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.

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of naïve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting naïve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

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.

Purification of the subcellular compartment in which exogenous antigens undergo endoplasmic reticulum-associated degradation from dendritic cells.

Dendritic cells (DCs) are capable of processing and presenting exogenous antigens using MHC class I molecules. This pathway is called antigen cross-presentation and plays an important role in the stimulation of naïve CD8(+) T cells for infectious and tumor immunity. Our previous studies in DC2.4 cells and bone marrow-derived DCs revealed that exogenously added ovalbumin (OVA) is processed through endoplasmic reticulum (ER)-associated degradation (ERAD) for cross-presentation. In this study, we aimed to further confirm these results by purification of the subcellular compartment in which exogenous antigens undergo ERAD from homogenates of DC2.4 cells pretreated with biotinylated OVA (bOVA). bOVA-containing vesicles were purified using streptavidin (SA)-magnetic beads from cell homogenates and were found to contain ER chaperones and ERAD components together with proteins for antigen presentation. In purified microsomes, bOVA was retained in membranous fractions and degraded by the ubiquitin proteasome system in presence reticulocyte lysates and ATP. These results strongly suggested that DCs processed and degraded exogenous antigens through ERAD for cross-presentation in this purified subcellular compartment.

Effects of chronic administration of bilobalide on amino acid levels in mouse brain.

We have previously demonstrated that 4-day-treatment of mice with bilobalide, a sesquiterpene of Ginkgo biloba L., increases GABA levels in mouse brain, but, effects of chronic treatment with it are not clear. To study effects of chronic treatment of mice with bilobalide on amino acid levels in the brain, we determined the levels of aspartate, glutamate, serine, glutamine, glycine, taurine and GABA in the hippocampus, striatum and cortex. Bilobalide (3 mg/kg/day) was administered orally to 4-week-old mice for 40 days. Bilobalide treatment resulted in a significant increase in the levels of glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine in the hippocampus of mice compared with the control. An increased level of glycine after bilobalide treatment was also detected in the striatum. In the cortex, bilobalide increased the GABA level, whereas it decreased the level of aspartate. These changes in the levels of various amino acids may be involved in the broad spectrum of pharmacological activities of the extract of Ginkgo biloba on the central nervous system.

Ion channels and diseases.

Ion channels play important roles in vital cellular signaling processes in both excitable and nonexcitable cells. Since 1987, a large number of channel genes have been cloned, and their biophysical properties, subunit stoichiometries, channel assemblies, and modulation by second messengers and ligands have been gradually elucidated. At present, more than ten ion channel genes have been identified as causing human hereditary diseases. Molecular techniques such as the positional cloning method are indispensable for finding new genes for channel-related diseases. Ion channels participate in the excitation-restoration of neurons and myocytes. Mutations of ion channels in these cells cause abnormal excitation and diseases such as long QT syndrome and ataxia. The second physiological function of ion channels, in addition to their regulation of cell excitability, is ion transport. Bartter's syndrome and Liddle's syndrome are due to abnormalities of ion transport. Most of these ion channel diseases are caused by loss of function, although some mutations are known to result in gain of function. The number of identified channel-related diseases is growing rapidly. Elucidation of the molecular basis of an ion channel disease not only provides new opportunities for early diagnosis and therapy for the disease but also provides clues to determine a previously unknown function of the ion channel.

Effects of extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver.

The effects of a standardized extract of Ginkgo biloba L. leaves (EGb) and its terpene constituents, bilobalide and ginkgolides, on the activities of detoxification enzymes, i.e., glutathione S-transferases (GSTs) and DT-diaphorase, and glutathione contents, were investigated in the mouse liver. Oral treatment with EGb (100-1,000 mg/kg) and bilobalide (10-30 mg/kg) once a day for 4 days caused a dose-dependent elevation in GST activity. Ginkgolide A (30 mg/kg, for 4 days) also significantly elevated GST activity, whereas ginkgolide B and ginkgolide C at the same dose had no effects. EGb significantly increased the protein level of GST pi, and bilobalide significantly increased those of GST alpha and GST mu Moreover, EGb-treatment and bilobalide-treatment caused significant elevations in DT-diaphorase activity and in hepatic glutathione contents.