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ABSTRACT: Within the liver, reactive oxygen species produced by infiltrating blood cells and Kupffer cells (resident macrophages) can injure hepatocytes. We hypothesized that hepatocyte survival is influenced by the relatively small juxtaposed population of hepatic stellate cells (HSCs). We used cultures of primary rat hepatocytes as targets for superoxide-induced damage, which was determined by crystal violet assay and lactate dehydrogenase release. An HSC-conditioned medium prevented the superoxide-induced death of hepatocytes, and the protective factor released by HSCs was a protein or proteins (apparent molecular weight > 100 kDa) resistant to heat (70°C) and pH (4.5-8.5). The protein or proteins were partially purified on DE52 cellulose, and the active fraction contained no detectable levels of superoxide dismutase: after separation by Sephadex G-100 gel filtration, the antioxidant activity could be reconstituted by the combination of 2 protein peaks, and this reconstituted activity was protective both in vitro and against liver ischemia/reperfusion injury in intact rats. Mass spectrometry proteomic studies confirmed that this activity could not be attributed to any previously identified antioxidant protein. Thus, HSCs protect hepatocytes against oxidative damage through the production of a novel protein, the further purification of which may lead to the isolation of a powerful oxygen radical scavenger with clinical applications.
Liver Transplantation 12/2010; 16(12):1400-9. · 3.39 Impact Factor
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ABSTRACT: Cardiovascular diseases (CVD) are one of the leading causes of global mortality for which cigarette smoking (CS) is a major and independent risk factor. Free radicals mediated oxidative stress has been implicated in the pathogenesis of smoking-related CVD and antioxidant nutrients are reported to prevent these cardiac damages. Therefore, the present study was conducted to evaluate the antioxidant role of (-)-epigallocatechin-gallate (EGCG) against chronic CS induced oxidative damage in circulation (rat blood) and heart of rats. Adult male albino rats were exposed to side stream CS for a period of 12 weeks and simultaneously administered with EGCG (20mg/kg b.w./day, p.o.). Exposure to CS significantly increased the cardiac and serum levels of oxidative stress markers such as lipid peroxidation, protein carbonyl, and decreased protein thiol, reduced glutathione, vitamin C and vitamin E, the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferases and glutathione reductase were found to decreased. Oral pretreatment with EGCG showed significant decrease in the levels of lipid peroxidation, protein carbonyl products and improved the antioxidant status by increasing the activities of enzymic antioxidants, and non-enzymic antioxidants. Histopathological alterations of the myocardial tissue were prevented by EGCG administration. These results suggest that chronic CS exposure enhances oxidative stress, thereby disturbing the tissue defense system and EGCG protects the heart from this oxidative damage through its antioxidant potential.
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 10/2010; · 2.24 Impact Factor
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ABSTRACT: Abstract
Cardiovascular diseases (CVD) are one of the leading causes of global mortality for which cigarette smoking is a major and independent risk factor. Free radicals mediated oxidative stress has been implicated in the pathogenesis of smoking-related cardiovascular diseases and antioxidant nutrients are reported to prevent these cardiac damages. Therefore, the present study was conducted to evaluate the antioxidant role of EGCG against chronic cigarette smoking (CS) induced oxidative damage in circulation (rat blood) and heart of rats. Adult male albino rats were exposed to side stream CS for a period of 12 weeks and simultaneously administered with EGCG (20 mg/kg b.w./day,p.o.). Exposure to CS significantly increased the cardiac and serum levels of oxidative stress markers such as lipid peroxidation, protein carbonyl, and decreased protein thiol, reduced glutathione, vitamin C, vitamin E, the activities of superoxide dismutase, catalase, glutathione peroxidase glutathione-S-transferases and glutathione reductase. Exposure to smoke also accelerated the histopathological and ultra structural alteration in cardiac tissue. Oral pretreatment with EGCG showed significant decrease in the levels of lipid peroxidation, protein carbonyl products and improved the antioxidant status by increasing the activities of enzymic-antioxidants, and non-enzymic antioxidants. Histopathological alterations of the myocardial tissue were prevented by EGCG administration. These results suggest that chronic CS exposure enhances oxidative stress, there by disturbing the tissue defense system and EGCG protects the heart from this oxidative damage through its antioxidant potential
Biomedecine [?] Pharmacotherapy 01/2010; · 2.00 Impact Factor
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ABSTRACT: Reactive oxygen species (ROS) activate retinoid-containing quiescent hepatic stellate cells (qHSCs) to retinoid-deficient fibrogenic myofibroblast-like cells (aHSCs). However, ROS also cause apoptosis of aHSCs, and apoptotic aHSCs are observed in inflammatory fibrotic liver. Here, we investigated mechanisms of the effects of oxidative stress on the survival of qHSCs and aHSCs. HSCs from normal rat liver were used after overnight culture (qHSCs), or in 3-5 passages (aHSCs). For in vivo induction of oxidative stress, tert-butylhydroperoxide was injected into control and CCl4-induced cirrhotic rats. Spontaneous caspase-3 activation and apoptosis, observed in cultured qHSCs, decreased with time and were unaffected by superoxide. In contrast, superoxide caused caspase-3 and p38-MAPK activation, reduction in Bcl-xL expression, and apoptosis in aHSCs. Inhibition of caspase-3 and p38-MAPK did not affect the viability of qHSCs in the absence or presence of superoxide, but inhibited superoxide-induced death of aHSCs. Glutathione (GSH) level and activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were lower in aHSCs than qHSCs. Superoxide increased GSH content, and activities of SOD, catalase and GPx in qHSCs but not in aHSCs. Incubation of 13-cis-retinoic acid (RA)-treated aHSCs with superoxide increased their GSH content significantly, and prevented superoxide-induced p38-MAPK and caspase-3 activation while dramatically reducing the extent of apoptosis. Finally, oxidative stress induced in vivo caused apoptosis of aHSCs in cirrhotic but not of qHSCs in control rats. These results suggest that the absence of retinoids render aHSCs susceptible to superoxide-induced apoptosis via caspase-3 and p38-MAPK activation.
Journal of Cellular Physiology 10/2008; 218(1):157-66. · 3.87 Impact Factor
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ABSTRACT: Augmenter of liver regeneration (ALR), a protein synthesized and stored in hepatocytes, is associated with mitochondria, and possesses sulfhydryl oxidase and cytochrome c reductase activities. We sought to determine the effects of ALR depletion in hepatocytes by antisense oligonucleotide transfection.
Rat hepatocytes in primary culture were transfected with antisense oligonucleotide for ALR mRNA (ALR-AS) or scrambled oligonucleotide. Various analyses were performed at times up to 24h after transfection.
Treatment with ALR-AS caused a decrease in ALR mRNA, cellular depletion of ALR protein primarily from mitochondria, and decreased viability. Flow cytometric analysis of ALR-AS-transfected hepatocytes stained with annexin-Vcy3 and 7-aminoactinomycin D revealed apoptosis as the predominant cause of death up to 6h; incubation beyond this time resulted in necrosis in addition to apoptosis. ALR-AS-transfection caused release of mitochondrial cytochrome c, activation of caspase-3, profound reduction in the ATP content, and cellular release of LDH. Inhibition of caspase-3 inhibited the early phase of ALR-AS-induced death but not the late phase that included ALR and LDH release.
These results suggest that ALR is critically important for the survival of hepatocytes by its association with mitochondria and regulation of ATP synthesis.
Journal of Hepatology 05/2008; 48(4):578-88. · 9.26 Impact Factor
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ABSTRACT: Compelling experimental evidence indicates that the interactions between endotoxin and hepatic stellate cells (HSCs) can play a significant role in the pathogenesis of liver disease. Endotoxin-induced release of a multifunctional mediator NO (via inducible NO synthase) and the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6 by HSCs could be an important mechanism of pathological changes in the liver. However, the signaling mechanisms of these effects are poorly understood. In this study, we found that endotoxin causes activation of mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated protein kinase [ERK] 1 and 2, p38, and c-Jun NH2-terminal kinase [JNK]) and nuclear factor kappaB (NF-kappaB) and production of H(2)O(2) in culture-activated HSCs. However, only p38 and NF-kappaB were found to be responsible for the synthesis of NO, IL-6, and TNF-alpha. Exogenous H(2)O(2) caused modest stimulation of TNF-alpha synthesis, did not affect the synthesis of NO or IL-6, and did not activate NF-kappaB or MAPKs. Inhibition of p38 and NF-kappaB activation by SB203580 and pyrrolidine dithiocarbamate, respectively, blocked endotoxin-induced H(2)O(2), NO, TNF-alpha, and IL-6 synthesis. Inhibition of ERK1/2 and JNK phosphorylation did not alter these effects of endotoxin. Whereas SB203580 inhibited endotoxin-induced NF-kappaB activation, pyrrolidine dithiocarbamate did not affect p38 phosphorylation in endotoxin-stimulated cells. In conclusion, endotoxin-induced synthesis of NO, TNF-alpha, and IL-6 in HSCs is mediated by p38 and NF-kappaB, with involvement of H(2)O(2) in TNF-alpha production.
Hepatology 09/2006; 44(2):389-98. · 11.66 Impact Factor
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ABSTRACT: Endotoxin is implicated in the pathology of acute liver failure. The mechanisms of its actions on quiescent hepatic stellate cells (qHSCs) and their implications in hepatocyte injury are incompletely understood. We investigated effects of endotoxin (bacterial lipopolysaccharide; LPS) on qHSCs and subsequently on hepatocytes. After overnight culture following their isolation, qHSCs were incubated with or without endotoxin for 24 h. The cells and the culture supernatant were analyzed for cytokines and nitric oxide (NO) synthesis. The effects of qHSC-conditioned media on hepatocytes were then determined. LPS increased inducible NO synthase expression, stimulated NO synthesis, and inhibited DNA synthesis in qHSCs. qHSC-conditioned medium inhibited DNA synthesis in hepatocytes without affecting NO synthesis, while LPS (1-1,000 ng/ml)-conditioned qHSC medium stimulated NO synthesis and caused further inhibition of DNA synthesis and apoptosis. These effects of LPS were more pronounced when qHSCs were incubated with serum, but not with LPS-binding protein (LBP) although CD14 (a receptor for LPS-LBP complex) was found in qHSCs. LPS stimulated the synthesis of TNF-alpha, interleukin (IL)-6, and IL-1beta but not of TGF-beta in qHSCs. Individually or together, L-N(G)-monomethylarginine and antibodies to IL-1beta, IL-6, and TNF-alpha only partly reversed qHSC + LPS-conditioned medium-induced inhibition of DNA synthesis in hepatocytes. These results suggest that the effects of LPS on qHSCs are novel, occurring without the aid of LBP/CD14. They also indicate that other factors, in addition to NO, TGF-beta, TNF-alpha, IL-1beta, and IL-6 are involved in the mechanisms of the growth inhibitory effects of qHSCs on hepatocytes.
Journal of Cellular Physiology 09/2005; 204(2):654-65. · 3.87 Impact Factor
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ABSTRACT: During liver injury, reactive oxygen species (ROS) are produced by the resident macrophages (Kupffer cells) and infiltrating blood cells such as neutrophils. ROS cause transformation of desmin-positive quiescent hepatic stellate cells (HSCs) into the proliferating activated phenotype that expresses alpha-smooth muscle actin (alpha-SMA). The highly fibrogenic and contractile activated HSCs (aHSCs) produce various cytokines and growth factors, and play important role in the pathophysiology of chronic liver disease. However, apoptotic aHSCs are also observed during active fibrogenesis in the injured liver. Therefore, we investigated the mechanisms of apoptosis of aHSCs in relation to ROS.
HSCs, isolated from normal rat liver, were activated in culture and effects of superoxide were determined between subcultures 3 and 5.
Treatment with superoxide caused apoptosis of aHSCs as determined by flow cytometry, TUNEL assay and DNA laddering analysis. The mechanisms of superoxide-induced apoptosis involved release of cytochrome c, increased Bax expression, increased caspase-3 activity, and hydrolysis of polyADP-ribose polymerase. Superoxide also increased the expression of antiapoptotic Bcl-xL and nuclear translocation of NFkappaB. Caspase-3 inhibitor (DEVD-fmk) and antioxidants (N-acetylcysteine, vitamin E and superoxide dismutase) inhibited superoxide-induced apoptosis.
Superoxide-induced apoptosis of aHSCs may be a novel mechanism of limiting chronic fibrotic liver injury.
Journal of Hepatology 11/2004; 41(4):567-75. · 9.26 Impact Factor
