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.

To evaluate the inhibitory effects of sulfasalazine (SSZ) on ionizing radiation (IR) induced epithelial-to-mesenchymal transition (EMT), migration, invasion, and other metastasis-related phenotypes in breast cancer cells in vitro, and to investigate its antagonistic effects as a therapeutic measure against breast cancer lung metastasis promoted by in vivo local IR.

Cultured breast cancer cell lines MCF-7 and 4T1 were used to study the morphological changes of cells after different doses of X-ray irradiation (2, 4, 6, 8 and 10 Gy) and the expression changes of EMT-related markers such as Vimentin and Snail were detected by Western blotting. A cell model with 4 Gy of X-ray irradiation was selected to evaluate the effect of SSZ (0.5 mmol/L) on metastasis-related phenotypes such as EMT, migration, and invasion of cells after IR, and on the protein expression of MMP2/9 and xCT after IR. After xenograft model of 4T1 cell breast cancer lung metastasis was established in BALB/c female mice, the mice were divided into control group, SSZ group (intraperitoneal injection of 8 mg SSZ, 2 times per day), IR group (X-ray 4 Gy irradiation) and IR+SSZ group (X-ray 4 Gy irradiation, intraperitoneal injection of 8 mg SSZ, 2 times per day). The tumor masses were harvested in 30 d after tumor loading, and the size of in situ tumor and the number of lung metastatic nodes were measured and calculated, and HE staining was used to observed in situ tumor and lung metastases.

Cultured breast cancer cells showed significant EMT morphological alterations after IR, and the expression of Vimentin and Snail was upregulated in a dose-related manner. SSZ inhibited IR-induced changes in EMT morphology and marker proteins, suppressed IR-induced phenotypes of enhanced migration and invasion of breast cancer cells (P<0.05), and down-regulated IR-induced enhanced expression of MMP2/9, xCT, and other proteins. In vivo animal model corresponded to the ex vivo cellular results that IR significantly inhibited in situ tumor growth. Compared with the control mice (1.70±0.54 cm³), the mean tumor volume was significantly smaller in the IR group (1.24±0.32 cm³) and IR+SSZ group (0.83±0.54 cm³) (P<0.05), and the reduction was more significant in the latter than in the former group (P<0.05). The number of lung metastases in the IR group (19.00±8.31) was significantly higher than that in the Con group (8.67±7.88) (P<0.05), and the number in the IR+SSZ group (6.10±7.78) was significantly lower than the IR group (P<0.05), while the number in the SSZ group (4.30±5.39) was not statistically different from the Con group. HE pathology showed that the number of metastases in the IR group was increased, the area was enlarged, the border was not clear, and the metastases had a tendency to diffuse. In contrast, the lung metastases in the IR+SSZ group had clearer borders and a weaker tendency to invade outward.

SSZ effectively inhibits IR-induced EMT, migration, and invasion of breast cancer cells and significantly antagonizes the lung metastasis-promoting effects of in vivo local irradiation of breast cancer.