Influence of epidermal growth factor on liver regeneration after partial hepatectomy in mice

The role of epidermal growth factor (EGF) in liver regeneration was studied in mice after partial hepatectomy. Two weeks before partial hepatectomy, mice were sham-operated (control) or sialoadenectomized (removal of submandibular glands) to reduce plasma EGF levels. Sialoadenectomized mice showed low plasma EGF levels (29.7 +/- 6.6 pmol/l; mean +/- S.E.M.) compared with controls (66.0 +/- 8.3 pmol/l). After partial hepatectomy, sialoadenectomized mice were treated with or without a daily s.c. injection of 5 micrograms EGF and the rate of DNA synthesis in the regenerating liver was monitored by [125I]iododeoxyuridine uptake. Control mice showed a sharp peak of DNA synthesis at 48 h after partial hepatectomy while sialoadenectomized mice showed a delayed and broad peak at 84 h. Treatment of sialoadenectomized mice with EGF (5 micrograms/mouse per day) completely restored the pattern of DNA synthesis so that a sharp peak appeared at 48 h. The total liver DNA content of the control mice (79.1 +/- 2.5% of the preoperative level; mean +/- S.E.M.) was significantly (P less than 0.01) higher than that of the sialoadenectomized mice (65.2 +/- 3.0%) 3 days after partial hepatectomy, but this difference disappeared on day 7 when liver regeneration was almost completed in both groups. Treatment of sialoadenectomized mice with EGF increased total liver DNA content (78.2 +/- 2.9%) to that of control mice on day 3 after partial hepatectomy. In addition, normal mice showed a rapid increase in plasma EGF levels at 1-8 h after partial hepatectomy, whereas sialoadenectomized mice showed low plasma EGF levels throughout the course of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of epidermal growth factor on liver regeneration after partial hepatectomy in rats

The role of epidermal growth factor on liver regeneration after partial hepatectomy in rats was investigated. After a 70% hepatectomy in rats, the concentration of epidermal growth factor in portal venous blood was unchanged compared with unoperated controls. However, small amounts of epidermal growth factor could be identified in portal venous blood after intestinal instillation of epidermal growth factor. Brunner's glands and the submandibular glands secrete epidermal growth factor. Extirpation of Brunner's glands decreased liver regeneration, whereas removal of the submandibular glands had no effect on liver regeneration. Epidermal growth factor antiserum reduced liver regeneration significantly. Oral or s.c. administration of epidermal growth factor had no effect on liver regeneration, whereas epidermal growth factor enhanced the effect of insulin and glucagon on liver regeneration. The results suggest that endogenous epidermal growth factor participates in stimulation of liver regeneration after partial hepatectomy in rats. Epidermal growth factor given together with insulin and glucagon had a synergistic effect on liver regeneration which suggests that liver regeneration in the rat is controlled by multiple regulatory peptides.     

Stimulatory effect of epidermal growth factor on liver regeneration after partial hepatectomy in rats.

The effect of epidermal growth factor (EGF) on liver regeneration was investigated in rats subjected to partial hepatectomy. In a dose-response study EGF in doses of 6 and 24 nmol/kg x day increased liver regeneration after treatment for 48 h compared with controls, whereas a dose of 48 nmol/kg x day had no effect. In a subsequent study EGF, 6 nmol/kg x day, accelerated liver regeneration significantly after 36, 48, and 72 h of treatment. A possible influence of EGF on other hepatotrophic factors was investigated. No changes in the concentration of gastrin, insulin, or glucagon was found in portal venous blood. This study has shown that EGF in small doses can stimulate liver regeneration, whereas higher doses are ineffective. The study suggests that EGF should be regarded as a hepatotrophic factor.     

Treatment of cirrhotic rats with epidermal growth factor and insulin accelerates liver DNA synthesis after partial hepatectomy

Prevention of postoperative hepatic failure is important after hepatic resection. In patients with cirrhosis, impaired liver function and regenerative capacity after major hepatic resection are associated with increased morbidity and mortality. In this study, a combination of epidermal growth factor (EGF) and insulin were used as hepatotrophic factors in an attempt to stimulate DNA synthesis after 70% hepatectomy (HTX). Regenerative capacity was evaluated in normal and cirrhotic rat liver by measuring DNA synthesis in vivo. Micronodular liver cirrhosis was established by the simultaneous oral administration of CCl₄ and phenobarbital. Epidermal growth factor plus insulin was injected subcutaneously immediately after and 12 h after HTX or sham operation was performed. Rats were killed 24 h after the operation and liver regeneration was estimated by [³H]-thymidine incorporation into DNA as well as an autoradiographic nuclear labelling index. Hepatectomy increased [³H]-thymidine incorporation significantly in both normal and cirrhotic rats. In cirrhotic rats, [³H]-thymidine incorporation after HTX was significantly lower than in normal rats and administration of a combination of EGF and insulin after HTX enhanced [³H]-thymidine incorporation. In conclusion, DNA synthesis 24 h after HTX is decreased in cirrhotic rats compared with normal rats and EGF supplementation with insulin accelerates DNA synthesis in hepatectomized cirrhotic rats. The data suggest that administration of combinations of exogenous hepatotrophic factors may play a useful role in the treatment of cirrhotic patients undergoing major hepatic resection.     

NF-kappaB activation in non-parenchymal liver cells after partial hepatectomy in rats: possible involvement in expression of heparin-binding epidermal growth factor-like growth factor

BACKGROUND/AIMS: Nuclear factor (NF) B activation plays a critical role in the initiation of liver growth after partial hepatectomy (PH). However, the issue of where specifically NF-B is activated is unclear. We previously reported that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a hepatotrophic factor. The aims of this study were to identify NF-B-activated cells and to clarify their involvement in HB-EGF expression after PH. METHODS: Using rats, a two-thirds PH was performed, after which NF-B-activated cells and HB-EGF-positive cells were identified by Southwestern histochemistry or immunohistochemistry. NF-B binding activity and HB-EGF gene expression were analyzed in rat hepatocytes and non-parenchymal cells (NPC) in primary culture. RESULTS: NF-B was activated in Kupffer cells and sinusoidal endothelial cells prior to activation in hepatocytes. HB-EGF immunoreactivity was detected after NF-B activation in the sinusoidal cells from those localized in the periportal zones of hepatic lobules. HB-EGF gene expression by tumor necrosis factor was accompanied by an increase in NF-B binding activity in NPC but not in hepatocytes in primary culture, which was abolished by pyrrolidine dithiocarbamate, an inhibitor of NF-B activation. CONCLUSIONS; NF-B was activated in NPC prior to activation in hepatocytes. NF-B activation may be involved in HB-EGF expression in NPC after PH.     

Expressed genes in regenerating rat liver after partial hepatectomy

AIM: To reveal the liver regeneration (LR) and its control as well as the occurrence of liver disease and to study the gene expression profiles of 551 genes after partial hepatectomy (PH) in regenerating rat livers. METHODS: Five hundred and fifty-one expressed sequence tags screened by suppression subtractive hybridization were made into an in-house cDNA microarray, and the expressive genes and their expressive profiles in regenerating rat livers were analyzed by microarray and bioinformatics. RESULTS: Three hundred of the analyzed 551 genes were up- or downregulated more than twofolds at one or more time points during LR. Most of the genes were up- or downregulated 2-5 folds, but the highest reached 90 folds of the control. One hundred and thirty-nine of them showed upregulation, 135 displayed downregulation, and up or down expression of 26 genes revealed a dependence on regenerating livers. The genes expressed in 24-h regenerating livers were much more than those in the others. Cluster analysis and generalization analysis showed that there were at least six distinct temporal patterns of gene expression in the. regenerating livers, that is, genes were expressed in the immediate early phase, early phase, intermediate phase, early-late phase, late phase, terminal phase. CONCLUSION: In LR, the number of down-regulated genes was almost similar to that of the upregulated genes; the successively altered genes were more than the rapidly transient genes. The temporal patterns of gene expression were similar 2 and 4 h, 12 and 16 h, 48 and 96 h, 72 and 144 h after PH. Microarray combined with suppressive subtractive hybridization can effectively identify the genes related to LR.     

Von Willebrand factor in plasma and in liver tissue after partial hepatectomy in the rat

BACKGROUND/AIMS: Von Willebrand factor (vWf) is found in high levels in plasma of patients with acute and chronic liver disease. The role of vWf in liver injury and repair is unknown. We studied the effect of liver mass and remodeling on plasma and tissue vWf after partial hepatectomy. METHODS: Rats were sacrificed postoperatively at intervals ranging from 60 min to 5 days, and vWf plasma levels were measured by enzyme-linked immunosorbent assay, using rabbit anti-human vWf, and by immunoperoxidase on cryosections, using rabbit anti-vWf/factor VIII. Northern blot hybridization was prepared with a complementary DNA specific to human vWf. RESULTS: vWf plasma levels increased early after sham operation and after 70% partial hepatectomy. The highest levels were reached at 24 h, remaining high for 5 days. Immunostaining showed intense staining of sinusoidal lining cells 4 h after partial hepatectomy, remaining so for 5 days. Non-significant changes in overall liver messenger RNA expression of vWf were seen over 5 days in sham operation and partial hepatectomy. CONCLUSIONS: After partial hepatectomy, plasma vWf is increased, probably due to both acute-phase reaction and decreased degradation. An increase in sinusoidal vWf immunostaining may suggest a role for this factor in tissue remodeling.     

Plasma-kallikrein clearance during liver regeneration after partial hepatectomy in the rat

Abstract: Aims/Background: The liver clears circulating plasma-kallikrein through a receptor-mediated endocytosis process: an initial fast phase is followed by a slow exponential phase. Methods: To determine whether the clearance rate of plasma-kallikrein is affected during liver regeneration, we perfused isolated rat livers with rat plasma-kallikrein (rPK) at 0, 1, 2, 3 and 7 days after partial hepatectomy or sham operation. Results: Liver regeneration was followed by the expression of the proliferating-cell nuclear antigen (PCNA) labeling index. The serum concentration of α₂-macroglobulin, an acute phase protein in rats, was measured. At day 1, the fast phase of rPK clearance rate increased in hepatectomized rats when compared with day 0 (4.9±0.4 and 3.7±0.4 mU/g liver · min, p<0.05). However, at day 2, the rPK fast phase clearance rate dropped significantly (2.6±0.2, p<0.05), when compared with day 1. No difference was found among the sham groups at different days of hepatectomy. These changes seem to be independent of the acute phase reaction. The regenerative liver weight increased continuously during the observation period. PCNA expression increased significantly after hepatectomy, with maximal PCNA-labeling indices at days 1 and 2, declining thereafter. Conclusion: The rPK fast phase clearance rate changes during liver regeneration, with a zenith occurring when PCNA labeling index is maximal (day 1) and a nadir occurring at the mitotic phase (day 2).     

Gene expression profile in the regenerating rat liver after partial hepatectomy

BACKGROUND/AIMS: When a loss of hepatic mass occurs, the expression of a large number of genes is either induced or altered, accompanying hepatocyte proliferation. In the present study, we made an in-house cDNA microarray containing 4608 elements (Liver chip), and analyzed extensively gene expression profiles of the regenerating liver after 70% partial hepatectomy (PHx) in rats. METHODS: RNAs were prepared from three rat livers at each time point (taken at 0, 6, 12, 18, 24, 48, 72 h, and 1 week after PHx). Using the liver chip, we performed large-scale analysis of gene expression during liver regeneration. Elements either up- or down-regulated more than twofold at one or more time points were selected. RESULTS: Among the 4608, 382 were identified. Using cluster analysis, we found great similarity between gene-expression profiles at 12 and 18 h after PHx as well as between 48 and 72 h after PHx. We also found that there are at least six distinct temporal patterns of gene expression in the regenerating rat liver after PHx. CONCLUSIONS: These results indicated that microarray analysis is a powerful approach for monitoring molecular events in the regenerating liver.     

Stimulation of DNA synthesis in hepatocytes by Kupffer cells after partial hepatectomy

The role of Kupffer cells during reparative regeneration of rat liver was investigated with an in vitro experimental model. Conditioned media from primary cultures of Kupffer cells isolated from intact and regenerating liver were added to primary cultures of hepatocytes, and [3H]thymidine incorporation into DNA was studied. Kupffer cell-conditioned media from intact liver and regenerating remnant liver significantly stimulated DNA synthesis in hepatocytes as compared with control media (p less than 0.05). Moreover, the stimulating activity of Kupffer cells prepared from regenerating liver at 6 and 12 hr after partial hepatectomy was significantly higher than that of Kupffer cells from untreated rats (p less than 0.05). The activity was found in serum-free conditioned media. This stimulating activity exponentially increased as the increase of the number of the cultured cells, indicating that the stimulating activity was released directly by cultured Kupffer cells. These results suggest that Kupffer cells stimulate DNA synthesis in hepatocytes by producing and releasing certain factor(s) at an early stage of liver regeneration after partial hepatectomy.     

生长激素对鼠部分肝切除术后肝再生影响

目的　探讨生长激素对 70 %肝切除后肝再生的影响。方法　 60只SD大鼠随机分为对照组及生长激素组 ,按Higgins方法行 70 %肝切除术 ,术后给药并分批于术后 6、2 4、48、72、96h处死 ,作如下比较 :①残肝肝重 ;②增殖细胞核抗原 (PCNA)标记指数 ;③图像定量分析法测量PCNA阳性产物面积及灰度值。结果　与对照组比较 ,生长激素组残肝肝重、PCNA标记指数、PCNA阳性产物面积在术后均显著增高 (P <0 .0 5 ) ,而灰度值则显著降低 (P <0 .0 5 )。结论　生长激素具有强烈促进肝细胞增殖和刺激肝再生的作用