Shin Jun-Kyu, Kim Jae-Sung
Department of Surgery, Washington University in St. Louis, St. Louis, MO, 63110, USA.
Department of Cell Biology & Physiology, Washington University in St. Louis, St. Louis, MO, 63110, USA.
Biochem Biophys Rep. 2021 Jul 15;27:101075. doi: 10.1016/j.bbrep.2021.101075. eCollection 2021 Sep.
We investigated the cytoprotective effect of desipramine (DMI) during simulated ischemia/reperfusion (I/R) of rat hepatocytes. Primary hepatocytes isolated from male Sprague-Dawley rats were subjected to 4 h of anoxia at pH 6.2 followed by normoxia at pH 7.4 for 2 h to simulate ischemia and reperfusion, respectively. During simulated reperfusion, some hepatocytes were reoxygenated using media containing 5 μM DMI. Necrotic cell death and the onset of mitochondrial permeability transition (MPT) were assessed using fluorometry and confocal microscopy. Changes in autophagic flux and autophagy-related proteins (ATGs) were analyzed by immunoblotting. DMI was shown to substantially delay MPT onset and suppress I/R related cell damage. Mechanistically, DMI treatment during reperfusion increased the expression level of the microtubule-associated protein 1A/1B-light chain 3 (LC3) processing enzymes, ATG4B and ATG7. Genetic knockdown of ATG4B abolished the cytoprotective effect of DMI. Together, these results indicate that DMI is a unique agent which enhances LC3 processing in an ATG4B-dependent way.
我们研究了去甲丙咪嗪（DMI）在大鼠肝细胞模拟缺血/再灌注（I/R）过程中的细胞保护作用。从雄性Sprague-Dawley大鼠分离的原代肝细胞分别在pH 6.2条件下进行4小时缺氧处理，随后在pH 7.4条件下进行2小时常氧处理，以分别模拟缺血和再灌注。在模拟再灌注期间，一些肝细胞使用含有5μM DMI的培养基进行复氧。使用荧光测定法和共聚焦显微镜评估坏死性细胞死亡和线粒体通透性转换（MPT）的发生。通过免疫印迹分析自噬通量和自噬相关蛋白（ATG）的变化。结果表明，DMI可显著延迟MPT的发生并抑制I/R相关的细胞损伤。机制上，再灌注期间DMI处理增加了微管相关蛋白1A/1B轻链3（LC3）加工酶ATG4B和ATG7的表达水平。ATG4B的基因敲低消除了DMI的细胞保护作用。总之，这些结果表明DMI是一种独特的药物，它以ATG4B依赖的方式增强LC3加工。
