Natural Hirudin Increases Rat Flap Viability by Anti-Inflammation via PARs/p38/NF-κB Pathway

Abstract

The present study aimed to evaluate the effect of natural hirudin on rat random skin flap viability and to determine the mechanism. Forty-eight rats were randomly divided into 2 groups. After the dorsal skin flap operation (3 cm × 10 cm in size), subcutaneous injections of 6 ATU hirudin were administered to group H (n = 24) every 12 h, while group C (n = 24) received an equal volume of 0.9% normal saline. Six rats from each group were euthanized 1, 2, 4, and 7 days after the operation. A full skin sample was collected from these rats to measure the p38-mitogen-activated protein kinase (p38-MAPK), phospho-p38- (Pp38-) MAPK, nuclear factor-κB (NF-κB) p65, phosphor-NF-κB (pNF-κB) p65, tumour necrosis factor- (TNF-) α, interleukin- (IL-) 6, and intercellular adhesion molecule- (ICAM-) 1 levels via western blot (WB) assays. The results showed that flap viability was significantly higher in the hirudin-treated group, which showed a reduced inflammatory response compared with the control group. The Pp38/p38, pNF-κB p65/NF-κB p65, TNF-α, IL-6, and ICAM-1 levels in the hirudin-treated group were lower than those in the control group. The results demonstrated that hirudin could improve random skin flap viability and suggested that this effect maybe occurs by blocking the thrombin/proteinase-activated receptors (PARs)/p38/NF-κB signalling pathway, thus decreasing the inflammatory response.

Figure 1

Images of the surgical procedures and flap evaluation. (a) A 3 cm × 9 cm flap was designed. (b) The flap was elevated. (c) The skin flaps were resutured to their original bed with 4-0 nylon sutures. (d) Injection of hirudin or 0.9% normal saline was performed. (e) A skin flap from group H on postoperative day 7 is shown. (f) A skin flap from group C on postoperative day 7 is shown.

Figure 2

TNF-α, IL-6, and ICAM-1 levels in the skin flaps. (a) WB images of TNF-α, IL-6, ICAM-1, and GAPDH. (b) IL-6 levels in groups C and H. The IL-6 levels in group C were significantly increased after the operation (^∗ P < 0.05 versus day 0). The IL-6 levels in group H demonstrated no significant changes and were lower than those in group C (^# P < 0.05 versus group C). (c) TNF-α levels in groups C and H. The TNF-α levels in the two groups were significantly increased after the operation (^∗ P < 0.05 versus day 0, ^△ P < 0.05 versus day 0). The TNF-α levels in group H were lower than those in group C (^# P < 0.05 versus group C). (d) ICAM-1 levels in groups C and H. The ICAM-1 levels in group C were significantly increased after the operation (^∗ P < 0.05 versus day 0). The ICAM-1 levels in group H were increased after the operation, but no significant differences were observed between the days, and the levels were lower than those in group C (^# P < 0.05 versus group C).

Figure 3

Pp38/p38 levels in groups C and H. The Pp38/p38 levels in groups C and H were significantly increased after the operation (^∗ P < 0.05 versus day 0, ^△ P < 0.05 versus day 0) and peaked on postoperative day 4. However, the levels in group H were lower than those in group C (^# P < 0.05 versus group C).

Figure 4

pNF-κB p65/NF-κB p65 levels in groups C and H. The pNF-κB P65/NF-κB p65 levels in group C were significantly increased after the operation (^∗ P < 0.05 versus day 0). The pNF-κB p65/NF-κB p65 levels in group H were significantly increased on postoperative days 2 and 4 (^△ P < 0.05 versus day 0). The pNF-κB p65/NF-κB p65 levels in group H were lower than those in group C after surgery (^# P < 0.05 versus group C).