Department of Anesthesiology (Hei Long Jiang Province Key Lab of Research on Anesthesiology and Critical Care Medicine), and the Second Affiliated Hospital, Harbin Medical University, Harbin, China.
J Heart Lung Transplant. 2013 Aug;32(8):823-31. doi: 10.1016/j.healun.2013.05.005. Epub 2013 Jun 6.
Hypoxia-reoxygenation of cultured macrovascular endothelial cells is used to study ischemia-reperfusion (IR)-related cellular and molecular changes; however, these models do not accurately depict events in pulmonary microvascular endothelial cells (PMVECs) during conventional lung retrieval and transplantation. We used rat PMVECs in a new non-hypoxic cell-based lung transplantation model to assess these events.
To simulate cold storage, rat PMVECs were preserved in 95% O2-5% CO2 at 4°C for 6 hours in low-potassium dextran solution. Dishes were warmed for 1 hour to room temperature for simulating implantation. Medium was added at 37°C in 50% O2-5% CO2-45% N2 to simulate reperfusion. Additional PMVECs were transfected with siRNA targeting mitogen-activated protein kinases (MAPKs) and then subjected to simulated IR.
MAPKs and NF-κB were activated during simulated reperfusion, and AP-1 was activated during ischemia and reperfusion. Increased malondialdehyde levels were found during cold ischemia, and apoptosis and production of IL-1β, IL-6, and TNF-α were observed during reperfusion. Silencing of MAPKs attenuated oxidative stress, inflammation and apoptosis. Silencing of JNK and p38 decreased NF-κB phosphorylation and increased inhibitor of NF-κB (IκB)α levels. Knockdown of ERK1/2 increased NF-κB phosphorylation but had no effect on IκBα expression. Silencing of JNK and ERK1/2, but not p38, decreased AP-1 phosphorylation.
Exposing rat PMVECs to simulated non-hypoxic IR caused lipid peroxidation, inflammation and apoptosis, which required MAPK-mediated NF-κB and AP-1 activation and distinct regulation of MAPKs by these 2 transcription factors. This model could be used to uncouple mechanisms of IR and evaluate potential therapeutics in alleviating IR injury.
培养的大血管内皮细胞的缺氧-复氧用于研究缺血-再灌注（IR）相关的细胞和分子变化；然而，这些模型并不能准确描述在常规肺获取和移植过程中肺微血管内皮细胞（PMVEC）中的事件。我们使用大鼠 PMVEC 在一种新的非缺氧细胞基础的肺移植模型中评估这些事件。
为了模拟冷藏，将大鼠 PMVEC 在低钾右旋糖酐溶液中于 4°C 下在 95% O2-5% CO2 中保存 6 小时。将培养皿加热 1 小时至室温以模拟植入。在 50% O2-5% CO2-45% N2 中于 37°C 下添加培养基以模拟再灌注。将另外的 PMVEC 用针对丝裂原活化蛋白激酶（MAPK）的 siRNA 转染，然后进行模拟 IR。
在模拟再灌注期间，MAPK 和 NF-κB 被激活，而在缺血和再灌注期间 AP-1 被激活。在冷缺血期间发现丙二醛水平升高，在再灌注期间观察到细胞凋亡和白细胞介素 1β（IL-1β）、白细胞介素 6（IL-6）和肿瘤坏死因子-α（TNF-α）的产生。MAPK 的沉默减弱了氧化应激、炎症和细胞凋亡。JNK 和 p38 的沉默降低了 NF-κB 磷酸化并增加了 NF-κB 抑制剂（IκB）α的水平。ERK1/2 的敲低增加了 NF-κB 磷酸化，但对 IκBα表达没有影响。JNK 和 ERK1/2 的沉默，但不是 p38 的沉默，降低了 AP-1 的磷酸化。
使大鼠 PMVEC 暴露于模拟非缺氧性 IR 导致脂质过氧化、炎症和细胞凋亡，这需要 MAPK 介导的 NF-κB 和 AP-1 激活以及这 2 个转录因子对 MAPK 的不同调节。该模型可用于分离 IR 机制并评估减轻 IR 损伤的潜在治疗方法。
