Sun Yu, Li Qi-Fang, Zhang Ying, Hu Rong, Jiang Hong
Department of Anesthesiology, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai.
Cell Physiol Biochem. 2013;31(4-5):579-91. doi: 10.1159/000350078. Epub 2013 Apr 26.
Survival of random-pattern skin flaps is important for the success of plastic and reconstructive surgeries. This study investigates isoflurane-induced protection against ischemia of skin flap and the underlying molecular mechanism in this process.
Human umbilical vein endothelial cells (HUVECs) and human skin fibroblast cells were exposed to isoflurane for 4 h. Expression of hypoxia inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) were analyzed up to 24 h post isoflurane exposure using qRT-PCR and western blot, or ELISA analyses. PI3K inhibitors--LY 294002 and wortmannin, mTOR inhibitor--rapamycin, and GSK3β inhibitor--SB 216763 were used respectively to assess the effects of isoflurane treatment and HIF-1α expression. Furthermore, 40 rats were randomly divided into 5 groups (control, isoflurane, scrambled siRNA plus isoflurane, HIF-1α siRNA plus isoflurane, and DMOG) and subjected to random-pattern skin flaps operation. Rats were prepared for evaluation of flap survival and full-feld laser perfusion imager (FLPI) (at 7 day) and microvessel density evaluation (at 10 day).
Isoflurane exposure induced expression of HIF-1α protein, HO-1 and VEGF mRNA and proteins in a time-dependent manner. Both LY 294002 and wortmannin inhibited phospho-Akt, phospho-mTOR, phospho-GSK 3β and HIF-1α expression after isoflurane exposure. Both wortmannin and rapamycin inhibited isoflurane-induced phospho-4E-BP1 (Ser 65) and phospho-P70(s6k) (Thr 389) and HIF-1α expression. SB 216763 pre-treatment could further enhance isoflurane-induced expression of phospho-GSK 3β (Ser 9) and HIF-1α protein compared to the isoflurane-alone cells. In animal experiments, isoflurane alone, scrambled siRNA plus isoflurane, or DMOG groups had significantly upregulated vascularity and increased survival of the skin flaps compared to the controls. However, HIF-1α knockdown abrogated the protective effect of isoflurane preconditioning in rats.
Isoflurane preconditioning improves survival of skin flaps by up the regulation of HIF-1α expression via Akt-mTOR and Akt-GSK 3β signaling pathways.
随意型皮瓣的存活对于整形和重建手术的成功至关重要。本研究调查异氟烷对皮瓣缺血的保护作用及其在此过程中的潜在分子机制。
将人脐静脉内皮细胞（HUVECs）和人皮肤成纤维细胞暴露于异氟烷4小时。使用qRT-PCR、蛋白质免疫印迹或酶联免疫吸附测定（ELISA）分析，在异氟烷暴露后长达24小时检测缺氧诱导因子-1α（HIF-1α）、血红素加氧酶-1（HO-1）和血管内皮生长因子（VEGF）的表达。分别使用磷脂酰肌醇-3激酶（PI3K）抑制剂LY 294002和渥曼青霉素、哺乳动物雷帕霉素靶蛋白（mTOR）抑制剂雷帕霉素以及糖原合成酶激酶3β（GSK3β）抑制剂SB 216763来评估异氟烷处理和HIF-1α表达的影响。此外，将40只大鼠随机分为5组（对照组、异氟烷组、乱序小干扰RNA加异氟烷组、HIF-1α小干扰RNA加异氟烷组和二甲基乙二酰甘氨酸（DMOG）组），并进行随意型皮瓣手术。对大鼠进行皮瓣存活评估，并使用全场激光灌注成像仪（FLPI）（在第7天）和微血管密度评估（在第10天）。
异氟烷暴露以时间依赖性方式诱导HIF-1α蛋白、HO-1和VEGF mRNA及蛋白的表达。LY 294002和渥曼青霉素均抑制异氟烷暴露后的磷酸化Akt、磷酸化mTOR、磷酸化GSK 3β和HIF-1α表达。渥曼青霉素和雷帕霉素均抑制异氟烷诱导的磷酸化4E结合蛋白1（Ser 65）和磷酸化P70核糖体蛋白S6激酶（Thr 389）以及HIF-1α表达。与单独使用异氟烷处理的细胞相比，SB 216763预处理可进一步增强异氟烷诱导的磷酸化GSK 3β（Ser 9）和HIF-1α蛋白的表达。在动物实验中，与对照组相比，单独使用异氟烷组、乱序小干扰RNA加异氟烷组或DMOG组的皮瓣血管生成显著上调，皮瓣存活率增加。然而，HIF-1α基因敲低消除了异氟烷预处理对大鼠的保护作用。
异氟烷预处理通过Akt-mTOR和Akt-GSK 3β信号通路上调HIF-1α表达，从而提高皮瓣存活率。
