Figure 5.

ADP activates myofibroblasts/fibroblasts and increases the amount of TGF-β⁺cells in the wounds of diabetic mice. Diabetic mice were subjected to excisional full-thickness wounding and then, topically treated with ADP (30 µM/mouse - 30 µL) or saline every day for 7 days. Some mice were treated by gavage with Clop (5 mg/kg) 1 h before ADP or saline administration, once a day for 7 days. (A) Wound tissues harvested at day 7 were stained for α-SMA (green) and DAPI (blue) and analyzed by immunofluorescence. (B) Gel bands and graphs depicting the semi-quantification of α-SMA by WB. Each bar represents a pool of skin-derived protein extracts obtained from at least 5 mice. ^*P<0.05 by Student’s t test compared to saline-treated mice; data are representative of two independent experiments. (C) Primary culture of neonate murine dermal fibroblasts was plated for 24 h, incubated with BrdU for more 24 h and the cell proliferation was evaluated by immunofluorescence. ^*P<0.05 by one-way ANOVA followed by Tukey post-test, compared to saline-treated mice; data are representative of three independent experiments. (D, E) Primary dermal murine fibroblasts were plated for 24 h, pre-incubated with mitomycin-C 5 μg/mL for 2 h and then incubated with different concentrations of ADP. The open ar9ea between the front edges of the scratch were evaluated at 0, 6, 12, 18 and 24 h after scratch and expressed as % of initial area. Fibroblast culture images represent only the first and last time points evaluated for cell migration. ^*P < 0.05 by two-way ANOVA followed by Bonferroni post-test, compared to saline-treated mice, the data are representative of three independent experiments. Photomicrographs and bar graphs of TGF-β⁺ cells determined by IHC in the (F) epidermis and (G) dermis obtained at day 7 after wounding. ^*P < 0.05 by one-way ANOVA followed by Tukey post-test, compared to saline-treated mice, n=8 per group.