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Ethanol administration is capable of inhibiting or delaying the partial hepatectomy (PH)-induced liver regeneration, probably altering liver metabolism by means of its oxidative metabolism. Since the regenerating liver has increased capacity for oxidizing ethanol, the present study was aimed to address the contribution of the ethanol-oxidizing metabolic pathways in the regenerating liver cells. Isolated hepatocytes were prepared from control livers and from animals subjected to two-thirds PH. In both preparations, ethanol oxidation was largely increased by incubation with glucose and was highly sensitive to inhibitors of ethanol-oxidizing enzymatic pathways (alcohol dehydrogenase, catalase and cytochrome P-4502E1 activities). The latter led to a total blockade of ethanol disposal by control hepatocytes, while liver cells from PH-rats only showed an early 70-75% inhibition of ethanol catabolism with the inhibitors used. In regenerating hepatocytes, the enhanced ethanol oxidation was blocked by scavengers of reactive oxygen species, an effect that correlated with enhanced cytoplasmic lipid peroxidation by-products. Both cell preparations showed similar sensitivity to inhibitors for the malate-aspartate shuttle and mitochondrial electron transport chain; the shift of the cytoplasmic redox state was also quite similar after ethanol oxidation. A more oxidized mitochondrial redox state was found in hepatocytes from PH-rats and more shifted to the reduced state during ethanol oxidation this effect was not abolished by inhibiting alcohol dehydrogenase activity. In conclusion, data clearly show that an important fraction of ethanol is metabolized through a non-enzymatic-mediated oxidative event, which could largely contribute to the deleterious effect of ethanol on the proliferating liver., (Copyright © 2019. Published by Elsevier Inc.)

Liver regeneration is a vital process of recovery after liver damage, which is a promising clinical strategy after partial hepatectomy (PHx). Schisandrol B (SolB), one of the bioactive ingredients from Schisandra sphenanthera, displays significant hepato-protection effects against drug-induced liver injure in mice. However, the effect of SolB on liver regeneration after PHx remains unclear. Here, we showed that SolB treatment promoted liver mass restoration and increased the number of proliferative hepatocytes following PHx. SolB treatment significantly improved the levels of growth factors (HGF and EGF) and cytokines (IL-6), which further activated STAT3/Akt/MAPK signaling pathways and induced the expression of several the protein of cell cycle core. Overall, this study is the first to demonstrate the role of SolB in promoting liver regeneration during PHx challenge, which provide a clinically relevant argument for using SolB to facilitate liver recovery after undergoing PHx or liver transplantation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Liver resection has become a common treatment for liver tumors and hepatocellular carcinoma over the past decades. However, after surgery, the remnant livers in some patients fail to regenerate. Therefore, there is an urgent medical need to develop drugs that can promote liver regeneration. The purpose of the current study is to investigate the promotive effect of alisol B 23-acetate (AB23A) on liver regeneration in mice following partial hepatectomy (PH), and further elucidate the involvement of farnesoid X receptor (FXR) in the liver regeneration-promotive effect using in vivo and in vitro experiments. The results showed that AB23A dose-dependently promoted hepatocyte proliferation via upregulating hepatocyte proliferation-related protein forkhead box M1b (FoxM1b), Cyclin D1 and Cyclin B1 expression, and attenuated liver injury via an inhibition in Cyp7a1 and an induction in efflux transporters Bsep expression resulting in reduced hepatic bile acid levels. These changes in the genes, as well as accelerated liver regeneration in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly showed the regulation of these genes by AB23A was abrogated when FXR was silenced. Luciferase reporter assay in HepG2 cells and molecular docking further demonstrated the effect of AB23A on FXR activation in vitro. In conclusions, AB23A produces promotive effect on liver regeneration, due to FXR-mediated regulation of genes involved in hepatocyte proliferation and hepato-protection. AB23A has the potential to be a novel therapeutic option for facilitating efficient liver regeneration in patients subjected to liver resection., (Copyright © 2014 Elsevier Inc. All rights reserved.)

All-trans retinoic acid (RA) is a potent inducer of regeneration. Because the liver is the principal site for storage and bioactivation of vitamin A, the current study examines the effect of RA in mouse hepatocyte proliferation and liver regeneration. Mice that received a single dose of RA (25μg/g) by oral gavage developed hepatomegaly with increased number of Ki67-positive cells and induced expression of cell cycle genes in the liver. DNA binding data revealed that RA receptors retinoic acid receptor β (RARβ) and retinoid x receptor α (RXRα) bound to cell cycle genes Cdk1, Cdk2, Cyclin B, Cyclin E, and Cdc25a in mice with and without RA treatment. In addition, RA treatment induced novel binding of RARβ/RXRα to Cdk1, Cdk2, Cyclin D, and Cdk6 genes. All RARβ/RXRα binding sites contained AGGTCA-like motifs. RA treatment also promoted liver regeneration after partial hepatectomy (PH). RA signaling was implicated in normal liver regeneration as the mRNA levels of RARβ, Aldh1a2, Crabp1, and Crbp1 were all induced 1.5 days after PH during the active phase of hepatocyte proliferation. RA treatment prior to PH resulted in early up-regulation of RARβ, Aldh1a2, Crabp1, and Crbp1, which was accompanied by an early induction of cell cycle genes. Western blotting for RARβ, c-myc, Cyclin D, E, and A further supported the early induction of retinoid signal and cell proliferation by RA treatment. Taken together, our data suggest that RA may regulate cell cycle progression and accelerates liver regeneration. Such effect is associated with an early induction of RA signaling, which includes increased expression of the receptor, binding proteins, and processing enzyme for retinoids., (Published by Elsevier Inc.)

Selenium in supra-nutritional doses is tumour-preventative in animal models and in humans. In this work, we have compared the effect of sodium selenite on tumour growth in a rat hepatocarcinogenesis model with the effect of sodium selenite on the regeneration of liver mass after partial hepatectomy. In the tumour model, 5 μg/mL sodium selenite in the drinking water reduced the rate of tumour growth for up to 12 months after initiation; the volume fraction of liver cancers was 14±4% with a mean bromodeoxyuridine-index of 19±11% in the treated rats compared to a volume fraction of 26±7% with a mean bromodeoxyuridine-index of 42±27% in the control rats. Despite its efficacy in reducing tumour growth, 5 μg/mL sodium selenite treatment did not affect the gain of liver mass or the rate of cell proliferation after partial hepatectomy. In the regenerating livers, the activity of the cytosolic selenoenzyme thioredoxin reductase (TrxR1) was briefly and transiently increased, an increase further potentiated by sodium selenite. TrxR1 was selectively over expressed in proliferating liver tumours in relation to the surrounding liver tissue in the tumour model, as shown using immunohistochemistry analyses. We suggest that sodium selenite is a suitable candidate for liver cancer prevention in patients with chronic liver diseases that are dependent on sustained liver regeneration due to its differential effects on neoplastic and regenerative cell proliferation. Furthermore, the over expression of TrxR1 in liver neoplasia can only partly be explained by increased growth., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The human placenta is an organ for fetus development and abundant reservoir of various bioactive molecules. Interest to human placenta extract (hPE) is growing, and application with trial of hPE is widening in oriental medicine including in liver diseases. However, underlying mechanisms for therapeutic effects are still unclear. Here, we investigated therapeutic effects of hPE in carbon tetrachloride (CCl(4))-injured rat liver model in vivo and in damaged rat hepatic cells exposed to CCl(4) in vitro. In addition, regulation of inflammatory responses by treatment of hPE was investigated. Serum levels of GOT/AST and GPT/ALT were significantly reduced (P<0.05), and uptake/excretion of indocyanine green in serum was significantly induced at 3 weeks after intravenous hPE administration in CCl(4)-injured rat model (P<0.05). Expression of type I collagen (Col I) and α-smooth muscle actin (α-SMA) was decreased, whereas that of matrix metalloproteinase-9 (MMP-9) was increased resulting in improvement of score for fibrotic grade in hPE group. Also, albumin, proliferation activities and molecules associated with liver regeneration (e.g. interleukin-6, gp130, ATP binding cassette transporters, cyclin A) were more increased in hPE administration group than Non-hPE group. hPE administration suppressed activated T-cell proliferation via increasing anti-inflammatory cytokines and decreasing pro-inflammatory cytokines. These results suggest that hPE could be effective for liver disease through reduction of fibrosis, induction of liver regeneration, and regulation of inflammatory responses. These findings are important for understanding the roles of hPE and provide evidences for therapeutic effects of hPE in hepatic diseases which could lead to potential clinical applications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Interferon (IFN)-γ is a cytokine known for its immunomodulatory and anti-proliferative action. In the liver, IFN-γ can induce hepatocyte apoptosis or inhibit hepatocyte cell cycle progression. This article reviews recent mechanistic reports that describe how IFN-γ may direct the fate of hepatocytes either towards apoptosis or a cell cycle arrest. This review also describes a probable role for IFN-γ in modulating hepatocyte fate during liver regeneration, transplantation, hepatitis, fibrosis and hepatocellular carcinoma, and highlights promising areas of research that may lead to the development of IFN-γ as a therapy to enhance recovery from liver disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Serine palmitoyltransferase (SPT) is a key enzyme in de novo sphingolipid biosynthesis. SPT activity in liver is up-regulated by pro-inflammatory cytokines, which play an important role in initiation of liver regeneration after partial hepatectomy (PH). The aim of the study was to investigate the impact of a high-fat diet or PPARα activation by bezafibrate on the activity and protein expression of SPT in rat liver after PH. The animals were divided into three groups: those fed a standard chow (SD), those fed a high-fat diet (HFD), and those treated with bezafibrate (BF). It has been found that the expression and activity of SPT increased in regenerating liver. This was accompanied by the elevation of plasma NEFA concentration. Moreover, in both diet groups, the content of sphinganine increased. Bezafibrate decreased protein expression of SPT at the 4th and 12th hour, and inhibited SPT activity at the 4th hour after PH. Both, the plasma NEFA concentration and sphinganine content decreased in the groups treated with bezafibrate. We conclude that partial hepatectomy stimulates de novo sphingolipid synthesis. Activation of PPARα by bezafibrate negatively affects this process, presumably by decreasing the availability of plasma-borne fatty acids., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Liver regeneration after partial hepatectomy (PH) is achieved by intense cells proliferation. Sphingosine-1-phosphate stimulates proliferation but ceramide and sphingosine induce apoptosis. The aim of the study was to investigate the influence of high-fat diet (HFD) on the sphingolipid metabolism during the first 24h of liver regeneration in rats. Rats were fed HFD or standard diet for 7 days prior to the PH. The content of sphingolipids and the activity of sphingomyelinases (n and aSMase), ceramidases (n and aCDase) and sphingosine kinase (SPHK) were measured. It has been found that HFD increased the activity of aSMase and nCDase at 4th hour after PH. The content of ceramide and sphingosine decreased in HFD group at each time point. This was accompanied by elevated content of sphingosine-1-phosphate and sphinganine-1-phosphate. Decrease in SPHK activity in cytosol after partial hepatectomy was inversely correlated (r=-0.7538) with increase in S1P, which suggest translocation of SPHK to plasma membrane. Shingosine-1-phosphate to ceramide ratio was higher in rats fed HFD. It is concluded that HFD stimulates the pro-mitotic action of the sphingolipid signaling in regenerating rat liver., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The effect of an acyclic synthetic retinoid analogue NIK-333, on the restoration of liver mass and recovery of liver function after 70% partial hepatectomy, was compared with natural retinoids in rats in vivo. NIK-333 (0.4 mg/kg/day, p.o.)- and all-trans-retinoic acid (ATRA: 4 mg/kg/day, p.o.)-treated rats showed an approximately 1.3- and 1.2-fold increase in liver-to-body weight ratio, respectively, compared to solvent-administered control rats on day 3 after 70% partial hepatectomy. Accordingly, 5-bromo-2'-deoxyuridine (BrdU)-labeling index in the regenerating liver was significantly higher in NIK-333- and ATRA-treated rats compared with control rats on days 0.5 and 1. However, retinol (40 mg/kg/day, p.o.) did not significantly increase either the liver-to-body weight ratio or the BrdU labeling index. In control rats, liver-related serum transaminase activities such as alanine aminotransferase and aspartate aminotransferase, were rapidly elevated on day 1 and then decreased to near pre-operative levels on day 5 following 70% partial hepatectomy. NIK-333 significantly lowered serum transaminases on days 1 and 3 after 70% partial hepatectomy compared with solvent-administered control rats. The transaminase-lowering effect of NIK-333 was more effective than that of ATRA. Retinol did not significantly decrease serum transaminases compared with the control. These results demonstrate that of the three retinoids, NIK-333 was the most potent in promoting the regeneration of liver mass and function with full recovery after 70% partial hepatectomy., (2010 Elsevier B.V. All rights reserved.)

The in vivo effects of glycyrrhizin on restoration of liver mass and recovery of liver function were compared with those of epidermal growth factor (EGF), ibuprofen and dexamethasone in 70% partially hepatectomized rats. Hepatic regenerative activity was assessed based on the ratio of liver weight to 100 g body weight, and 5-bromo-2'-deoxyuridine (BrdU) incorporation into hepatocyte DNA in the remnant liver. Glycyrrhizin (50 mg/kg/day, i.p.)- or EGF (1.0 microg/kg/day, i.p.)-treated rats showed an approx. 1.4-fold increase in liver weight/100 g body weight ratio over saline-treated control rats on days 2 and 3 after 70% partial hepatectomy. BrdU labeling index in the remnant regenerating liver was significantly higher in glycyrrhizin- or EGF-treated rats when compared with saline-treated control rats on days 0.5 and 1. Ibuprofen (100 mg/kg/day, i.p.) and dexamethasone (0.1 mg/kg/day, i.p.) did not significantly increase either liver weight/100 g body weight ratio or BrdU labeling index. Serum activity of liver-related transaminases, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), elevated rapidly on day 1 and decreased to near pre-operative levels on day 5 after 70% partial hepatectomy in saline-treated control rats. Injection of glycyrrhizin or EGF significantly decreased the elevated serum ALT and AST activities on days 2 and 3 after hepatectomy when compared with saline-treated control rats. The transaminase-lowering effects of glycyrrhizin or EGF were smaller than those of ibuprofen and dexamethasone. These results demonstrate that injection of glycyrrhizin or EGF significantly enhances regeneration of liver mass and function, as well as recovery from the liver damage induced by surgical resection.

Previously we reported that an ordinarily nonlethal dose of thioacetamide (300 mg/kg) causes liver failure and 90% mortality in type 1 diabetic rats, primarily because of inhibited tissue repair. On the other hand, the diabetic rats receiving 30 mg thioacetamide/kg exhibited equal initial liver injury and delayed tissue repair compared to nondiabetic rats receiving 300 mg thioacetamide/kg, resulting in a delay in recovery from that liver injury and survival. These data indicate that impaired tissue repair in diabetes is a dose-dependent function of diabetes. The objective of the present study was to test the hypothesis that disrupted nuclear factor-kappaB (NF-kappaB)-regulated cyclin D1 signaling may explain dose-dependent impaired tissue repair in the thioacetamide-treated diabetic rats. Administration of 300 mg thioacetamide/kg to nondiabetic rats led to sustained NF-kappaB-regulated cyclin D1 signaling, explaining prompt compensatory tissue repair and survival. For the first time, we report that NF-kappaB-DNA binding is dependent on the dose of thioacetamide in the liver tissue of the diabetic rats. Administration of 300 mg thioacetamide/kg to diabetic rats inhibited NF-kappaB-regulated cyclin D1 signaling, explaining inhibited tissue repair, liver failure and death, whereas remarkably higher NF-kappaB-DNA binding but transient down regulation of cyclin D1 expression explains delayed tissue repair in the diabetic rats receiving 30 mg thioacetamide/kg. These data suggest that dose-dependent NF-kappaB-regulated cyclin D1 signaling explains inhibited versus delayed tissue repair observed in the diabetic rats receiving 300 and 30 mg thioacetamide/kg, respectively.

The effects of a specific inhibitor of the Na+/H+ exchanger, 5-(N,N-hexamethylene)-amiloride (HMA), on liver regeneration after partial hepatectomy were investigated. A single injection of HMA inhibited DNA synthesis and caused apoptosis in regenerating liver. Characteristic DNA fragmentation was observed at 4 h after partial hepatectomy with HMA-injection. The activity of Jun N-terminal kinase (JNK) increased to a maximal level at 15 min after partial hepatectomy in HMA-injected rats, while it was not detected until 30 min in the control. Western blot analysis revealed that the injection of HMA markedly increased c-Jun and phosphorylated c-Jun protein levels at 30 min after partial hepatectomy. An increase in p53 was also observed at 30 min after the HMA-injection and was followed by the upregulation of p21WAF1/CIP1 protein expression at 1 h after partial hepatectomy. These results suggested that HMA induced apoptosis accompanied by the activation of JNK and the upregulation of c-Jun, p53 and p21WAF1/CIP1 expression at an early stage of liver regeneration.

Owing to its powers of regeneration, the liver is capable of in vivo "tissue engineering" which enables complete restoration of liver architecture and re-establishment of the specific functions of the liver after various types of liver injury. Our current understanding of liver regeneration forms the basis of modern liver surgery and is now taken into consideration in the treatment of many liver diseases, in liver transplantation and hepatic tissue engineering. These advances have been achieved primarily by studies of liver regeneration in animal models after partial hepatectomy, attention being focused on the general mechanisms of cell proliferation. In recent years, however, toxin-induced models of liver regeneration have assumed growing importance, and by studying the interaction between cell damage and cell regeneration have made possible an investigation of liver regeneration of greater clinical relevance. However, the mechanisms of liver regeneration in patients with pre-existing chronic liver damage such as liver cirrhosis are still largely unexplored. This review examines and critically appraises the various approaches to the study of liver regeneration in animal models, including both surgical and pharmacological approaches.

Glutamine promotes hepatic regeneration in nourished (N) rats. The aim of the present study was to evaluate the effects of glutamine-enriched total parenteral nutrition (TPN) on liver regeneration in malnourished (MN) rats.Seventy-two male Wistar rats ( congruent with 270 g) were assigned to one of two groups: N and MN. Each group was then subdivided into three groups: the first underwent partial hepatectomy (PH) and received standard TPN enriched with L-alanyl-L-glutamine (Ala-Gln); the second also underwent PH and received standard TPN, but enriched with a solution containing proline and alanine (Ala-Pro); and the third underwent no surgical procedure (control group). All experimental groups received isocaloric (188 kcal. kg(-1). d(-1)) and isonitrogenous (1.12 g of nitrogen. kg(-1). d(-1)) TPN for 96 h. All animals were injected with bromodeoxyuridine 2 h before death. The hepatic regeneration index (HRI), hepatic growth percentage (HG) and hepatic morphology were analyzed. In MN rats, HRI and HG were higher with glutamine enrichment (HRI = 81 and HG = 190) than with proline-containing TPN (HRI = 66 and HG = 154; P < 0.05) and HRI was 100 times higher in animals that underwent PH than in control animals. Morphologic analysis of hepatic tissue showed no difference among the six groups.Glutamine-enriched TPN promoted growth of the remnant liver in MN rats after PH, maintained cellular proliferation in the various experimental groups after surgery, and maintained hepatic morphology of MN rats after surgery.

To investigate apoptosis induced by selenite in hepatocytes in vivo, rats received a single injection of sodium selenite immediately after partial hepatectomy. Characteristic DNA fragmentation in gel electrophoresis and in situ end-labeling and the increase in caspase-3 activity were observed at 4 h after partial hepatectomy with selenite injection. The activation of Jun N-terminal kinase (JNK) was observed as early as 15 min and increased to about 10-fold the maximal level of the control at 1 and 2 h after partial hepatectomy in selenite-injected rats, while a transient increase was observed at 1 h in the control. Western blot analysis revealed that the c-Jun and the phosphorylated c-Jun protein markedly increased after 30 min and reached a maximal level at 1 and 2 h after partial hepatectomy with selenite injection, although c-Jun and a faint band of the phosphorylated c-Jun were observed after 1 h in the control. The levels of c-jun mRNA and c-Fos protein and mRNA in selenite-injected rats also increased more than in the control. The rise in the p53 protein level after partial hepatectomy with selenite injection was followed by the upregulation of p21(WAF1/CIP1) mRNA and protein expression. These results suggested that selenite induced apoptosis accompanied by the activation of caspase-3 and JNK and the upregulation of c-jun, c-fos, p53 and p21(WAF1/CIP1) at the early stage of liver regeneration.

Objectives: Liver regeneration is influenced by cholesterol and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA-reductase). HMG-CoA-reductase is a key enzyme for cholesterol synthesis. Recent studies have shown that inhibitors of HMG-CoA-reductase improve liver functions after 67% partial hepatectomy (PH)., Methods: Male Wistar rats (W) and Prague hereditary hypercholesterolemic rats (PHHC) were used. Aqua pro injectione (AI) or pravastatin (prava; 1 mg/kg) was administered orally once daily. Group 1: W, standard diet (SD) + AI; group 2: W, SD + prava; group 3: W, cholesterol-enriched diet (chol) + AI; group 4: PHHC, chol + AI; group 5: PHHC, chol + prava. After 27 d, PH was performed in all groups., Results: Groups fed chol before PH had significantly higher liver triacylglycerol content (group 3: 25.8 +/- 2.6 mg/g of liver weight; group 4: 16.0 +/- 1.0 of liver weight; group 5: 22.0 +/- 1.0 of liver weight) than did the groups fed SD (group 1: 6.1 +/- 0.5; group 2: 5.9 +/- 0.7). Liver DNA synthesis after PH was significantly lower in chol-fed groups (group 3: 561 +/- 78; group 4: 472 +/- 92) than in SD-fed groups (group 1: 1645 +/- 574; group 2: 2935 +/- 1298), except the chol-fed PHHC given prava (group 5: 3230 +/- 527)., Conclusions: In prava-treated rats, the induction of HMG-CoA activity overcame the inhibitory capability of pravastatin. The induction of HMG-CoA-reductase activity had a stimulatory effect on the initiation of liver regeneration.

Polyamines are key factors in macromolecule synthesis during liver regeneration. It has been postulated that interferon-alpha (IFNalpha) decreases putrescine levels in regenerating liver by inhibiting ornithine decarboxylase (ODC) activity, the main enzyme in polyamine biosynthesis. In the present study, we analysed the effects of a pharmacological dose of IFNalpha on polyamine and ODC levels during the regenerative process following partial hepatectomy in rats. Synthesis of ODC by isolated hepatocytes from IFN-treated rats with regenerating livers was also assessed. Furthermore, we investigated the effect of IFNalpha-2b on DNA and total protein synthesis in 24-hr regenerating livers. No effect on DNA synthesis was observed at the dose of IFNalpha-2b used, but total protein synthesis decreased significantly in IFNalpha-2b-treated rats undergoing liver regeneration (7.0 +/- 2.0 and 12.1 +/- 1.7%. min(-1) in hepatectomized rats treated with IFNalpha-2b and saline, respectively). ODC levels were also reduced significantly (by 50%) in hepatectomized rats treated with IFNalpha-2b versus saline. In parallel with the ODC decrease, the concentrations of putrescine and spermidine (63 +/- 25 vs 101 +/- 15 nmol/g liver and 1.08 +/- 0.35 vs 2.14 +/- 0.22 micromol/g liver, respectively, in IFNalpha-2b- and saline-treated hepatectomized rats) showed similar, significant diminutions. Moreover, the incorporation of [35S]methionine into ODC was decreased dramatically in isolated hepatocytes from IFNalpha-2b-treated hepatectomized rats 12 hr after surgery. In conclusion, the protein synthesis rate in regenerating liver was impaired by therapeutic doses of IFNalpha-2b. In addition, the results presented in this study suggest that IFNalpha-2b negatively regulates ODC synthesis, causing a reduction in polyamine levels during liver regeneration.

Recent studies have shown that many factors orchestrate liver regeneration after a two-thirds partial hepatectomy (PH). However, the termination mechanism in liver regeneration has not been thoroughly studied. In this paper, we report that the activity of liver caspase-3-like protease, which is specifically activated in apoptosis, increases 18, 36, and 48 hr after PH during maximal hepatocyte proliferative activity. This is the first study that shows the activation of an apoptosis-executing enzyme during physiological liver regeneration. These results suggest that apoptosis is induced in each surge of DNA synthesis as the termination mechanism. When phenoxybenzamine, an alpha-blocker that has been reported to inhibit DNA synthesis during liver regeneration, was injected 8 hr after PH, the caspase-3-like activity in the liver peaked at 15 hr after PH and the enzyme activity also increased in plasma at 18 and 24 hr after PH in sharp contrast to the case of normal regeneration. These results indicate that extensive apoptosis is caused by phenoxybenzamine and that the secondary necrosis of apoptotic cells results in the increase of caspase-3-like protease activity in the plasma.

Cell death following ischemia-reperfusion injury is a major concern in clinical issues such as organ transplantation and trauma. The need to identify agents with a potential for preventing such damage has assumed great importance. We have evaluated the efficacy of picroliv, a potent antioxidant derived from the plant Picrorhiza kurrooa, in protecting against hepatic ischemia-reperfusion injury in vivo. Picroliv was fed to male Sprague Dawley rats in a dose of 12 mg/kg once daily by oral gavage for 7 days prior to hepatic ischemia. Ischemia was induced by occluding the hepatic pedicel with a microaneurysm clip for 30 min and reperfusion was allowed thereafter for varying period (15-120 min) by releasing the microaneurysm clip. Picroliv pretreatment resulted in better hepatocyte glycogen preservation and reduced apoptosis. Reduction in apoptosis was associated with decreased mRNA expression of caspase-3 and Fas. Oxidant induced cellular damage as measured by tissue malondialdehyde (MDA) levels was significantly less following picroliv pretreatment. Both a reduction in neutrophil infiltration and an increased level of intracellular antioxidant enzyme superoxide dismutase possibly contributed to the reduction in tissue lipid peroxidation. Tissue inflammatory cytokines level of interleukin-1alpha (IL-1alpha) and interleukin-1beta (IL-1beta) was also lower in picroliv group. Furthermore, picroliv pretreatment resulted in enhanced proliferating cell nuclear antigen (PCNA) immunoreactivity. These studies strongly suggest picroliv to be a promising agent for ameliorating injury following ischemia-reperfusion.

Ethanol metabolism can induce modifications in liver metabolic pathways that are tightly regulated through the availability of cellular energy and through the redox state. Since partial hepatectomy (PH)-induced liver proliferation requires an oversupply of energy for enhanced syntheses of DNA and proteins, the present study was aimed at evaluating the effect of acute ethanol administration on the PH-induced changes in cellular redox and energy potentials. Ethanol (5 g/kg body weight) was administered to control rats and to two-thirds hepatectomized rats. Quantitation of the liver content of lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, and adenine nucleotides led us to estimate the cytosolic and mitochondrial redox potentials and energy parameters. Specific activities in the liver of alcohol-metabolizing enzymes also were measured in these animals. Liver regeneration had no effect on cellular energy availability, but induced a more reduced cytosolic redox state accompanied by an oxidized mitochondrial redox state during the first 48 hr of treatment; the redox state normalized thereafter. Administration of ethanol did not modify energy parameters in PH rats, but this hepatotoxin readily blocked the PH-induced changes in the cellular redox state. In addition, proliferating liver promoted decreases in the activity of alcohol dehydrogenase (ADH) and of cytochrome P4502E1 (CYP2E1); ethanol treatment prevented the PH-induced diminution of ADH activity. In summary, our data suggest that ethanol could minimize the PH-promoted metabolic adjustments mediated by redox reactions, probably leading to an ineffective preparatory event that culminates in compensatory liver growth after PH in the rat.

Recently, it has been suggested that ethanol-induced inhibition of liver regeneration results from decreases in hepatic putrescine levels and/or increases in hepatic gamma-aminobutyric acid (GABA)ergic activity. Because putrescine can be metabolized by diamine (DAO) and monoamine (MAO) oxidases to GABA, we documented the effects of acute ethanol exposure on hepatic MAO or DAO activity following partial hepatectomy (PHx) in rats. We also documented the effects of ethanol on GABA transaminase (GABA-T), the enzyme responsible for GABA metabolism in the liver, and tissue putrescine and GABA levels. Adult, male Sprague-Dawley rats (200-250 g) were treated with either ethanol (3 g/kg) or equal volumes of saline by gastric gavage 1 h prior to a 70% PHx or sham surgery. Rats were then sacrificed (n = 5-7/group) at various times (0-72 h) post-PHx. Enzymatic activity and putrescine/GABA levels were determined by standard isotopic techniques and high-performance liquid chromatography respectively. Hepatic DAO activities in ethanol-treated rats were transiently higher than in saline-treated controls (30% increases at 6 h, p < 0.05). Hepatic MAO and GABA-T activities in acute ethanol-treated rats were essentially identical to saline-treated controls. Although hepatic putrescine levels were similar in ethanol- and saline-treated rats, hepatic GABA levels were approximately three times higher in ethanol-treated rats at 12 and 24 h post-PHx (p < 0.0001). In conclusion, the results of this study indicate that acute ethanol exposure has a limited effect on the enzymatic conversion of putrescine to GABA following partial hepatectomy in the liver. The results also indicate that increased GABAergic inhibition rather than decreased putrescine stimulation is more likely to play a role in ethanol-induced inhibition of hepatic regeneration.

The expression of asialoglycoprotein receptor (ASGP-R) on human hepatocarcinoma cells might be exploited to reduce the extrahepatic toxicity of DNA synthesis inhibitors by their conjugation with galactosyl- terminating peptides. We conjugated 2',2'-difluorodeoxycytidine (dFdC), an inhibitor of DNA synthesis active on solid tumors, with lactosaminated poly-L-lysine (L-poly(LYS)). In experiments in vitro, L-poly(LYS)-dFdC inhibited proliferation of Hep G2 cells, a human hepatocarcinoma cell line which maintains the ASGP-R. Inhibition was rescued by asialofetuin. To study the pharmacological action of the conjugate in vivo, we used rats 18-24 hr after 2/3 hepatectomy and observed that regenerating hepatocytes expressed ASGP-R on their surface and internalized L-poly(LYS)-dFdC. Conjugate uptake by bone marrow, spleen, and intestine was negligible. We also found that L-poly(LYS)-dFdC inhibited [3H]thymidine incorporation into DNA of regenerating liver. These results indicated that hepatectomized rats were a suitable animal model to study the pharmacological action, on DNA-synthesizing hepatocytes, of conjugates binding to ASGP-R and carrying inhibitors of DNA synthesis. L-poly(LYS)-dFdC also inhibited [3H]thymidine incorporation in bone marrow, spleen, and intestine. Evidence was obtained that inhibition of DNA synthesis in extrahepatic tissues was a consequence of drug release from hepatocytes into blood-stream after the bond with the carrier has been broken down within liver cells. Possible ways of reducing the exit of dFdC from liver cells, thereby obtaining an inhibition of DNA synthesis restricted to dividing hepatocytes, were discussed.

The intestine is now known to be an important site of protein production in the body, and glutamine (GLN) stimulates both secreted and non-secreted protein synthesis in the small bowel. The purpose of the present study was to evaluate the effect of GLN-supplemented parenteral nutrition on liver regeneration after hepatectomy. Animals were divided into two groups: a sham-operated control group (Group A) and a 70%-hepatectomy group (Group B). Postoperatively, one-third of the animals in each group were maintained on intravenous 10% glucose solution, on 10% glucose with 2% standard amino acid solution, or 10% glucose supplemented with 2% glutamine for 24 h. GLN administration after hepatectomy significantly promoted liver regeneration. In addition, assessment of amino acid metabolism showed that GLN administration activated GLN metabolism in the intestine and promoted alanine uptake by the remnant liver. This metabolic response also enhanced both secreted and non-secreted protein synthesis in intestinal epithelial cells, especially in cells isolated from the crypts. The proteins produced are important as a portal production factor for liver regeneration and intestinal cell proliferation. Bacterial and endotoxin translocation, on the other hand, was significantly reduced. Thus, the results of this study suggest that intravenous administration of GLN after hepatectomy significantly promoted liver regeneration.

Purine and pyrimidine metabolism is a key process after hepatic surgery. To evaluate the effect of purine and pyrimidine supplementation on hepatic regeneration, the following clinical in vitro and in vivo experiments were performed. Changes in blood nucleotides, nucleosides and nucleobase were analyzed by high performance liquid chromatography in patients and rats after partial hepatectomy. The effect of supplementation of nucleotide-nucleoside solution (OG-VI) or their components on nucleic acids syntheses in primary monolayer cultured hepatocytes and preoperative intraperitoneal injection of OG-VI on hepatic regeneration in the partially hepatectomized rats were evaluated. Blood purine and pyrimidine levels in patients change after hepatectomy and their changes indicate increased salvage synthesis of purine and pyrimidine in the regenerating liver. Addition of appropriate amounts of inosine, GMP, uridine, or thymidine, the substrates for salvage purine and pyrimidine syntheses, to primary cultures of hepatocytes enhanced both DNA and RNA syntheses by the salvage and de novo pathways. The OG-VI mixture also enhanced the syntheses of DNA and RNA. Preoperative administration of OG-VI to partially-hepatectomized rats enhances hepatic DNA synthesis in a way similar to the in vitro study.

After hepatectomy, purine and pyrimidine metabolism is a key process in the synthesis of DNA and RNA and maintaining cellular energy metabolism. The purpose of this study is to evaluate changes in blood purine and pyrimidine levels after partial hepatectomy and the effect of purine and pyrimidine nucleoside solution injection on hepatic regeneration under the hypothesis that the rat after partial hepatectomy requires substrates for salvage nucleotide synthesis and changes blood nucleoside and nucleobase levels. Blood levels of nucleotides, nucleosides, and nucleobase by high-performance liquid chromatography method and liver ATP level by enzymatic analysis, and the effect of preoperative injection of nucleoside solution (OG-VI) on hepatic regeneration ratio and hepatocytes DNA synthesis, were assessed in rats after 70% partial hepatectomy. Decreased liver adenosine triphosphate and increased plasma xanthine and hypoxanthine after partial hepatectomy indicated an increase in catabolism of purine nucleotides in regenerating liver. Plasma thymidine and cytidine levels increased, then returned to the prevalue, suggesting that the thymidine and cytidine pool was enlarged. OG-VI increased labeling indices of hepatocytes at postoperative d 1 (POD) and hepatic regeneration ratio at POD 14. Blood purine nucleobase and pyrimidine nucleoside levels change after partial hepatectomy and preoperative supply of nucleoside solution is effective for increasing hepatocytes DNA synthesis and hepatic regeneration after partial hepatectomy.

We studied the effects of lipid emulsions for total parenteral nutrition (TPN) on hepatic regeneration after partial hepatectomy in rats. Daily energy intake was maintained at 1172 kJ.kg-1.day-1 while the percentage of nonprotein energy sources was changed. Animals were divided into four groups: lipid-free, 10%-lipid, 20%-lipid, and 40%-lipid. TPN was continued for up to 1 wk. The content of proteins, the ratio of proteins to triglycerides, and the yield of mitochondrial protein in the remnant liver 7 days after partial hepatectomy were larger in animals receiving TPN with lipids than in those receiving lipid-free TPN, whereas the amounts of triglycerides and cholesterol in the liver of the latter animals were larger. The degree of fatty infiltration of the hepatic lobule was most distinct in the lipid-free group. Furthermore, activities of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and alkaline phosphatase in the serum tended to be higher in the lipid-free group. Phosphorylating ability of mitochondria in the regenerating liver 7 days after partial hepatectomy was not different among the four groups; however, the highest value for the respiratory control index was obtained in the 40%-lipid group. The application of a lipid emulsion to TPN is useful for hepatic regeneration after partial hepatectomy; however, the ideal concentration of lipids in TPN awaits further investigation.

The effect of orally administered 17(S),20-dimethyl-6-oxo prostaglandin E1 (PGE1) methyl ester (OP) on liver regeneration was examined in 66% hepatectomized rats. Administration of OP increased the mitotic index of the hepatocyte 3 days after hepatectomy, and the mitotic index response due to OP was dose dependent. The OP administration had no effect on food intake, but reduced water intake. The serum scores relative to nutrition and hepatic function showed transient change after OP administration, whereas the serum blood urea nitrogen level indicated a slight renal dysfunction with OP. The fat store in the body was transiently reduced. These observations lead us to conclude that orally administered OP is capable of stimulating liver regeneration without serious systemic effects.

Several investigations have been suggesting that lymphocytes can be responsible for growth regulation of liver. The interleukin-2 (IL-2) production by spleen cells was noted to be increased 2 days after resection of 70% of the volume of the liver in C3H/He mice. The effects of IL-2 production and its antagonists: cyclosporine A (CsA) and FK506 on liver regeneration were thus studied by measuring in vivo incorporation of Bromodeoxyuridine (BrdU). By intraperitoneal injection of IL-2 5 times every 8 h after hepatectomy. BrdU labeling indices at 36 h after the operation were suppressed in a dose-dependent manner. On the other hand, treatment with CsA and FK506, which inhibit IL-2 production, increased the mitotic indices of the regenerating livers. Serum levels of aminotransferases were not affected by FK506 at a dose sufficient for the mitotic stimulation. Based on these results, CsA and FK506 would appear to stimulate liver cell proliferation by suppressing IL-2 production and inhibiting of NK cell activity.

In order to elucidate the effects of acute ethanol on compensatory liver growth (regeneration), the steady state c-myc mRNA levels were studied following two-thirds partial hepatectomy. After surgery, control rat livers exhibited two peaks of c-myc transcripts, at 0.5-2 h and at 8-10 h. Sham surgery did not induce c-myc mRNA expression. Ethanol (3 g/kg), administered by gavage at 1 hour prehepatectomy, had no effect on the initial peak of c-myc mRNA; however, the second peak was eliminated. Control gavage of isocaloric glucose prior to partial hepatectomy had no effects on either of the subsequent c-myc mRNA peaks. Blood alcohol levels were found to be elevated throughout the prereplicative phase. These results suggest that ethanol may disrupt proto-oncogene expression near the restriction point at the G1/S boundary of the cell cycle in hepatocytes.

1. delta-Aminolevulinic acid synthetase (ALA-S), rhodanese and microsomal heme oxygenase (MHO), were quantitated in Cl4C induced regenerating mouse liver. 2. Maximal hepatomegalia was observed at 48 hr after i.p. injection of a single dose of the toxin. 3. ALA-S activity decreased on day 2, and then significantly increased (50%) between days 3 and 7, returning afterwards to control values. 4. Cytoplasmic rhodanese, as well as MHO activities, exhibited a clear correlation as compared with the ALA-S activity profile. 5. Porphyrin biosynthesis from precursor delta-aminolevulinic acid (ALA) was significantly increased even after 15 days of intoxication. 6. Present results would indicate that Cl4C is acting in a dual fashion.

The present investigation was undertaken to test our hypothesis that the slow responses of hepatocellular regeneration and tissue repair after CCl4-induced liver injury are responsible for the high sensitivity of gerbils to the hepatotoxic and lethal effects of CCl4. These studies were conducted in normal and actively regenerating livers using male gerbils 5 or 15 days after partial (2/3) hepatectomy (PH5 and PH15, respectively), or those undergoing sham operation (SH). An LD50 dose of CCl4 (80 microL/kg, i.p.) resulted in a mortality (21%) significantly (P less than 0.05) less than 50% in PH5 gerbils 48 hr after CCl4 administration, whereas the mortality observed in PH15 or SH gerbils was not significantly different from 50%. The elevations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly (P less than 0.05) less in PH5 gerbils than in PH15 or SH groups after the administration of either the LD50 dose or a low dose (15 microL/kg) of CCl4. Histopathological and histomorphometric examinations also indicated that CCl4-induced liver injury was less severe in PH5 gerbils than in the PH15 and SH groups. The hepatic microsomal cytochrome P450 content measured before CCl4 administration in the PH5 gerbils was decreased (26%) significantly (P less than 0.05) as compared with the SH group, but was not significantly different from that of PH15 gerbils. In vivo metabolism of 14CCl4 and lipid peroxidation in liver tissue were not significantly different among the various groups. Therefore, the protection against CCl4 toxicity observed in PH5 gerbils is unlikely to be due to decreased bioactivation of CCl4 or lipid peroxidation in that group. [3H]Thymidine incorporation into hepatocellular nuclear DNA was 4- to 5-fold higher in PH5 gerbils than in the PH15 and SH groups, indicating active hepatocellular proliferation in PH5 gerbils. [3H]Thymidine incorporation was further increased significantly (P less than 0.05) 24 hr after challenge with a low dose of CCl4 in PH5 gerbils, whereas it remained low until 48 hr after the CCl4 injection in the PH15 or SH group. The protection against CCl4 toxicity afforded by partial hepatectomy was closely associated with active hepatocellular regeneration. The overall results confirm the concept that the high sensitivity of gerbils to CCl4 is due to very sluggish hepatocellular regeneration and tissue repair response to the CCl4-induced liver injury.

We examined the effect of long-chain triglyceride (LCT) and medium-chain triglyceride (MCT) emulsions on hepatic regeneration. After approximately 70% hepatectomy, Sprague-Dawley rats were maintained for 96 hours on total parenteral nutrition (TPN) (250 kcal/kg per day; nonprotein calories-nitrogen 160:1) with LCT or MCT as 30% of nonprotein calories. There were no significant differences in the body weight, cumulative nitrogen balance, urinary 3-methylhistidine excretion, or changes in the energy stores between the two groups; but the fatty acid composition of the phospholipid fraction of the regenerating liver differed significantly between the LCT and the MCT groups. The extent of hepatic regeneration by weight was 88.7 +/- 10.5% in the MCT group and 99.1 +/- 10.6% in the LCT group by 96 hours after hepatectomy. Furthermore, the incorporation of 3H-orotic acid into DNA and RNA of regenerating liver cells in the LCT group was higher than in the MCT group 24 hours after hepatectomy. These observations indicate that essential fatty acids--components of the cell membrane and precursors as functional mediators--are very important to hepatic regeneration.

Partial hepatectomy (PH) (70% resection) causes within 4 hr an accumulation of ornithine decarboxylase (EC 4.1.1.17, ODC) mRNAs concomitant with an increase in ODC activity, maximum values being observed at 8 and 16 hr, respectively. In the early hours of hepatic regeneration, enhancement of transcriptional-rate of ODC gene, demonstrated by nuclear run-on analysis, can account for the accumulation of ODC mRNAs. The involvement of catecholamines in these processes is demonstrated by using prazosin and propranolol, specific antagonists of alpha 1 and beta adrenoceptors, respectively. Prazosin reduces almost completely the rise of ODC activity at 4 hr, without affecting mRNA levels. At 16 hr, enzyme activity and mRNAs increase, however, over the values observed in regenerating liver of prazosin-untreated animals. These findings suggest that alpha 1-receptor activation triggers positive control signals for ODC gene expression at the early time of liver regeneration and, on the contrary, negative signals at later times by mainly post-transcriptional and transcriptional mechanisms, respectively. Propranolol reduces similarly the initial 4 hr-rise of ODC activity. These results indicate that activation of both alpha 1- and beta-adrenoceptors causes the large increase in ODC activity. Pharmacological manipulation of intracellular Ca2+ levels by verapamil, a Ca2(+)-channel blocker, or neomycin, an inhibitor of Ca2+ release from endogenous stores, diminishes ODC activity at 4 and 16 hr after PH. ODC mRNA levels, which are not modified at 4 hr, increase over the values of partially hepatectomized rat liver at 16 hr. Trifluoperazine inhibits both ODC activity and mRNA accumulation at the times studied. As a working hypothesis it is proposed that Ca2(+)-mediated processes induced by catecholamines are involved in ODC gene expression during the prereplicative phase of liver regeneration.

The effect of a nucleoside-based mixture (OG-VI) on protein synthesis in partially hepatectomized rats was evaluated. Twenty male Wistar rats weighing about 200 g underwent 70% hepatectomy by Higgins and Anderson's method, and then were subjected to total parenteral nutrition (TPN) for 3 days: the control group received only standard TPN (S-TPN group) and the experiment group received standard TPN supplemented with a nucleoside-nucleotide mixture (13% W/V, OG-VI) (OG-TPN group). The rates of whole body protein turnover and protein synthesis and breakdown were determined for 3 days after hepatectomy by the method of Picou and Taylor-Roberts with [15N] glycine. The nitrogen balances on days 1 and 3 and the cumulative nitrogen balance were significantly higher in the OG-TPN group than in the control group. Moreover, the rates of protein turnover and protein synthesis were significantly higher in the OG-TPN group than in the control group. Thus, increase in protein synthesis, rather than change in protein breakdown, was concluded to be responsible for the better nitrogen balance of the OG-TPN group. These findings suggest that the nucleoside-nucleotide mixture was used for the syntheses of RNA and DNA through the salvage pathway in regenerating liver and that this resulted in enhanced protein turnover with simultaneous increase in protein synthesis.

In the past, it has been necessary for pharmacological intervention of epithelial proliferation to mostly be limited to non-specifically arresting or killing actively proliferating cells. As our understanding of the mechanisms involved in mediating the processes of growth and differentiation increases, we can hope to see the development of a new pharmacology, in which proliferation of individual systems may be regulated in a less drastic manner.

Contrary to a recent report by Okazaki et al. (Biochem. biophys. Res. Commun., 1978, 81, 512-520) we show that high concentrations of biliverdin (100 mg/kg, i.p.) slightly reduce the mitotic rate in rat liver. Adenylate cyclase and both the "high Km" and "low Km" phosphodiesterase of rat liver plasma membranes are inhibited by high concentrations of biliverdin. Biliverdin has no effect on cyclic AMP production in isolated hepatocytes. An intracellular binding protein (glutathione-S-transferase B) is shown to bind biliverdin providing a mechanism for maintaining a low free intracellular concentration of the tetrapyrrole analogous to that of albumin in plasma. In conclusion, these results are not consistent with a role for biliverdin in stimulating liver regeneration in the rat via a mechanism involving elevated cyclic AMP levels.

The increases in activity of hepatic thymidylate synthetase and of thymidine kinase, which catalyze the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed during liver regeneration in rats which had been given alpha-adrenoceptor antagonists (phenoxybenzamine and phentolamine) or adrenergic neuron blockers (guanethidine and reserpine). These suppressions were not observed with a beta-adrenoceptor antagonist (propranolol), or an anticholinergic agent (atropine methyl nitrate). The rise in the activity of the thymidylate-synthesizing enzymes was closely correlated with the increase in the DNA content of the liver. It is concluded that catecholamine regulates the increase in the activity of thymidylate synthetase and thymidine kinase, which are key enzymes in DNA synthesis in regenerating liver. It is also suggested that sympathetic nerves play an important role in liver regeneration.