Indomethacin, a non-steroidal anti-inflammatory drug, develops gastropathy by inducing reactive oxygen species-mediated mitochondrial pathology and associated apoptosis in gastric mucosa: a novel role of mitochondrial aconitase oxidation

Abstract

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 (ΔΨ_m). Mitochondrial pathology is associated with increased generation of intra-mitochondrial-reactive oxygen species, such as O₂, H₂O₂ and ·OH, leading to oxidative stress. Formula 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 H₂O₂, 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 α-phenyl-n-tert-butylnitrone, a spin-trap, prevents indomethacin-induced mitochondrial ultrastructural changes, oxidative stress, collapse of ΔΨm, 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 Formula-mediated mitochondrial aconitase inactivation to release intra-mitochondrial iron, which by generating ·OH promotes gastric mucosal cell apoptosis and gastropathy during indomethacin treatment.