Figure 1.
β-catenin status, wound healing and stem cell markers during the healing process in small wounds.
Full-thickness wounds (diameter = 0.5 cm) were generated on the backs of 8-week-old C3H mice. Wounded tissues were excised at 1, 4, 7, and 10 d post-wounding, and subjected to H&E staining, immunohistochemical analysis, and western blotting as described in the Materials and Methods. (A) H&E staining (first row panels) (original magnification ×100) and immunohistochemical staining for β-catenin, keratin 14, α-SMA, collagen I, and collagen III (other row panels) in the wounds (original magnification ×200). EP, epidermis. (B) Western blot analyses of β-catenin, keratin 14, α-SMA, collagen I, collagen III, and ERK in large and small wounds. (C) Immunohistochemical analysis of Nestin or CD34 in the wounds at 1, 4, 7, 10 d post-wounding (original magnification ×635).
Figure 2.
Effects of VPA on cutaneous wound healing.
After four full-thickness skin excisions (diameter = 0.5 cm) were made on the backs of 8-week-old C3H mice, 500 mM VPA was topically applied to the wounds daily. Tissues were excised from the wounded area and fixed in paraformaldehyde for H&E staining. (A) Gross images of representative wounds after the 7-d VPA treatment. (B) Relative healing of wounds by VPA treatment. Wound sizes were measured at 1, 3, 5, and 7 d after wounding. Asterisks denote the significant differences between control and test groups as measured by t-test with two asterisks being p<0.005 (n = 10). (C) H&E stained sections of wounded skin treated with or without VPA (original magnification ×100).
Figure 3.
Effects of VPA on levels of β-catenin, wound healing, proliferation, and apoptosis markers in small wounds.
The wounded skin of 8-week-old male C3H mice was treated daily with 500 mM VPA for 7 days. A portion of the wounded tissues was excised and frozen in liquid nitrogen for Western blot analysis, and the remaining tissue was fixed in paraformaldehyde. (A) Immunohistochemical analysis of β-catenin, filaggrin, loricrin, or keratin 14 in the neo-epidermis of control and VPA-treated wounds (original magnification ×635). (B) Western blot analysis of β-catenin, filaggrin, loricrin, keratin 14, α-tubulin, α-SMA, collagen I, collagen III, caspase 8, Mcl-1, or PCNA in the control and VPA-treated wounds. (C) Immunohistochemical analysis of α-SMA collagen I, or collagen III in the control and VPA-treated wounds (original magnification ×635). (D) The control and VPA-treated wounds were stained for Ki67 and evaluated using the TUNEL assay. The nuclei were stained with DAPI (original magnification ×635).
Figure 4.
Effects of VPA on cutaneous wound healing in large wounds.
A full-thickness skin excision (diameter = 1.5 cm) was made on the backs of 8-week-old C3H mice, and 500 mM VPA was topically applied to the wounds daily. Tissues were excised from the wounded area and fixed in paraformaldehyde for immunohistochemistry or frozen in liquid nitrogen for Western blotting. (A) Representative gross images of wounded skin after 10-d VPA treatment. (B) Representative H&E stained tissues of wounded skin treated with or without VPA. Arrows represent the wound edge (original magnification ×40). (C) Immunohistochemical analysis of β-catenin, filaggrin, loricrin, keratin 14, or PCNA in the neo-epidermis of control and VPA-treated wounds (original magnification ×635). (D) Immunohistochemical analysis of α-SMA, collagen I, or collagen III in the control and VPA-treated wounds (original magnification ×635). (E) Western blot analysis of β-catenin, filaggrin, loricrin, keratin 14, PCNA, α-SMA, collagen I, collagen III, or α-tubulin in the control and VPA-treated wounds.
Figure 5.
Effects of VPA on the expression of stem cell markers in wounds.
A full-thickness skin excision (diameter = 0.5 cm or 1.5 cm) was made on the backs of 8-week-old C3H mice, and 500 mM VPA was topically applied to the wounds daily. (A) Immunohistochemical analysis of Nestin or CD34 in the control and VPA-treated small wounds (original magnification ×635). (B) Immunohistochemical analysis of Nestin or CD34 in the control and VPA-treated large wounds (original magnification ×635).
Figure 6.
Effects of VPA, β-catenin siRNA, U0126, or LY294002 on HaCaT keratinocyte migration.
HaCaT keratinocytes were used to determine the effect of VPA on human keratinocyte migration. Cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, streptomycin (100 µg/ml), and penicillin (100 µg/ml) in 5% CO2 at 37°C. After scratch wounding with sterile pipette tips, HaCaT keratinocytes were incubated with medium containing 2% serum with or without 100 µM VPA for 24 h. (A) Cells treated with or without 100 µM VPA were stained with crystal violet for 24 h (first row panel; original magnification ×40). Immunocytochemical analyses of phalloidin, β-catenin, or E-cadherin in control and VPA treated HaCaT cells (second, third, and fourth row panel; original magnification ×400). (B) The relative wound closure rate of HaCat cells treated with or without 100 µM VPA. The wound closure rate was measured using NIS-Elements imaging software. Asterisks denote the significant differences between control and test groups as measured by t-test with one asterisk being p<0.05 (n = 3). (C) Western blot analysis of β-catenin, E-cadherin, p-ERK, p-Akt, or α-tubulin in control and VPA-treated HaCaT cells. (D) HaCaT cells were transfected with 100 nM β-catenin siRNA before VPA treatment. The wound closure rate was measured using NIS-Elements imaging software. Asterisks indicate the statistically significant differences as measured by t-test with one asterisk being p<0.05 and two asterisks being p<0.005 (n = 3). (E) 10 µM U0126 or LY294002 was pre-treated for 1 h before VPA treatment, and the wound closure rate was measured after 24-h VPA treatment. One asterisk means p<0.05 and asterisks being p<0.005 (n = 3).