Cui Chao, Lin Huang, Shi Yanyu, Pan Ruiyan
Department of Aesthetic Plastic Surgery and Laser Medicine, Beijing Anzhen Hospital, Capital Medical University, Anzhen Road #2, Chaoyang District, Beijing 100029, China.
Beijing Institute of Heart Lung and Blood Vessel Diseases, Anzhen Road #2, Chaoyang District, Beijing 100029, China.
Biochem Biophys Res Commun. 2017 Sep 16;491(2):277-284. doi: 10.1016/j.bbrc.2017.07.112. Epub 2017 Jul 23.
In recent years, many studies have demonstrated that endogenous adenosine induced by ischemia postconditioning reduces apoptosis in animal and cell models, but no study has clearly elucidated the effects of hypoxia postconditioning (HPC) in human dermal microvascular endothelial cells (HDMECs) of flaps, and the subtype of adenosine receptors involved remains unknown. In our study, we sought to identify the roles of adenosine A receptor, NDRG3 (N-myc downstream-regulated gene 3) and Raf-ERK pathway in the anti-apoptotic effects of hypoxia postconditioning.
Human dermal microvascular endothelial cells were put into a hypoxic incubator (94% N + 5% CO + 1% O) for 8 h (hypoxia), and followed 24 h of normoxic culture with 95% air and 5% CO (reoxygenation). Hypoxia postconditioning model of HDMECs was achieved as follows: Before HDMECs were put into a normoxic incubator, HDMECs were treated by three cycles of 5 min of brief reoxygenation and 5 min of re-hypoxia. Opening level of mitochondrial permeability transition pore and change of mitochondrial membrane potential were detected with related Kit. The protein expressions of mitochondrion apoptosis, adenosine A receptor and NDRG3-Raf-ERK pathway were measured by western blot.
Hypoxia/reoxygenation (H/R) resulted in injury in HDMRCs as evidenced by an increase in apoptosis percentage, mitochondrial membrane permeability and an increase in expression of pro-apoptosis proteins (Bax, c-caspase-3 and cytochrom C), meanwhile, hypoxia/reoxygenation increased expression of A receptor, NDRG3, p-c-Raf, p-ERK, which was significantly attenuated by hypoxia postconditioning treatment. Moreover, Hypoxia/reoxygenation (H/R) resulted in a decrease in expression of anti-apoptotic protein (Bcl-2). However, the protective effect of hypoxia postconditioning treatment could be inhibited by adding CGS21680, a selective adenosine A receptor agonist (all P values < 0.05).
近年来，许多研究表明，缺血后处理诱导的内源性腺苷可减少动物和细胞模型中的细胞凋亡，但尚无研究明确阐明缺氧后处理（HPC）对皮瓣人真皮微血管内皮细胞（HDMECs）的影响，且所涉及的腺苷受体亚型仍不清楚。在我们的研究中，我们试图确定腺苷A受体、NDRG3（N-myc下游调控基因3）和Raf-ERK通路在缺氧后处理抗凋亡作用中的作用。
将人真皮微血管内皮细胞置于缺氧培养箱（94%N + 5%CO + 1%O）中8小时（缺氧），然后在95%空气和5%CO的常氧培养条件下培养24小时（复氧）。HDMECs的缺氧后处理模型如下：在HDMECs放入常氧培养箱之前，对其进行3个循环的5分钟短暂复氧和5分钟再缺氧处理。用相关试剂盒检测线粒体通透性转换孔的开放水平和线粒体膜电位的变化。通过蛋白质免疫印迹法检测线粒体凋亡、腺苷A受体和NDRG3-Raf-ERK通路的蛋白表达。
缺氧/复氧（H/R）导致HDMRCs损伤，表现为凋亡百分比增加、线粒体膜通透性增加以及促凋亡蛋白（Bax、c-caspase-3和细胞色素C）表达增加，同时，缺氧/复氧增加了A受体、NDRG3、p-c-Raf、p-ERK的表达，而缺氧后处理显著减弱了这些表达。此外，缺氧/复氧（H/R）导致抗凋亡蛋白（Bcl-2）表达降低。然而，添加选择性腺苷A受体激动剂CGS21680可抑制缺氧后处理的保护作用（所有P值<0.05）。
