Butylphthalide Inhibits Autophagy and Promotes Multiterritory Perforator Flap Survival

Abstract

Multiterritory perforator flap is an important plastic surgery technique, yet its efficacy can be limited by partial necrosis at the choke Ⅱ zone. Butylphthalide (NBP) has been used for many diseases but has not been studied in the multiterritory perforator flap. With the effect of NBP, we observed increasing in capillary density, inhibition of autophagy and oxidative stress, and a reduction in apoptosis of cells, all consistent with increased flap survival. However, the protective effect of NBP on multiterritory perforator flap was lost following administration of the autophagy agonist rapamycin (Rap). Through the above results, we assumed that NBP promotes flap survival by inhibiting autophagy. Thus, this study has found a new pharmacological effect of NBP on the multiterritory perforator by inhibiting autophagy to prevent distal postoperative necrosis and exert effects on angiogenesis, oxidative stress, and apoptosis within the flap.

Keywords: Angiogenesis; Autophagy; apoptosis; butylphthalide; multiterritory perforator flap.

FIGURE 1

NBP improves survival of multiterritory perforator flap. (A) Determine the best dose of the drug by the survival area of rat skin flap. (B) Digital images of control and NBP-treated rats on POD 7. (C) Percentage of survival area on POD 7. (D) LDBF in each group on POD 7. (E) Percentage of blood flow signal intensity within the flap. (F) H&E staining of blood vessel density in the control and NBP groups (200X); scale bar, 50 μm. (G) IHC staining for CD34 in the choke Ⅱ zone of the control and NBP groups (200X); scale bar, 50 μm. (H) Percentage of microvascular density (MVD) in each group. (I) Percentage of CD34 positive vessels. Values are shown as mean ± SEM, n = 6 per group. *p < 0.05 and **p < 0.01 vs. control group.

FIGURE 2

NBP improves angiogenesis in multiterritory perforator flap. (A) and (B) IHC of VEGF and CDH5 in the ischemic flap of the control and NBP-treated rats. (C) and (D) Optical density values of VEGF and CDH5. (E) Western blotting of MMP9, VEGF, and CDH5 in control and NBP-treated groups. (F) Optical density values of MMP9, VEGF, and CDH5 from western blot. Gels were run under similar experimental conditions and cropped edited only for clarity. Values are shown as mean ± SEM, n = 6 per group. *p < 0.05 and **p < 0.01 vs. control group.

FIGURE 3

NBP attenuates apoptosis in multiterritory perforator flap. (A) IHC for CASP3 in the control and NBP groups (200X); scale bar, 50 μm. (B) CASP3 optical density for each group. (C) Western blotting of CYC, Bax, and CASP3. (D) Optical density of CYC, Bax, and CASP3 in each group. Gels were run under similar experimental conditions and cropped edited only for clarity. Values are shown as mean ± SEM, n = 6 per group. *p < 0.05 and **p < 0.01 vs. control group.

FIGURE 4

NBP attenuates oxidative stress in multiterritory perforator flap. (A) IHC of SOD1 in each group (200X); scale bar, 50 μm.(B) Optical density values of SOD1 in each group. (C) Western blotting for SOD1, HO1, and eNOS in each group. (D) Optical density values of SOD1, HO1, and eNOS in each group. Gels were run under similar experimental conditions and cropped edited only for clarity. Values are shown as mean ± SEM, n = 6 per group. *p < 0.05 and **p < 0.01 vs. control group.

FIGURE 5

NBP inhibits autophagy in multiterritory perforator flap. (A) IHC for CTSD in the ischemic flap of the control and NBP-treated animals (200X); scale bar, 50 μm. (B) Autophagosome (LC3, red) immunofluorescent staining of cells in the choke Ⅱ zone in the control and NBP groups. Nuclei are counterstained with DAPI (blue) (scale bar, 20 μm). (C) Optical density values of CTSD in each group. (D) Percentage of LC3-positive cells in each group. (E) and (F) Western blot for Beclin1, CTSD, SQSTM1/p62, and LC3 in the ischemic flap of the control and NBP groups. Gels were run under similar experimental conditions and edited only for clarity. Values are shown as mean ± SEM, n = 6 per group. *p < 0.05 and **p < 0.01 vs. control group.

FIGURE 6

Rapamycin reverses effects of NBP on angiogenesis, oxidative stress, and apoptosis in multiterritory perforator flap. (A) Autophagosomes (LC3, red) in cells in the control, NBP, NBP + rapamycin, and rapamycin groups. Nuclei are counterstained with DAPI (blue) (scale bar, 20 μm). (B) Digital images of the control, NBP, NBP + rapamycin, and rapamycin groups on POD 7. (C) LDBF in each group on POD 7. (D) Percentage of survival area on POD 7. (E) Percentage of the signal intensity of blood flow within the flap in each group. (F) LC3 positive cells in each group. (G) Autophagy-related protein expression (LC3, CTSD, Beclin1, and SQSTM1/p62) and angiogenesis-related proteins (VEGF, MMP9, and CDH5). (H) Optical density of LC3, CTSD, Beclin1, SQSTM1/p62, VEGF, MMP9, and CDH5 in each group. (L) Apoptosis-related protein expression (CYC, Bax, and CASP3) and oxidative stress-related protein expression (SOD1, HO1, and eNOS) in each group. (M) Optical density of CYC, Bax, CASP3, SOD1, HO1, and eNOS expressions in each group. Gels were run under similar experimental conditions and edited only for clarity. Values are shown as mean ± SEM, n = 6 per group. *p < 0.05 and **p < 0.01 vs. control group; #p < 0.05 and ##p < 0.01 vs. NBP group.

FIGURE 7

Multiterritory perforator flap procedure. (A) A 2.5 × 11.0 cm skin flap at the back area was marked. (B) Approximate distribution of involved arteries. (C) Exposed arteries: dorsal thoracic (TD), posterior intercostal (IC), and deep circumflex (DCI) arteries. (D) Excision of the TD, PIC, and preservation of the DCI. (E) The flap is sutured back, and its approximate blood supply range is shown. (F) Multiterritory perforator flap areas: anatomical, dynamic, and potential. The choke Ⅰ zone is between the anatomical and dynamic areas. The choke Ⅱ zone is between the dynamic and the potential areas.