Roxadustat promotes hypoxia-inducible factor-1α/vascular endothelial growth factor signalling to enhance random skin flap survival in rats

Abstract

Random skin flaps have limited clinical application as a broad surgical reconstruction treatment because of distal necrosis. The prolyl hydroxylase domain-containing protein inhibitor roxadustat (RXD) enhances angiogenesis and reduces oxidative stress and inflammation. This study explored the function of RXD in the survival of random skin flaps. Thirty-six male Sprague-Dawley rats were randomly divided into low-dose RXD group (L-RXD group, 10 mg/kg/2 day), high-dose RXD group (H-RXD group, 25 mg/kg/2 day), and control group (1 mL of solvent, 1:9 DMSO:corn oil). The proportion of surviving flaps was determined on day 7 after surgery. Angiogenesis was assessed by lead oxide/gelatin angiography, and microcirculation blood perfusion was evaluated by laser Doppler flow imaging. Specimens in zone II were obtained, and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as indicators of oxidative stress. Histopathological status was evaluated with haematoxylin and eosin staining. The levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and the inflammatory factors interleukin (IL)-1β, IL-6, and tumour necrosis factor-α (TNF-α) were detected by immunohistochemistry. RXD promoted flap survival and microcirculatory blood perfusion. Angiogenesis was detected distinctly in the experimental group. SOD activity increased and the MDA level decreased in the experimental group. Immunohistochemistry indicated that the expression levels of HIF-1α and VEGF were increased while the levels of IL-6, IL-1β, and TNF-α were decreased after RXD injection. RXD promoted random flap survival by reinforcing vascular hyperplasia and decreasing inflammation and ischaemia-reperfusion injury.

Keywords: HIF-1α; VEGF; angiogenesis; inflammation; necrosis; random skin flap; roxadustat.

FIGURE 1

(A) Modified McFarlane (3 × 9 cm) flap on each rat. The area was divided into Zone I, Zone II, and Zone III. (B) Disconnection of the two iliac vessels.

FIGURE 2

(A) Records by digital photographs of flaps in three different zones. General observations of the surviving and necrotic parts of the flaps. (B) Flap survival rate on day 7. Roxadustat promoted the survival rate of skin flaps.**P < .01.

FIGURE 3

(A) Laser Doppler flowmetry angiography demonstrating blood perfusion on day 7. (B) Records of blood perfusion in Zone II. Roxadustat enhanced blood perfusion.**P < .01.

FIGURE 4

X‐ray angiography of flaps in three groups on day 7. Roxadustat increased the number of flap vessels.

FIGURE 5

(A) Histopathological features of the flaps were examined with haematoxylin and eosin staining on day 7. Roxadustat (RXD) decreased histopathological damage. (B) Neutrophil density in Zone II. RXD reduced the infiltration of neutrophil. (C) The microvascular density in Zone II on day 7. RXD promoted microvessel density. **P < .01,*P < .1.

FIGURE 6

(A) Mean activity of superoxide dismutase (SOD) on day 7. (B) Mean activity of malondialdehyde (MDA) on day 7. Roxadustat promoted SOD activity and inhibited MDA activity. **P < .01.

FIGURE 7

(A) Magnification after immunohistochemical staining with different pro‐inflammatory cytokines on day 7. (B) Expression of interleukin (IL)‐6, IL‐1β, and tumour necrosis factor‐α (TNF‐α). Roxadustat attenuated the expression of IL‐6, IL‐1β, and TNF‐α.**P < .01, *P < .1.

FIGURE 8

(A) Magnification after immunohistochemical staining of hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) on day 7. (B) Expression of HIF‐1α on day 7. Roxadustat (RXD) improved the expression of HIF‐1α. (C) Expression of VEGF on day 7. RXD improved the expression of VEGF. **P < .01,***P < .001.

FIGURE 9

Roxadustat promotes hypoxia‐inducible factor‐1α/vascular endothelial growth factor signalling to enhance random skin flap survival.