Glycosylation and ligand-binding activities of rat plasma fibronectin during liver regeneration after partial hepatectomy

Fibronectin (FN) is a multifunctional glycoprotein present in the extracellular matrix (ECM) and plasma. We previously reported that the glycosylation and ligand-binding of vitronectin (VN) change markedly after partial hepatectomy (PH). Here we show the changes of FN during liver regeneration. The yields of purified sham-operated (SH-) and PH-FN were higher than that of non-operated (NO)-FN, while binding activities of FNs to ECM ligands were changed only slightly by hepatectomy. The carbohydrate concentration of PH-FN decreased to 66% of that of NO- and SH-FN. By using LC/MS(n), eight kinds of complex-type N-glycan structures were found to be present in all FNs, and bi- and trisialobiantennary glycans were the major structures. Fucosylation was markedly increased, while O-acetylation of sialic acid was found to be decreased in PH-FN. The alterations in glycosylation and biological activities of FN after PH are different from those of VN, suggesting that these glycoproteins play different biological functions in tissue remodeling.     

Chronobiology of the proliferative events related to angiogenesis in mice liver regeneration after partial hepatectomy

In liver regeneration the formation of new capillary blood vessels is a fundamental requirement for cellular proliferation. Vascular endothelial growth factor (VEGF) is involved in the events of angiogenesis, the mRNA of which is expressed in both hepatocytes and non-parenchymal cells. In this experimental design we try to establish if during liver regeneration in mouse, the expression of VEGF is produced before or after the hepatocytes proliferation. C3H/S adult male mice were divided in three groups in order to study: VEGF expression; S-phase index (SI); and mitotic activity (MA) of hepatocytes. The results that were analyzed by ANOVA, show that VEGF expression starts to increase 26 h after PH with a peak at 28 h. Furthermore, the DNA synthesis (DNAs) reaches maximal level 42 h after pH, meanwhile the MA of the hepatocytes shows an increase 8h after the DNAs peak. In conclusion, it could be argued that the chronobiology of the events related to liver regeneration in mice started with a release of VEGF by the hepatocytes, followed by its DNAs and mitosis.     

Effects of carvacrol on defects of ischemia-reperfusion in the rat liver

Many plants found in nature have been used to treat various illnesses. One such plant is oregano (Kekik in Turkish). Health beneficial effects of carvacrol obtained from oregano oil have been shown scientifically. We have investigated the comparative effects of carvacrol in the liver of rats subjected to ischemia-reperfusion defect, with silymarin. To test the effects we formed four groups using male Wistar albino rats. Group I was control. The other three groups of animals were administered 60 min prior to surgical operation single doses of physiological serum, carvacrol and silymarin, respectively. Group II, III and IV animal were subjected to 45 min long liver ischemia and 60 min reperfusion. Blood and tissue samples were collected for biochemical and histological analysis following the test.AST and ALT values obtained after biochemical analysis of the serums showed statistically significant difference in group II than the other three groups. A statistical evaluation of the serum AST levels among the groups II, III and IV showed that both groups III and IV which had no difference in between were significantly different in a positive way from group II (p<0.001). As to the serum ALT levels, difference between group II and group III (p<0.001) and group II and group IV (p<0.01) was found significant. No statistical difference was observed in groups I, III and IV for GSH, MDA and CAT levels of the liver. A statistical evaluation of the GSH level in group III and group IV was found to be significantly different from group II (p<0.001) without any difference between them. A similar evaluation for MDA and CAT levels among the revealed no difference between group III and group IV, however, group II showed difference with group II and group IV (p<0.05).Histological findings were in harmony with the biochemical results. We conclude that carvacrol protects the liver against defects caused by ischemia and reperfusion, and carvacrol is not hepatotoxic at the applied dosage.     

Liver regeneration after partial hepatectomy in rat is more impaired in a steatotic liver induced by dietary fructose compared to dietary fat

Hepatic steatosis (HS) has a negative effect on liver regeneration, but different pathophysiologies of HS may lead to different outcomes. Male Sprague-Dawley rats were fed a high fructose (66% fructose; H-fruc), high fat (54% fat; H-fat), or control chow diet for 4 weeks. Based on hepatic triglyceride content and oil red O staining, HS developed in the H-fruc group, but was less severe compared to the H-fat group. Hepatic mRNA expression levels of fatty acid synthase and fructokinase were increased and those of carnitine palmitoyltransferase-1 and peroxisome proliferator-activated receptor-α were decreased in the H-fruc group compared to the H-fat group. Liver regeneration after 70% partial hepatectomy (PHx) was evaluated by measuring the increase in postoperative liver mass and PCNA-positive hepatocytes, and was impaired in the H-fruc group compared to the H-fat and control groups on days 3 and 7. Serum levels of tumor necrosis factor-α, interleukin-6 and hepatocyte growth factor did not change significantly after PHx. In contrast, serum TGF-β1 levels were slightly but significantly lower in the control group on day 1 and in the H-fat group on day 3 compared to the level in each group on day 0, and then gradually increased. However, the serum TGF-β1 level did not change after PHx in the H-fruc group. These results indicate that impairment of liver regeneration after PHx in HS is related to the cause, rather than the degree, of steatosis. This difference may result from altered metabolic gene expression profiles and potential dysregulation of TGF-β1 expression.     

Partial hepatectomy induced liver proteome changes in mice

Acceleration of liver regeneration could be of great clinical benefit in various liver-associated diseases. However, at present little is known about therapeutic interventions to enhance this regenerative process. Our limited understanding and the complexity of the mechanisms involved have prevented the identification of new targets for treatment. Here we propose a broad-range proteomic approach to this problem that makes possible the simultaneous study of different signaling and metabolic pathways on the liver proteome. Changes in protein expression in mouse livers (n = 5 per group) at 6 h and 12 h after partial hepatectomy and sham operation, as compared to untreated controls, were analyzed using two-dimensional gel electrophoresis, mass spectrometry (MS), and mass fingerprinting. Twelve proteins, identified by MS, were up-regulated by at least 2-fold after partial hepatectomy. These included adipose differentiation-related protein, gamma-actin, enoyl coenzyme A hydratase 1, serum amyloid A and eukaryotic translation initiation factor 3. These results indicate that liver regeneration following partial hepatectomy affects various signaling and metabolic pathways.     

Liver proteome analysis of adaptive response in rat immediately after partial hepatectomy

The extraordinary ability of the liver to regenerate after resection continues to be an important fascination to mammalian liver researchers. However, at present, there are still several central questions regarding the process of liver regeneration that are not clear. In our study, we try to clarify how the liver is able to maintain its functions as well as to initiate liver regeneration after a significant loss of two-thirds. Here differentially expressed proteins in rat livers at 1 h after partial hepatectomy (PHx) and sham operation were analyzed using 2-DE combined with MALDI-TOF/TOF MS. After the analysis, 24 significantly changed spots (ratio> or =2, p<0.05) were identified. Those proteins are involved in important liver functions such as metabolism, detoxification, and inflammation. Based on the changes in the protein levels found in our data, we identified two aspects of remnant liver immediately after PHx, which focused on the hepatic adaptation and the inflammatory response associated with the initiation of liver regeneration after PHx. For the first time, the differential expression of pyruvate dehydrogenase complex (PDHX), paraoxonase 1 (PON1), thyroid hormone receptor beta, GAP43 (where GAP stands for growth-associated protein), and interleukin-2 (IL2), after PHx, were validated by Western blot.     

Alrp,a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats

Abstract

Augmenter of Liver Regeneration (Alrp) enhances, through unknown mechanism/s, hepatocyte proliferation only when administered to partially hepatectomized (PH) rats. Liver resection, besides stimulating hepatocyte proliferation, induces reactive oxygen species (ROS), triggering apoptosis. To clarify the role of Alrp in the process of liver regeneration, hepatocyte proliferation, apoptosis, ROS-induced parameters and morphological findings of regenerating liver were studied from PH rats Alrp-treated for 72 h after the surgery. The same parameters, evaluated on regenerating liver from albumin-treated PH rats, were used as control. The results demonstrated that Alrp administration induces the anti-apoptotic gene expression, inhibits hepatocyte apoptosis and reduces ROS-induced cell damage. These and similar data from in vitro studies and the presence of ‘Alrp homologous proteins’ in viruses as well as in mammals (i) allow to hypothesize that Alrp activity/ies may not be exclusive for regenerating liver and (ii) suggest the use of Alrp in the treatment of oxidative stress-related diseases.     

Effects of partial hepatectomy on microtubules and hepatocellular transport of indocyanine green in rats

The effects of partial hepatectomy on plasma disappearance and biliary excretion of indocyanine green (ICG) have been studied in rats and correlated with morphometric changes of hepatocellular microtubules. The plasma disappearance rate of ICG was in good accord with recovery of liver weight after partial hepatectomy. Biliary excretion of ICG per 100 g liver significantly increased between 3 h and 7 days postoperatively. Colchicine significantly reduced plasma disappearance and biliary excretion of ICG, with no reduction in bile flow, in both intact and hepatectomized rats. Morphometrically, microtubules significantly increased from 3 h following partial hepatectomy and reached a maximum at 24 h with a gradual return to preoperative values at 5 days. These observations suggest that the increased hepatocellular transport of ICG after partial hepatectomy is related to an increase in the number of microtubules.     

Oxygenation alters ganglioside expression in rat liver following partial hepatectomy

Gangliosides from livers of weanling rats were analyzed after 15% partial hepatectomy (PH) and different pre- and post-operative hyberbaric oxygenation (pre- and postHBO). Neu5Ac was the predominant ganglioside-derived sialic acid (>85%) compared to Neu5Gc. Almost identical low total sialic acid content (Neu5Ac+Neu5Gc) of the control and operated nonHBO animals opposed a 6.4- to 7.6-fold increase in pre- and postHBO animals (69.26 and 81.64pmol/mg wet weight, respectively). NanoESI-QTOF mass spectrometry combined with HPTLC immunostaining revealed GM3(Neu5Ac) and GM3(Neu5Gc) as major gangliosides, correlating with the respective sialic acid concentrations. Minor neolacto-series gangliosides were enhanced in preHBO and postHBO, but GM1-core gangliosides only in preHBO rats. GM2 and GalNAc-GM1b were clearly detectable in oxygenated rats compared to traces in the control and nonHBO animals. These results point at a functional role of gangliosides in liver growth regulation and reconstitution after PH combined with pre- and post-operative HBO treatment.     

In vitro stimulation of rat liver cells by homologous partial hepatectomy serum

Summary A low passage rat liver cell line demonstrated in vitro growth stimulation when cultured in the presence of serum of homologous, partially hepatectomized rats. After 4-day incubation a 3.25-fold increase in the cell population was observed in cultures supplemented with posthepatectomy serum at a dilution of 1∶10. No response was observed with sham-operated animal serum. Continous cultures of Chang human liver and Don hamster lung cells were not responsive to the posthepatectomy serum. The limitations of tetraphenylboron as a dispersing agent for primary rat liver cells are discussed. Supported by Grant 67-7 from the Illinois Division of the American Cancer Society.     

EGF responsiveness of hepatocytes after partial hepatectomy

We investigate the effect of EGF on IP₃ production, PLCγ phosphorylation, calcium transients in rat hepatocytes isolated in quiescent liver (G₀ phase of cell cycle) and at 4 h (G₁ phase of cell cycle) and 24 h (M phase of cell cycle) after partial hepatectomy. Our results show that EGF does not utilize IP₃ and calcium as its signal transduction molecules when the hepatocytes are in vivo stimulated to entry in the cell cycle. In particular the growth factor does not phosphorylate PLCγ and induces a decrease in IP₃ content. These data suggest that EGF utilizes different signal transduction to send information from receptor to nucleus during PH with respect to the quiescent liver.     

Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice

We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) α and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.     

Vascular endothelial growth factor and nitric oxide in rat liver regeneration

In this work we investigated the role of nitric oxide (NO) in the angiogenesis mediated by vascular endothelial growth factor (VEGF) during rat liver regeneration after two-thirds partial hepatectomy. Sham operated (Sh) and partially hepatectomized (PH) male Wistar rats were randomized in three experimental groups: control (treated with vehicle); pre-treated with sodium nitroprusside (SNP: 0.25 mg/kg body weight, i.v. at a rate of 1 ml/h) and pre-treated with the preferential iNOS inhibitor, aminoguanidine (AG, 100 mg/kg body weight, i.p.). Animals were killed at 5, 24 and 72 h after surgery. At 5 h post-surgery, NO production was estimated by EPR (Sh-Control: 37.65+/-10.70; PH-Control: 88.13+/-1.60(); Sh-SNP: 90.35+/-3.11(); PH-SNP: 119.5+/-12.10()(#); Sh-AG: 33.27+/-5.23, PH-AG: 36.80+/-3.40(#)) (p<0.05 vs Sh-Control; (#)p<0.05 vs PH-Control). At 24 h after PH, VEGF levels showed no difference between PH-Control and PH-SNP animals. However, after 72 h, VEGF protein levels in PH-SNP animals were found to be increased (above 300%) with respect to PH-Control. On the other hand, aminoguanidine (AG) pre-treatment blocked the rise of inhibition of NO generation and decreased VEGF expression. Our results demonstrated that NO plays a role in modulating VEGF protein expression after hepatectomy in rats.     

Carvacrol partially reverses symptoms of diabetes in STZ-induced diabetic rats

Little is known about the protective effects of carvacrol on the symptoms of streptozotocin induced diabetes in rats. Hence, this present study was designed to evaluate the protective effect of the strong antioxidant, carvacrol, on the symptoms of streptozotocin induced diabetes in rats. Carvacrol at the doses of 25 and 50 mg/kg body weight were orally administered to diabetic rats for a period of 7 days after the onset of diabetes. Food-water intake and body weight changes were daily recorded. Biochemical parameters such as serum glucose, insulin, total cholesterol, alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase were measured. Although treatment of diabetic rats with oral administration of carvacrol resulted in a slight reduction in serum glucose level and significant reduction in serum total cholesterol, alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase in comparison with diabetic control rats, there were no significant differences in serum insulin levels, food-water intake values and body weight changes. Despite the inadequacy of carvacrol on diabetes treatments, it was determined to have at least a partially protective role on liver enzymes.