Effects of the Cytoplasm and Mitochondrial Specific Hydroxyl Radical Scavengers TA293 and mitoTA293 in Bleomycin-Induced Pulmonary Fibrosis Model Mice.

Lung fibrosis is the primary pathology in idiopathic pulmonary fibrosis and is considered to result from an increase in reactive oxygen species (ROS) levels in alveolar epithelial cells. However, the exact mechanism underlying lung fibrosis remains unclear and there is no effective therapy. The hydroxyl radical (•OH) has the strongest oxidizing potential among ROS. Recently, •OH localized to the cytoplasm (cyto •OH) was reported to induce cellular senescence, while mitochondria-localized •OH (mt •OH) was reported to induce apoptosis. We developed the cyto •OH- and mt •OH-scavenging antioxidants TA293 and mitoTA293 to evaluate the effects of cyto •OH and mt •OH in a bleomycin (BLM)-induced pulmonary fibrosis model. Treatment of BLM-induced pulmonary fibrosis mice with TA293 suppressed the induction of cellular senescence and fibrosis, as well as inflammation in the lung, but mitoTA293 exacerbated these. Furthermore, in BLM-stimulated primary alveolar epithelial cells, TA293 suppressed the activation of the p-ATMser1981/p-p53ser15/p21, p-HRI/p-eIF2ser51/ATF4/p16, NLRP3 inflammasome/caspase-1/IL-1ß/IL1R/p-p38 MAPK/p16, and p21 pathways and the induction of cellular senescence. However, mitoTA293 suppressed the induction of mitophagy, enhanced the activation of the NLRP3 inflammasome/caspase-1/IL1ß/IL1R/p-p38 MAPK/p16 and p21 pathways, and exacerbated cellular senescence, inflammation, and fibrosis. Our findings may help develop new strategies to treat idiopathic pulmonary fibrosis.

Cytoplasmic OH scavenger TA293 attenuates cellular senescence and fibrosis by activating macrophages through oxidized phospholipids/TLR4.

AIMS: Elucidation of the biological roles of the mitochondrial and cytoplasmic hydroxyl radical (cyto OH) is hampered by the absence of site-specific OH scavengers. Earlier findings using cyto OH scavenger, TA293, indicated that cyto OH causes cellular senescence, and senescence-associated secretory phenotype (SASP) factors secreted from cells cause macrophage infiltration, inflammation, and apoptosis. However, we found that macrophage infiltration occurs before senescent cells appear. We therefore aimed to elucidate how cyto OH-induces macrophage activation and investigate the mechanism by which activated macrophages cause oxidative stress, inflammation, and apoptosis. MAIN METHODS: In vivo imaging of pyocyanin- and TA293-treated, macrophage-depleted Toll-like receptor 4-knockout (TLR4-/-) OKD48- and IDOL-Tg mouse models were used to visualize oxidative stress and inflammation. SA-ß-gal and TUNEL staining were used to detect cellular senescence and apoptosis. The mRNA expression of SASP factors were quantified by qRT-PCR. Activation mechanism of cyto OH-mediated macrophages was studied by an ex vivo analysis that created macrophage-activated oxidized phospholipids (OxPLs) using TLR4-/- mice. KEY FINDINGS: Cyto OH produced OxPLs that acted as TLR4 ligands, resulting in macrophage activation. Macrophages were not involved in oxidative stress in tissues or with oxidative damage caused by cyto OH, but significantly exacerbated cellular senescence, inflammation, apoptosis, and fibrosis. SIGNIFICANCE: We present a novel mechanism by which cyto OH-induced macrophage activation exacerbates cellular senescence, inflammation, apoptosis, and fibrosis independently from the known cyto OH-induced cellular senescence pathway. Notably, through suppression of this pathway, TA293 shows promise as a therapeutic agent to prevent fibrosis caused by cyto OH-induced oxidative stress.

The novel antioxidant TA293 reveals the role of cytoplasmic hydroxyl radicals in oxidative stress-induced senescence and inflammation.

The hydroxyl radical (OH) possesses the strongest oxidation potential among reactive oxygen species (ROS). Hydroxyl radicals react nonpreferentially with proteins, lipids, and nucleic acids. Additionally, mitochondrial localization of OH causes dysfunction in the mitochondria. The cytoplasmic targets of OH-induced oxidation are unknown. No cytoplasm-specific OH scavenger is available; thus, elucidating the cytoplasmic targets of OH-induced oxidation has proven difficult. Accordingly, we developed a cytoplasm-specific OH-targeted scavenger, TA293, and a mitochondrion-specific scavenger, mitoTA293. Both TA293 and mitoTA293 scavenged OH but not O2- or H2O2. We then examined the intracellular localization of both scavengers in vitro and in vivo. TA293 scavenged pyocyanin-induced cytoplasmic OH but not antimycin A-induced mitochondrial oxidation. mitoTA293 scavenged antimycin A-induced mitochondrial OH but not cytoplasmic OH. TA293 but not mitoTA293 suppressed pyocyanin-induced oxidative damage in the lungs and kidneys of mice. Additionally, TA293 suppressed the expression of inflammatory signaling pathway components and mediators and suppressed OH-induced cellular senescence and apoptosis. These data suggested that TA293 could be used as a novel tool for studying the effects of hydroxyl radical damage within the cytoplasm.

Effect of TA-270, a novel quinolinone derivative, on antigen-induced nasal blockage in a guinea pig model of allergic rhinitis.

TA-270 (4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone) is a novel quinolinone derivative that has been demonstrated to possess an anti-oxidative activity against peroxynitrite, a potent oxidant, that is generated by the reaction of nitric oxide with superoxide anions. The current study describes the inhibitory effect of TA-270 on the biphasic nasal blockage induced by repeated antigen challenge in an allergic rhinitis guinea pig model. In the present in vitro study, TA-270 potently inhibited the oxidative reaction induced by peroxynitrite (IC(50)=79 nM). In addition, TA-270 (0.3-30 mg/kg, p.o.) dose-dependently inhibited peroxynitrite (3 mM, 10 mul/nostril)-induced nasal blockage in guinea pigs. In the antigen-induced allergic rhinitis model, TA-270 (0.3, 3, and 30 mg/kg, p.o.) given 1 h before the antigen challenge suppressed early phase nasal blockage by 36%, 42%, and 63%, respectively. Furthermore, TA-270 (0.3, 3, and 30 mg/kg, p.o.) showed a relatively strong suppression of late phase nasal blockage (39%, 62%, and 72%, respectively). The late phase nasal blockage was significantly inhibited (61%) even when TA-270 (30 mg/kg, p.o.) was administered 18 h before the antigen challenge. In conclusion, TA-270 improved antigen-induced nasal blockage, probably through its peroxynitrite scavenging action, and the effect was sustained for at least 18 h. Thus, TA-270 would be expected to relieve nasal blockage in allergic rhinitis patients.

TA-270 [4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone], an anti-asthmatic agent, inhibits leukotriene production induced by IgE receptor stimulation in RBL-2H3 cells.

A novel quinolinone derivative, TA-270 [4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone], has been shown to inhibit antigen-induced asthmatic responses including the early-phase bronchoconstriction in actively sensitized guinea pigs. Here we characterized the action mechanisms of TA-270 in cellular level in vitro. In RBL-2H3 mast cells sensitized with dinitrophenol (DNP)-specific IgE, the antigen exhibited several mast cell functions, including hexosaminidase release as a marker of degranulation, production of tumor necrosis factor-alpha, and production of immunologically detective leukotrienes. These antigen-induced actions were associated with the activation of several early signaling events, including inositol phosphate production reflecting phospholipase C activation and extracellular signal-regulated kinase activation. When the cells were treated with TA-270, the antigen-induced leukotriene production was almost completely suppressed, but other antigen-induced actions listed above were hardly affected. This drug also failed to affect the antigen-induced phospholipase A2 activation as evaluated by the total release of arachidonic acid and its metabolites from the cells prelabeled with radioactive arachidonic acid. However, TA-270 clearly changed the arachidonic acid metabolic pathway. It suppressed the accumulation of 5-lipoxygenase products, including leukotrienes, but hardly affected the accumulation of cyclooxygenase products. The inhibitory action of TA-270 on leukotriene production was also observed in human neutrophils and eosinophils. We conclude that TA-270 inhibits 5-lipoxygenase activity and, thereby, suppresses the antigen-induced leukotriene production.

Effects of radical scavengers, TA248 and TA276, on stunned myocardium in dogs: involvement of K ATP channels.

TA248 (7-(beta-D-glucopyranosyloxy)-4-hydroxy-3-octyloxy-2H-1-benzopyran-2-one) and TA276 (sodium 7-hydroxy-3-octyloxy-2H-1-benzopyran-2-one-4-oxide) were newly developed as radical scavengers. In vitro, TA276 scavenged both superoxide anions (. O(2)(-)) and hydroxyl radicals (. OH). TA248 also trapped. O(2)(-), but had less activity on. OH. In vivo, left ventricular contractile functions were determined in pentobarbital-anesthetized open-chest dogs. A regional portion of the left ventricular wall was made ischemic for 20 min by ligating the left anterior descending coronary artery and then reperfused for 60 min. TA248 (3 mg/kg) and TA276 (3 mg/kg) injected i.v. 10 min before occlusion significantly improved myocardial stunning that is contractile dysfunction observed after reperfusion following brief ischemia. Glibenclamide (1 mg/kg) injected i.v. 20 min before occlusion significantly worsened the myocardial stunning. Pretreatment with glibenclamide completely abolished the beneficial effect of TA276 on myocardial stunning, whereas it only partially attenuated that of TA248, showing some improvement even in the presence of glibenclamide. Because of the incomplete scavenging activity of TA248, residual. OH may play some roles in improvement of myocardial stunning with TA248 in the presence of glibenclamide. We speculate that the. OH may eject glibenclamide from its binding site on K(ATP) channels, leading to opening of the channels.