Integrative Transcriptomic Analysis Reveals Upregulated Apoptotic Signaling in Wound-Healing Pathway in Rat Liver Fibrosis Models.

Liver fibrosis, defined by the aberrant accumulation of extracellular matrix proteins in liver tissue due to chronic inflammation, represents a pressing global health issue. In this study, we investigated the transcriptomic signatures of three independent liver fibrosis models induced by bile duct ligation, carbon tetrachloride, and dimethylnitrosamine (DMN) to unravel the pathological mechanisms underlying hepatic fibrosis. We observed significant changes in gene expression linked to key characteristics of liver fibrosis, with a distinctive correlation to the burn-wound-healing pathway. Building on these transcriptomic insights, we further probed the p53 signaling pathways within the DMN-induced rat liver fibrosis model, utilizing western blot analysis. We observed a pronounced elevation in p53 protein levels and heightened ratios of BAX/BCL2, cleaved/pro-CASPASE-3, and cleaved/full length-PARP in the livers of DMN-exposed rats. Furthermore, we discovered that orally administering oligonol-a polyphenol, derived from lychee, with anti-oxidative properties-effectively countered the overexpressions of pivotal apoptotic genes within these fibrotic models. In conclusion, our findings offer an in-depth understanding of the molecular alterations contributing to liver fibrosis, spotlighting the essential role of the apoptosis pathway tied to the burn-wound-healing process. Most importantly, our research proposes that regulating this pathway, specifically the balance of apoptosis, could serve as a potential therapeutic approach for treating liver fibrosis.

Kaempferol Suppresses Carbon Tetrachloride-Induced Liver Damage in Rats via the MAPKs/NF-κB and AMPK/Nrf2 Signaling Pathways.

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-1ß, 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.

Protective Effect of Oligonol on Dimethylnitrosamine-Induced Liver Fibrosis in Rats via the JNK/NF-κB and PI3K/Akt/Nrf2 Signaling Pathways.

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-1ß, 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-ß1, 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.

Is it worth expending energy to convert biliverdin into bilirubin?

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.

Oligonol Ameliorates CCl4-Induced Liver Injury in Rats via the NF-Kappa B and MAPK Signaling Pathways.

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-1ß, 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 activity in vitro and in vivo, and hepatoprotective activity against t-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.

Hepatoprotective effects of zingerone on carbon tetrachloride- and dimethylnitrosamine-induced liver injuries in rats.

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-1ß, 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.

Salicylideneamino-2-thiophenol modulates nuclear factor-κB through redox regulation during the aging process.

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.

Effect of sinapic acid against carbon tetrachloride-induced acute hepatic injury in rats.

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-1ß 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.

Effect of sinapic acid against dimethylnitrosamine-induced hepatic fibrosis in rats.

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-ß1 (TGF-ß1), 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-ß1 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.

Identification and analysis of expressed genes using a cDNA library from rat thymus during regeneration following cyclophosphamide-induced T cell depletion.

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-ß1, 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.

Aspirin enhances doxorubicin-induced apoptosis and reduces tumor growth in human hepatocellular carcinoma cells in vitro and in vivo.

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.

In vivo detection of glutathione disulfide and oxidative stress monitoring using a biosensor.

A highly sensitive in vivo biosensor for glutathione disulfide (GSSG) is developed using covalently immobilized-glutathione reductase (GR) and -ß-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.

Aspirin induces apoptosis in vitro and inhibits tumor growth of human hepatocellular carcinoma cells in a nude mouse xenograft model.

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.

Hypoxia-inducible factor-1α regulates the expression of genes in hypoxic hepatic stellate cells important for collagen deposition and angiogenesis.

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Effect of dietary supplementation of grape skin and seeds on liver fibrosis induced by dimethylnitrosamine in rats.

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.

Resveratrol inhibits dimethylnitrosamine-induced hepatic fibrosis in rats.

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.

Hypoxia-inducible factor-dependent production of profibrotic mediators by hypoxic Kupffer cells.

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.

The proanthocyanidins inhibit dimethylnitrosamine-induced liver damage in rats.

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.

Upregulation of early growth response factor-1 by bile acids requires mitogen-activated protein kinase signaling.

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 factor-dependent production of profibrotic mediators by hypoxic hepatocytes.

BACKGROUND/AIMS: During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B and plasminogen activator inhibitor-1 (PAI-1) in the liver, during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1alpha in liver cell types. Accordingly, the hypothesis was tested that HIF-1alpha is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells. METHODS: In this study, hepatocytes were isolated from the livers of control and HIF-1alpha- or HIF-1beta-deficient mice and exposed to hypoxia. RESULTS: Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1alpha and upregulated PAI-1, vascular endothelial cell growth factor and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, the levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1alpha-deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2alpha, may also regulate these genes. In support of this, HIF-2alpha was activated in hypoxic hepatocytes, and exposure of HIF-1beta-deficient hepatocytes to 1% oxygen completely prevented upregulation of PAI-1, vascular endothelial cell growth factor and ADM-1, suggesting that HIF-2alpha may also contribute to upregulation of these genes in hypoxic hepatocytes. CONCLUSIONS: Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.