Modulation of Cellular NAD+ Attenuates Cancer-Associated Hypercoagulability and Thrombosis via the Inhibition of Tissue Factor and Formation of Neutrophil Extracellular Traps.

Cancer-associated thrombosis is the second-leading cause of mortality in patients with cancer and presents a poor prognosis, with a lack of effective treatment strategies. NAD(P)H quinone oxidoreductase 1 (NQO1) increases the cellular nicotinamide adenine dinucleotide (NAD+) levels by accelerating the oxidation of NADH to NAD+, thus playing important roles in cellular homeostasis, energy metabolism, and inflammatory responses. Using a murine orthotopic 4T1 breast cancer model, in which multiple thrombi are generated in the lungs at the late stage of cancer development, we investigated the effects of regulating the cellular NAD+ levels on cancer-associated thrombosis. In this study, we show that dunnione (a strong substrate of NQO1) attenuates the prothrombotic state and lung thrombosis in tumor-bearing mice by inhibiting the expression of tissue factor and formation of neutrophil extracellular traps (NETs). Dunnione increases the cellular NAD+ levels in lung tissues of tumor-bearing mice to restore the declining sirtuin 1 (SIRT1) activity, thus deacetylating nuclear factor-kappa B (NF-κB) and preventing the overexpression of tissue factor in bronchial epithelial and vascular endothelial cells. In addition, we demonstrated that dunnione abolishes the ability of neutrophils to generate NETs by suppressing histone acetylation and NADPH oxidase (NOX) activity. Overall, our results reveal that the regulation of cellular NAD+ levels by pharmacological agents may inhibit pulmonary embolism in tumor-bearing mice, which may potentially be used as a viable therapeutic approach for the treatment of cancer-associated thrombosis.

Augmentation of NAD+ levels by enzymatic action of NAD(P)H quinone oxidoreductase 1 attenuates adriamycin-induced cardiac dysfunction in mice.

BACKGROUND: Adriamycin (ADR) is a powerful chemotherapeutic agent extensively used to treat various human neoplasms. However, its clinical utility is hampered due to severe adverse side effects i.e. cardiotoxicity and heart failure. ADR-induced cardiomyopathy (AIC) has been reported to be caused by myocardial damage and dysfunction through oxidative stress, DNA damage, and inflammatory responses. Nonetheless, the remedies for AIC are even not established. Therefore, we illustrate the role of NAD+/NADH modulation by NAD(P)H quinone oxidoreductase 1 (NQO1) enzymatic action on AIC. METHODS AND RESULTS: AIC was established by intraperitoneal injection of ADR in C57BL/6 wild-type (WT) and NQO1 knockout (NQO1-/-) mice. All Mice were orally administered dunnione (named NQO1 substrate) before and after exposure to ADR. Cardiac biomarker levels in the plasma, cardiac dysfunction, oxidative biomarkers, and mRNA and protein levels of pro-inflammatory mediators were determined compared the cardiac toxicity of each experimental group. All biomarkers of Cardiac damage and oxidative stress, and mRNA levels of pro-inflammatory cytokines including cardiac dysfunction were increased in ADR-treated both WT and NQO1-/- mice. However, this increase was significantly reduced by dunnione in WT, but not in NQO1-/- mice. In addition, a decrease in SIRT1 activity due to a reduction in the NAD+/NADH ratio by PARP-1 hyperactivation was associated with AIC through increased nuclear factor (NF)-κB p65 and p53 acetylation in both WT and NQO1-/- mice. While an elevation in NAD+/NADH ratio via NQO1 enzymatic action using dunnione recovered SIRT1 activity and subsequently deacetylated NF-κB p65 and p53, however not in NQO1-/- mice, thereby attenuating AIC. CONCLUSION: Thus, modulation of NAD+/NADH by NQO1 may be a novel therapeutic approach to prevent chemotherapy-associated heart failure, including AIC.

The inhibitory effect of beta-lapachone on RANKL-induced osteoclastogenesis.

ß-lapachone (ß-L) is a substrate of reduced nicotinamide adenine dinucleotide (NADH): quinone oxidoreductase 1 (NQO1). NQO1 reduces quinones to hydroquinones using NADH as an electron donor and consequently increases the intracellular NAD+/NADH ratio. The activation of NQO1 by ß-L has beneficial effects on several metabolic syndromes, such as obesity, hypertension, and renal injury. However, the effect of ß-L on bone metabolism remains unclear. Here, we show that ß-L might be a potent inhibitor of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. ß-L inhibited osteoclast formation in a dose-dependent manner and also reduced the expression of osteoclast differentiation marker genes, such as tartrate-resistant acid phosphatase (Acp5 or TRAP), cathepsin K (CtsK), the d2 isoform of vacuolar ATPase V0 domain (Atp6v0d2), osteoclast-associated receptor (Oscar), and dendritic cell-specific transmembrane protein (Dc-stamp). ß-L treatment of RANKL-induced osteoclastogenesis significantly increased the cellular NAD+/NADH ratio and resulted in the activation of 5' AMP-activated protein kinase (AMPK), a negative regulator of osteoclast differentiation. In addition, ß-L treatment led to significant suppression of the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and peroxisome proliferator-activated receptor gamma coactivator 1ß (PGC1ß), which can stimulate osteoclastogenesis. ß-L treatment downregulated c-Fos and nuclear factor of activated T-cells 1 (NFATc1), which are master transcription factors for osteoclastogenesis. Taken together, the results demonstrated that ß-L inhibits RANKL-induced osteoclastogenesis and could be considered a potent inhibitor of RANKL-mediated bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis.

Dunnione ameliorates cisplatin-induced small intestinal damage by modulating NAD(+) metabolism.

Although cisplatin is a widely used anticancer drug for the treatment of a variety of tumors, its use is critically limited because of adverse effects such as ototoxicity, nephrotoxicity, neuropathy, and gastrointestinal damage. Cisplatin treatment increases oxidative stress biomarkers in the small intestine, which may induce apoptosis of epithelial cells and thereby elicit damage to the small intestine. Nicotinamide adenine dinucleotide (NAD(+)) is a cofactor for various enzymes associated with cellular homeostasis. In the present study, we demonstrated that the hyper-activation of poly(ADP-ribose) polymerase-1 (PARP-1) is closely associated with the depletion of NAD(+) in the small intestine after cisplatin treatment, which results in downregulation of sirtuin1 (SIRT1) activity. Furthermore, a decrease in SIRT1 activity was found to play an important role in cisplatin-mediated small intestinal damage through nuclear factor (NF)-κB p65 activation, facilitated by its acetylation increase. However, use of dunnione as a strong substrate for the NADH:quinone oxidoreductase 1 (NQO1) enzyme led to an increase in intracellular NAD(+) levels and prevented the cisplatin-induced small intestinal damage correlating with the modulation of PARP-1, SIRT1, and NF-κB. These results suggest that direct modulation of cellular NAD(+) levels by pharmacological NQO1 substrates could be a promising therapeutic approach for protecting against cisplatin-induced small intestinal damage.

Pharmacological activation of NQO1 increases NAD⁺ levels and attenuates cisplatin-mediated acute kidney injury in mice.

Cisplatin is a widely used chemotherapeutic agent for the treatment of various tumors. In addition to its antitumor activity, cisplatin affects normal cells and may induce adverse effects, such as ototoxicity, nephrotoxicity, and neuropathy. Various mechanisms, such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammatory responses, are critically involved in cisplatin-induced adverse effects. As NAD(+) is a cofactor for various enzymes associated with cellular homeostasis, we studied the effects of increased NAD(+) levels by means of NAD(P)H: quinone oxidoreductase 1 (NQO1) activation using a known pharmacological activator (ß-lapachone) in wild-type and NQO1(-/-) mice on cisplatin-induced renal dysfunction in vivo. The intracellular NAD(+)/NADH ratio in renal tissues was significantly increased in wild-type mice co-treated with cisplatin and ß-lapachone compared with the ratio in mice treated with cisplatin alone. Inflammatory cytokines and biochemical markers for renal damage were significantly attenuated by ß-lapachone co-treatment compared with those in the cisplatin alone group. Notably, the protective effects of ß-lapachone in wild-type mice were completely abrogated in NQO1(-/-) mice. Moreover, ß-lapachone enhanced the tumoricidal action of cisplatin in a xenograft tumor model. Thus, intracellular regulation of NAD(+) levels through NQO1 activation might be a promising therapeutic target for the protection of cisplatin-induced acute kidney injury.

Activation of lipopolysaccharide-TLR4 signaling accelerates the ototoxic potential of cisplatin in mice.

Dysfunction in immune surveillance during anticancer chemotherapy of patients often causes weakness of the host defense system and a subsequent increase in microbial infections. However, the deterioration of organ-specific function related to microbial challenges in cisplatin-treated patients has not yet been elucidated. In this study, we investigated cisplatin-induced TLR4 expression and its binding to LPS in mouse cochlear tissues and the effect of this interaction on hearing function. Cisplatin increased the transcriptional and translational expression of TLR4 in the cochlear tissues, organ of Corti explants, and HEI-OC1 cells. Furthermore, cisplatin increased the interaction between TLR4 and its microbial ligand LPS, thereby upregulating the production of proinflammatory cytokines, such as TNF-α, IL-1ß, and IL-6, via NF-κB activation. In C57BL/6 mice, the combined injection of cisplatin and LPS caused severe hearing impairment compared with that in the control, cisplatin-alone, or LPS-alone groups, whereas this hearing dysfunction was completely suppressed in both TLR4 mutant and knockout mice. These results suggest that hearing function can be easily damaged by increased TLR expression and microbial infections due to the weakened host defense systems of cancer patients receiving therapy comprising three to six cycles of cisplatin alone or cisplatin combined with other chemotherapeutic agents. Moreover, such damage can occur even though patients may not experience ototoxic levels of cumulative cisplatin concentration.

Augmentation of NAD+ by Dunnione Ameliorates Imiquimod-Induced Psoriasis-Like Dermatitis in Mice.

Background: Dunnione has anti-inflammatory properties arising from its ability to alter the ratio of NAD+/NADH through NAD(P)H quinone oxidoreductase 1 (NQO1) enzymatic action, followed by subsequent inhibition of NF-κB and inflammatory cytokines. Psoriasis is a chronic, inflammatory skin disorder in which the IL-23/Th17 axis plays an important role in inflammation. However, it is unclear whether modulation of NAD+ levels affects psoriasis, such as skin inflammation. Therefore, in this study, we investigated the effect of NAD+/NADH ratio modulation on imiquimod (IMQ)-induced, psoriasis-like skin inflammation in mice. Methods: Psoriasis-like skin inflammation was generated by daily topical application of IMQ cream. The severity of dermatitis was assessed using the Psoriasis Area Severity Index (PASI) and histochemistry. Expression of inflammatory cytokines was detected by enzyme-linked immunosorbent assay and quantitative PCR. Acetylation of NF-κB p65 and STAT3 was determined by Western blotting. Results: Dunnione improved IMQ-induced epidermal hyperplasia and inflammation, consistent with decreased levels of inflammatory cytokines (IL-17, IL-22, and IL-23) in skin lesions. Moreover, we found that an increase in the NAD+/NADH ratio by dunnione restored SIRT1 activity, thereby reduced imiquimod-induced STAT3 acetylation, which modulates the expression of psoriasis-promoting inflammatory cytokines, such as IL-17, IL-22, and IL-23. Conclusion: Pharmacological modulation of cellular NAD+ levels could be a promising therapeutic approach for psoriasis-like skin disease.

Roles of NADPH oxidases in cisplatin-induced reactive oxygen species generation and ototoxicity.

In our previous study, we clearly demonstrated the roles of pro-inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1beta (IL-1beta), and IL-6, and subsequent reactive oxygen species (ROS) generation on the pathogenesis of cisplatin ototoxicity in vitro and in vivo. ROS generation in cisplatin-treated HEI-OC1 auditory cells was also correlated with changing mitochondrial membrane potential. However, the roles of NADPH oxidase in cisplatin-induced ROS generation and ototoxicity have not been fully elucidated. Herein, immunohistochemical studies demonstrated that treatment of cisplatin induced the expression of NADPH oxidase isoforms NOX-1 and NOX-4 in HEI-OC1 auditory cells. Expression of mRNA for NOX-1, NOX-4, NOXO1, NOXA1, p47(phox), and p67(phox) was also increased. Inhibition of NADPH oxidase with diphenyleniodonium chloride or apocynin abolished ROS production and the subsequent apoptotic cell death in cisplatin-treated cells. Furthermore, suppression of NOX1 and NOX4 expression by small interfering RNA transfection markedly abolished the cytotoxicity and ROS generation by cisplatin. Together, our data suggest that ROS generated, in part, through the activation of NADPH oxidase plays an essential role in cisplatin ototoxicity.

Mutation of a self-processing site in caspase-8 compromises its apoptotic but not its nonapoptotic functions in bacterial artificial chromosome-transgenic mice.

Caspase-8, the proximal enzyme in the death-induction pathway of the TNF/nerve growth factor receptor family, is activated upon juxtaposition of its molecules within the receptor complexes and is then self-processed. Caspase-8 also contributes to the regulation of cell survival and growth, but little is known about the similarities or the differences between the mechanisms of these nonapoptotic functions and of the enzyme's apoptotic activity. In this study, we report that in bacterial artificial chromosome-transgenic mice, in which the aspartate residue upstream of the initial self-processing site in caspase-8 (D387) was replaced by alanine, induction of cell death by Fas is compromised. However, in contrast to caspase-8-deficient mice, which die in utero at mid-gestation, the mice mutated at D387 were born alive and seemed to develop normally. Moreover, mice with the D387A mutation showed normal in vitro growth responses of T lymphocytes to stimulation of their Ag receptor as well as of B lymphocytes to stimulation by LPS, normal differentiation of bone marrow macrophage precursors in response to M-CSF, and normal generation of myeloid colonies by the bone marrow hematopoietic progenitors, all of which are compromised in cells deficient in caspase-8. These finding indicated that self-processing of activated caspase-8 is differentially involved in the different functions of this enzyme: it is needed for the induction of cell death through the extrinsic cell death pathway but not for nonapoptotic functions of caspase-8.

A chalcone glycoside from the fruits of Sorbus commixta Hedl.

Sorbus commixta Hedl. (Rosaceae) has been traditionally used in oriental countries for the treatment of asthma and other bronchial disorders. In this study, a chalcone glycoside was isolated from the ethyl acetate extract of the fruits of this plant. The compound was identified as neosakuranin based on the spectroscopic analysis and comparison with literature data. This is the first report of isolation of neosakuranin from Sorbus commixta.

Dimethoxycurcumin, a Synthetic Curcumin Analogue, Induces Heme Oxygenase-1 Expression through Nrf2 Activation in RAW264.7 Macrophages.

Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] induces heme oxygenase-1 (HO-1) expression via activation of the nuclear factor-erythroid-2-related factor 2 (Nrf2), whereas tetrahydrocurcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione], one of curcumin in vivo metabolites, has no effect on HO-1 expression and Nrf2 activation. The aim of this study was to investigate whether dimethoxycurcumin [1,7-bis(4,3-dimethoxyphenyl)-1,6-heptadiene-3,5-dione], a synthetic curcumin analogue with higher metabolic stability over curcumin, could induce HO-1 expression to the same extent as curcumin in RAW264.7 macrophages. Dimethoxycurcumin and curcumin, but not tetrahydrocurcumin, induced HO-1 expression and Nrf2 nuclear translocation, suggesting that the unsaturated nature of the diarylheptanoid chain of the compounds are crucial for HO-1 expression and Nrf2 activation. Blockage of Nrf2 synthesis by small interfering RNA abolished HO-1 expression by dimethoxycurcumin, indicating that dimethoxycurcumin may induce HO-1 expression via Nrf2 activation. In comparison, dimethoxycurcumin and curcumin had about the same effect on HO-1 expression, suggesting that dimethoxycurcumin retains the HO-1-inducing activity of its parent compound curcumin in RAW264.7 macrophages.

Augmentation of cellular NAD+ by NQO1 enzymatic action improves age-related hearing impairment.

Age-related hearing loss (ARHL) is a major neurodegenerative disorder and the leading cause of communication deficit in the elderly population, which remains largely untreated. The development of ARHL is a multifactorial event that includes both intrinsic and extrinsic factors. Recent studies suggest that NAD+ /NADH ratio may play a critical role in cellular senescence by regulating sirtuins, PARP-1, and PGC-1α. Nonetheless, the beneficial effect of direct modulation of cellular NAD+ levels on aging and age-related diseases has not been studied, and the underlying mechanisms remain obscure. Herein, we investigated the effect of ß-lapachone (ß-lap), a known plant-derived metabolite that modulates cellular NAD+ by conversion of NADH to NAD+ via the enzymatic action of NADH: quinone oxidoreductase 1 (NQO1) on ARHL in C57BL/6 mice. We elucidated that the reduction of cellular NAD+ during the aging process was an important contributor for ARHL; it facilitated oxidative stress and pro-inflammatory responses in the cochlear tissue through regulating sirtuins that alter various signaling pathways, such as NF-κB, p53, and IDH2. However, augmentation of NAD+ by ß-lap effectively prevented ARHL and accompanying deleterious effects through reducing inflammation and oxidative stress, sustaining mitochondrial function, and promoting mitochondrial biogenesis in rodents. These results suggest that direct regulation of cellular NAD+ levels by pharmacological agents may be a tangible therapeutic option for treating various age-related diseases, including ARHL.

Pharmacological stimulation of NQO1 decreases NADPH levels and ameliorates acute pancreatitis in mice.

Reactive oxygen species (ROS) regulates the activation of inflammatory cascades and tissue damage in acute pancreatitis. NADPH oxidase (NOX) is upregulated in pancreatitis and is one of the major enzymes involved in ROS production using NADPH as a general rate-limiting substrate. Dunnione, a well-known substrate of NAD(P)H:quinone oxidoreductase 1 (NQO1), reduces the ratio of cellular NADPH/NADP+ through the enzymatic action of NQO1. This study assessed whether a reduction in cellular NADPH/NADP+ ratio can be used to regulate caerulein-induced pancreatic damage associated with NOX-induced ROS production in animal models. Dunnione treatment significantly reduced the cellular NADPH/NADP+ ratio and NOX activity through the enzymatic action of NQO1 in the pancreas of the caerulein-injection group. Similar to these results, total ROS production and expressions of mRNA and protein for NOX subunits Nox1, p27phox, p47phox, and p67phox also decreased in the dunnione-treated group. In addition, caerulein-induced pancreatic inflammation and acinar cell injury were significantly reduced by dunnione treatment. This study is the first to demonstrate that modulation of the cellular NADPH:NADP+ ratio by enzymatic action of NQO1 protects acute pancreatitis through the regulation of NOX activity. Furthermore, these results suggest that modulation of the NADPH:NADP+ ratio in cells by NQO1 may be a novel therapeutic strategy for acute pancreatitis.

NAD+ augmentation ameliorates acute pancreatitis through regulation of inflammasome signalling.

Acute pancreatitis (AP) is a complicated disease without specific drug therapy. The cofactor nicotinamide adenine dinucleotide (NAD+) is an important regulator of cellular metabolism and homeostasis. However, it remains unclear whether modulation of NAD+ levels has an impact on caerulein-induced AP. Therefore, in this study, we investigated the effect of increased cellular NAD+ levels on caerulein-induced AP. We demonstrated for the first time that the activities and expression of SIRT1 were suppressed by reduction of intracellular NAD+ levels and the p53-microRNA-34a pathway in caerulein-induced AP. Moreover, we confirmed that the increase of cellular NAD+ by NQO1 enzymatic action using the substrate ß-Lapachone suppressed caerulein-induced AP with down-regulating TLR4-mediated inflammasome signalling, and thereby reducing the inflammatory responses and pancreatic cell death. These results suggest that pharmacological stimulation of NQO1 could be a promising therapeutic strategy to protect against pathological tissue damage in AP.

Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide.

Hydrogen sulfide (H(2)S), a regulatory gaseous molecule that is endogenously synthesized by cystathionine gamma-lyase (CSE) and/or cystathionine beta-synthase (CBS) from L-cysteine (L-Cys) metabolism, is a putative vasodilator, and its role in nitric oxide (NO) production is unexplored. Here, we show that at noncytotoxic concentrations, H(2)S was able to inhibit NO production and inducible NO synthase (iNOS) expression via heme oxygenase (HO-1) expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Both H(2)S solution prepared by bubbling pure H(2)S gas and NaSH, a H(2)S donor, dose dependently induced HO-1 expression through the activation of the extracellular signal-regulated kinase (ERK). Pretreatment with H(2)S or NaHS significantly inhibited LPS-induced iNOS expression and NO production. Moreover, NO production in LPS-stimulated macrophages that are expressing CSE mRNA was significantly reduced by the addition of L-Cys, a substrate for H(2)S, but enhanced by the selective CSE inhibitor beta-cyano-L-alanine but not by the CBS inhibitor aminooxyacetic acid. While either blockage of HO activity by the HO inhibitor, tin protoporphyrin IX, or down-regulation of HO-1 expression by HO-1 small interfering RNA (siRNA) reversed the inhibitory effects of H(2)S on iNOS expression and NO production, HO-1 overexpression produced the same inhibitory effects of H(2)S. In addition, LPS-induced nuclear factor (NF)-kappaB activation was diminished in RAW264.7 macrophages preincubated with H(2)S. Interestingly, the inhibitory effect of H(2)S on NF-kappaB activation was reversed by the transient transfection with HO-1 siRNA, but was mimicked by either HO-1 gene transfection or treatment with carbon monoxide (CO), an end product of HO-1. CO treatment also inhibited LPS-induced NO production and iNOS expression via its inactivation of NF-kappaB. Collectively, our results suggest that H(2)S can inhibit NO production and NF-kappaB activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.

3-Hydroxyanthranilic acid, one of L-tryptophan metabolites, inhibits monocyte chemoattractant protein-1 secretion and vascular cell adhesion molecule-1 expression via heme oxygenase-1 induction in human umbilical vein endothelial cells.

Heme oxygenase (HO)-1 is important in the vascular system, and its genetic or pharmacological induction in endothelium would be effective for the prevention and treatment of atherosclerosis. The naturally occurring antioxidant 3-hydroxyanthranilic acid (HA), one of l-tryptophan metabolites formed in vivo along the metabolic route known as the kynurenine pathway during inflammation or infection, was found to induce HO-1 expression and to stimulate nuclear translocation of NF-E2 related factor 2 (Nrf2) in human umbilical vein endothelial cells (HUVECs). Pre-treatment with HA inhibited the secretion of monocyte chemoattractant protein (MCP)-1, the expression of vascular cell adhesion molecule (VCAM)-1 and the activation of transcriptional nuclear factor (NF)-kappaB in HUVECs stimulated with tumor necrosis factor-alpha, the major pro-inflammatory cytokine causing endothelial inflammation. Interestingly, the observed anti-inflammatory effects of HA were mimicked by a HO-1 inducer, cobalt protoporphyrin, and bilirubin, one of HO-1 enzymatic products, but abolished in the presence of a HO-1 inhibitor, tin protoporphyrin. Based on our findings, we suggest that Nrf2-dependent HO-1 expression induced by HA inhibits MCP-1 secretion, VCAM-1 expression and NF-kappaB activation associated with vascular injury and inflammation in atherosclerosis.

Comparative effects of curcuminoids on endothelial heme oxygenase-1 expression: ortho-methoxy groups are essential to enhance heme oxygenase activity and protection.

Recently, it has been reported that curcumin, which is known as a potent antioxidant, acts as a non- stressful and non-cytotoxic inducer of the cytoprotective heme oxygenase (HO)-1. In this study, naturally occurring curcuminoids, such as pure curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), were compared for their potential ability to modulate HO-1 expression and cytoprotective activity in human endothelial cells. All three curcuminoids could induce HO-1 expression and HO activity with differential levels. The rank order of HO activity was curcumin, DMC and BDMC. In comparison with endothelial protection against H2O2-induced cellular injury, cytoprotective capacity was found to be highest with curcumin, followed by DMC and BDMC. Interestingly, cytoprotective effects afforded by curcuminoids were considerably associated with their abilities to enhance HO activity. Considering that the main difference among the three curcuminoids is the number of methoxy groups (none for BDMC, one for DMC, and two for curcumin), the presence of methoxy groups in the ortho position on the aromatic ring was suggested to be essential to enhance HO-1 expression and cytoprotection in human endothelial cells. Our results may be useful in designing more efficacious HO-1 inducers which could be considered as promising pharmacological agents in the development of therapeutic approaches for the prevention or treatment of endothelial diseases caused by oxidative damages.

1,2,3,4,6-penta-O-galloyl-beta-D-glucose up-regulates heme oxygenase-1 expression by stimulating Nrf2 nuclear translocation in an extracellular signal-regulated kinase-dependent manner in HepG2 cells.

AIM: To examine the potency of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) as a hepatic heme oxygenase-1 (HO-1) inducer and its regulation in HepG2 cells. METHODS: Expression of HO-1 and NF-E2-related factor 2 (Nrf2) and activation of mitogen-activated protein (MAP) kinases were analyzed by Western blot, immunofluorescence assay, and flow cytometry. Transfections of HO-1 gene, small interfering RNAs for HO-1 and Nrf2, and dominant-negative gene for MAP/extracellular signal-regulated kinase (ERK) were carried out to dissect the signaling pathways leading to HO-1 expression in HepG 2 cells. RESULTS: PGG up-regulated HO-1 expression and this expression conferred cytoprotection against oxidative injury induced by t-butyl hydroperoxide. Moreover, PGG induced Nrf2 nuclear translocation, which was found to be an upstream step of PGG-induced HO-1 expression, and ERK activation, of which pathway was involved in PGG-induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. CONCLUSION: PGG up-regulates HO-1 expression by stimulating Nrf2 nuclear translocation in an ERK-dependent manner, and HO-1 expression by PGG may serve as one of the important mechanisms for its hepatoprotective effects.

Increased Cellular NAD+ Level through NQO1 Enzymatic Action Has Protective Effects on Bleomycin-Induced Lung Fibrosis in Mice.

BACKGROUND: Idiopathic pulmonary fibrosis is a common interstitial lung disease; it is a chronic, progressive, and fatal lung disease of unknown etiology. Over the last two decades, knowledge about the underlying mechanisms of pulmonary fibrosis has improved markedly and facilitated the identification of potential targets for novel therapies. However, despite the large number of antifibrotic drugs being described in experimental pre-clinical studies, the translation of these findings into clinical practices has not been accomplished yet. NADH:quinone oxidoreductase 1 (NQO1) is a homodimeric enzyme that catalyzes the oxidation of NADH to NAD+ by various quinones and thereby elevates the intracellular NAD+ levels. In this study, we examined the effect of increase in cellular NAD+ levels on bleomycin-induced lung fibrosis in mice. METHODS: C57BL/6 mice were treated with intratracheal instillation of bleomycin. The mice were orally administered with ß-lapachone from 3 days before exposure to bleomycin to 1-3 weeks after exposure to bleomycin. Bronchoalveolar lavage fluid (BALF) was collected for analyzing the infiltration of immune cells. In vitro, A549 cells were treated with transforming growth factor ß1 (TGF-ß1) and ß-lapachone to analyze the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT). RESULTS: ß-Lapachone strongly attenuated bleomycin-induced lung inflammation and fibrosis, characterized by histological staining, infiltrated immune cells in BALF, inflammatory cytokines, fibrotic score, and TGF-ß1, α-smooth muscle actin accumulation. In addition, ß-lapachone showed a protective role in TGF-ß1-induced ECM expression and EMT in A549 cells. CONCLUSION: Our results suggest that ß-lapachone can protect against bleomycin-induced lung inflammation and fibrosis in mice and TGF-ß1-induced EMT in vitro, by elevating the NAD+/NADH ratio through NQO1 activation.

The fatty acid chain elongase, Elovl1, is required for kidney and swim bladder development during zebrafish embryogenesis.

Very long chain fatty acids are required for sphingolipid synthesis, lipid homeostasis, myelin formation, epidermal permeability, and retinal function. Seven different enzymes are known to be involved in the elongation cycle of fatty acids, with different chain-length specificities. Elovl1 is one of those enzymes whose function has been linked mainly to the synthesis of sphingolipids and the epidermal barrier. However, the role of Elovl1 in organogenesis is not clear. In zebrafish, 2 Elovl1 genes, elovl1a and elovl1b, are highly expressed in the swim bladder, and elovl1b is also expressed in the kidney. We found that both elovl1 knockdown embryos contain increased levels of long chain fatty acids from carbon number 14 to 20 as compared to control embryos. Oil-Red-O staining shows that yolk lipid consumption is greatly reduced, whereas lipid droplets accumulate within the swim bladder. Notably, knockdown of either elovl1a or elovl1b affects the expression of genes involved in swim bladder development and impairs inflation of the swim bladder. Consistent with its expression in the pronephros, knockdown of elovl1b alone affects the expression of genes required for kidney development and reduces renal clearance. Our findings strongly suggest that both elovl1 genes are a key determinant of swim bladder and kidney development in zebrafish, which may be comparatively applicable to lung and kidney development in humans.