Maity Pallab, Bindu Samik, Dey Sumanta, Goyal Manish, Alam Athar, Pal Chinmay, Mitra Kalyan, Bandyopadhyay Uday
Department of Infectious Diseases and Immunology, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Rd., Jadavpur, Kolkata 700032, West Bengal, India.
Electron Microscopy Unit, Central Drug Research Institute, Chatter Manzil Palace, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India.
J Biol Chem. 2009 Jan 30;284(5):3058-3068. doi: 10.1074/jbc.M805329200. Epub 2008 Dec 2.
We have investigated the role of mitochondria on the development of indomethacin (a non-steroidal anti-inflammatory drug)-induced gastric mucosal apoptosis and associated gastropathy in rat. Transmission electron microscopic studies indicate that indomethacin damages mitochondrial ultrastructure and causes mitochondrial dysfunction as evident from decreased stage-3 respiration, dehydrogenase activity, and transmembrane potential (DeltaPsi(m)). Mitochondrial pathology is associated with increased generation of intra-mitochondrial-reactive oxygen species, such as O(2)(*), H(2)O(2) and OH, leading to oxidative stress. O(2)() is the most effective to damage mitochondrial aconitase, leading to the release of iron from its iron-sulfur cluster. The released iron, by interacting with intra-mitochondrial H(2)O(2), forms *OH. Immunoprecipitation of mitochondrial aconitase and subsequent Western immunoblotting indicate carbonylation of aconitase along with the loss of activity in vivo after indomethacin treatment. The release of iron has been documented by fluorescence imaging of mucosal cells by using Phen Green SK, a specific probe for chelatable iron. Interestingly, intra-mitochondrial *OH generation is crucial for the development of mitochondrial pathology and activation of mitochondrial death pathway by indomethacin. Scavenging of OH by dimethyl sulfoxide or alpha-phenyl-n-tert-butylnitrone, a spin-trap, prevents indomethacin-induced mitochondrial ultrastructural changes, oxidative stress, collapse of DeltaPsi(m), and mitochondrial dysfunction. The scavengers also restore indomethacin-induced activation of caspase-9 and caspase-3 to block mitochondrial pathway of apoptosis and gastric mucosal damage. This study, thus, reveals the critical role of O(2)()-mediated mitochondrial aconitase inactivation to release intra-mitochondrial iron, which by generating *OH promotes gastric mucosal cell apoptosis and gastropathy during indomethacin treatment.
我们研究了线粒体在吲哚美辛（一种非甾体抗炎药）诱导的大鼠胃黏膜细胞凋亡及相关胃病发生过程中的作用。透射电子显微镜研究表明，吲哚美辛会损害线粒体超微结构并导致线粒体功能障碍，这从第三阶段呼吸、脱氢酶活性及跨膜电位（ΔΨm）降低可明显看出。线粒体病理改变与线粒体内活性氧（如超氧阴离子（O₂⁻）、过氧化氢（H₂O₂）和羟自由基（·OH））生成增加相关，从而导致氧化应激。超氧阴离子对破坏线粒体乌头酸酶最有效，导致铁从其铁硫簇中释放出来。释放出的铁与线粒体内的过氧化氢相互作用，形成羟自由基。对线粒体乌头酸酶进行免疫沉淀，随后进行蛋白质免疫印迹分析表明，吲哚美辛处理后，乌头酸酶发生羰基化且在体内活性丧失。利用可螯合铁的特异性探针Phen Green SK对黏膜细胞进行荧光成像，记录了铁的释放。有趣的是，线粒体内羟自由基的生成对于吲哚美辛诱导的线粒体病理改变及线粒体死亡途径的激活至关重要。用二甲基亚砜或自旋捕获剂α-苯基-N-叔丁基硝酮清除羟自由基，可防止吲哚美辛诱导的线粒体超微结构改变、氧化应激、ΔΨm崩溃及线粒体功能障碍。这些清除剂还能恢复吲哚美辛诱导的半胱天冬酶-9和半胱天冬酶-3的激活，从而阻断凋亡的线粒体途径及胃黏膜损伤。因此，本研究揭示了超氧阴离子介导的线粒体乌头酸酶失活在释放线粒体内铁过程中的关键作用，释放出的铁通过生成羟自由基促进吲哚美辛治疗期间胃黏膜细胞凋亡及胃病的发生。
