Orthopedic Department, First Affiliated Hospital, Fujian Medical University, Fuzhou, China.
Department of pharmacology, Fujian Medical University, Fuzhou, China.
Int Wound J. 2022 Dec;19(8):2000-2011. doi: 10.1111/iwj.13800. Epub 2022 Mar 22.
Fasudil is reported to be effective at protecting against ischaemic diseases, and at augmenting axon growth. In this study, we aim to evaluate its efficacy in promoting flap survival and reinnervation. Ninety-two Institute of Cancer Research (ICR) mice were used and divided into the control, Fasudil, LY294002, Fasudil+LY294002 groups, receiving a daily intraperitoneal injection of normal saline, Fasudil (10 mg/kg), LY294002 (5 mg/kg), and Fasudil (10 mg/kg) + LY294002 (5 mg/kg), respectively. On days 0 and 5, the blood perfusion and diameter of the iliolumbar artery in the pedicle of the flaps in the four groups were evaluated using laser speckling contrast imaging (LSCI). On day 5, the flaps were photographed and the necrosis rate of the flaps was calculated using Photoshop CS6. In addition, tissues were harvested from the flaps and divided into two parts. One part underwent routine cryosection and immunofluorescent staining using the antibody against CD31 for evaluation of the microvascular density in the four groups. In the other part, the expression of RhoA, ROCK1+2, p-CPI-17, p-MYPT, p-PTEN, p-PI3K, p-Akt, and vascular endothelial growth factor (VEGF) within the flaps were determined using western blotting. Moreover, at days 0, 7, 15, and 30 after flap surgery, the axons within the flaps were evaluated using immunofluorescent staining with the antibody against Neurofilament-200. It turned out that the necrosis rate was (24.4 ± 7.7)%, (5.2 ± 1.6)%, (29.8 ± 4.2)%, and (30.9 ± 7.1)%, respectively, in the control, Fasudil, LY294002, LY294002+Fasudil groups. There was a significant reduction in the necrosis rate of the flaps in the Fasudil group (P < .001). The LSCI and immunofluorescent staining demonstrated that Fasudil could significantly expand the diameter of the iliolumbar artery in the pedicle, boost the overall blood perfusion, and increase the microvascular density of the flaps in the Fasudil group (P < .05), which could all be abolished by PI3K inhibitor LY294002. On day 5, the expression of p-CPI-17, p-MYPT, and p-PTEN were downregulated, whereas pPI3K, p-Akt, and VEGF were upregulated in the Fasudil group (P < .001). As for reinnervation, Neurofilament-200 fluorescent staining revealed that at days 15 and 30 after flap harvest, only in the Fasudil group could new axons be observed. It can be concluded that Fasudil could simultaneously improve the survival and axon growth after flap harvest, a dual efficacy achieved by inhibition of the RhoA/ROCK pathway, which in turn activates /PI3K/AKT pathway.
法舒地尔被报道具有预防缺血性疾病和促进轴突生长的作用。在本研究中，我们旨在评估其促进皮瓣存活和再神经支配的疗效。使用 92 只 ICR 小鼠，并将其分为对照组、法舒地尔组、LY294002 组和法舒地尔+LY294002 组，分别接受生理盐水、法舒地尔（10mg/kg）、LY294002（5mg/kg）和法舒地尔（10mg/kg）+LY294002（5mg/kg）的每日腹腔内注射。在第 0 天和第 5 天，使用激光散斑对比成像（LSCI）评估四组皮瓣蒂部髂腰动脉的血流灌注和直径。第 5 天，对皮瓣进行拍照，并使用 Photoshop CS6 计算皮瓣的坏死率。此外，从皮瓣中采集组织，并分为两部分。一部分进行常规冷冻切片和用抗 CD31 抗体进行免疫荧光染色，以评估四组的微血管密度。在另一部分中，使用 Western blot 检测皮瓣内 RhoA、ROCK1+2、p-CPI-17、p-MYPT、p-PTEN、p-PI3K、p-Akt 和血管内皮生长因子（VEGF）的表达。此外，在皮瓣手术后第 0、7、15 和 30 天，使用抗神经丝-200 抗体进行免疫荧光染色评估皮瓣内的轴突。结果显示，对照组、法舒地尔组、LY294002 组和 LY294002+法舒地尔组的皮瓣坏死率分别为（24.4±7.7）%、（5.2±1.6）%、（29.8±4.2）%和（30.9±7.1）%。法舒地尔组皮瓣坏死率明显降低（P<0.001）。LSCI 和免疫荧光染色表明，法舒地尔可显著扩大皮瓣蒂部髂腰动脉的直径，增加整体血流灌注，并增加法舒地尔组皮瓣的微血管密度（P<0.05），而这些作用均可被 PI3K 抑制剂 LY294002 所阻断。第 5 天，p-CPI-17、p-MYPT 和 p-PTEN 的表达下调，而 p-PI3K、p-Akt 和 VEGF 的表达上调（P<0.001）。至于再神经支配，神经丝-200 荧光染色显示，在皮瓣切除后第 15 天和第 30 天，只有法舒地尔组才能观察到新的轴突。可以得出结论，法舒地尔可以同时改善皮瓣切除后的存活和轴突生长，这种双重作用是通过抑制 RhoA/ROCK 通路实现的，进而激活/PI3K/AKT 通路。
