Topical knockdown of HO-1 through siRNA improves skin wound healing in mice with radiation-wound combined injury

To detect the expression profile of heme oxygenase-1 (HO-1) during the process of wound repair in radiation-wound combined injury (R-W-CI), and evaluate its wound healing improving effects of R-W-CI by HO-1 knockdown with siRNA.

A total of 36 male C57BL/6J mice (8 weeks old) were randomly and equally divided into a simple skin wound group (W group) and a skin wound group combined with whole-body radiation (6 Gy) injury (R-W-CI group). During the wound healing process, the wounds were photographed and recorded, and the residual areas were quantified by Image J. Wound tissues were sampled and stained with HE staining for pathological and histological observation, and the damage to the hematopoietic system was assessed by dynamic examination of the peripheral blood. The expression and changes of HO-1 in wound tissues were detected by q-PCR and Western blotting. Then, 26 male C57BL/6J mice (8 weeks old) were randomly and equally divided into siRNA knockdown HO-1 group (si-HO-1 group) and siRNA negative control group (si-NC group). After radiation combined injury was inflicted, 60 μL of F127 gel loaded with si-HO-1 (5 μm/L) was applied to each wound in the si-HO-1 group, and an equal amount of F127 gel loaded with negative control si-NC was applied to the wound in the si-NC group. The knockdown of HO-1 in wound tissues was detected by Western blotting, and the changes in wound area were observed. In the wound tissues harvested in 3 d after wounding, the expression of cytokines IL-1β, IL-6 and TNF-α was examined by q-PCR and the proliferation of granulation tissues was evaluated by Ki67 immunohistochemical staining. HE staining was performed on wound tissues on day 3 and day 9 post-injury to assess the improvement effect of knockdown of HO-1 on wound healing of radiation combined injuries.

Compared with the W group, semi-quantitative analysis of the residual wound area showed that healing was significantly delayed in the R-W-CI group on days 7 and 10 post-injury (P<0.01). HE staining on day 7 showed that in the R-W-CI group, the re-epithelialization was delayed, and the growth of granulation tissues was poor; and at the same time, peripheral blood leukocytes and their classified counts showed a significant decrease in the early period after injury (P<0.05). Further tests indicated that the expression of HO-1 protein was slightly higher in the wound of the R-W-CI group than that of the W group in 3 and 7 d after injury, though no significant difference (P>0.05), whereas statistical difference was seen in 10 d (P<0.05), accompanied by the distribution of the full-length and truncated forms of HO-1 protein. Quantitative PCR obtained similar results in the mRNA expression of HO-1 in wounds in both 7 and 10 d after injury (P<0.05). siRNA intervention could effectively knock down the HO-1 protein level of the wounds (P<0.05), promote wound contraction (P<0.05), reduce the width of the wound (P<0.01), up-regulate the inflammatory cytokines IL-6 and TNF-α in 3 d, enhance the proliferation of repair cells in wound margin, and improve the growth of the granulation tissue in the R-W-CI model when compared with the conditions after si-NC intervention.

There exists a sustained high expression level of HO-1 during wound repair, and wound knockdown of HO-1 by siRNA can improve the lack of inflammation status and promote wound healing in R-W-CI mice.