Athar Alam

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

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 (ΔΨm). Mitochondrial pathology is associated with increased generation of intra-mitochondrial-reactive oxygen species, such as \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document}, H2O2 and ·OH, leading to oxidative stress. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} 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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document}-mediated mitochondrial aconitase inactivation to release intra-mitochondrial iron, which by generating ·OH promotes gastric mucosal cell apoptosis and gastropathy during indomethacin treatment.

Gallic Acid Prevents Nonsteroidal Anti-Inflammatory Drug-Induced Gastropathy in Rat by Blocking Oxidative Stress and Apoptosis

Nonsteroidal anti-inflammatory drug (NSAID)-induced oxidative stress plays a critical role in gastric mucosal cell apoptosis and gastropathy. NSAIDs induce the generation of hydroxyl radical ((*)OH) through the release of free iron, which plays an important role in developing gastropathy. Thus, molecules having both iron-chelating and antiapoptotic properties will be beneficial in preventing NSAID-induced gastropathy. Gallic acid (GA), a polyphenolic natural product, has the capacity to chelate free iron. Here, we report that GA significantly prevents, as well as heals, NSAID-induced gastropathy. In vivo, GA blocks NSAID-mediated mitochondrial oxidative stress by preventing mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. In vitro, GA scavenges free radicals and blocks (*)OH-mediated oxidative damage. GA also attenuates gastric mucosal cell apoptosis in vivo as well as in vitro in cultured gastric mucosal cells as evident from the TUNEL assay. GA prevents NSAID-induced activation of caspase-9, a marker for the mitochondrial pathway of apoptosis, and restores NSAID-mediated collapse of the mitochondrial transmembrane potential and dehydrogenase activity. Thus, the inhibition of mitochondrial oxidative stress by GA is associated with the inhibition of NSAID-induced mitochondrial dysfunction and activation of apoptosis in gastric mucosal cells, which are responsible for gastric injury or gastropathy.

Translocation of Heme Oxygenase-1 to Mitochondria Is a Novel Cytoprotective Mechanism against Non-steroidal Anti-inflammatory Drug-induced Mitochondrial Oxidative Stress, Apoptosis, and Gastric Mucosal Injury

Background: The inherent cytoprotective mechanism involved in repair of injured gastric mucosa is not clear. Results: HO-1 is induced and translocated to mitochondria to favor repair of gastric mucosal injury induced by non-steroidal anti-inflammatory drug-mediated mitochondrial oxidative stress (MOS). Conclusion: Mitochondrial localization of HO-1 is a novel cytoprotective mechanism against MOS-mediated gastric mucosal injury. Significance: Induction of HO-1 in gastric mucosa is beneficial for gastroprotection. The mechanism of action of heme oxygenase-1 (HO-1) in mitochondrial oxidative stress (MOS)-mediated apoptotic tissue injury was investigated. MOS-mediated gastric mucosal apoptosis and injury were introduced in rat by indomethacin, a non-steroidal anti-inflammatory drug. Here, we report that HO-1 was not only induced but also translocated to mitochondria during gastric mucosal injury to favor repair mechanisms. Furthermore, mitochondrial translocation of HO-1 resulted in the prevention of MOS and mitochondrial pathology as evident from the restoration of the complex I-driven mitochondrial respiratory control ratio and transmembrane potential. Mitochondrial translocation of HO-1 also resulted in time-dependent inhibition of apoptosis. We searched for the plausible mechanisms responsible for HO-1 induction and mitochondrial localization. Free heme, the substrate for HO-1, was increased inside mitochondria during gastric injury, and mitochondrial entry of HO-1 decreased intramitochondrial free heme content, suggesting that a purpose of mitochondrial translocation of HO-1 is to detoxify accumulated heme. Heme may activate nuclear translocation of NF-E2-related factor 2 to induce HO-1 through reactive oxygen species generation. Electrophoretic mobility shift assay and chromatin immunoprecipitation studies indicated nuclear translocation of NF-E2-related factor 2 and its binding to HO-1 promoter to induce HO-1 expression during gastric injury. Inhibition of HO-1 by zinc protoporphyrin aggravated the mucosal injury and delayed healing. Zinc protoporphyrin further reduced the respiratory control ratio and transmembrane potential and enhanced MOS and apoptosis. In contrast, induction of HO-1 by cobalt protoporphyrin reduced MOS, corrected mitochondrial dysfunctions, and prevented apoptosis and gastric injury. Thus, induction and mitochondrial localization of HO-1 are a novel cytoprotective mechanism against MOS-mediated apoptotic tissue injury.

Malarial infection develops mitochondrial pathology and mitochondrial oxidative stress to promote hepatocyte apoptosis

Activation of the mitochondrial apoptosis pathway by oxidative stress has been implicated in hepatocyte apoptosis during malaria. Because mitochondria are the source and target of reactive oxygen species (ROS), we have investigated whether hepatocyte apoptosis is linked to mitochondrial pathology and mitochondrial ROS generation during malaria. Malarial infection induces mitochondrial pathology by inhibiting mitochondrial respiration, dehydrogenases, and transmembrane potential and damaging the ultrastructure as evident from transmission electron microscopic studies. Mitochondrial GSH depletion and formation of protein carbonyl indicate that mitochondrial pathology is associated with mitochondrial oxidative stress. Fluorescence imaging of hepatocytes documents intramitochondrial superoxide anion (O(2)(-)) generation during malaria. O(2)(-) inactivates mitochondrial aconitase to release iron from iron-sulfur clusters, which forms the hydroxyl radical ((.)OH) interacting with H(2)O(2) produced concurrently. Malarial infection inactivates mitochondrial aconitase, and carbonylation of aconitase is evident from Western immunoblotting. The release of iron has been documented by fluorescence imaging of hepatocytes using Phen Green SK, and mitochondrial (.)OH generation has been confirmed. During malaria, the depletion of cardiolipin and formation of the mitochondrial permeability transition pore favor cytochrome c release to activate caspase-9. Interestingly, mitochondrial (.)OH generation correlates with the activation of both caspase-9 and caspase-3 with the progress of malarial infection, indicating the critical role of (.)OH.

Novel Anti-inflammatory Activity of Epoxyazadiradione against Macrophage Migration Inhibitory Factor INHIBITION OF TAUTOMERASE AND PROINFLAMMATORY ACTIVITIES OF MACROPHAGE MIGRATION INHIBITORY FACTOR

Background: Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in many infectious and non-infectious diseases. Results: Epoxyazadiradione, a limonoid, inhibits the tautomerase activity of both human and malarial MIF and prevents MIF-induced proinflammatory reactions. Conclusion: Epoxyazadiradione bears therapeutic potential against MIF-induced proinflammatory reactions. Significance: This novel molecule is a significant addition in the discovery of anti-inflammatory drugs. Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in various infectious and non-infectious diseases. We have investigated the mechanism of anti-inflammatory activity of epoxyazadiradione, a limonoid purified from neem (Azadirachta indica) fruits, against MIF. Epoxyazadiradione inhibited the tautomerase activity of MIF of both human (huMIF) and malaria parasites (Plasmodium falciparum (PfMIF) and Plasmodium yoelii (PyMIF)) non-competitively in a reversible fashion (Ki, 2.11–5.23 μm). Epoxyazadiradione also significantly inhibited MIF (huMIF, PyMIF, and PfMIF)-mediated proinflammatory activities in RAW 264.7 cells. It prevented MIF-induced macrophage chemotactic migration, NF-κB translocation to the nucleus, up-regulation of inducible nitric-oxide synthase, and nitric oxide production in RAW 264.7 cells. Epoxyazadiradione not only exhibited anti-inflammatory activity in vitro but also in vivo. We tested the anti-inflammatory activity of epoxyazadiradione in vivo after co-administering LPS and MIF in mice to mimic the disease state of sepsis or bacterial infection. Epoxyazadiradione prevented the release of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α when LPS and PyMIF were co-administered to BALB/c mice. The molecular basis of interaction of epoxyazadiradione with MIFs was explored with the help of computational chemistry tools and a biological knowledgebase. Docking simulation indicated that the binding was highly specific and allosteric in nature. The well known MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) inhibited huMIF but not MIF of parasitic origin. In contrast, epoxyazadiradione inhibited both huMIF and plasmodial MIF, thus bearing an immense therapeutic potential against proinflammatory reactions induced by MIF of both malaria parasites and human.

Lansoprazole Protects and Heals Gastric Mucosa from Non-steroidal Anti-inflammatory Drug (NSAID)-induced Gastropathy by Inhibiting Mitochondrial as Well as Fas-mediated Death Pathways with Concurrent Induction of Mucosal Cell Renewal

We have investigated the mechanism of antiapoptotic and cell renewal effects of lansoprazole, a proton pump inhibitor, to protect and heal gastric mucosal injury in vivo induced by indomethacin, a non-steroidal anti-inflammatory drug (NSAID). Lansoprazole prevents indomethacin-induced gastric damage by blocking activation of mitochondrial and Fas pathways of apoptosis. Lansoprazole prevents indomethacin-induced up-regulation of proapoptotic Bax and Bak and down-regulation of antiapoptotic Bcl-2 and BclxL to maintain the normal proapoptotic/antiapoptotic ratio and thereby arrests indomethacin-induced mitochondrial translocation of Bax and collapse of mitochondrial membrane potential followed by cytochrome c release and caspase-9 activation. Lansoprazole also inhibits indomethacin-induced Fas-mediated mucosal cell death by down-regulating Fas or FasL expression and inhibiting caspase-8 activation. Lansoprazole favors mucosal cell renewal simultaneously by stimulating gene expression of prosurvival proliferating cell nuclear antigen, survivin, epidermal growth factor, and basic fibroblast growth factor. The up-regulation of Flt-1 further indicates that lansoprazole activates vascular epidermal growth factor-mediated controlled angiogenesis to repair gastric mucosa. Lansoprazole also stimulates the healing of already formed ulcers induced by indomethacin. Time course study of healing indicates that it switches off the mitochondrial death pathway completely but not the Fas pathway. However, lansoprazole heals mucosal lesions almost completely after overcoming the persisting Fas pathway, probably by favoring the prosurvival genes expression. This study thus provides the detailed mechanism of antiapoptotic and prosurvival effects of lansoprazole for offering gastroprotection against indomethacin-induced gastropathy.

Identification and molecular characterization of an Alba-family protein from human malaria parasite Plasmodium falciparum

We have investigated the DNA-binding nature as well as the function of a putative Alba (Acetylation lowers binding affinity) family protein (PfAlba3) from Plasmodium falciparum. PfAlba3 possesses DNA-binding property like Alba family proteins. PfAlba3 binds to DNA sequence non-specifically at the minor groove and acetylation lowers its DNA-binding affinity. The protein is ubiquitously expressed in all the erythrocytic stages of P. falciparum and it exists predominantly in the acetylated form. PfAlba3 inhibits transcription in vitro by binding to DNA. Plasmodium falciparum Sir2 (PfSir2A), a nuclear localized deacetylase interacts with PfAlba3 and deacetylates the lysine residue of N-terminal peptide of PfAlba3 specific for DNA binding. PfAlba3 is localized with PfSir2A in the periphery of the nucleus. Fluorescence in situ hybridization studies revealed the presence of PfAlba3 in the telomeric and subtelomeric regions. ChIP and ChIP ReChIP analyses further confirmed that PfAlba3 binds to the telomeric and subtelomeric regions as well as to var gene promoter.

Impact of Intravascular Hemolysis in Malaria on Liver Dysfunction INVOLVEMENT OF HEPATIC FREE HEME OVERLOAD, NF-κB ACTIVATION, AND NEUTROPHIL INFILTRATION

Background: Multiorgan failure is evident in conditions of intravascular hemolysis. Results: Persistent intravascular hemolysis in malaria causes liver damage because of excess hepatic free heme accumulation, TNFα release, NF-κB activation, and neutrophil infiltration. Conclusion: Intravascular hemolysis may result in hepatic failure as result of oxidative stress. Significance: Intravascular hemolysis in any condition may damage liver or other vascular organs. We have investigated the impact of persistent intravascular hemolysis on liver dysfunction using the mouse malaria model. Intravascular hemolysis showed a positive correlation with liver damage along with the increased accumulation of free heme and reactive oxidants in liver. Hepatocytes overinduced heme oxygenase-1 (HO-1) to catabolize free heme in building up defense against this pro-oxidant milieu. However, in a condition of persistent free heme overload in malaria, the overactivity of HO-1 resulted in continuous transient generation of free iron to favor production of reactive oxidants as evident from 2′,7′-dichlorofluorescein fluorescence studies. Electrophoretic mobility shift assay documented the activation of NF-κB, which in turn up-regulated intercellular adhesion molecule 1 as evident from chromatin immunoprecipitation studies. NF-κB activation also induced vascular cell adhesion molecule 1, keratinocyte chemoattractant, and macrophage inflammatory protein 2, which favored neutrophil extravasation and adhesion in liver. The infiltration of neutrophils correlated positively with the severity of hemolysis, and neutrophil depletion significantly prevented liver damage. The data further documented the elevation of serum TNFα in infected mice, and the treatment of anti-TNFα antibodies also significantly prevented neutrophil infiltration and liver injury. Deferoxamine, which chelates iron, interacts with free heme and bears antioxidant properties that prevented oxidative stress, NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Furthermore, the administration of N-acetylcysteine also prevented NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Thus, hepatic free heme accumulation, TNFα release, oxidative stress, and NF-κB activation established a link to favor neutrophil infiltration in inducing liver damage during hemolytic conditions in malaria.

Nonsteroidal anti-inflammatory drug induces proinflammatory damage in gastric mucosa through NF-κB activation and neutrophil infiltration: Anti-inflammatory role of heme oxygenase-1 against nonsteroidal anti-inflammatory drug

Nonsteroidal anti-inflammatory drug (NSAID)-induced mitochondrial oxidative stress (MOS) is an important prostaglandin (PG)-independent pathway of the induction of gastric mucosal injury. However, the molecular mechanism behind MOS-mediated gastric pathology is still obscure. In various pathological conditions of tissue injury oxidative stress is often linked with inflammation. Here we report that MOS induced by indomethacin (an NSAID) induces gastric mucosal inflammation leading to proinflammatory damage. Indomethacin, time dependently stimulated the expression of proinflammatory molecules such as intercellular adhesion molecule 1(ICAM-1), vascular cell adhesion molecule 1(VCAM-1), interleukin1β (IL-1β), and monocyte chemotactic protein-1 (MCP-1) in gastric mucosa in parallel with the increase of neutrophil infiltration and injury of gastric mucosa in rat. Western immunoblotting and confocal microscopic studies revealed that indomethacin induced nuclear translocation of nuclear factor kappa-B (NF-κB) in gastric mucosal cells, which resulted in proinflammatory signaling. The prevention of MOS by antioxidant tryptamine-gallic acid hybrid (SEGA) inhibited indomethacin-induced expression of ICAM-1, VCAM-1, IL-1β, and MCP-1. SEGA also prevented indomethacin-induced NF-κB activation and neutrophil infiltration as documented by chromatin immunoprecipitation studies and neutrophil migration assay, respectively. Heme oxygenase-1 (HO-1), a cytoprotective enzyme associated with tissue repair mechanisms is stimulated in response to oxidative stress. We have investigated the role of HO-1 against MOS and MOS-mediated inflammation in recovering from gastropathy. Indomethacin stimulated the expression of HO-1 and indomethacin-stimulated HO-1 expression was reduced by SEGA, an antioxidant, which could prevent MOS. Thus, the data suggested that the induction of HO-1 was a protective response against MOS developed by indomethacin. Moreover, the induction of HO-1 by cobalt protoporphyrin inhibited inflammation and chemical silencing of HO-1 by zinc protoporphyrin aggravated the inflammation by indomethacin. Thus, NSAID by promoting MOS-induced proinflammatory response damaged gastric mucosa and HO-1 protected NSAID-induced gastric mucosal damage by preventing NF-κB activation and proinflammatory activity.

Synthesis and bio-evaluation of human macrophage migration inhibitory factor inhibitor to develop anti-inflammatory agent

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is involved in the development of an array of inflammatory disorders including rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis and sepsis. The synthesis of MIF-inhibitor is a rationale approach to develop novel anti-inflammatory agent to treat multitude of inflammatory diseases. In this work, we have synthesized and evaluated MIF-inhibitory activity of a series of small molecules containing isoxazoline skeleton. Mode of binding of this inhibitor to human MIF (huMIF) was determined by docking studies. The synthesized molecules inhibit tautomerase activity of huMIF. The anti-inflammatory activity of the most active inhibitor, 4-((3-(4-hydroxy-3-methoxyphenyl)-4, 5-dihydroisoxazol-5-yl) methoxy) benzaldehyde (4b) was evaluated against huMIF-induced inflammation in a cellular model (RAW 264.7 cell). Compound 4b significantly inhibits huMIF-mediated NF-κB translocation to the nucleus, up-regulation of inducible nitric oxide synthase and nitric oxide production in RAW 264.7 cell which are the markers for inflammation. The compound 4b is not cytotoxic as evident from cell viability assay. Hence, the compound 4b has potential to be a novel anti-inflammatory agent.