Herrera Blanca, Murillo Miguel M, Alvarez-Barrientos Alberto, Beltrán Jesús, Fernández Margarita, Fabregat Isabel
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Free Radic Biol Med. 2004 Jan 1;36(1):16-26. doi: 10.1016/j.freeradbiomed.2003.09.020.
Transforming growth factor-beta (TGF-beta) induces an oxidative stress process in hepatocytes that mediates its apoptotic activity. To determine the cellular source of the early reactive oxygen species (ROS) generated by fetal rat hepatocytes in response to TGF-beta, we used inhibitors that block different ROS-producing systems. Diphenyleneiodonium, which inhibits NADPH oxidase and other flavoproteins, completely blocked the increase in ROS induced by TGF-beta, coincidently with an impairment of caspase-3 activation and cell death. Rotenone, an inhibitor of the NADH dehydrogenase in mitochondrial complex I, attenuated, but did not completely inhibit, ROS-production, caspase activation, and cell death mediated by TGF-beta. No significant protection was observed with inhibitors of other ROS-producing systems, such as cytochrome P450 (metyrapone), cyclooxygenase (indomethacin), and xanthine oxidase (allopurinol). Additional experiments have indicated that two different mechanisms could be involved in the early ROS production by TGF-beta. First, an inducible (cycloheximide-inhibited) NADPH oxidase-like system could account for the extramitochondrial production of ROS. Second, TGF-beta could increase ROS by a rapid downregulation of antioxidant genes. In particular, intramitochondrial ROS would increase by depletion of MnSOD. Finally, glutathione depletion is a late event and it would be more the consequence than the cause of the increase in ROS induced by TGF-beta.
转化生长因子-β（TGF-β）在肝细胞中诱导氧化应激过程，介导其凋亡活性。为了确定胎鼠肝细胞对TGF-β产生的早期活性氧（ROS）的细胞来源，我们使用了阻断不同ROS产生系统的抑制剂。抑制NADPH氧化酶和其他黄素蛋白的二苯基碘鎓完全阻断了TGF-β诱导的ROS增加，同时伴有caspase-3激活和细胞死亡的受损。鱼藤酮是线粒体复合物I中NADH脱氢酶的抑制剂，可减弱但不能完全抑制TGF-β介导的ROS产生、caspase激活和细胞死亡。对于其他ROS产生系统的抑制剂，如细胞色素P450（甲吡酮）、环氧化酶（吲哚美辛）和黄嘌呤氧化酶（别嘌呤醇），未观察到明显的保护作用。额外的实验表明，TGF-β早期产生ROS可能涉及两种不同机制。首先，一种可诱导的（环己酰亚胺抑制的）NADPH氧化酶样系统可能负责线粒体外ROS的产生。其次，TGF-β可能通过快速下调抗氧化基因来增加ROS。特别是，线粒体内ROS会因锰超氧化物歧化酶的消耗而增加。最后，谷胱甘肽耗竭是一个晚期事件，它更可能是TGF-β诱导的ROS增加的结果而非原因。
