Multifaceted effects of astragaloside IV on promotion of random pattern skin flap survival in rats

Abstract

Random pattern skin flap transplantation is frequently applied in plastic and reconstructive surgery, but the distal part of skin flaps often suffers necrosis due to ischemia. Astragaloside IV (AS-IV), a natural saponin purified from Astragalus membranaceus, may have beneficial functions for flap survival. In this study, rats were divided into a control group and an AS-IV treatment group, and underwent surgery using a modified "McFarlane flap" model. After intragastric administration of vehicle control or AS-IV for their respective groups, flap survival area and water content were measured 7 days after surgery. Flap tissue was separated to test protein expressions related to angiogenesis, inflammation, oxidative stress and autophagy via western blot, immunohistochemistry, and immunofluorescence. Results showed that AS-IV improved flap survival area and reduced tissue edema. AS-IV also increased mean vessel densities and upregulated levels of VEGF protein, both of which indicate increased angiogenesis. Furthermore, AS-IV depressed leukocyte infiltration, decreased expressions of inflammatory proteins TNF-α, IL1β and IL6, increased SOD activity, decreased MDA content, and stimulated autophagy. Overall, our results suggest that AS-IV promotes skin flap survival via inducing angiogenesis, depressing inflammation and dampening oxidative stress; it also activates autophagy, which may be an underlying mechanism for oxidative stress depression.

Keywords: Astragaloside IV; angiogenesis; autophagy; inflammation; oxidative stress; random skin flap survival.

Figure 1

Astragaloside IV improves flap survival area and ameliorates tissue edema. The appearance in the exterior and the degree of edema of the random skin flap were assessed after a modified “McFarlane flap” model was established. A. Digital photographs of flap survival in the Control and AS-IV groups were taken on day 3 and day 7. B. Flap survival percentages for each group were then quantified and analyzed. C. Digital photographs of the inner side of the skin flap were taken from each group to show the degree of tissue edema. D. Quantification and analysis of the tissue water content percentages in each group are shown. Significance: *p < 0.05 versus the Control group. Data represent Mean values ± SEM, n=6 per group.

Figure 2

Astragaloside IV promoted angiogenesis in the ischemic areas of the flap. On day 7, angiogenesis in the ischemic area (Area II) of the flap was assessed via H&E staining, Immunohistochemistry for CD34, and Western blotting for VEGF expressions. A. H&E staining to observe the neovascularization in Area II of the Control group and the AS-IV group (original magnification, × 200; scan bar, 50 μm). B. The mean vessel densities (MVDs) in each group were quantified and analyzed. C. Immunohistochemistry for CD34 to visualize vessels in Area II of the Control and AS-IV groups (original magnification, × 200; scan bar, 50 μm). D. CD34-positive vessel densities in each group are quantified and analyzed. E. Immunohistochemistry for VEGF protein expression in the Area II of the Control group and the AS-IV group (original magnification, × 200; scan bar, 100 μm). F. Optical density values of VEGF expressions are quantified and analyzed for each group. G. Western blotting for VEGF protein expressions in Area II tissue in the Control and AS-IV groups. Gels were run under the same experimental conditions, and cropped blots are used here. The original images are available in Figure S1A. H. Optical density values of VEGF expressions in each group are quantified and analyzed for each group. Significance: *p < 0.05 versus Control group. Data presented as Mean ± SEM, n=6 per group.

Figure 3

Astragaloside IV depressed inflammation in the ischemic area of flap. On day 7, the inflammation in Area II was assessed via Immunofluorescence for CD45 and Western blotting for TNF-α, IL1β, and IL6 expressions. A. Immunofluorescence showing leukocyte infiltration in Area II of the Control and AS-IV groups; leukocytes were labeled with CD45 (green), nuclei was labeled with DAPI (blue) (original magnification, × 200; scan bar, 50 μm). B. CD45-positive cell densities were quantified and analyzed in ischemic flaps for each group. C. Western blot results for TNF-α, the IL1β, and IL6 protein expression in Area II tissue of the Control group and the AS-IV group. Gels were run under the same experimental conditions, and cropped blots are used here. The original images are available in Figure S1B, S1C. D-F. Optical density values of TNF-α, IL1β, and IL6 expressions are quantified and analyzed in each group. Significance: *p < 0.05 versus Control group. Data presented as Mean ± SEM, n=6 per group.

Figure 4

Astragaloside IV dampened oxidative stress in the ischemic area of flap. The oxidative stress in Area II of flaps was assessed via immunohistochemistry for SOD, SOD level, and MDA content test 7 days after surgery. A. Immunohistochemistry for SOD protein expression in Area II of flaps in the Control and AS-IV groups (original magnification, × 200; scan bar, 100 μm). B. Optical density values of SOD expressions are quantified and analyzed in each group. C. The level of SOD activity in Area II of the Control group and the AS-IV group measured via the xanthine oxidase method is quantified and analyzed. D. The values of MDA content in Area II in each group was quantified and analyzed via the modified TBA test. Significance: *p < 0.05 versus Control group. Data presented as Mean ± SEM, n=6 per group.

Figure 5

Astragaloside IV activated autophagy in the ischemic areas of flaps. The autophagy activity in Area II of flap was assessed via Immunofluorescence for LC3 and western blotting for LC3II/I, Beclin1, CTSD, and P62 protein expressions 7 days after surgery. A. Immunofluorescence for the autophagosomes in the cells in Area II of the Control and the AS-IV groups; autophagosomes were labeled via LC3II punctate dots (green), nuclei were labeled with DAPI (blue) (scan bar, 15 μm). B, C. Western blotting was performed for LC3II/I, Beclin1, CTSD and P62 protein expressions in Area II tissue from the Control group and the AS-IV group. Gels were run under the same experimental conditions, and cropped blots are used here. The original images are available in Figure S1D. D-G. Optical density values of LC3II/I, Beclin1, CTSD, and p62 expressions were quantified and analyzed in each group. Significance: *p < 0.05 versus Control group. Data presented as Mean ± SEM, n=6 per group.