1.南京特殊教育师范学院 康复科学学院;2.东南大学附属中大医院 心血管内科;3.东南大学附属中大医院 康复医学科
目的 探讨静脉注射脑源性神经营养因子（BDNF）对有氧运动促进心肌梗死后心肌血管生成、改善心功能的协同效应及其机制。 方法 细胞试验中，我们利用平行板流动小室建立12dyn/cm2的层流剪切力（LSS）以模拟运动对血管壁的生理效应。分别观察非循环流体、循环流体、循环流体加入人重组BDNF蛋白（50ng/ml）所制造的LSS对人脐静脉内皮细胞（HUVECs）上BDNF蛋白表达、BDNF高亲和力受体TrkB及其下游Akt通路磷酸化水平的影响，并进一步观察其对HUVECs体外血管生成能力的作用。动物试验中，60只12周龄雄性Sprague-Dawley（SD）大鼠按随机数字表抽取10只作为假手术组（Sham），余下大鼠永久性结扎左冠状动脉前降支后，随机分为心肌梗死组（MIC）、心肌梗死+BDNF组（MICB）、心肌梗死+运动组（MIE）、心肌梗死+运动+ BDNF组（MIEB）；造模术前及术后1周分别进行心彩超检查，随后开始为期4周的有氧运动训练，MIEB组大鼠在运动前10分钟尾静脉注射人重组BDNF蛋白（0.4μg/kg）, MICB组仅注射等量BDNF；训练结束行心彩超检查后腹腔麻醉取材，免疫组化检测心肌梗死周围区血管密度。 结果 循环流体产生的LSS可诱发HUVECs上BDNF蛋白表达水平增加，TrkB及其下游Akt通路磷酸化程度持续增强，循环流体中BDNF水平随干预时间的延长而增高。非循环流体产生的LSS可诱发HUVECs上BDNF蛋白表达增加，但TrkB及其下游Akt通路均处于未激活状态。增加循环流体中BDNF浓度可扩大TrkB及其下游Akt通路活化程度，进一步提高HUVECs体外血管生成能力。心肌梗死大鼠有氧运动结合尾静脉注射 BDNF较单纯注射BDNF和单纯运动组大鼠心肌血管密度显著增高、心功能改善。 结论 运动通过LSS以BDNF浓度依赖性模式诱发HUVECs上TrkB持续磷酸化并激活其下游Akt信号通路，提高细胞迁移及小管形成能力；外源性补充BDNF可协同有氧训练强化运动的促心肌血管生成效应、进一步改善心功能。
Objective To explore the synergistic effects and related mechanisms of brain-derived neurotrophic factor (BDNF) on aerobic exercise-induced angiogenesis and improved cardiac function. Methods In the in vitro experiments, human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress (LSS) of 12 dyn/cm2 to mimic the effects of exercise training on vascular tissue. non?circulating flow, circulating flow, circulating flow with BDNF (50ng/ml) patterns were used to produce LSS. BDNF/TrkB protein expression levels were examined, meanwhile phosphorylated TrkB and Akt protein levels were also observed under the intervention of LSS induced by the three different flow patterns respectively; HUVEC were divided into five groups: control group, non?circulating flow group, circulating flow group, BDNF group, circulating flow with BDNF group; after intervention for 6 h, the capacities of cell migration and tube formation were analysed. In the in vivo experiments, fifty-eight rats were assigned to Sham, myocardial infarction (MIC), myocardial infarction + BDNF (MICB), myocardial infarction + exercise (MIE) and myocardial infarction + exercise + BDNF (MIEB) groups, The exercise groups were subjected to 4 weeks of treadmill running; The MIEB group included rats subjected to exercise and daily tail intravenous injection of rhBDNF at a dose of 0.4μg/kg 10 minutes before exercise, the MICB group included rats subjected to the same dose of rhBDNF only; The rats underwent cardiac functional evaluations, prior to exercise training, at 1 week after cardiac surgery and following the 4?week period of exercise training; The blood vessel density in the surrounding area of myocardial infarction was detected by immunohistochemistry after the 4?week period of training. Results An increased BDNF protein expression, as well as phosphorylated of TrkB and Akt levels were observed in HUVECs exposed to LSS generated by circulating fluid. LSS elicited sustained increase BDNF level in the circulating medium. Circulating fluid with BDNF could increase the TrkB and Akt phosphorylated degree and improve the angiogenesis ability of HUVECs. In HUVECs exposed to LSS generated by non-circulating fluid, an increased BDNF protein expression were also observed, while the TrkB and its downstream Akt pathway were inactive. The in vivo experiments confirmed that BDNF administration in association with aerobic exercise increased myocardial angiogenesis and improved cardiac function more significantly compared with that in rats receiving only exercise intervention. Conclusions Exercise activates BDNF/TrkB axis and its downstream Akt pathway through LSS in a BDNF?dependent manner; exogenous supplementation of BDNF in association with aerobic exercise may enhance the pro-myocardial angiogenesis effect of exercise and further improve cardiac function.