Jung-Ran Noh

Fenofibrate Differentially Regulates Plasminogen Activator Inhibitor-1 Gene Expression via Adenosine Monophosphate-Activated Protein Kinase-Dependent Induction of Orphan Nuclear Receptor Small Heterodimer Partner

Plasminogen activator inhibitor type I (PAI‐1) is a marker of the fibrinolytic system and serves as a possible predictor for hepatic metabolic syndromes. Fenofibrate, a peroxisome proliferator‐activated receptor α (PPARα) agonist, is a drug used for treatment of hyperlipidemia. Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of crucial genes involved in various metabolic pathways. In this study, we show that fenofibrate increased SHP gene expression in cultured liver cells and in the normal and diabetic mouse liver by activating the adenosine monophosphate–activated protein kinase (AMPK) signaling pathway in a PPARα‐independent manner. Administration of transforming growth factor beta (TGF‐β) or a methionine‐deficient and choline‐deficient (MCD) diet to induce the progressive fibrosing steatohepatitis model in C57BL/6 mice was significantly reversed by fenofibrate via AMPK‐mediated induction of SHP gene expression with a dramatic decrease in PAI‐1 messenger RNA (mRNA) and protein expression along with other fibrotic marker genes. No reversal was observed in SHP null mice treated with fenofibrate. Treatment with another PPARα agonist, WY14643, showed contrasting effects on these marker gene expressions in wild‐type and SHP null mice, demonstrating the specificity of fenofibrate in activating AMPK signaling. Fenofibrate exhibited a differential inhibitory pattern on PAI‐1 gene expression depending on the transcription factors inhibited by SHP. Conclusion: By demonstrating that a PPARα‐independent fenofibrate‐AMPK‐SHP regulatory cascade can play a key role in PAI‐1 gene down‐regulation and reversal of fibrosis, our study suggests that various AMPK activators regulating SHP might provide a novel pharmacologic option in ameliorating hepatic metabolic syndromes. (HEPATOLOGY 2009.)

Antioxidant effects of the chestnut (Castanea crenata) inner shell extract in t-BHP-treated HepG2 cells, and CCl4- and high-fat diet-treated mice

The antioxidant effects of chestnut inner shell extract (CISE) were investigated in a tert-butylhydroperoxide (t-BHP)-treated HepG2 cells, and in mice that were administered carbon tetrachloride (CCl(4)) and fed a high-fat diet (HFD). Pre-incubation with CISE significantly blocked the oxidative stress induced by t-BHP treatment in HepG2 cells (P<0.05) and preserved the activities of catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase compared to group treated with t-BHP only. Similarly, the CCl(4)- and HFD-induced reduction of antioxidant enzymes activities in liver was prevented by CISE treatment compared to control groups. Furthermore, hepatic lipid peroxidation were remarkably lower (P<0.05) in the CISE-treated groups with t-BHP or HFD. To determine the active compound of CISE, the fractionation of CISE has been conducted and scoparone and scopoletin were identified as main compounds. These compounds were also shown to inhibit the t-BHP-induced ROS generation and reduction in antioxidant enzyme activity in an in vitro model system. From these results, it was demonstrated that CISE has the ability to protect against damage from oxidative stressors such as t-BHP, CCl(4) and HFD in in vitro and in vivo models. The CISE might be useful for the prevention of oxidative damage in liver cells and tissues.

Protective Effect of Korean Red Ginseng against Aflatoxin B1-Induced Hepatotoxicity in Rat.

Korean red ginseng (KRG), the steamed root of Panax ginseng Meyer, has a variety of biological properties, including anti-inflammatory, antioxidant and anticancer effects. Aflatoxin B1 (AFB1) produced by the Aspergillus spp. causes acute hepatotoxicity by lipid peroxidation and oxidative DNA damage, and induces liver carcinoma in humans and laboratory animals. This study was performed to examine the protective effects of KRG against hepatotoxicity induced by AFB1 using liver-specific serum marker analysis, histopathology, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. In addition, to elucidate the possible mechanism of hepatoprotective effects, superoxide dismutase, catalase, glutathione peroxidase, and malondialdehyde were analyzed. Rats were treated with 250 mg/kg of KRG (KRG group) or saline (AFB1 group) for 4 weeks and then received 150 μg/kg of AFB1 intraperitoneally for 3 days. Rats were sacrificed at 12 h, 24 h, 48 h, 72 h, or 1 wk after AFB1 treatment. In the KRG pre-treatment group, serum alanine aminotransferase, aspartate aminotransferase, and malondialdehyde levels were low, but superoxide dismutase, catalase, and glutathione peroxidase activities were high as compared to the AFB1 alone group. Histopathologically, AFB1 treatment induced necrosis and apoptosis in hepatocytes, and led to inflammatory cells infiltration in the liver. KRG pre-treatment ameliorated these changes. These results indicate that KRG may have protective effects against hepatotoxicity induced by AFB1 that involve the antioxidant properties of KRG.

Activation of NAD(P)H:quinone Oxidoreductase Ameliorates Spontaneous Hypertension in an Animal Model via Modulation of eNOS Activity

AIMS Hypertension is one of the most common human diseases worldwide, and extensive research efforts are focused upon the identification and utilizing of novel therapeutic drug targets. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is an important regulator of blood pressure (BP). β-Lapachone (βL), a well-known substrate of NAD(P)H:quinone oxidoreductase (NQO1), increases the cellular NAD(+)/NADH ratio via the activation of NQO1. In this study, we evaluated whether βL-induced activation of NQO1 modulates BP in an animal model of hypertension. METHODS AND RESULTS Spontaneously hypertensive rats (SHR), primary human aortic endothelial cells (HAEC), and endothelial cell lines were used to investigate the hypotensive effect of βL and its mode of action. βL treatment stimulated endothelium-dependent vascular relaxation in response to acetylcholine in aorta of SHR and dramatically lowered BP in SHR, but the hypotensive effect was completely blocked by eNOS inhibition with ω-nitro-l-arginine methyl ester. Aortic eNOS phosphorylation and eNOS protein expression were significantly increased in βL-treated SHR. In vitro studies revealed that βL treatment elevated the intracellular NAD(+)/NADH ratio and concentration of free Ca(2+) ([Ca(2+)]i), and resulted in Akt/AMP-activated protein kinase/eNOS activation. These effects were abolished by NQO1 siRNA and [Ca(2+)]i inhibition through a ryanodine receptor blockade. CONCLUSION This study is the first to demonstrate that NQO1 activation has a hypotensive effect mediated by eNOS activation via cellular NAD(+)/NADH ratio modulation in an animal model. These results provide strong evidence suggesting NQO1 might be a new therapeutic target for hypertension.

Prevention of salt-induced renal injury by activation of NAD(P)H:quinone oxidoreductase 1, associated with NADPH oxidase

NADPH oxidase (NOX) is a predominant source of reactive oxygen species (ROS), and the activity of NOX, which uses NADPH as a common rate-limiting substrate, is upregulated by prolonged dietary salt intake. β-Lapachone (βL), a well-known substrate of NAD(P)H:quinone oxidoreductase 1 (NQO1), decreases the cellular NAD(P)H/NAD(P)(+) ratio via activation of NQO1. In this study, we evaluated whether NQO1 activation by βL modulates salt-induced renal injury associated with NOX-derived ROS regulation in an animal model. Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet were used to investigate the renoprotective effect of NQO1 activation. βL treatment significantly lowered the cellular NAD(P)H:NAD(P)(+) ratio and dramatically reduced NOX activity in the kidneys of HS diet-fed DS rats. In accordance with this, total ROS production and expression of oxidative adducts also decreased in the βL-treated group. Furthermore, HS diet-induced proteinuria and glomerular damage were markedly suppressed, and inflammation, fibrosis, and apoptotic cell death were significantly diminished by βL treatment. This study is the first to demonstrate that activation of NQO1 has a renoprotective effect that is mediated by NOX activity via modulation of the cellular NAD(P)H:NAD(P)(+) ratio. These results provide strong evidence that NQO1 might be a new therapeutic target for the prevention of salt-induced renal injury.

Sulforaphane protects against acetaminophen-induced hepatotoxicity

Oxidative stress is closely associated with acetaminophen (APAP)-induced toxicity. Heme oxygenase-1 (HO-1), an antioxidant defense enzyme, has been shown to protect against oxidant-induced tissue injury. This study investigated whether sulforaphane (SFN), as a HO-1 inducer, plays a protective role against APAP hepatotoxicity in vitro and in vivo. Pretreatment of primary hepatocyte with SFN induced nuclear factor E2-factor related factor (Nrf2) target gene expression, especially HO-1 mRNA and protein expression, and suppressed APAP-induced glutathione (GSH) depletion and lipid peroxidation, which eventually leads to hepatocyte cell death. A comparable effect was observed in mice treated with APAP. Mice were treated with 300 mg/kg APAP 30 min after SFN (5 mg/kg) administration and were then sacrificed after 6 h. APAP alone caused severe liver injuries as characterized by increased plasma AST and ALT levels, GSH depletion, apoptosis, and 4-hydroxynonenal (4-HNE) formations. This APAP-induced liver damage was significantly attenuated by pretreatment with SFN. Furthermore, while hepatic reactive oxygen species (ROS) levels were increased by APAP exposure, pretreatment with SFN completely blocked ROS formation. These results suggest that SFN plays a protective role against APAP-mediated hepatotoxicity through antioxidant effects mediated by HO-1 induction. SFN has preventive action in oxidative stress-mediated liver injury.

Susceptibility to gold nanoparticle-induced hepatotoxicity is enhanced in a mouse model of nonalcoholic steatohepatitis.

Although the safety of gold nanoparticle (AuNP) use is of growing concern, most toxicity studies of AuNPs had focused on their chemical characteristics, including their physical dimensions, surface chemistry, and shape. The present study examined the susceptibility of rodents with healthy or damaged livers to AuNP-induced hepatotoxicity. To induce a model of liver injury, mice were fed a methionine- and choline-deficient (MCD) diet for 4 weeks. Sizes and biodistribution of 15-nm PEGylated AuNPs were analyzed by transmission electron microscopy. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated with an automatic chemical analyzer, and liver sections were subjected to pathological examination. Activities of antioxidant enzymes were determined by biochemical assay. Lateral tail vein injection of MCD diet-fed mice with 5 mg kg(-1) AuNPs significantly elevated the serum ALT and AST levels compared to MCD diet-fed mice injected with mPEG (methylpolyethylene glycol). Similarly, severe hepatic cell damage, acute inflammation, and increased apoptosis and reactive oxygen species (ROS) production were observed in the livers of AuNP-injected mice on the MCD diet; these liver injuries were attenuated in mice fed a normal chow diet. The results suggest that AuNPs display toxicity in a stressed liver environment by stimulating the inflammatory response and accelerating stress-induced apoptosis. These conclusions may point to the importance of considering health conditions, including liver damage, in medical applications of AuNPs.

Metformin ameliorates acetaminophen hepatotoxicity via Gadd45β-dependent regulation of JNK signaling in mice

BACKGROUND & AIMS Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure. Prolonged c-Jun N-terminal kinase (JNK) activation plays a central role in APAP-induced liver injury and growth arrest, and DNA damage-inducible 45 beta (Gadd45β) is known to inhibit JNK phosphorylation. Metformin has recently been shown to have hepatoprotective effects. The aim of the present study is to investigate whether metformin mitigates APAP-induced hepatotoxicity and to ascertain the molecular basis of this effect. METHODS We used APAP- and/or metformin-treated Gadd45β knockout (KO) mice and wild type (WT) C57BL/6J control mice. Primary mouse hepatocytes were isolated from WT and Gadd45β KO mice were used for in vitro study. RESULTS Metformin pretreatment protected against APAP toxicity with decreased liver damage, and inhibited APAP-induced prolonged hepatic JNK phosphorylation in WT mice. Gadd45β expression was increased after APAP treatment, and the expression of Gadd45β was further enhanced by metformin. The effects of metformin on APAP-induced liver injury and JNK phosphorylation were abolished in Gadd45β KO mice. Notably, subtoxic doses of APAP caused cell death and sustained JNK phosphorylation in Gadd45β-deficient primary hepatocytes. In parallel, APAP increased mortality, severe liver injury, and JNK activation in Gadd45β KO mice. Interestingly, metformin administered after APAP treatment protected against APAP-evoked hepatotoxicity in WT mice, but not in Gadd45β KO mice. CONCLUSIONS This study is the first to demonstrate that metformin shows protective and therapeutic effects against APAP overdose-evoked hepatotoxicity via Gadd45β-dependent JNK regulation. Metformin would be a promising therapeutic strategy for treatment of APAP overdose.

Protection of NAD(P)H:quinone oxidoreductase 1 against renal ischemia/reperfusion injury in mice.

UNLABELLED Ischemia/reperfusion (I/R) is the most common cause of acute renal injury. I/R-induced reactive oxygen species (ROS) are thought to be a major factor in the development of acute renal injury by promoting the initial tubular damage. NAD(P)H quinone oxidoreductase 1 (NQO1) is a well-known antioxidant protein that regulates ROS generation. The purpose of this study was to investigate whether NQO1 modulates the renal I/R injury (IRI) associated with NADPH oxidase (NOX)-derived ROS production in an animal model. We analyzed renal function, oxidative stress, and tubular apoptosis after IRI. NQO1(-/-) mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In the kidneys of NQO1(-/-) mice, the cellular NADPH/NADP(+) ratio was significantly higher and NOX activity was markedly higher than in those of NQO1(+/+) mice. The activation of NQO1 by β-lapachone (βL) significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis by renal I/R. Moreover, the βL treatment significantly lowered the cellular NADPH/NADP(+) ratio and dramatically reduced NOX activity in the kidneys after IRI. From these results, it was concluded that NQO1 has a protective role against renal injury induced by I/R and that this effect appears to be mediated by decreased NOX activity via cellular NADPH/NADP(+) modulation. These results provide convincing evidence that NQO1 activation might be beneficial for ameliorating renal injury induced by I/R.

Protective role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in cisplatin-induced nephrotoxicity

Although cisplatin is widely used as an anti-cancer agent, its use is significantly limited because of its tendency to induce nephrotoxicity through poorly understood mechanisms. NAD(P)H:quinone oxidoreductase 1 (NQO1) is well known to regulate ROS generation. The purpose of this study was to investigate whether NQO1 modulates cisplatin-induced renal failure associated with NADPH oxidase (NOX)-derived ROS production in an animal model. NQO1-/- mice were treated with cisplatin (18 mg/kg) and renal function, oxidative stress, and tubular apoptosis were assessed. NQO1-/- mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In accordance with these results, the cellular NADPH/NADP ratio and NOX activity were markedly increased in the kidneys of NQO1-/- mice compared to NQO1+/+ mice. In addition, activation of NQO1 by βL treatment significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis. This study demonstrates that NQO1 protects cells against renal failure induced by cisplatin, and that this effect is mediated by decreased NOX activity via cellular NADPH/NADP modulation. These results provide convincing evidence that NQO1 might be beneficial for ameliorating renal failure induced by cisplatin.