APPA Increases Lifespan and Stress Resistance via Lipid Metabolism and Insulin/IGF-1 Signal Pathway in Caenorhabditis elegans.

Animal studies have proven that 1-acetyl-5-phenyl-1H-pyrrol-3-yl acetate (APPA) is a powerful antioxidant as a novel aldose reductase inhibitor independently synthesized by our laboratory; however, there is no current information on APPA's anti-aging mechanism. Therefore, this study examined the impact and mechanism of APPA's anti-aging and anti-oxidation capacity using the Caenorhabditis elegans model. The results demonstrated that APPA increases C. elegans' longevity without affecting the typical metabolism of Escherichia coli OP50 (OP50). APPA also had a non-toxic effect on C. elegans, increased locomotor ability, decreased the levels of reactive oxygen species, lipofuscin, and fat, and increased anti-stress capacity. QRT-PCR analysis further revealed that APPA upregulated the expression of antioxidant genes, including sod-3, gst-4, and hsp-16.2, and the critical downstream transcription factors, daf-16, skn-1, and hsf-1 of the insulin/insulin-like growth factor (IGF) receptor, daf-2. In addition, fat-6 and nhr-80 were upregulated. However, the APPA's life-prolonging effects were absent on the daf-2, daf-16, skn-1, and hsf-1 mutants implying that the APPA's life-prolonging mechanism depends on the insulin/IGF-1 signaling system. The transcriptome sequencing also revealed that the mitochondrial route was also strongly associated with the APPA life extension, consistent with mev-1 and isp-1 mutant life assays. These findings aid in the investigation of APPA's longevity extension mechanism.

Chronic cough caused by choledochoduodenal fistula: a case report.

BACKGROUND: Chronic cough is characterized by cough as the only or main symptom, with a duration of more than 8 weeks and no obvious abnormality in chest X-ray examination. Its etiology is complex, including respiratory disease, digestive system disease, circulation system disease, and psychological disease. Although a set of etiological diagnosis procedures for chronic cough have been established, it is still difficult to diagnose chronic cough and there are still some patients with misdiagnosis. CASE PRESENTATION: We present a case of a 54-year-old female patient who had chronic cough for 28 years. Physical examination had no positive signs and she denied any illness causing cough like tuberculosis, rhinitis. Recurrent clinic visits and symptomatic treatment didn't improve the condition. Finally, gastroscopy identified the possible etiology of choledochoduodenal fistula that was proved by surgery. And after surgery, the patient's cough symptoms were significantly improved. CONCLUSION: We report a rare case of chronic cough caused by choledochoduodenal fistula which demonstrates our as yet inadequate recognition of the etiology and pathogenesis. Written informed consent was obtained from the patient.

Early growth response factor 1 is essential for cigarette smoke-induced MUC5AC expression in human bronchial epithelial cells.

Early growth response factor 1 (Egr-1) is a zinc finger transcription factor which responses rapidly to a variety of extracellular stimuli. Previous studies have suggested that Egr-1 exerts pathological functions in chronic obstructive pulmonary disease (COPD) by regulation of cigarette smoking-induced autophagy, cell death, and inflammation. However, little is known about the role of Egr-1 in regulation of mucus production in airway epithelium. In this study, we observed that cigarette smoke extract (CSE) induced a successive expression of Egr-1 and MUC5AC in human bronchial epithelial (HBE) cells. Knockdown of Egr-1 markedly attenuated CSE-induced MUC5AC production, and chromatin immunoprecipitation revealed that Egr-1 transcriptionally bound to MUC5AC promoter upon CSE stimulation. Concurrently, CSE increased the expression of c-Jun and c-Fos, two subunits of activator protein 1 (AP-1) which also critically regulates CSE-induced MUC5AC in HBE cells. CSE also induced a physical interaction of Egr-1 and AP-1, and knockdown of Egr-1 significantly decreased CSE-induced expression of c-Fos and c-Jun. Furthermore, knockdown of c-Fos remarkably attenuated the CSE-induced Egr-1 binding to MUC5AC promoter. These data taken together demonstrate that Egr-1 is essential for CSE-induced MUC5AC production in HBE cells likely through interaction with and modulation of AP-1, and re-emphasize targeting Egr-1 as a novel therapeutic strategy for COPD.

Interaction of caveolin-1 with ATG12-ATG5 system suppresses autophagy in lung epithelial cells.

Autophagy plays a pivotal role in cellular homeostasis and adaptation to adverse environments, although the regulation of this process remains incompletely understood. We have recently observed that caveolin-1 (Cav-1), a major constituent of lipid rafts on plasma membrane, can regulate autophagy in cigarette smoking-induced injury of lung epithelium, although the underlying molecular mechanisms remain incompletely understood. In the present study we found that Cav-1 interacted with and regulated the expression of ATG12-ATG5, an ubiquitin-like conjugation system crucial for autophagosome formation, in lung epithelial Beas-2B cells. Deletion of Cav-1 increased basal and starvation-induced levels of ATG12-ATG5 and autophagy. Biochemical analyses revealed that Cav-1 interacted with ATG5, ATG12, and their active complex ATG12-ATG5. Overexpression of ATG5 or ATG12 increased their interactions with Cav-1, the formation of ATG12-ATG5 conjugate, and the subsequent basal levels of autophagy but resulted in decreased interactions between Cav-1 and another molecule. Knockdown of ATG12 enhanced the ATG5-Cav-1 interaction. Mutation of the Cav-1 binding motif on ATG12 disrupted their interaction and further augmented autophagy. Cav-1 also regulated the expression of ATG16L, another autophagy protein associating with the ATG12-ATG5 conjugate during autophagosome formation. Altogether these studies clearly demonstrate that Cav-1 competitively interacts with the ATG12-ATG5 system to suppress the formation and function of the latter in lung epithelial cells, thereby providing new insights into the molecular mechanisms by which Cav-1 regulates autophagy and suggesting the important function of Cav-1 in certain lung diseases via regulation of autophagy homeostasis.

Caveolin-1 inhibits expression of antioxidant enzymes through direct interaction with nuclear erythroid 2 p45-related factor-2 (Nrf2).

The Nrf2 (nuclear erythroid 2 p45-related factor-2) signaling pathway is known to play a pivotal role in a variety of oxidative stress-related human disorders. It has been reported recently that the plasma membrane resident protein caveolin-1 (Cav-1) can regulate expression of certain antioxidant enzymes and involves in the pathogenesis of oxidative lung injury, but the detailed molecular mechanisms remain incompletely understood. Here, we demonstrated that Cav-1 inhibited the expression of antioxidant enzymes through direct interaction with Nrf2 and subsequent suppression of its transcriptional activity in lung epithelial Beas-2B cells. Cav-1 deficiency cells exhibited higher levels of antioxidant enzymes and were more resistant to oxidative stress induced cytotoxicity, whereas overexpression of Cav-1 suppressed the induction of these enzymes and further augmented the oxidative cell death. Cav-1 constitutively interacted with Nrf2 in both cytosol and nucleus. Stimulation of 4-hydroxynonenol increased the Cav-1-Nrf2 interaction in cytosol but disrupted their association in the nucleus. Knockdown of Cav-1 also disassociated the interaction between Nrf2 and its cytoplasmic inhibitor Keap1 (Kelch-like ECH-associated protein 1) and increased the Nrf2 transcription activity. Mutation of the resembling Cav-1 binding motif on Nrf2 effectively attenuated their interaction, which exhibited higher transcription activity and induced higher levels of antioxidant enzymes relative to the wild-type control. Altogether, these studies clearly demonstrate that Cav-1 inhibits cellular antioxidant capacity through direct interaction with Nrf2 and subsequent suppression of its activity, thereby implicating in certain oxidative stress-related human pathologies.

Retinoic acid-related orphan receptor-α is induced in the setting of DNA damage and promotes pulmonary emphysema.

RATIONALE: The discovery that retinoic acid-related orphan receptor (Rora)-α is highly expressed in lungs of patients with COPD led us to hypothesize that Rora may contribute to the pathogenesis of emphysema. OBJECTIVES: To determine the role of Rora in smoke-induced emphysema. METHODS: Cigarette smoke extract in vitro and elastase or cigarette smoke exposure in vivo were used to model smoke-related cell stress and airspace enlargement. Lung tissue from patients undergoing lung transplantation was examined for markers of DNA damage and Rora expression. MEASUREMENTS AND MAIN RESULTS: Rora expression was induced by cigarette smoke in mice and in cell culture. Gene expression profiling of Rora-null mice exposed to cigarette smoke demonstrated enrichment for genes involved in DNA repair. Rora expression increased and Rora translocated to the nucleus after DNA damage. Inhibition of ataxia telangiectasia mutated decreased the induction of Rora. Gene silencing of Rora attenuated apoptotic cell death in response to cigarette smoke extract, whereas overexpression of Rora enhanced apoptosis. Rora-deficient mice were protected from elastase and cigarette smoke induced airspace enlargement. Finally, lungs of patients with COPD showed evidence of increased DNA damage even in the absence of active smoking. CONCLUSIONS: Taken together, these findings suggest that DNA damage may contribute to the pathogenesis of emphysema, and that Rora has a previously unrecognized role in cellular responses to genotoxicity. These findings provide a potential link between emphysema and features of premature ageing, including enhanced susceptibility to lung cancer.

Autophagy protein microtubule-associated protein 1 light chain-3B (LC3B) activates extrinsic apoptosis during cigarette smoke-induced emphysema.

Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by chronic exposure to cigarette smoke (CS), which involves airway obstruction and alveolar loss (i.e., emphysema). The mechanisms of COPD pathogenesis remain unclear. Our previous studies demonstrated elevated autophagy in human COPD lung, and as a cellular and tissue response to CS exposure in an experimental model of emphysema in vivo. We identified the autophagic protein microtubule-associated protein 1 light chain-3B (LC3B) as a positive regulator of CS-induced lung epithelial cell death. We now extend these initial observations to explore the mechanism by which LC3B mediates CS-induced apoptosis and emphysema development in vivo. Here, we observed that LC3B(-/-) mice had significantly decreased levels of apoptosis in the lungs after CS exposure, and displayed resistance to CS-induced airspace enlargement, relative to WT littermate mice. We found that LC3B associated with the extrinsic apoptotic factor Fas in lipid rafts in an interaction mediated by caveolin-1 (Cav-1). The siRNA-dependent knockdown of Cav-1 sensitized epithelial cells to CS-induced apoptosis, as evidenced by enhanced death-inducing signaling complex formation and caspase activation. Furthermore, Cav-1(-/-) mice exhibited higher levels of autophagy and apoptosis in the lung in response to chronic CS exposure in vivo. In conclusion, we demonstrate a pivotal role for the autophagic protein LC3B in CS-induced apoptosis and emphysema, suggestive of novel therapeutic targets for COPD treatment. This study also introduces a mechanism by which LC3B, through interactions with Cav-1 and Fas, can regulate apoptosis.

Caveolin-1 regulates the secretion and cytoprotection of Cyr61 in hyperoxic cell death.

Cysteine-rich 61 (Cyr61) belongs to the CCN family and mediates cell proliferation, survival, and apoptosis. Our previous studies showed that Cyr61 protected against hyperoxia-induced lung cell death via Akt phosphorylation. Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. Emerging data show that cav-1 regulates signal transduction-associated proteins that reside in the caveolae. Numerous integrin-related pathways, including PI3K/Akt-induced cell survival are controlled by cav-1-mediated signaling. Our data showed that recombinant Cyr61 promoted cell proliferation and resistance to hyperoxia-induced cell death in vitro. Neutralizing antibodies reversed the above effects, indicating functional role of secreted Cyr61 in response to hyperoxic stress. While deletion of cav-1 protected cells from hyperoxia-induced cell death, Cyr61-neutralizing antibodies abolished this protective effect. Furthermore, Cyr61 and cav-1 colocalized and physically interacted via integrins in bronchial epithelial cells. Deletion of cav-1 increased extracellular and decreased cytosolic Cyr61, both in vitro and in vivo. Pretreatment with Brefeldin A increased intracellular Cyr61 in cav-1(-/-) cells, while decreasing extracellular Cyr61. Taken together, Cav-1/Cyr61 interaction via integrins represents a novel pathway of Cyr61 signaling involving cav-1-dependent processes, which play a critical role in regulating hyperoxia-induced cell death.

Hydroxyoctadecadienoic acid as a potential biomarker for oxidative stress in patients with chronic hepatitis C.

BACKGROUND AND AIM: The possible involvement of oxidative stress in hepatitis C virus (HCV)-induced liver damage and hepatocarcinogenesis has been reported. We have recently developed a novel method to measure total hydroxyoctadecadienoic acid (tHODE) and have proposed its usefulness as a biomarker for lipid peroxidation. The present study was undertaken to evaluate oxidative stress in HCV-infected liver diseases by several potential oxidative stress markers including tHODE and further to validate the biomarkers for evaluating the efficacy of iron reduction therapy. METHODS: Total hydroxyoctadecadienoic acid, total 8-iso-prostagrandin F(2alpha) (t8-iso-PGF(2alpha)), selenoprotein P and other antioxidant compounds were measured in the plasma and erythrocytes obtained from 42 healthy controls and 78 HCV patients. Plasma levels of biomarkers and antioxidants were also assessed during the iron reduction therapy for 16 weeks in 12 HCV patients. RESULTS: The concentrations of tHODE in the plasma and erythrocytes and t8-iso-PGF(2alpha) in the plasma of chronic HCV-infected patients were significantly higher than those of healthy controls. Plasma levels of vitamin E and vitamin C of HCV-infected patients were lower than those of the controls. Furthermore, the plasma tHODE significantly correlated with serum aminotransferases and type IV collagen-7S domain in chronic HCV-infected patients. During the iron reduction therapy, the plasma levels of tHODE but not t8-iso-PGF(2alpha) decreased and inversely its stereo-isomer ratio (ZE/EE) increased in parallel with the decreases of serum alanine aminotransferase, ferritin and alpha-fetoprotein. CONCLUSION: The levels of tHODE in chronic HCV-infected patients can be a useful biomarker for the evaluation of oxidative stress in chronic hepatitis C.

Deletion of caveolin-1 protects hyperoxia-induced apoptosis via survivin-mediated pathways.

Hyperoxia-induced lung injury is an established model that mimics human acute respiratory distress syndrome. Cell death is a prominent feature in lungs following prolonged hyperoxia. Caveolae are omega-shaped invaginations of the plasma membrane. Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. We have recently shown that deletion of cav-1 (cav-1-/-) protected against hyperoxia-induced cell death and lung injury both in vitro and in vivo; however, the mechanisms remain unclear. Survivin, a member of the inhibitor of apoptosis protein family, inhibits apoptosis in tumor cells. Although emerging evidence suggests that survivin is involved in wound healing, especially in vascular injuries, its role in hyperoxia-induced lung injury has not been investigated. Our current data demonstrated that hyperoxia induced apoptosis via suppressing survivin expression. Deletion of cav-1 abolished this suppression and subsequently protected against hyperoxia-induced apoptosis. Using "gain" and "loss" of function assays, we determined that survivin protected lung cells from hyperoxia-induced apoptosis via the inhibition of apoptosis executor caspase-3. Overexpression of survivin by deletion of cav-1 was regulated by Egr-1. Egr-1 functioned as a negative regulator of survivin expression. Deletion of cav-1 upregulated survivin via decreased Egr-1 binding of the survivin promoter region. Together, this study illustrates the effect of hyperoxia on survivin expression and the role of survivin in hyperoxia-induced apoptosis. We also demonstrate that deletion of cav-1 protects hyperoxia-induced apoptosis via modulation of survivin expression.

Early growth response gene 1 is essential for urban particulate matter-induced inflammation and mucus hyperproduction in airway epithelium.

Particulate matter (PM) has been implicated as a risk factor for human airway disorders. However, the biological mechanisms underlying the correlation between PM exposure and adverse airway effects have not yet been fully clarified. The objective of this study was to explore the possible role of early growth response gene 1 (Egr-1) in PM-induced toxic effects in pulmonary inflammation and mucus hyperproduction in vitro and in vivo. Particulate matter exposure induced a rapid Egr-1 expression in human bronchial epithelial (HBE) cells and in mouse lungs. Genetic blockage of Egr-1 markedly reduced PM-induced inflammatory cytokines, e.g., IL6 and IL8, and MUC5AC in HBE cells, and these effects were mechanistically mediated by the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) pathways, respectively. Egr-1-knockout mice displayed significantly reduced airway inflammation and mucus hyperproduction in response to PM exposure in vivo. Moreover, polycyclic aromatic hydrocarbons (PAHs) contained in the PM also induced Egr-1 expression, and also played a role in the inflammatory responses and mucus production. Taken together, our data reveal novel Egr-1 signaling that mediates the NF-κB and AP-1 pathways to orchestrate PM-induced pulmonary inflammation and mucus hyperproduction, suggesting that Egr-1 inhibition could be an effective therapeutic approach for airway disorders or disease exacerbations induced by airborne particulate pollution.

Antioxidant effects of 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran and alpha-tocopherol in hyperlipidemic mice as evaluated by hydroxyoctadecadienoic acid and 7-hydroxycholesterol.

It has been hypothesized that oxidative modification of low density lipoprotein plays a key role in the pathogenesis of atherosclerosis. In order to elucidate the role of lipid oxidation and its inhibition in vivo, apolipoprotein E and alpha-tocopherol (alphaT) transfer protein double knockout (ApoE(-/-)alpha-TTP(-/-)) and apolipoprotein E knockout (ApoE(-/-)) mice fed with a vitamin E-depleted diet and a diet containing 0.002 wt.% alphaT, respectively, were used with or without the treatment of a synthetic antioxidant 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653, 0.2 wt.%). The lipid oxidation markers of total hydroxylinoleic acid (tHODE), 8-iso-prostaglandin F(2alpha), and 7-hydroxycholesterol (t7-OHCh) in the blood, liver, and brain were inclusively measured with or without an excessive cholesterol-feeding (Ch-diet). The tHODE levels were elevated by Ch-diet in the plasma and brain of ApoE(-/-)alpha-TTP(-/-) mice and in the liver of ApoE(-/-) mice without BO-653. The levels of t7-OHCh in the liver were also increased due to the Ch-diet, and the ratio of t7-OHCh to the parent compound cholesterol was reduced to the control levels by BO-653. In summary, it was demonstrated by biomarkers, tHODE and t7-OHCh, that the added BO-653 in their diets exerted antioxidative effects in vivo under the condition of reduced vitamin E.

Lipid peroxidation in mice fed a choline-deficient diet as evaluated by total hydroxyoctadecadienoic acid.

OBJECTIVE: The relevance of oxidative stress in mice fed a choline-deficient diet (CDD) was investigated in relation to the oxidative stress marker, hydroxyoctadecadienoic acid (HODE) in comparison with F2-isoprostanes. Further, the protective effects of antioxidants against oxidative damage were assessed by using HODE. METHODS: We recently proposed total HODE as a biomarker for oxidative stress in vivo. Biological samples such as plasma, urine, and tissues were first reduced and then saponified to convert various oxidation products of linoleates to HODE. In the present study, this method was applied to measure oxidative damage in mice induced by CDD for 1 mo. RESULTS: CDD, when compared with choline-controlled diet (CCD), increased liver weight and fatty acid accumulation but the increase in body weight was less significant. Remarkable increases in HODE and 8-iso-prostaglandin F(2alpha) in liver and plasma were observed when mice were fed with the CDD for 1 mo compared with the CCD. The HODE level was about two to three orders higher than the F2-isoprostane level. This increase was decreased to the level of the CCD when alpha-tocopherol or 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran, a potent synthetic antioxidant, was mixed with the CDD. The stereoisomer ratio of HODE (9-and-13 (Z,E)-HODE/9-and-13 (E,E)-HODE) was decreased by CDD compared with CCD, which was spared by the addition of alpha-tocopherol and 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran. However, the increase in plasma glutamic-pyruvic transaminase and fatty acids in liver induced by the CDD was not recovered by any antioxidant. CONCLUSIONS: This study clearly demonstrated that oxidative stress was involved in fatty liver formation induced by the CDD and that HODE was a good biomarker for an oxidative stress in vivo.

Autophagy in idiopathic pulmonary fibrosis.

BACKGROUND: Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis. METHODS: Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-ß(1) on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model. RESULTS: Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-ß(1) inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-ß(1). In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin. CONCLUSION: Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-ß(1) may represent a mechanism for the promotion of fibrogenesis in IPF.

Advantages and limitation of BODIPY as a probe for the evaluation of lipid peroxidation and its inhibition by antioxidants in plasma.

Oxidative stress and the role of antioxidants are currently one of the most important subjects in the field of life science. In the present study, we assessed the oxidation of plasma lipids induced by free radicals and its inhibition by antioxidants with a fluorescence probe BODIPY. Vitamin E and C-depleted plasma was used to evaluate the inherent action of several antioxidants. BODIPY reacted with free radicals in plasma to emit fluorescence (ex. 510 nm, em. 520 nm), which was suppressed by the antioxidants in a concentration-dependent manner. However, the suppression of fluorescence emission by antioxidants did not always correlate quantitatively with the suppression of lipid peroxidation. For example, alpha-tocopherol suppressed BODIPY fluorescence but enhanced the peroxidation of plasma lipids in the absence of ascorbic acid. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without a phytyl side chain, almost completely suppressed both fluorescence emission and lipid peroxidation in the plasma. These results show that BODIPY can be used as a convenient probe for radical scavenging, but that care should be taken for the evaluation of antioxidant capacity.