香青兰总黄酮通过VEGF-B/AMPK通路缓解氧化应激抗H9c2细胞缺血再灌注损伤

Total flavonoids of Dracocephalum moldavica L.inhibit oxidative stress against H9c2 cell ischemia-reperfusion injury via VEGF-B/AMPK pathway

香青兰总黄酮(total flavonoids of Dracocephalum moldavica L.,TFDM)是从维吾尔医药传统药材香青兰中提取分离出来的有效部位,香青兰具有补益心脑、活血化瘀等功效,长期广泛用于治疗心脑血管疾病。本研究旨在确定香青兰总黄酮对H9c2(大鼠心肌细胞)细胞缺氧复氧(hypoxia/re-oxygenation,H/R)损伤的影响及其作用机制。采用缺氧缺糖9 h合并复氧复糖2 h建立H9c2细胞缺氧/复氧损伤模型,模拟心肌缺血再灌注心肌损伤,考察香青兰总黄酮对细胞活力、心肌细胞损伤标志物、氧化应激水平、活性氧自由基(reactive oxygen radical,ROS)含量的影响,并应用Western blot法检测血管内皮生长因子B(vascular endothelial growth factor B,VEGF-B)和磷酸腺苷激活蛋白激酶[adenosine 5'-monophosphate(AMP)-activated protein kinase,AMPK]通路相关蛋白表达。结果显示,香青兰总黄酮显著提高H/R损伤后心肌细胞活力,减少细胞上清中乳酸脱氢酶和肌酸激酶同工酶含量。显著降低丙二醛,增高超氧化物歧化酶和谷胱甘肽过氧化物酶活性、降低细胞内ROS和一氧化氮含量。Western blot分析显示,香青兰总黄酮降低H/R损伤H9c2细胞的BCL-2关联X蛋白水平,上调B淋巴细胞瘤-2基因表达。香青兰总黄酮上调VEGF-B/AMPK通路相关蛋白VEGF-B、血管内皮生长因子受体1、神经纤毛蛋白1、过氧化物酶体增殖物活化受体γ共激活因子1α、磷酸化AMPK、磷酸化的哺乳动物雷帕霉素靶蛋白水平。上述研究结果表明,香青兰总黄酮能够明显减轻心肌细胞H/R损伤,其机制可能与上调VEGF-B/AMPK通路抑制氧化应激反应有关。

Total flavonoids of Dracocephalum moldavica L.(TFDM)is an effective component extracted and isolated from the traditional Uighur medicinal herb Cymbidium fragrans.Cymbidium fragrans has the effects of tonifying the heart and brain,promoting blood circulation and resolving blood stasis,and has been widely used in the treatment of cardiovascular and cerebrovascular diseases for a long time.The purpose of this study was to determine the effect of total flavonoids from Cymbidium fragrans on hypoxia/re-oxygenation(H/R)injury in H9c2(rat cardiomyocytes)cells and its mechanism.A model(H/R)of hypoxia/re-oxygenation injury in H9c2 cells was established using hypoxia and glucose deprivation for 9 h combined with re-oxygenation and rehydration for 2 h to simulate myocardial ischemia-reperfusion injury.The effects of total flavonoids from Cymbidium fragrans on cell viability,markers of myocardial cell damage,oxidative stress levels,and reactive oxygen radical(ROS)content were investigated,Western blot was used to detect the expression of vascular endothelial growth factor B(VEGFB)and adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)pathway related proteins.The results showed that the total flavonoids of Cymbidium fragrans significantly increased the viability of myocardial cells after H/R injury,and decreased the content of lactate dehydrogenase(LDH)and creatine kinase isozyme(CK-MB)in the cell supernatant.It significantly reduced malondialdehyde(MDA),increased superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)activities,and decreased intracellular ROS and nitric oxide(NO)content.Western blot analysis showed that the total flavonoids of Cymbidium fragrans decreased Bax levels in H9c2 cells damaged by H/R and increased Bcl-2 expression.Total flavones of Cymbidium fragrans upregulate VEGF-B/AMPK pathway related proteins VEGF-B,vascular endothelial growth factor receptor 1(VEGFR-1),neuropilin 1(NRP-1),peroxisome-proliferator-activated receptorγcoactivator-1α(PGC-1α),phosphorylated adenosine monophosphate activated protein(p-AMPK)and phospho mechanistic target of rapamycin(p-MTOR)levels.The above research results indicate that the total flavonoids of Cymbidium can significantly reduce the H/R injury of myocardial cells,which may be related to the upregulation of VEGF-B/AMPK pathway and inhibition of oxidative stress response.