Figure 1.
HE staining showing the histological changes of mouse skin after deep second-degree burn injury.
(A) Normal skin. (B) Burned skin from mice suffering from hot steam for 4 s. Deep partial-thickness burn injury was observed and hair follicles were not damaged. Yellow arrowed lines indicated the thickness of skin in unburned and burned mice. Red arrows indicated hair follicles.
Figure 2.
RT-PCR and IB assays showing the changes on Hsp90α expression after burn injury at the wound edge in mice.
(A) mRNA level of Hsp90α was measured at each time point after burn injury by real-time PCR and normalized against GAPDH. Results showed that the mRNA level in burned skin peaked at 3∼6 h and was 20 times higher than that in control mice (p<0.05). (B) The change of Hsp90α protein level after burn injury was analyzed by western blotting (p<0.05). (C) Histogram quantified the relative Hsp90α level in (B).
Figure 3.
IHC assay showing the changes of Hsp90α immunostaining in burned mouse skin.
(A) Unwound normal skin showed a few positive Hsp90α stainings. (C, E) Epidermal and dermal tissues after burn injury appeared more positive brownish stainings, indicating that Hsp90α was induced after the burn stimulation. In addition, Hsp90α level was the highest at 12 h post-treatment (C) and somewhat decreased at 48 h (E). Magnification of red boxed areas in (A), (C) and (E) was shown as (B), (D)and (F), respectively.
Figure 4.
An in vitro scratch assay showing the effects of Hsp90α on the migration of heat-shocked cells.
Images were taken at the indicated time of incubation. Hsp90α-treated group showed more rapid reduction in the gap size at each time point tested than that in the control group, while 17-DMAG group showed slower gap closure even than the control (p<0.05). AG, average gap, normalized to the gap size at 0 h.
Figure 5.
Flow cytometry assay showing the effects of Hsp90α on cell cycle progression and apoptosis following heat shock.
Heat-shocked cells treated with (A) saline, (B) Hsp90α, and (C) 17-DMAG for 24 h were assessed by flow cytometry. Hsp90α increased the number of cells in G1 phase, while 17-DMAG induced cell cycle arrest at G0–G1 phases. These results were represented histogramatically in (D). FITC- Annexin V/propidium iodide (PI) was used to measure the apoptosis rate of HaCaT cells following three treatments: (E) saline, (F) Hsp90α, and (G)17-DMAG. The percentage of apoptotic cell population was decreased in Hsp90α group and increased in 17-DMAG group (H) (p<0.05).
Figure 6.
An in vivo study showing the effects of Hsp90α on burn wound healing.
(A) Deep second-degree burn wounds (20 mm in diameter) on Balb/c mice were treated topically with saline or Hsp90α (1 µg/ml) daily for 5 days following burn injury. In 17-DMAG group, 17-DMAG at 25 mg/kg b.w. was injected intraperitoneally three times per week for two weeks prior to burn treatment, and then saline was applied topically. (B) Histogram quantified the wound size on day 0, 5, 9, 13 and 21 (n = 5 in each group). (C) Hsp90α promoted the re-epithelialization of deep second-degree burn wound. On day 7 after burn injury, biopsies of burned and unburned skins were excised from control, Hsp90α, and 17-DMAG-treated groups. Samples were then HE stained and photographed with an FSX100 microscope. Images were reconstituted to show the whole healed and unhealed areas. Area between two vertical red lines indicated unhealed skin, while area with black dotted lines indicated newly formed epidermis. Green arrows marked the advancing migrating epithelial tongues on each side of the wounds.