Figure 1.
Tiger17 promotes HaCat cells proliferation and migration.
(A) Keratinocytes, fibroblasts or macrophages (2×104 cells/ml) were separately cultured in wells of 96-well plate, and cells were incubated with various concentration tiger17 (2.5, 5, 10, 20 μg/ml) or sterile H2O alone for 24 h. After incubation with 5 mg/ml MTT for 4 h, absorbance was determined. It was very obviously that both HaCat keratinocytes and HSFs were increased by tiger17 treated, in a concentration dependent manner. Tiger17 had little effect on macrophages proliferation. Values are the mean ± SE of three independent experiments. *P<0.05, **P<0.01 as compared between tiger17-stimulated and non-stimulated cells. (B) HaCat keratinocytes cells (1×106 cells/ml) were cultured in wells of 6-well plate; cells were scratched and incubated with tiger17 (20 μg/ml) or vehicle. (B-1) Cells migration was recorded by photomicrograph at post-scratched 0 hour, 24th hour and 36th hour. The black line denoted the margin of gap. (B-2) The repair rate of scarification was calculated by Image J. According to the formula, repair rate of scarification % = (the gap width of 0 hour - temporal gap width)/the gap width of 0 hour ×100%. Scarification width was measured in triplicates wells. The distance of matched 6 pairs of points was separately measured in each well for averaging the gap width. Values are the mean ± SE of three independent experiments. **P<0.01 as compared between Tiger17-treated and untreated cells.
Figure 2.
Topical application of tiger17 accelerated healing of full-thickness skin wounds in mice.
Two full-thickness wounds were made on the back of a mouse. Mice bearing wound were randomly divided into two groups, T group and E group, 10 mice each. T group was separately provided 20 μl of tiger17 (20 μg/ml) or vehicle; and E group was respectively applied 20 μl of tiger17 (20 μg/ml) or 20 μl of EGF (100 μg/ml) solution. (A) Images of a representative mouse from each group taken on post-operative day 0, 2, 4, 7 and 9 were shown. (B) Evaluation of wound closure by morphometrical analysis of wound areas (Image J, NIH). Wound residual area was calculated (n = 10) from three independent experiments. *P<0.05; **P<0.01 (red asterisk represented tiger 17 treatment vs EGF treatment, and blue asterisk represented tiger 17 treatment vs control). (C) Post-operative day 8, healing skin was examined by H&E staining sections. The gap between neo-epithelial tongue was much narrower in tiger17-treated group (white arrows pointed) than control (white arrows pointed); and neo-epithelial tongue, marked by yellow dotted line, was much longer in the same magnitude of enlargement in tiger17 treatment mice. NE: neo-epithelium; GT: granulation tissue; Es: eschar. (D) Reepithelialization rate. Wound reepithelialization rate was calculated from the H&E stain sections. Bars = mean ± SEM. *P<0.05; **P<0.01 (tiger17 treatment vs control). Average and standard error of the mean were derived from three independent experiments and 6 different sections per experiment.
Figure 3.
Tiger17 promoted macrophages recruitment, TGF-β1 upexpression and myofibroblasts differentiation.
(A, B) Concentration of TGF-β1 or IL-6 (pg/ml) in the control and various concentration tiger17 (2.5, 5, 10, 20 μg/ml)-treated supernatant. Values are expressed as mean ± SE of triplicate, three independent experiments. *P<0.05; **P<0.01 (tiger17 treatment vs. control). (C, E, G) Immunohistochemical results for anti-F4/80, anti-TGF-β1 and anti-α-SMA. (D, F, H) F4/80, TGF-β1, or α-SMA positive cell numbers of per high power field were significant different between tiger17 treatment and control, **P<0.01. Average and standard error of the mean were derived from three independent experiments and 6 different fields each section (×100).
Figure 4.
Effects of tiger17 on the Smads signal pathway.
(A) Raw 264.7 cells were treated with the indicated concentrations of tiger17 (2.5, 5, 10, 20 μg/ml) or vehicle; Western blotted for total-Smad2, p-Smad2, total-Smad3, p-Smad3 or toal-Smad7 was performed. β-Actin was used as the internal control. (B, C, D) The band intensity of various inducer of Smads pathway = each phosphate-contents/homologous total-contents. The band intensity of inhibitory Smad7 = each Smad7-contents/the same concentration tiger17-stimulated β-Actin. (NIH, Image J). Values are the mean ± SE of three independent experiments, *P<0.05, **P<0.01.
Figure 5.
Effects of tiger17 on the MAPKs signal pathway.
(A) Raw 264.7 cells were treated with the indicated concentrations of tiger17 (2.5, 5, 10, 20 μg/ml) or vehicle; Western blotted for total or phospho-JNK, Erk or P38 was performed. β-Actin was used as the internal control. (B, C, D) The band intensity of various inducer of MAPKs pathway = each phosphate-contents/homologous total-contents. (NIH, Image J). (E) After MAPKs pathway inhibitors were used, TGF-β1 expression was decreased, even the same way tiger17 treatment. Values are the mean ± SE of three independent experiments, *P<0.05, **P<0.01.