Structure-function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum.

Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the ß-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.

Plasmodium falciparum Alba6 exhibits DNase activity and participates in stress response.

Alba domain proteins, owing to their functional plasticity, play a significant role in organisms. Here, we report an intrinsic DNase activity of PfAlba6 from Plasmodium falciparum, an etiological agent responsible for human malignant malaria. We identified that tyrosine28 plays a critical role in the Mg2+ driven 5'-3' DNase activity of PfAlba6. PfAlba6 cleaves both dsDNA as well as ssDNA. We also characterized PfAlba6-DNA interaction and observed concentration-dependent oligomerization in the presence of DNA, which is evident from size exclusion chromatography and single molecule AFM-imaging. PfAlba6 mRNA expression level is up-regulated several folds following heat stress and treatment with artemisinin, indicating a possible role in stress response. PfAlba6 has no human orthologs and is expressed in all intra-erythrocytic stages; thus, this protein can potentially be a new anti-malarial drug target.

NSAID targets SIRT3 to trigger mitochondrial dysfunction and gastric cancer cell death.

Gastric cancer (GC) is a deadly malignancy that demands effective therapeutic intervention capitalizing unique drug target/s. Here, we report that indomethacin, a cyclooxygenase non-selective non-steroidal anti-inflammatory drug, arrests GC cell growth by targeting mitochondrial deacetylase Sirtuin 3 (SIRT3). Interaction study revealed that indomethacin competitively inhibited SIRT3 by binding to nicotinamide adenine dinucleotide (NAD)-binding site. The Cancer Genome Atlas data meta-analysis indicated poor prognosis associated with high SIRT3 expression in GC. Further, transcriptome sequencing data of human gastric adenocarcinoma cells revealed that indomethacin treatment severely downregulated SIRT3. Indomethacin-induced SIRT3 downregulation augmented SOD2 and OGG1 acetylation, leading to mitochondrial redox dyshomeostasis, mtDNA damage, respiratory chain failure, bioenergetic crisis, mitochondrial fragmentation, and apoptosis via blocking the AMPK/PGC1α/SIRT3 axis. Indomethacin also downregulated SIRT3 regulators ERRα and PGC1α. Further, SIRT3 knockdown aggravated indomethacin-induced mitochondrial dysfunction as well as blocked cell-cycle progression to increase cell death. Thus, we reveal how indomethacin induces GC cell death by disrupting SIRT3 signaling.

Nuclease activity of Plasmodium falciparum Alba family protein PfAlba3.

Plasmodium falciparum Alba domain-containing protein Alba3 (PfAlba3) is ubiquitously expressed in intra-erythrocytic stages of Plasmodium falciparum, but the function of this protein is not yet established. Here, we report an apurinic/apyrimidinic site-driven intrinsic nuclease activity of PfAlba3 assisted by divalent metal ions. Surface plasmon resonance and atomic force microscopy confirm sequence non-specific DNA binding by PfAlba3. Upon binding, PfAlba3 cleaves double-stranded DNA (dsDNA) hydrolytically. Mutational studies coupled with mass spectrometric analysis indicate that K23 is the essential residue in modulating the binding to DNA through acetylation-deacetylation. We further demonstrate that PfSir2a interacts and deacetylates K23-acetylated PfAlba3 in favoring DNA binding. Hence, K23 serves as a putative molecular switch regulating the nuclease activity of PfAlba3. Thus, the nuclease activity of PfAlba3, along with its apurinic/apyrimidinic (AP) endonuclease feature identified in this study, indicates a role of PfAlba3 in DNA-damage response that may have a far-reaching consequence in Plasmodium pathogenicity.

Honokiol, an inducer of sirtuin-3, protects against non-steroidal anti-inflammatory drug-induced gastric mucosal mitochondrial pathology, apoptosis and inflammatory tissue injury.

BACKGROUND AND PURPOSE: Mitochondrial oxidative stress, inflammation and apoptosis primarily underlie gastric mucosal injury caused by the widely used non-steroidal anti-inflammatory drugs (NSAIDs). Alternative gastroprotective strategies are therefore needed. Sirtuin-3 pivotally maintains mitochondrial structural integrity and metabolism while preventing oxidative stress; however, its relevance to gastric injury was never explored. Here, we have investigated whether and how sirtuin-3 stimulation by the phytochemical, honokiol, could rescue NSAID-induced gastric injury. EXPERIMENTAL APPROACH: Gastric injury in rats induced by indomethacin was used to assess the effects of honokiol. Next-generation sequencing-based transcriptomics followed by functional validation identified the gastroprotective function of sirtuin-3. Flow cytometry, immunoblotting, qRT-PCR and immunohistochemistry were used measure effects on oxidative stress, mitochondrial dynamics, electron transport chain function, and markers of inflammation and apoptosis. Sirtuin-3 deacetylase activity was also estimated and gastric luminal pH was measured. KEY RESULTS: Indomethacin down-regulated sirtuin-3 to induce oxidative stress, mitochondrial hyperacetylation, 8-oxoguanine DNA glycosylase 1 depletion, mitochondrial DNA damage, respiratory chain defect and mitochondrial fragmentation leading to severe mucosal injury. Indomethacin dose-dependently inhibited sirtuin-3 deacetylase activity. Honokiol prevented mitochondrial oxidative damage and inflammatory tissue injury by attenuating indomethacin-induced depletion of both sirtuin-3 and its transcriptional regulators PGC1α and ERRα. Honokiol also accelerated gastric wound healing but did not alter gastric acid secretion, unlike lansoprazole. CONCLUSIONS AND IMPLICATIONS: Sirtuin-3 stimulation by honokiol prevented and reversed NSAID-induced gastric injury through maintaining mitochondrial integrity. Honokiol did not affect gastric acid secretion. Sirtuin-3 stimulation by honokiol may be utilized as a mitochondria-based, acid-independent novel gastroprotective strategy against NSAIDs.

Rab7 of Plasmodium falciparum is involved in its retromer complex assembly near the digestive vacuole.

BACKGROUND: Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate. METHODS: The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined. RESULTS: PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg2+ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly. CONCLUSION: Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum. GENERAL SIGNIFICANCE: This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.

Indomethacin impairs mitochondrial dynamics by activating the PKCζ-p38-DRP1 pathway and inducing apoptosis in gastric cancer and normal mucosal cells.

The subcellular mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastric cancer and normal mucosal cells is elusive because of the diverse cyclooxygenase-independent effects of these drugs. Using human gastric carcinoma cells (AGSs) and a rat gastric injury model, here we report that the NSAID indomethacin activates the protein kinase Cζ (PKCζ)-p38 MAPK (p38)-dynamin-related protein 1 (DRP1) pathway and thereby disrupts the physiological balance of mitochondrial dynamics by promoting mitochondrial hyper-fission and dysfunction leading to apoptosis. Notably, DRP1 knockdown or SB203580-induced p38 inhibition reduced indomethacin-induced damage to AGSs. Indomethacin impaired mitochondrial dynamics by promoting fissogenic activation and mitochondrial recruitment of DRP1 and down-regulating fusogenic optic atrophy 1 (OPA1) and mitofusins in rat gastric mucosa. Consistent with OPA1 maintaining cristae architecture, its down-regulation resulted in EM-detectable cristae deformity. Deregulated mitochondrial dynamics resulting in defective mitochondria were evident from enhanced Parkin expression and mitochondrial proteome ubiquitination. Indomethacin ultimately induced mitochondrial metabolic and bioenergetic crises in the rat stomach, indicated by compromised fatty acid oxidation, reduced complex I- associated electron transport chain activity, and ATP depletion. Interestingly, Mdivi-1, a fission-preventing mito-protective drug, reversed indomethacin-induced DRP1 phosphorylation on Ser-616, mitochondrial proteome ubiquitination, and mitochondrial metabolic crisis. Mdivi-1 also prevented indomethacin-induced mitochondrial macromolecular damage, caspase activation, mucosal inflammation, and gastric mucosal injury. Our results identify mitochondrial hyper-fission as a critical and common subcellular event triggered by indomethacin that promotes apoptosis in both gastric cancer and normal mucosal cells, thereby contributing to mucosal injury.

Hydrazonophenol, a Food Vacuole-Targeted and Ferriprotoporphyrin IX-Interacting Chemotype Prevents Drug-Resistant Malaria.

The rapid emergence of resistance against frontline antimalarial drugs essentially warrants the identification of new-generation antimalarials. Here, we describe the synthesis of ( E)-2-isopropyl-5-methyl-4-((2-(pyridin-4-yl)hydrazono)methyl)phenol (18), which binds ferriprotoporphyrin-IX (FeIII-PPIX) ( Kd = 33 nM) and offers antimalarial activity against chloroquine-resistant and sensitive strains of Plasmodium falciparum in vitro. Structure-function analysis reveals that compound 18 binds FeIII-PPIX through the -C═N-NH- moiety and 2-pyridyl substitution at the hydrazine counterpart plays a critical role in antimalarial efficacy. Live cell confocal imaging using a fluorophore-tagged compound confirms its accumulation inside the acidic food vacuole (FV) of P. falciparum. Furthermore, this compound concentration-dependently elevates the pH in FV, implicating a plausible interference with FeIII-PPIX crystallization (hemozoin formation) by a dual function: increasing the pH and binding free FeIII-PPIX. Different off-target bioassays reduce the possibility of the promiscuous nature of compound 18. Compound 18 also exhibits potent in vivo antimalarial activity against chloroquine-resistant P. yoelii and P. berghei ANKA (causing cerebral malaria) in mice with negligible toxicity.

Macrophage migration inhibitory factor regulates mitochondrial dynamics and cell growth of human cancer cell lines through CD74-NF-κB signaling.

The indispensable role of macrophage migration inhibitory factor (MIF) in cancer cell proliferation is unambiguous, although which specific roles the cytokine plays to block apoptosis by preserving cell growth is still obscure. Using different cancer cell lines (AGS, HepG2, HCT116, and HeLa), here we report that the silencing of MIF severely deregulated mitochondrial structural dynamics by shifting the balance toward excess fission, besides inducing apoptosis with increasing sub-G0 cells. Furthermore, enhanced mitochondrial Bax translocation along with cytochrome c release, down-regulation of Bcl-xL, and Bcl-2 as well as up-regulation of Bad, Bax, and p53 indicated the activation of a mitochondrial pathway of apoptosis upon MIF silencing. The data also indicate a concerted down-regulation of Opa1 and Mfn1 along with a significant elevation of Drp1, cumulatively causing mitochondrial fragmentation upon MIF silencing. Up-regulation of Drp1 was found to be further coupled with fissogenic serine 616 phosphorylation and serine 637 dephosphorylation, thus ensuring enhanced mitochondrial translocation. Interestingly, MIF silencing was found to be associated with decreased NF-κB activation. In fact, NF-κB knockdown in turn increased mitochondrial fission and cell death. In addition, the silencing of CD74, the cognate receptor of MIF, remarkably increased mitochondrial fragmentation in addition to preventing cell proliferation, inducing mitochondrial depolarization, and increasing apoptotic cell death. This indicates the active operation of a MIF-regulated CD74-NF-κB signaling axis for maintaining mitochondrial stability and cell growth. Thus, we propose that MIF, through CD74, constitutively activates NF-κB to control mitochondrial dynamics and stability for promoting carcinogenesis via averting apoptosis.

Detection of retromer assembly in Plasmodium falciparum by immunosensing coupled to Surface Plasmon Resonance.

Retromer complex plays a crucial role in intracellular protein trafficking and is conserved throughout the eukaryotes including malaria parasite, Plasmodium falciparum, where it is partially conserved. The assembly of retromer complex in RBC stages of malarial parasite is extremely difficult to explore because of its complicated physiology, small size, and intra-erythrocytic location. Nonetheless, understanding of retromer assembly may pave new ways for the development of novel antimalarials targeting parasite-specific protein trafficking pathways. Here, we investigated the assembly of retromer complex in P. falciparum, by an immunosensing method through highly sensitive Surface Plasmon Resonance (SPR) technique. After taking leads from the bioinformatics search and literature, different interacting proteins were identified and specific antibodies were raised against them. The sensor chip was prepared by covalently linking antibody specific to one component and the whole cell lysate was passed through it in order to trap the interacting complex. Antibodies raised against other interacting components were used to detect them in the trapped complex on the SPR chip. We were able to detect three different components in the retromer complex trapped by the immobilized antibody specific against a different component on a sensor chip. The assay was reproduced and validated in a different two-component CD74-MIF system in mammalian cells. We, thus, illustrate the assembly of retromer complex in P. falciparum through a bio-sensing approach that combines SPR with immunosensing requiring a very small amount of sample from the native source.

Acute mental stress induces mitochondrial bioenergetic crisis and hyper-fission along with aberrant mitophagy in the gut mucosa in rodent model of stress-related mucosal disease.

Psychological stress, depression and anxiety lead to multiple organ dysfunctions wherein stress-related mucosal disease (SRMD) is common to people experiencing stress and also occur as a side effect in patients admitted to intensive care units; however the underlying molecular aetiology is still obscure. We report that in rat-SRMD model, cold restraint-stress severely damaged gut mitochondrial functions to generate superoxide anion (O2•-), depleted ATP and shifted mitochondrial fission-fusion dynamics towards enhanced fission to induce mucosal injury. Activation of mitophagy to clear damaged and fragmented mitochondria was evident from mitochondrial translocation of Parkin and PINK1 along with enhanced mitochondrial proteome ubiquitination, depletion of mitochondrial DNA copy number and TOM 20. However, excess and sustained accumulation of O2•--generating defective mitochondria overpowered the mitophagic machinery, ultimately triggering Bax-dependent apoptosis and NF-κB-intervened pro-inflammatory mucosal injury. We further observed that stress-induced enhanced serum corticosterone stimulated mitochondrial recruitment of glucocorticoid receptor (GR), which contributed to gut mitochondrial dysfunctions as documented from reduced ETC complex 1 activity, mitochondrial O2•- accumulation, depolarization and hyper-fission. GR-antagonism by RU486 or specific scavenging of mitochondrial O2•- by a mitochondrially targeted antioxidant mitoTEMPO ameliorated stress-induced mucosal damage. Gut mitopathology and mucosal injury were also averted when the perception of mental stress was blocked by pre-treatment with a sedative or antipsychotic. Altogether, we suggest the role of mitochondrial GR-O2•--fission cohort in brain-mitochondria cross-talk during acute mental stress and advocate the utilization of this pathway as a potential target to prevent mitochondrial unrest and gastropathy bypassing central nervous system.

Selective scavenging of intra-mitochondrial superoxide corrects diclofenac-induced mitochondrial dysfunction and gastric injury: A novel gastroprotective mechanism independent of gastric acid suppression.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat multiple inflammatory diseases and pain but severe gastric mucosal damage is the worst outcome of NSAID-therapy. Here we report that mitoTEMPO, a mitochondrially targeted superoxide (O2-) scavenger protected as well as healed gastric injury induced by diclofenac (DCF), the most commonly used NSAID. Common existing therapy against gastric injury involves suppression of gastric acid secretion by proton pump inhibitors and histamine H2 receptor antagonists; however, dyspepsia, vitamin B12 deficiency and gastric microfloral dysbalance are the major drawbacks of acid suppression. Interestingly, mitoTEMPO did not inhibit gastric acid secretion but offered gastroprotection by preventing DCF-induced generation of O2- due to mitochondrial respiratory chain failure and by preventing mitochondrial oxidative stress (MOS)-mediated mitopathology. MitoTEMPO even restored DCF-stimulated reduced fatty acid oxidation, mitochondrial depolarization and bioenergetic crisis in gastric mucosa. MitoTEMPO also prevented the activation of mitochondrial pathway of apoptosis and MOS-mediated proinflammatory signaling through NF-κB by DCF. Furthermore, mitoTEMPO when administered in rats with preformed gastric lesions expedited the healing of gastric injury and the healed stomach exhibited its normal physiology as evident from gastric acid and pepsin secretions under basal or stimulated conditions. Thus, in contrast to the existing antiulcer drugs, mitochondrially targeted O2- scavengers like mitoTEMPO may represent a novel class of gastroprotective molecules that does not affect gastric acid secretion and may be used in combination with DCF, keeping its anti-inflammatory action intact, while reducing its gastrodamaging effects.

Antimalarial Activity of Small-Molecule Benzothiazole Hydrazones.

We synthesized a new series of conjugated hydrazones that were found to be active against malaria parasite in vitro, as well as in vivo in a murine model. These hydrazones concentration-dependently chelated free iron and offered antimalarial activity. Upon screening of the synthesized hydrazones, compound 5f was found to be the most active iron chelator, as well as antiplasmodial. Compound 5f also interacted with free heme (KD [equilibrium dissociation constant] = 1.17 ± 0.8 µM), an iron-containing tetrapyrrole released after hemoglobin digestion by the parasite, and inhibited heme polymerization by parasite lysate. Structure-activity relationship studies indicated that a nitrogen- and sulfur-substituted five-membered aromatic ring present within the benzothiazole hydrazones might be responsible for their antimalarial activity. The dose-dependent antimalarial and heme polymerization inhibitory activities of the lead compound 5f were further validated by following [(3)H]hypoxanthine incorporation and hemozoin formation in parasite, respectively. It is worth mentioning that compound 5f exhibited antiplasmodial activity in vitro against a chloroquine/pyrimethamine-resistant strain of Plasmodium falciparum (K1). We also evaluated in vivo antimalarial activity of compound 5f in a murine model where a lethal multiple-drug-resistant strain of Plasmodium yoelii was used to infect Swiss albino mice. Compound 5f significantly suppressed the growth of parasite, and the infected mice experienced longer life spans upon treatment with this compound. During in vitro and in vivo toxicity assays, compound 5f showed minimal alteration in biochemical and hematological parameters compared to control. In conclusion, we identified a new class of hydrazone with therapeutic potential against malaria.

The Alba protein family: Structure and function.

Alba family proteins are small, basic, dimeric nucleic acid-binding proteins, which are widely distributed in archaea and a number of eukaryotes. This family of proteins bears the distinct features of regulation through acetylation/deacetylation, hence named as acetylation lowers binding affinity (Alba). Alba family proteins bind DNA cooperatively with no apparent sequence specificity. Besides DNA, Alba proteins also interact with diverse RNA species and associate with ribonucleo-protein complexes. Initially, Alba proteins were recognized as chromosomal proteins and supposed to be involved in the maintenance of chromatin architecture and transcription repression. However, recent studies have shown increasing evidence of functional plasticity among Alba family of proteins that widely range from genome packaging and organization, transcriptional and translational regulation, RNA metabolism, and development and differentiation processes. In recent years, Alba family proteins have attracted growing interest due to their widespread occurrence in large number of organisms. Presence in multiple copies, functional crosstalk, differential binding affinity, and posttranslational modifications are some of the key factors that might regulate the biological functions of Alba family proteins. In this review article, we present an overview of the Alba family proteins, their salient features and emphasize their functional role in different organisms reported so far.

Expression, purification and characterization of Plasmodium falciparum vacuolar protein sorting 29.

Translocation of various proteins to the subcellular organelles is an essential mechanism to regulate the metabolic pathways and often vacuolar protein sorting (VPS) proteins are involved in this transportation. Plasmodium falciparum VPS29 (PfVPS29) is predicted to be a functional component in the assembly of the retromer complex; however, so far detailed characterization of PfVPS29 in its native form is not yet done. We report the successful expression and purification of tag-free recombinant PfVPS29 with a yield of 5.6 mg from 1 L of Escherichia coli culture. PfVPS29 was purified by combined anion-exchange and size exclusion chromatography. The protein showed a single band in SDS-PAGE and it exhibited molecular mass of 21.7 kDa as measured by MALDI-TOF mass spectrometry. Secondary structure was elucidated by circular dichroism spectroscopy. It was found to be a monomeric protein in solution as evident from dynamic light scattering studies, chemical cross-linking experiments and size exclusion chromatography. Subsequently, polyclonal anti-PfVPS29 antibody was generated and used for evaluating protein expression by western blot and following subcellular localization in P. falciparum by confocal immunofluoroscence microscopy. PfVPS29 was found to be located in cytoplasm and expressed from early trophozoite to schizont stages with maximum expression in trophozoite stage. This study provides purification, biophysical characterization and subcellular localization of PfVPS29 in different asexual stages of P. falciparum.

Ellagic Acid, a Dietary Polyphenol, Inhibits Tautomerase Activity of Human Macrophage Migration Inhibitory Factor and Its Pro-inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

Ellagic acid (EA), a phenolic lactone, inhibited tautomerase activity of human macrophage migration inhibitory factor (MIF) noncompetitively (Ki = 1.97 ± 0.7 µM). The binding of EA to MIF was determined by following the quenching of tryptophan fluorescence. We synthesized several EA derivatives, and their structure-activity relationship studies indicated that the planar conjugated lactone moiety of EA was essential for MIF inhibition. MIF induces nuclear translocation of NF-κB and chemotaxis of peripheral blood mononuclear cells (PBMCs) to promote inflammation. We were interested in evaluating the effect of EA on nuclear translocation of NF-κB and chemotactic activity in human PBMCs in the presence of MIF. The results showed that EA inhibited MIF-induced NF-κB nuclear translocation in PBMCs, as evident from confocal immunofluorescence microscopic data. EA also inhibited MIF-mediated chemotaxis of PBMCs. Thus, we report MIF-inhibitory activity of EA and inhibition of MIF-mediated proinflammatory responses in PBMCs by EA.

Association of heme oxygenase 1 with the restoration of liver function after damage in murine malaria by Plasmodium yoelii.

The liver efficiently restores function after damage induced during malarial infection once the parasites are cleared from the blood. However, the molecular events leading to the restoration of liver function after malaria are still obscure. To study this, we developed a suitable model wherein mice infected with Plasmodium yoelii (45% parasitemia) were treated with the antimalarial α/ß-arteether to clear parasites from the blood and, subsequently, restoration of liver function was monitored. Liver function tests clearly indicated that complete recovery of liver function occurred after 25 days of parasite clearance. Analyses of proinflammatory gene expression and neutrophil infiltration further indicated that hepatic inflammation, which was induced immediately after parasite clearance from the blood, was gradually reduced. Moreover, the inflammation in the liver after parasite clearance was found to be correlated positively with oxidative stress and hepatocyte apoptosis. We investigated the role of heme oxygenase 1 (HO-1) in the restoration of liver function after malaria because HO-1 normally renders protection against inflammation, oxidative stress, and apoptosis under various pathological conditions. The expression and activity of HO-1 were found to be increased significantly after parasite clearance. We even found that chemical silencing of HO-1 by use of zinc protoporphyrin enhanced inflammation, oxidative stress, hepatocyte apoptosis, and liver injury. In contrast, stimulation of HO-1 by cobalt protoporphyrin alleviated liver inflammation and reduced oxidative stress, hepatocyte apoptosis, and associated tissue injury. Therefore, we propose that selective induction of HO-1 in the liver would be beneficial for the restoration of liver function after parasite clearance.

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), interleukin1ß (IL-1ß), 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-1ß, 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.