction and activity of MMP-1 in nasal fibroblasts.

<p>Nasal fibroblasts were stimulated with IL-1β (10 ng/ml) alone or in conjunction with baicalin (50 μM) for 72 hours. (A) Contractile activity was assessed by a collagen gel contraction assay and the contraction area was measured. (B) Collagenase activity, matrix-metalloproteinase (MMP)-1 secretion was measured using collagen zymography. Values are the mean ± SEM of independent samples. *<i>p</i> < 0.05 vs. control; ^†<i>p</i> < 0.05 vs. IL-1β alone.</p

Baicalin Down-Regulates IL-1β-Stimulated Extracellular Matrix Production in Nasal Fibroblasts

<div><p>Purpose</p><p>Baicalin, a Chinese herbal medicine, has anti-fibrotic and anti-inflammatory effects. The aims of present study were to investigate the effects of baicalin on the myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction of interleukin (IL)-1β-stimulated nasal fibroblasts and to determine the molecular mechanism of baicalin in nasal fibroblasts.</p><p>Methods</p><p>Nasal fibroblasts were isolated from the inferior turbinate of patients. Baicalin was used to treat IL-1β-stimulated nasal fibroblasts. To evaluate cytotoxicity, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. The expression levels of α-smooth muscle actin (SMA), fibronectin, phospho-mitogen-activated protein kinase (p-MAPK), p-Akt, p-p50, p-p65, and p-IκBα were measured by western blotting, reverse transcription-polymerase chain reaction (RT—PCR),or immunofluorescence staining. Fibroblast migration was analyzed with scratch assays and transwell migration assays. Total collagen was evaluated with the Sircol collagen assay. Contractile activity was measured with a collagen gel contraction assay.</p><p>Results</p><p>Baicalin (0–50 μM) had no significant cytotoxic effects in nasal fibroblasts. The expression of α<i>–</i>SMA and fibronectin were significantly down-regulated in baicalin-treated nasal fibroblasts. Migration, collagen production, and contraction of IL-1β-stimulated nasal fibroblasts were significantly inhibited by baicalin treatment. Baicalin also significantly down-regulated p-MAPK, p-Akt, p-p50, p-p65, and p-IκBα in IL-1β-stimulated nasal fibroblasts.</p><p>Conclusions</p><p>We showed that baicalin down-regulated myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction via the MAPK and Akt/ NF-κB pathways in IL-1β-stimulated nasal fibroblasts.</p></div

Effect of baicalin on IL-1β-stimulated migration in nasal fibroblasts.

<p>Nasal fibroblasts were stimulated with IL-1β (10 ng/ml) alone or in conjunction with baicalin (50 μM) up to 48 hours. (A) Phase-contrast images (200x) of the migration scratch assay at 48 hours. (B) The distance of cell migration was measured from the phase-contrast images (200x) taken at 0 to 48 hours. (C) Phase-contrast images (200x) of the transwell migration assay at 48 hours. (D) The number of cell migration was counted from phase-contrast images (400x) taken at 0 to 48 hours. Values are the mean ± SEM of independent samples. *<i>p</i> < 0.05 vs. control; ^†<i>p</i> < 0.05 vs. IL-1β alone.</p

Effect of baicalin on IL-1β-stimulated myofibroblast differentiation and extracellular matrix production in nasal fibroblasts.

<p>(A) After treatement with IL-1β (10 ng/ml) with or without baicalin (0–50 μM) for 24 hours, expression level of <i>α-SMA</i>, <i>fibronectin</i> and <i>collagen type I</i> mRNA was determined by RT—PCR. (B) After treatement with IL-1β (10 ng/ml) with or without baicalin (0–50 μM) for 72 hours, level of α-SMA and fibronectin protein was measured by western blot and (C) the total amount of collagen was measured by Sircol assay. (D) After treatement with IL-1β (10 ng/ml) with or without baicalin (0–50 μM) for 72 hours, localization of α-SMA and fibronectin protein were observed by immunocytochemical staining. Values are the means ± SEM of three independent experiments. Images were acquired by confocal laser scanning microscopy. *<i>p</i> < 0.05 vs. control; ^†<i>p</i> < 0.05 vs. IL-1β alone. Scale bar = 50 μm.</p

Effect of baicalin on IL-1β-stimulated NF-κB signaling in nasal fibroblasts.

<p>(A) Nasal fibroblasts were stimulated with IL-1β (10 ng/ml) alone or in conjunction with baicalin (50 μM) for 60 minutes. p-p50, p50, p-p65 and p65 protein production were determined by western blotting and (B) protein expression of p-IκBα and IκBα were assessed by western blotting. (C) Nasal fibroblasts were stimulated with IL-1β (10 ng/ml), with or without baicalin (50 μM) or an NF-κB inhibitor (BAY-117082, 1 μM) for 72 hours. α-SMA and fibronectin protein expression was examined by western blotting and (D) total collagen was measured by the Sircol assay. Values are the means ± SEM of three independent experiments. *<i>p</i> < 0.05 vs. control; ^†<i>p</i> < 0.05 vs. IL-1β alone.</p

Signaling pathway of DEP-induced IL-6 and IL-8 expression in nasal fibroblasts.

<p>(A) The expression level of phosphorylated p38 (p-p38) and Akt (p-Akt) was determined by western blot in nasal fibroblast treated by DEP (50 μg/mL), in the in the presence or absence of SB203580 (p38 inhibitor, 10 μmol/L) or LY294002 (Akt inhibitor, 10 μmol/L). β-actin was used as an internal control. (B) Expression levels of IL-6 and IL-8 were measured by ELISA after treatment with DEP with or without SB203580 or LY294002. The graphic data represents the means ± SEM of three independent experiments. * p<0.05, ** p<0.01 compared to control; † p<0.05 compared to treatment with DEP alone. </p