A Study of the Wound Healing Mechanism of a Traditional Chinese Medicine, Angelica sinensis, Using a Proteomic Approach
Figure 5
Functional confirmation of the effects of AS and FA treatment on human fibroblasts. (a) Collagen secretion was significantly increased after AS extract treatment. After cells were seeded in a 24 well plate and treated with 0.1% DMSO, 300 μg/mL AS extract or 3.5 μM FA for 24 hr, the medium were collected to analyze the collagen concentration. The graph represents the ratio of collagen secretion compared to the DMSO control. (b) Fibroblast migration is promoted after treatment with AS extract or FA. A total of 2 × 104 cells were seeded into the top of Boyden chamber with 0.1% DMSO, 300 μg/mL AS extract or 3.5 μM FA and added fresh culture medium into the bottom. After 6 hr, the cells were stained by 5% Giemsa dissolved in water and the upper cells on Boyden chamber were scraped with cotton swabs then the cells counted using an inverted microscope. (c) TGF-β expression was increased after AS extract treatment and was correlated with collagen secretion (a). A total of 1 × 106 fibroblasts were seeded onto a 10 cm culture dish and then treated with DMSO, 300 μg/mL AS extract or 3.5 μM FA for 24 hr. After washing with 1 × PBS, the cells were lysed by NP-40 lysis buffer with protease and phosphatase inhibitor. TGF-β expression was determined by immunoblotting. The expression of phospho-Akt (d) and phospho-Erk (e) were also examined by immunoblotting and it was found that AS extract was able to significantly increase expression of these proteins. The relative expression levels were detected by Fujifilm Multigauge software and normalized with β-tubulin. The results are expressed as mean ± SD (). *, **, compared with the control.
