Search

Oxidative stress plays a critical role in the development of liver disease, making antioxidants a promising therapeutic approach for the prevention and management of liver injuries. The aim of this study was to investigate the hepatoprotective effects of kaempferol, an antioxidant flavonoid found in various edible vegetables, and its underlying mechanism in male Sprague-Dawley rats with carbon tetrachloride (CCl4)-induced acute liver damage. Oral administration of kaempferol at doses of 5 and 10 mg/kg body weight resulted in the amelioration of CCl4-induced abnormalities in hepatic histology and serum parameters. Additionally, kaempferol decreased the levels of pro-inflammatory mediators, TNF-α and IL-1β, as well as COX-2 and iNOS. Furthermore, kaempferol suppressed nuclear factor-kappa B (NF-κB) p65 activation, as well as the phosphorylation of Akt and mitogen-activated protein kinase members (MAPKs), including extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 in CCl4-intoxicated rats. In addition, kaempferol improved the imbalanced oxidative status, as evidenced by the reduction in reactive oxygen species levels and lipid peroxidation, along with increased glutathione content in the CCl4-treated rat liver. Administering kaempferol also enhanced the activation of nuclear factor-E2-related factor (Nrf2) and heme oxygenase-1 protein, as well as the phosphorylation of AMP-activated protein kinase (AMPK). Overall, these findings suggest that kaempferol exhibits antioxidative, anti-inflammatory, and hepatoprotective effects through inhibiting the MAPK/NF-κB signaling pathway and activating the AMPK/Nrf2 signaling pathway in CCl4-intoxicated rats.

Oligonol is a low molecular weight polyphenol product derived from lychee fruit by a manufacturing process. We investigated oligonol’s anti-fibrotic effect and the underlying mechanism in dimethylnitrosamine (DMN)-induced chronic liver damage in male Sprague–Dawley rats. Oral administration of oligonol (10 and 20 mg/kg body weight) ameliorated the DMN-induced abnormalities in liver histology and serum parameters in rats. Oligonol prevented the DMN-induced elevations of TNF-α, IL-1β, IL-6, cyclooxygenase-2, and inducible nitric oxide synthase expressions at the mRNA level. NF-κB activation and JNK phosphorylation in DMN-treated rats were ablated by oligonol. Oligonol reduced the enhanced production of hepatic malondialdehyde and reactive oxygen species and recovered protein SH, non-protein SH levels, and catalase activity in the DMN treated liver. Nrf2 translocation into the nucleus was enhanced, and PI3K and phosphorylated Akt levels were increased by administering oligonol. The level of hepatic fibrosis-related factors such as α-smooth muscle actin, transforming growth factor-β1, and type I collagen was reduced in rats treated with oligonol. Histology and immunohistochemistry analysis showed that the accumulation of collagen and activation of hepatic stellate cells (HSCs) in liver tissue were restored by oligonol treatment. Taken together, oligonol showed antioxidative, hepatoprotective, and anti-fibrotic effects via JNK/NF-κB and PI3K/Akt/Nrf2 signaling pathways in DMN-intoxicated rats. These results suggest that antioxidant oligonol is a potentially useful agent for the protection against chronic liver injury.

In this study, we investigated the hepatoprotective and anti-fibrotic effects of zingerone, one of the active components of ginger, against carbon tetrachloride (CCl4)- and dimethylnitrosamine (DMN)-induced liver injuries in rats, respectively. Oral administration of zingerone (10 mg/kg) reduced CCl4-induced abnormalities in liver histology, serum alanine aminotransferase and aspartate aminotransferase levels, and liver malondialdehyde levels. Zingerone treatment attenuated CCl4-induced increases in inflammatory mediators, including tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, and inducible nitric oxide synthase mRNA levels. Western blot analysis showed that zingerone suppressed activation of nuclear factor-kappa B (NF-κB) p65 and phosphorylation of extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinases (MAPKs). Liver fibrosis induced by DMN (10 mg/kg, intraperitoneally) was ameliorated by administration of zingerone (10 and 20 mg/kg, orally). Zingerone treatment reduced DMN-induced elevation of hydroxyproline content and hepatic stellate cell activation. In conclusion, zingerone showed antioxidative and anti-inflammatory effects in CCl4-intoxicated rats by inhibiting oxidative stress and NF-κB activation via blockade of the activation of upstream MAPKs. Moreover, zingerone had hepatoprotective and anti-fibrotic effects against DMN-induced liver injury suggesting its usefulness in the prevention of liver inflammation and the development of hepatic fibrosis.

Bilirubin (BR) is generated by the reduction of biliverdin (BV), a metabolite that results from the catalytic degradation of heme by the isoforms of heme oxygenase (HO). BV is nontoxic and water-soluble but BR is potentially toxic and lipophilic. Therefore, a further metabolic step is required for BR before excretion is possible. The reductive conversion of BV to BR costs energy and is evolutionarily conserved in human physiology. There must be a compelling reason for this apparently nonsensical evolutionary conservation. In addition to the differences between BR and BV-such as water solubility, antioxidant activity, and participation as a receptor ligand-in the present study, we focused on the chemistry of the two metabolites with regard to an electrophilic functional group called a Michael reaction acceptor (MRA). Our data reveal that the BR reacts with thiol compounds forming adducts, whereas no reaction occurs with BV. Furthermore, the binding of biotin-tagged BR to Kelch-like ECH-associated protein 1 (KEAP1)-a biological electrophile sensor-was prevented by pretreatment with BR or a thiol compound, but was not by pretreatment with BV. In cells, BR could bind to KEAP1 to release and activate nuclear factor-erythroid 2 (NF-E2) p45-related factor 2, a cytoprotective transcription factor, leading to the induction of HO-1. These findings may provide a physiological rationale for the energy-consuming conversion of BV to BR.

Aim Many intracellular components have been implicated in the regulation of redox homeostasis, but homeostasis can be unbalanced by reactive species (RS), which also probably contribute to underlying inflammatory processes. Nuclear factor-κB (NF-κB) is a well-known redox-sensitive transcription factor that controls the genes responsible for regulating inflammation. Methods In the present study, the authors investigated the effect of short-term salicylideneamino-2-thiophenol (SAL-2) administration on the modulation of pro-inflammatory NF-κB through redox regulation in aged rats. In addition, we compared the effects of SAL-2 and caloric restriction (CR) on inflammation and redox balance. The subjects were 24-month-old (old) Fischer 344 rats administered SAL-2 (10 mg/kg/day) by dietary supplementation or placed on a 30% restricted diet for 10 days, and 6-month-old (young) rats fed ad libitum for 10 days. Results We found that NF-κB activation and the expressions of its related genes (vascular cell adhesion molecule-1, intercellular adhesion molecule-1, cyclooxygenase-2 and inducible nitric oxide synthase) were suppressed by SAL-2 supplementation in old CR rats to the levels observed in young rats. In addition, our molecular studies showed that the inhibitory effect of SAL-2 on the activation of NF-κB was mediated by the ability of SAL-2 to block the nuclear translocations of cytosolic thioredoxin and redox factor-1. Conclusion These findings strongly indicate that SAL-2 stabilizes age-related redox imbalance and modulates the signal transduction pathway involved in the age-associated molecular inflammatory process. Geriatr Gerontol Int 2015; 15: 211–219.

Acute hepatic inflammation is regarded as a hallmark of early stage fibrosis, which can progress to extensive fibrosis and cirrhosis. Sinapic acid is a phenylpropanoid compound that is abundant in cereals, nuts, oil seeds, and berries and has been reported to exhibit a wide range of pharmacological properties. In this study, we investigated the anti-inflammatory effect of sinapic acid in carbon tetrachloride (CCl4)-induced acute hepatic injury in rats. Sinapic acid was administered orally (10 or 20 mg/kg) to rats at 30 min and 16 h before CCl4 intoxication. Sinapic acid treatment of rats reduced CCl4-induced abnormalities in liver histology, serum alanine transaminase and aspartate transaminase activities, and liver malondialdehyde levels. In addition, sinapic acid treatment significantly attenuated the CCl4-induced production of inflammatory mediators, including tumor necrosis factor-alpha and interleukin-1β mRNA levels, and increased the expression of nuclear factor-kappa B (NF-κB p65). Sinapic acid exhibited strong free radical scavenging activity in vitro. Thus, sinapic acid protected the rat liver from CCl4-induced inflammation, most likely by acting as a free radical scavenger and modulator of NF-κB p65 activation and proinflammatory cytokine expression. Sinapic acid may thus have potential as a therapeutic agent for suppressing hepatic inflammation.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells, including colon, prostate, breast and leukemia. Among them, aspirin, a classical NSAID, shows promise in cancer therapy in certain types of cancers. We hypothesized that aspirin might affect the growth of liver cancer cells since liver is the principal site for aspirin metabolism. Therefore, we investigated the effects of aspirin on the HepG2 human hepatocellular carcinoma cell line in vitro and the HepG2 cell xenograft model in BALB/c nude mice. We found that treatment with aspirin inhibited cell growth and induced apoptosis involving both extrinsic and intrinsic pathways as measured by DNA ladder formation, alteration in the Bax/Bcl-2 ratio, activation of the caspase activities and related protein expressions. In vivo antitumor activity assay also showed that aspirin resulted in significant tumor growth inhibition compared to the control. Oral administration of aspirin (100 mg/kg/day) caused a significant reduction in the growth of HepG2 tumors in nude mice. These findings suggest that aspirin may be used as a promising anticancer agent against liver cancer.

Hepatic stellate cells in the liver play a key role in the development of fibrosis (1). During chronic liver injury caused by alcohol, hepatitis virus, genetic disorders, among others, mediators are released that stimulate hepatic stellate cells to differentiate into myofibroblasts and stimulate them to proliferate and produce collagen, a key component of fibrosis (2). In addition to soluble mediators as important activators of hepatic stellate cells, alterations in the hepatic microenvironment may modulate hepatic stellate cell function. For example, alterations in matrix stiffness and changes in interactions between integrins on hepatic stellate cells and the extracellular matrix may affect the activation state of these cells (3, 4). In addition to these microenvironmental influences, exposure of hepatic stellate cells to hypoxia during chronic injury may be an important stimulus of hepatic stellate cell activation. Several studies have shown that regions of hypoxia develop in the liver during chronic injury (5–7). Furthermore, it has been demonstrated that hypoxia stimulates the release of a variety of mediators from hepatic stellate cells that may affect the progression of fibrosis. For example, hepatic stellate cells exposed to hypoxia synthesize and release the proangiogenic mediators, vascular endothelial growth factor (VEGF) and angiopoietin-1 (8, 9). Studies have demonstrated that angiogenesis may be important for liver fibrosis development, and hepatic stellate cells may be an important source of proangiogenic mediators (10). In addition, exposure of the human hepatic stellate cell line, LX-2 cells, to hypoxia increased levels of α-smooth muscle actin and type I collagen, suggesting that hypoxia also promotes activation of these cells (11). Similarly exposure of culture activated rat hepatic stellate cells to hypoxia stimulated synthesis of type I collagen (7). Although these studies have demonstrated that hypoxia affects hepatic stellate cell function, what remains unknown is the mechanism by which hypoxia modulates gene expression in these cells. One mechanism by which hypoxia modulates gene expression is by activating a number of transcription factors, including hypoxia-inducible factors (HIFs). HIFs are composed of an alpha subunit, typically HIF-1α or HIF-2α, and a beta subunit, HIF-1β also called the arylhydrocarbon receptor nuclear translocator (12, 13). In normoxic cells, HIFα subunits are constitutively produced and immediately targeted for proteolytic degradation. When cells become hypoxic, however, the mechanisms that target HIFα subunits for degradation are inhibited allowing HIFα protein levels to increase (14). HIFα subunits then translocate to the nucleus where they heterodimerize with HIF-1β and regulate expression of genes involved in glycolysis, angiogenesis, iron metabolism, pH control, and others functions. Studies have demonstrated that HIF-1α is activated in the transformed hepatic stellate cell line, LX-2 cells (11). Whether HIF-1α is activated in primary hepatic stellate cells and regulates expression of genes important for hepatic stellate cell function and fibrogenesis, however, has not been investigated. Accordingly, in the present studies the hypothesis was tested that HIF-1α is activated in hepatic stellate cells and regulate expression of genes important for hepatic stellate cell activation and liver fibrosis.

Resveratrol, a phytoalexin found in grapes and red wines, has been reported to exhibit a wide range of pharmacological properties. In this study, we investigated the protective effect of resveratrol on hepatic injury induced by dimethylnitrosamine (DMN) in rats. Oral administration of resveratrol (20 mg/kg daily for 4 weeks) remarkably prevented the DMN-induced loss in body and liver weight, and inhibited the elevation of serum alanine transaminase, aspartate transaminase, alkaline phosphatase and bilirubin levels. Resveratrol also increased serum albumin and hepatic glutathione levels and reduced the hepatic level of malondialdehyde due to its antioxidant effect. Furthermore, DMN-induced elevation of hydroxyproline content was reduced in the resveratrol treated rats, the result of which was consistent with the reduction in type I collagen mRNA expression and the histological analysis of liver tissue stained with Sirius red. The reduction in hepatic stellate cell activation, as assessed by alpha-smooth muscle actin staining, and the reduction in transforming growth factor-beta1 mRNA expression were associated with resveratrol treatment. In conclusion, resveratrol exhibited in vivo hepatoprotective and antifibrogenic effects against DMN-induced liver injury, suggesting that resveratrol may be useful in the prevention of the development of hepatic fibrosis.

Aim Liver fibrosis develops when chronic liver injury stimulates cells in the liver to produce mediators that activate hepatic stellate cells and stimulate them to secrete collagen. Recent studies suggest that the hypoxia-regulated transcription factor, hypoxia-inducible factor-1alpha, is essential for upregulation of profibrotic mediators, such as platelet-derived growth factor, in the liver during the development of liver fibrosis. What remains unknown, however, is the cell type-specific regulation of profibrotic mediators by hypoxia-inducible factors. Accordingly, in the present study the hypothesis tested was that hypoxia-inducible factors regulate production of profibrotic mediators by hypoxic Kupffer cells. Methods Kupffer cells were isolated from control mice and hypoxia-inducible factor-1beta-deficient mice and exposed to room air or 1% oxygen (i.e. hypoxia). Levels of profibrotic mediators were quantified by real-time polymerase chain reaction. Results Exposure of Kupffer cells isolated from control mice to 1% oxygen activated hypoxia-inducible factor-1alpha, and increased mRNA levels of platelet-derived growth factor-B, vascular endothelial growth factor, angiopoietin-1 and monocyte chemotactic protein-1. Upregulation of all of these mediators by hypoxia was prevented in Kupffer cells isolated from hypoxia-inducible factor-1beta-deficient mice. Conclusion RESULTS from these studies suggest that hypoxia-inducible factors are critical regulators of profibrotic mediator production by hypoxic Kupffer cells.

Cholestasis results when excretion of bile acids from the liver is interrupted. Liver injury occurs during cholestasis, and recent studies showed that inflammation is required for injury. Our previous studies demonstrated that early growth response factor-1 (Egr-1) is required for development of inflammation in liver during cholestasis, and that bile acids upregulate Egr-1 in hepatocytes. What remains unclear is the mechanism by which bile acids upregulate Egr-1. Bile acids modulate gene expression in hepatocytes by activating the farnesoid X receptor (FXR) and through activation of mitogen-activated protein kinase (MAPK) signaling. Accordingly, the hypothesis was tested that bile acids upregulate Egr-1 in hepatocytes by FXR and/or MAPK-dependent mechanisms. Deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) stimulated upregulation of Egr-1 to the same extent in hepatocytes isolated from wild-type mice and FXR knockout mice. Similarly, upregulation of Egr-1 in the livers of bile duct-ligated (BDL) wild-type and FXR knockout mice was not different. Upregulation of Egr-1 in hepatocytes by DCA and CDCA was prevented by the MEK inhibitors U0126 and SL-327. Furthermore, pretreatment of mice with U0126 prevented upregulation of Egr-1 in the liver after BDL. Results from these studies demonstrate that activation of MAPK signaling is required for upregulation of Egr-1 by bile acids in hepatocytes and for upregulation of Egr-1 in the liver during cholestasis. These studies suggest that inhibition of MAPK signaling may be a novel therapy to prevent upregulation of Egr-1 in liver during cholestasis.

Hypoxia-inducible factors (HIFs) are a group of transcription factors rapidly activated in hypoxic cells. Once activated, these transcription factors regulate expression of genes that allow cells to adapt to a hypoxic environment. HIFs are composed of an α subunit, either HIF-1α or HIF-2α, and a β subunit, HIF-1β (1, 2). HIF-1α and HIF-2α protein subunits are constitutively produced in cells. In normoxic cells, these subunits are immediately targeted for proteosomal degradation. In cells that are hypoxic, however, the mechanisms that target HIFs for degradation are inhibited, allowing HIF-1α and HIF-2α to translocate to the nucleus (3). In the nucleus both HIF-1α and HIF-2α heterodimerize with HIF-1β and regulate expression of genes involved in angiogenesis, iron homeostasis, glycolysis, and others (2). Although HIFs are essential for survival, several studies have implicated HIFs in the pathogenesis of several disease processes, including liver fibrosis. Liver fibrosis is characterized by excessive deposition of extracellular matrix in the liver during chronic injury. This disease has many causes including those of known etiology, such as chronic alcohol consumption, hepatitis virus, nonalcoholic steatohepatitis, and genetic disorders, and those of unknown etiology, such as primary biliary cirrhosis, sclerosing cholangitis, and others. Liver fibrosis is initiated when chronic liver injury stimulates numerous cells types, including hepatocytes, bile duct epithelial cells, Kupffer cells, and other inflammatory cells to produce protein and nonprotein mediators (e.g., growth factors, chemokines, reactive oxygen species). These mediators cause cells in the liver, such as hepatic stellate cells, peribiliary fibroblasts, hepatocytes, bile duct epithelial cells, and bone marrow-derived cells to differentiate into myofibroblasts (4). In addition, these mediators stimulate myofibroblast proliferation (5), and stimulate these cells to migrate to injured regions of the liver (i.e., chemotaxis) (6, 7). Once the myofibroblasts accumulate in these regions, they are stimulated to produce collagen and other components of extracellular matrix causing fibrosis. Recent studies have shown that HIF-1α is an important regulator of profibrotic mediator production in the liver during the development of fibrosis (8). In this study, regions of hypoxia developed in the livers of bile duct-ligated mice prior to the development of liver fibrosis (8). Furthermore, HIF-1α was activated in hepatocytes and Kupffer cells within these hypoxic regions. Mice deficient in HIF-1α developed less fibrosis after bile duct ligation, compared to control mice with normal levels of HIF-1α, indicating that HIF-1α is important for the development of liver fibrosis. The mechanism by which HIF-1α promoted fibrosis appeared to be related to HIF-1α-dependent regulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B, and plasminogen activator inhibitor-1 (PAI-1) (8). Although these studies demonstrated an important role for HIF-1α in regulating production of profibrotic mediators during the development of fibrosis, the cell type specific regulation of profibrotic genes by HIF-1α was not examined. Accordingly, in the present study, the hypothesis was tested that HIF-1α is activated in hypoxic hepatocytes and regulates expression of genes, such as PDGF-A, PDGF-B, and PAI-1 that promote fibrosis.

Liver fibrosis is characterized by excessive deposition of extracellular matrix in the liver during chronic injury. During early stages of this disease, cells begin to synthesize and secrete profibrotic proteins that stimulate matrix production and inhibit matrix degradation. Although it is clear that these proteins are important for development of fibrosis, what remains unknown is the mechanism by which chronic liver injury stimulates their production. In the present study, the hypothesis was tested that hypoxia-inducible factor-1alpha (HIF-1alpha) is activated in the liver during chronic injury and regulates expression of profibrotic proteins. To investigate this hypothesis, mice were subjected to bile duct ligation (BDL), an animal model of liver fibrosis. HIF-1alpha protein was increased in the livers of mice subjected to BDL by 3 days after surgery. To test the hypothesis that HIF-1alpha is required for the development of fibrosis, control and HIF-1alpha-deficient mice were subjected to BDL. Levels of type I collagen and alpha-smooth muscle actin mRNA and protein were increased in control mice by 14 days after BDL. These levels were significantly reduced in HIF-1alpha-deficient mice. Next, the levels of several profibrotic mediators were measured to elucidate the mechanism by which HIF-1alpha promotes liver fibrosis. Platelet-derived growth factor (PDGF)-A, PDGF-B, and plasminogen activator inhibitor-1 mRNA levels were increased to a greater extent in control mice subjected to BDL compared with HIF-1alpha-deficient mice at 7 and 14 days after BDL. Results from these studies suggest that HIF-1alpha is a critical regulator of profibrotic mediator production during the development of liver fibrosis.

Oxidative stress is thought to be a key risk factor in the development of hepatic diseases. Blocking or retarding the reactions of oxidation and the inflammatory process by antioxidants could be a promising therapeutic intervention for prevention or treatment of liver injuries. Oligonol is a low molecular weight polyphenol containing catechin-type monomers and oligomers derived from lychee fruit. In this study, we investigated the anti-inflammatory effect of oligonol on carbon tetrachloride- (CCl4-) induced acute hepatic injury in rats. Oral administration of oligonol (10 or 50 mg/kg) reduced CCl4-induced abnormalities in liver histology and serum AST and serum ALT levels. Oligonol treatment attenuated the CCl4-induced production of inflammatory mediators, including TNF-α, IL-1β, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) mRNA levels. Western blot analysis showed that oligonol suppressed proinflammatory nuclear factor-kappa B (NF-κB) p65 activation, phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) as well as Akt. Oligonol exhibited strong antioxidative activityin vitroandin vivo, and hepatoprotective activity againstt-butyl hydroperoxide-induced HepG2 cells. Taken together, oligonol showed antioxidative and anti-inflammatory effects in CCl4-intoxicated rats by inhibiting oxidative stress and NF-κB activation via blockade of the activation of upstream kinases including MAPKs and Akt.

Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. Nerve growth factor (NGF) is increasingly recognized as a potent immunomodulator, promoting "cross-talk" between various types of immune system cells. The present study describes the expression of NGF during thymus regeneration following acute involution induced by cyclophosphamide in the rat. Immunohistochemical stain demonstrated not only the presence of NGF but also its upregulated expression mainly in the subcapsular, paraseptal, and perivascular epithelial cells, and medullary epithelial cells including Hassall's corpuscles in both the normal and regenerating thymus. Biochemical data obtained using Western blot and RT-PCR supported these results and showed that thymic extracts contain NGF protein and mRNA, at higher levels during thymus regeneration. Thus, our results suggest that NGF expressed in these thymic epithelial cells plays a role in the T lymphopoiesis associated with thymus regeneration during recovery from acute thymic involution.

Hepatocyte injury during cholestasis depends in part on the release of proinflammatory mediators that cause neutrophils to accumulate in the liver and become activated to damage hepatocytes. The mechanism by which cholestasis stimulates production of proinflammatory mediators in the liver is not completely understood. The studies presented here tested the hypothesis that the transcription factor early growth response factor-1 (Egr-1) is required for inflammation to occur in the liver during cholestasis. The results of these studies show that Egr-1 was rapidly upregulated, primarily in hepatocytes, in mice subjected to bile duct ligation, an animal model of cholestasis. To determine whether Egr-1 was required for inflammation and hepatocyte injury during cholestasis, bile duct ligation was performed in wild-type and Egr-1 knockout mice. Hepatocyte injury, neutrophil accumulation, and upregulation of macrophage inflammatory protein-2 (MIP-2) and intercellular adhesion molecule-1 (ICAM-1) in the liver were significantly reduced in Egr-1 knockouts. By contrast, levels of tumor necrosis factor-alpha (TNF-alpha) and collagen (i.e., a biomarker of liver fibrosis) were not different between wild-types and Egr-1 knockouts subjected to bile duct ligation. Because hepatocytes are exposed to elevated concentrations of bile acids during cholestasis, it was determined that bile acids upregulate Egr-1 in primary mouse hepatocytes. Deoxycholic acid dose-dependently increased Egr-1 protein in hepatocytes. Results from these studies suggest a scenario in which elevated concentrations of bile acids during cholestasis increase expression of Egr-1 in hepatocytes. Egr-1 then upregulates proinflammatory mediators that cause neutrophils to accumulate in the liver and become activated to damage hepatocytes.

A sensor for cytochrome c (cyt c) was developed using a carbon paste electrode (CPE) modified with cellulose-DNA. Cyt c was adsorbed on the cellulose-DNA modified CPE through the electrostatic interaction between them. Owing to this process, a pair of well-defined peaks appeared at +48 mV/85 mV (Epc/Epa vs. Ag/AgCl). This property of the cellulose-DNA modified CPE was utilized for the analysis of cyt c in a biological sample. The optimum experimental conditions for analysis were investigated and a calibration plot was obtained between 1.0×10−4 M and 1.0×10−6 M (±5% at n=5) at the optimized condition. The detection limit for cyt c at the optimized experimental condition was 5.0×10−7 M (S/N=3) for 30 min of deposition time with differential pulse voltammetry (DPV). The real sample analysis was carried out with the standard addition method to evaluate the developed method. The content of cyt c in total proteins of 80.0 mg/mL in rat mitocondria fractions was determined to be 0.12 (±0.02) mg/mL.

Naringenin, a phytoalexin found in grapefruits and tomatoes, has been reported to exhibit a wide range of pharmacological properties. In this study, we investigated the protective effect of naringenin on hepatic injury induced by dimethylnitrosamine (DMN) in rats. Oral administration of naringenin (20 and 50 mg/kg daily for 4 weeks) remarkably prevented the DMN-induced loss in body and liver weights and inhibited the elevation of serum alanine transaminase, aspartate transaminase, alkaline phosphatase, and bilirubin levels. Naringenin also restored serum albumin and total protein levels, and reduced the hepatic level of malondialdehyde. Furthermore, DMN-induced collagen accumulation, as estimated by histological analysis of liver tissue stained with Sirius red, was reduced in the naringenin-treated rats. A reduction in hepatic stellate cell activation, as assessed by alpha-smooth muscle actin staining, was associated with naringenin treatment. In conclusion, these results demonstrate that naringenin exhibited in vivo hepatoprotective and anti-fibrogenic effects against DMN-induced liver injury. It suggests that naringenin may be useful in preventing the development of hepatic fibrosis.

The antioxidative effects of mulberroside A and oxyresveratrol obtained from Mori Cortex were examined. Mulberroside A and oxyresveratrol showed an inhibitory effect against FeSO4/H2O2-induced lipid peroxidation in rat microsomes and a scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radical. The anti-inflammatory effects of mulberroside A and oxyresveratrol using the carrageenin-induced model of inflammation were investigated in rats. Mulberroside A and oxyresveratrol significantly reduced paw edema. To investigate the mechanism of the anti-inflammatory action of these compounds, we examined the effects of oxyresveratrol on lipopolysaccharide (LPS)-induced responses in murine macrophage cell line RAW 264.7. Exposure of LPS-stimulated cells to oxyresveratrol inhibited nitrite accumulation in the culture medium. Oxyresveratrol also inhibited the LPS-stimulated increase of inducible nitric oxide synthase (iNOS) expression in a concentration-dependent manner; however, it had little effect on iNOS enzyme activity, suggesting that the inhibitory activity of oxyresveratrol is mainly due to the inhibition of iNOS expression rather than iNOS enzyme activity. Oxyresveratrol significantly inhibited LPS-evoked nuclear translocation of NF-kB and cyclooxygenase-2 (COX-2) activity in RAW 264.7 cells. The results suggest that the anti-inflammatory properties of oxyresveratrol might be correlated with inhibition of the iNOS expression through down-regulation of NF-kB binding activity and significant inhibition of COX-2 activity.

Quercetin, one of the most abundant flavonoids in human diet has been reported to exhibit a wide range of pharmacological properties. In this study, we investigated the protective effect of quercetin on hepatic injury induced by dimethylnitrosamine (DMN) in rats. Treatment with DMN caused a significant decrease in body and liver weight. Oral administration of quercetin (10 mg kg−1 daily for 4 weeks) remarkably prevented this DMN-induced loss in body and liver weight and inhibited the elevation of serum alanine transaminase, aspartate transaminase and bilirubin levels. Quercetin also increased serum albumin and hepatic glutathione levels and reduced the hepatic level of malondialdehyde. Furthermore, DMN-induced elevation of hydroxyproline content was reduced in the quercetin treated rats, the result of which was consistent with a reduction in type I collagen mRNA production and histological analysis of liver tissue stained with Sirius red. A reduction in hepatic stellate cell activation, as assessed by α-smooth muscle actin staining, was associated with quercetin treatment as well as a reduction in transforming growth factor-β1 expression. In conclusion, these results demonstrate that quercetin exhibited in-vivo hepatoprotective and anti-fibrogenic effects against DMN-induced liver injury and suggest that quercetin may be useful in the preventing the development of hepatic fibrosis.

Two new isoflavone glycosides together with glycitein, tectoridin and glycitin,were isolated from the flowers of Pueraria thunbergiana . These structures were determined as4′,7-dihydroxy-6-methoxyisoflavone 7- O - β - d -xylopyranosyl- (1→6)- β - d -glucopyranoside and4′,5,7-trihydroxy-6-methoxyisoflavone 7- O - β - d -xylopyranosyl- (1→6)- β - d -glucopyranoside.

Fe(II)-tetrakis-N,N,N',N'(2-pyridylmethyl) ethylenediamine (Fe-TPEN) catalyzes the dismutation of superoxide, and blocks the toxic effect of paraquat on Escherichia coli growth and survival. We examined antioxidative effects of Fe-TPEN on lipid peroxidation and t-butyl hydroperoxide induced cell damage. Fe-TPEN inhibited the FeSO4/H2O2 induced lipid peroxidation in the rat liver homogenates with an IC50 value of 30.2 microM, and protected Ac2F cell damage by t-butyl hydroperoxide in a dose-dependent manner (EC50 value is 2.6 microM). Also, hepatoprotective effect of Fe-TPEN (5 mg/kg, i.p.) was investigated using CCl4 induced liver injury in rats. This complex inhibited the elevation of serum alanine aminotransferase (AST) and aspartate aminotransferase (ALT) levels in CCl4 induced liver injuries, and improved submassive necrosis and fatty degeneration of the hepatocytes. Fe-TPEN also prevented the loss of total and nonprotein SH contents, glutathione peroxidase and glutathione-S-transferase activity in cytosol of rat liver. Although the exact mechanism of action is not clear, antioxidative properties as well as attenuation of hepatocellular defense systems by Fe-TPEN seem to be important on its potent hepatoprotective effect in CCl4-intoxicated rat.

Many intracellular components have been implicated in the regulation of redox homeostasis, but homeostasis can be unbalanced by reactive species (RS), which also probably contribute to underlying inflammatory processes. Nuclear factor-κB (NF-κB) is a well-known redox-sensitive transcription factor that controls the genes responsible for regulating inflammation.In the present study, the authors investigated the effect of short-term salicylideneamino-2-thiophenol (SAL-2) administration on the modulation of pro-inflammatory NF-κB through redox regulation in aged rats. In addition, we compared the effects of SAL-2 and caloric restriction (CR) on inflammation and redox balance. The subjects were 24-month-old (old) Fischer 344 rats administered SAL-2 (10 mg/kg/day) by dietary supplementation or placed on a 30% restricted diet for 10 days, and 6-month-old (young) rats fed ad libitum for 10 days.We found that NF-κB activation and the expressions of its related genes (vascular cell adhesion molecule-1, intercellular adhesion molecule-1, cyclooxygenase-2 and inducible nitric oxide synthase) were suppressed by SAL-2 supplementation in old CR rats to the levels observed in young rats. In addition, our molecular studies showed that the inhibitory effect of SAL-2 on the activation of NF-κB was mediated by the ability of SAL-2 to block the nuclear translocations of cytosolic thioredoxin and redox factor-1.These findings strongly indicate that SAL-2 stabilizes age-related redox imbalance and modulates the signal transduction pathway involved in the age-associated molecular inflammatory process.

Miniaturization and stabilization of a sensing device has been essential in the development of implantable and in vivo electrochemical sensors. Smaller electrodes provide improved spatial and temporal resolution as well as reduced tissue damage [1]. To achieve long-time stability and miniaturization, we tried to make a thin film dual sensor combined of two screen printed electrodes to form a microfluidic channel. To examine the performance of the microfluidic duel senor (MDS), we tested it to simultaneously monitor glutamate and superoxide anion (O2 •-) as a model case. Glutamate, which is a major excitatory neurotransmitter in the central nervous system and highly expressed in the mammalian body and brain [2-3].O2 •-is the most intracellular reactive oxygen species which is the primary oxygen free radical generated through the electron transport chain in mitochondria. In the present study, O2 •- was monitored with a sensor using co-immobilization of a lipid (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n-dodecanylamine, DGPD) and cytochrome C-copper nanoflowers (Cyt C-CuNFs) onto a conducting polymer (CP) layer through covalent bonding (SPCE/PtNPs/DGPD/pTTBA/Cyt C-CuNFs/PEI). Otherwise, the glutamate sensor was prepared with a glutamate oxidase attached-CP layer coated on Pt nanoparticles (PtNPs) (SPCE/PtNPs/nanoCP-GlOx), where they were prepared and used as a catalyst to oxidize H2O2 generated by the enzyme reaction. Where, to immobilize the Cyt C-CuNFs and glutamate oxidase, separately, poly-[2,2′:5′,2″-terthiophene-3′-(p-benzoic acid)] (pTTBA) was formed electrochemically on the catalytic electrode surface. Male Sprague-Dawley rats (150.0 - 180.0 g) were obtained from Samtako (Osan, Korea). Animals were provided standard rat chow with free access to tap water and were maintained at a controlled temperature (23 ± 3 oC) and humidity (50 ± 20%) with a 12 h light-dark cycle. With respect to ethical issues and scientific care, the animal protocol used in this study was reviewed and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval number PNU 2008-0541). The sensor was placed in rat liver for the measurements (inset of Figure 1(A)). The performance of sensing device was evaluated before and after in vivo experiments, and chronoamperograms were recorded for each of the sensor at different applied potentials, simultaneously. For O2 •- detection, the applied potential was -0.40 V for monitoring the reduction current of H2O2 at the SPCE/PtNPs/DGPD/pTTBA/Cyt C-CuNFs/PEI electrode, whereas, glutamate was detected through the monitoring of oxidation current of H2O2 at a SPCE/ PtNPs/CP-GlOx micro film electrode at the applied potential of +0.45 V. Figure 1(A) shows the typical current-time plots that were obtained for O2 •- in a 0.1 M phosphate buffer solution (PBS, pH 7.0) and calibration plots for (i) before and (ii) after an in vivo experiment. Under the optimized conditions, the calibration plot showed a linear relationship with the O2 •- concentration in the range of 0.2 - 7.0 nM for before and after in vivo experiments. The relative standard deviations of O2 •- concentrations were found to be 2.5 and 4.1%, before and after an in vivo experiment. The detection limit of O2 •- was determined to be 25.0 ± 0.5 pM by SPCE/PtNPs/DGPD/pTTBA/Cyt C-CuNFs/PEI modified electrode. The amperometric response of the SPCE/PtNPs/nanoCP-GlOx sensor was carried out using various concentrations of glutamate. Figure 1(B) shows typical current-time plots for the addition of glutamate in a 0.1 M PBS (i) before and after (ii) in vivo experiments. Under the optimized conditions, the steady state currents showed a linear relationship with the glutamate concentrations in the range of 10.0 nM - 150.0 μM and 150.0 μM - 15.0 mM. The relative standard deviations at 1.0 μM glutamate concentration were found to be 2.2 and 3.6%. The detection limit of glutamate was determined to be 2.5 ± 0.5 nM by SPCE/PtNPs/nanoCP-GlOx sensor based on five-times measurement for the standard deviation of the blank noise (95% confidence level, k = 3, n = 5). [1] Noh, H.-B.; Shim, Y.-B.; et al., Chem. Comm ., 2015, 51, 6659-6662. [2] Rahman, Md.A.; Shim, Y.-B.; et al., Anal. Chem ., 2005, 77, 4854-4860. [3] Rahman, Md.A.; Shim, Y.-B.; et al., Anal. Chem ., 2012, 84, 6654-6660. Figure 1

Sinapic acid is a member of the phenylpropanoid family and is abundant in cereals, nuts, oil seeds, and berries. It exhibits a wide range of pharmacological properties. In this study, we investigated the hepatoprotective and antifibrotic effects of sinapic acid on dimethylnitrosamine (DMN)-induced chronic liver injury in rats. Sinapic acid remarkably prevented DMN-induced loss of body weight. This was accompanied by a significant increase in levels of serum alanine transaminase, aspartate transaminase, and liver malondialdehyde content. Furthermore, sinapic acid reduced hepatic hydroxyproline content, which correlated with a reduction in the expression of type I collagen mRNA and histological analysis of collagen in liver tissue. Additionally, the expression of hepatic fibrosis-related factors such as α-smooth muscle actin and transforming growth factor-β1 (TGF-β1), were reduced in rats treated with sinapic acid. Sinapic acid exhibited strong scavenging activity. In conclusion, we find that sinapic acid exhibits hepatoprotective and antifibrotic effects against DMN-induced liver injury, most likely due to its antioxidant activities of scavenging radicals, its capacity to suppress TGF-β1 and its ability to attenuate activation of hepatic stellate cells. This suggests that sinapic acid is a potentially useful agent for the protection against liver fibrosis and cirrhosis.

Understanding the mechanisms of thymus regeneration is necessary for designing strategies to enhance host immunity when immune function is suppressed due to T cell depletion. In this study, expressed sequence tag (EST) analysis was performed following generation of a regenerating thymus cDNA library to identify genes expressed in thymus regeneration. A total of 1,000 ESTs were analyzed, of which 770 (77%) matched to known genes, 178 matched to unknown genes (17.8%) and 52 (5.2%) did not match any known sequences. The ESTs matched to known genes were grouped into eight functional categories: gene/protein synthesis (28%), metabolism (24%), cell signaling and communication (17%), cell structure and motility (6%), cell/organism defense and homeostasis (6%), cell division (3%), cell death/apoptosis (2%), and unclassified genes (14%). Based on the data of RT-PCR analysis, the expression of TLP, E2IG2, pincher, Paip2, TGF-β1, 4-1BB and laminin α3 genes was increased during thymus regeneration. These results provide extensive molecular information, for the first time, on thymus regeneration indicating that the regenerating thymus cDNA library may be a useful source for identifying various genes expressed during thymus regeneration.

Combined therapy with multiple drugs is a common practice in the treatment of cancer, which can achieve better therapeutic effects than a single drug, and can reduce the side effects as well as drug resistance. This study aimed to determine whether aspirin (ASA) shows synergism with doxorubicin (DOX) in HepG2 human hepatocellular carcinoma cells in vitro and in a HepG2 cell xenograft model in BALB/c nude mice. When treated in combination, DOX (0.25 nmol/ml) and ASA (5 µmol/ml) produced strong synergy in growth inhibition, cell cycle arrest and importantly, apoptosis in vitro in comparison to single treatments. Moreover, ASA (100 mg/kg/day orally) and DOX (1.2 mg/kg biweekly ip) induced synergistic antitumor activity in the HepG2 cell xenograft model in nude mice. Therefore, the combination of ASA and DOX could be used as a novel combination regimen which provides a strong anticancer synergy in the treatment of hepatocellular carcinoma.

A highly sensitive in vivo biosensor for glutathione disulfide (GSSG) is developed using covalently immobilized-glutathione reductase (GR) and -β-nicotinamide adenine dinucleotide phosphate (NADPH) on gold nanoparticles deposited on poly[2,2':5',2″-terthiophene-3'-(p-benzoic acid)] (polyTTBA). The fabricated biosensor was characterized with SEM, TEM, XPS, and QCM. Analytical parameters affecting the biosensor performance were optimized in terms of applied potential, NADPH:GR ratio, temperature, and pH. A linear calibration plot is obtained using chronoamperometry in the dynamic range between 0.1 μM and 2.5 mM of GSSG, with a detection limit of 12.5 ± 0.5 nM. The developed biosensor is applied to detect GSSG in a real plasma sample. A microbiosensor was applied to detect the in vivo GSSG concentration to monitor the oxidative stress caused by diquat and t-butyl hydroperoxide. The results obtained are reliable, implying a promising approach for a GSSG biosensor in clinical diagnostics and oxidative stress monitoring.

Several studies have shown that regions of hypoxia develop in the liver during chronic injury. Furthermore, it has been demonstrated that hypoxia stimulates the release of mediators from hepatic stellate cells (HSCs) that may affect the progression of fibrosis. The mechanism by which hypoxia modulates gene expression in HSCs is not known. Recent studies demonstrated that the hypoxia-activated transcription factor, hypoxia-inducible factor (HIF)-1α, is critical for the development of fibrosis. Accordingly, the hypothesis was tested that HIF-1α is activated in HSCs and regulates the expression of genes important for HSC activation and liver fibrosis.Hepatic stellate cells were isolated from mice and exposed to hypoxia. HIF-1α and HIF-2α activation were measured, and gene expression was analysed by gene array analysis. To identify the genes regulated by HIF-1α, HSCs were isolated from control and HIF-1α-deficient mice.Exposure of primary mouse HSCs to 0.5% oxygen activated HIF-1α and HIF-2α. mRNA levels of numerous genes were increased in HSCs exposed to 0.5% oxygen, many of which are important for HSC function, angiogenesis and collagen synthesis. Of the mRNAs increased, chemokine receptor (Ccr) 1, Ccr5, macrophage migration inhibitory factor, interleukin-13 receptor α1 and prolyl-4-hydroxylase α2 (P4h α2) were completely HIF-1α dependent. Upregulation of the vascular endothelial growth factor and the placental growth factor was partially HIF-1α dependent and upregulation of angiopoietin-like 4 and P4h α1 was HIF-1α independent.Results from these studies demonstrate that hypoxia, through activation of HIF-1α, regulates the expression of genes that may alter the sensitivity of HSCs to certain activators and chemotaxins, and regulates the expression of genes important for angiogenesis and collagen synthesis.

Liver fibrosis develops when chronic liver injury stimulates cells in the liver to produce mediators that activate hepatic stellate cells and stimulate them to secrete collagen. Recent studies suggest that the hypoxia-regulated transcription factor, hypoxia-inducible factor-1alpha, is essential for upregulation of profibrotic mediators, such as platelet-derived growth factor, in the liver during the development of liver fibrosis. What remains unknown, however, is the cell type-specific regulation of profibrotic mediators by hypoxia-inducible factors. Accordingly, in the present study the hypothesis tested was that hypoxia-inducible factors regulate production of profibrotic mediators by hypoxic Kupffer cells.Kupffer cells were isolated from control mice and hypoxia-inducible factor-1beta-deficient mice and exposed to room air or 1% oxygen (i.e. hypoxia). Levels of profibrotic mediators were quantified by real-time polymerase chain reaction.Exposure of Kupffer cells isolated from control mice to 1% oxygen activated hypoxia-inducible factor-1alpha, and increased mRNA levels of platelet-derived growth factor-B, vascular endothelial growth factor, angiopoietin-1 and monocyte chemotactic protein-1. Upregulation of all of these mediators by hypoxia was prevented in Kupffer cells isolated from hypoxia-inducible factor-1beta-deficient mice.RESULTS from these studies suggest that hypoxia-inducible factors are critical regulators of profibrotic mediator production by hypoxic Kupffer cells.

Proanthocyanidins are naturally occurring compounds widely available in fruits, vegetables, nuts and seeds. They are a class of phenolic compounds and have been reported to exhibit a wide range of biological effects. In this study, we investigated the protective effect of grape seed proanthocyanidins on hepatic injury induced by dimethylnitrosamine (DMN) in rats. Treatment with DMN caused a significant increase in levels of serum alanine transaminase, aspartate transaminase, alkaline phosphatase, and bilirubin. Oral administration of proanthocyanidins (20 mg/kg daily for 4 weeks) remarkably prevented these elevations. Proanthocyanidins also restored serum albumin and total protein levels, and reduced the hepatic level of malondialdehyde. Furthermore, DMN-induced collagen accumulation, as estimated by histological analysis of liver tissue stained with Sirius red, was reduced in the proanthocyanidins-treated rats. A reduction in hepatic stellate cell activation, as assessed by alpha-smooth muscle actin staining, was associated with proanthocyanidins treatment. In conclusion, these results demonstrate that proanthocyanidins exhibited in vivo hepatoprotective and anti-fibrogenic effects against DMN-induced liver injury. It suggests that grape seed proanthocyanidins may be useful in preventing the development of hepatic fibrosis.

In many clinical situations which cause thymic involution and thereby result in immune deficiency, T cells are the most often affected, leading to a prolonged deficiency of T cells. Since only the thymic-dependent T cell production pathway secures stable regeneration of fully mature T cells, seeking strategies to enhance thymic regeneration should be a key step in developing therapeutic methods for the treatment of these significant clinical problems. This study clearly shows that receptor activator of NF-kappaB ligand (RANKL) stimulates mouse thymic epithelial cell activities including cell proliferation, thymocyte adhesion to thymic epithelial cells, and the expression of cell death regulatory genes favoring cell survival, cell adhesion molecules such as ICAM-1 and VCAM-1, and thymopoietic factors including IL-7. Importantly, RANKL exhibited a significant capability to facilitate thymic regeneration in mice. In addition, this study demonstrates that RANKL acts directly on the thymus to activate thymus regeneration regardless of its potential influences on thymic regeneration through an indirect or systemic effect. In light of this, the present study provides a greater insight into the development of novel therapeutic strategies for effective thymus repopulation using RANKL in the design of therapies for many clinical conditions in which immune reconstitution is required.

Thymic epithelial cells, which constitute a major component of the thymic microenvironment, provide a crucial signal for intrathymic T cell development and selection. Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. NGF is increasingly recognized as a potent immunomodulator, promoting "cross-talk" between various types of immune system cells. The present study clearly shows that NGF stimulates mouse thymic epithelial cell activities in vitro including cell proliferation, thymocyte adhesion to thymic epithelial cells, and the expression of cell adhesion molecules such as ICAM-1 and VCAM-1, and thymopoietic factors including IL-7, GM-CSF, SDF-1, TARC and TECK. Thus, our data are of considerable clinical importance showing that trophic NGF activity could be used to enhance the thymus regeneration and develop methods to improve host immunity when the immune function is depressed due to thymic involution.

Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, is refractory to most anticancer drugs. Doxorubicin offers a survival benefit to patients with intermediate hepatocellular carcinoma. However, the favorable therapeutic response to doxorubicin is often associated with severe toxicity. The present research was aimed at developing a strategy of increasing doxorubicin sensitivity so that lower doses may be used without compromising efficacy. Recently, aspirin (ASA) and some other nonsteroidal anti-inflammatory drugs (NSAIDs) have drawn much attention for their protective effects against colon cancer and cardiovascular disease. Several studies also found that regular ASA use is significantly associated with reduced risk of lung cancer. We investigated the effects of ASA on HepG2 human hepatocellular carcinoma cell line. Growth inhibition effect of aspirin was measured by MTT assay. Flow cytometry analysis revealed that aspirin can induce G2/M phase arrest which we observed by the level of cell cycle regulatory proteins and DNA ladder formation indicates apoptosis dose dependently. We found that ASA treatment followed both extrinsic and intrinsic pathways for apoptosis induction. In addition to this, when administered in combination, doxorubicin (DOX) and ASA produced synergistic effect in growth inhibition in human hepatocellular carcinoma cells and induced apoptosis in caspase-dependent pathway. Therefore, clinically achievable concentrations of DOX and ASA used in combination may produce a strong anticancer synergy that warrants investigation as a therapeutic strategy to enhance the efficacy of doxorubicin for treating HCC. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R01-2006-000-11117-0)]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 226.

Grape is one of the most popular and widely cultivated fruits in the world. Although grape skin and seeds are waste product of the winery and grape juice industry, these wastes contain large amounts of phytochemicals such as flavonoids, phenolic acids, and anthocyanidins, which play an important role as chemopreventive and anticancer agents. We evaluated efficacies of grape skin and seeds on hepatic injury induced by dimethylnitrosamine (DMN) in rats. Treatment with DMN significantly increased levels of serum alanine transaminase, aspartate transaminase, alkaline phosphatase, and bilirubin. Diet supplementation with grape skin or seeds (10% daily for 4 weeks) prevented these elevations. The grape skin and seeds also restored serum albumin and total protein levels, and reduced the hepatic level of hydroxyproline and malondialdehyde. Furthermore, grape skin and seeds reduced DMN-induced collagen accumulation, as estimated by histological analysis of liver tissue stained with Sirius red. Grape skin and seeds also reduced hepatic stellate cell activation, as assessed by α-smooth muscle actin staining. In conclusion, grape skin and seeds exhibited in vivo hepatoprotective and antifibrogenic effects against DMN-induced liver injury, suggesting that grape skin and seeds may be useful in preventing the development of hepatic fibrosis.