Liver regeneration after partial hepatectomy in rats with defective bilirubin conjugation or biliary excretion

The role of conjugated bilirubin in liver regeneration after partial hepatectomy (PH) was studied in Gunn rats, in transport-mutant (TR^–) rats, and in rats with extrahepatic biliary obstruction. Ornithine decarboxylase (ODC) and thymidine kinase (TK) activities in liver homogenates and immunohistochemistry ofin vivo bromodeoxyuridine (BrdU) incorporation in hepatic DNA were followed as regeneration parameters at 24 and 48 hr after PH. The results relative to TK activity and BrdU incorporation were consistent with significantly delayed hepatic DNA synthesis in Gunn rats in comparison to control Wistar and TR^– rats. This delay in DNA synthesis was not reflected in the hepatic ODC activity. After one week of complete common bile duct obstruction (CBO), an increased TK activity and BrdU incorporation was seen. PH following CBO resulted in a further increase in ODC activity and BrdU incorporation. TK activity did not change, however. These data relative to the regulation of hepatic DNA synthesis after PH in Gunn rats and in rats with extrahepatic biliary obstruction suggest a possible stimulatory role for conjugated bilirubin in hepatic regeneration; however, the normal hepatic DNA synthesis in TR^– rats studied before PH and the subnormal DNA synthesis seen 24hr after PH in TR^– rats and in rats with CBO indicate that conjugated bilirubin does not stimulate hepatic DNA synthesis.Presented at the Proceedings of the International Meeting on Normal and Neoplastic Growth in Hepatology, Bari, Italy, June 1989.     

Liver-targeted doxorubicin: effects on rat regenerating hepatocytes.

BACKGROUND/AIMS: The conjugate of doxorubicin (DOXO) with lactosaminated human albumin (L-HSA) has the potential of improving DOXO efficacy in the treatment of hepatocellular carcinomas (HCCs) expressing the asialoglycoprotein receptor (ASGP-R). In view of an adjuvant chemotherapy with L-HSA-DOXO after the surgical removal of the tumour, in the present experiments we verified whether DOXO accumulation produced by the conjugate can impair the liver regeneration following hepatic resection in non-cirrhotic liver. METHODS: Using saline-injected hepatectomised rats as controls, we studied the effects of the conjugate on the ultrastructure of regenerating hepatocytes and evaluated [3H]thymidine incorporation, mitotic index and rate of DNA recovery in the liver remnant. RESULTS: L-HSA-DOXO caused a selective drug accumulation in liver remnant, with low DOXO levels in extra-hepatic tissues. It did not change the ultrastructure of hepatocytes and did not increase serum alanine aminotransferase. It decreased [3H]thymidine incorporation and mitotic index, causing a moderate delay in hepatic DNA recovery. CONCLUSIONS: The experiments indicate a substantial resistance of rat regenerating hepatocytes to high intracellular concentrations of DOXO. They support the possibility of using L-HSA-DOXO in an adjuvant chemotherapy after the surgical removal of HCCs which maintain the ASGP-R.     

Regulatory effects of corticosterone on ornithine decarboxylase activity during liver regeneration in rats

AIMS: The regulation of ornithine decarboxylase (ODC) gene expression and enzyme activity by corticosterone during rat liver regeneration induced by partial hepatectomy (PH) was evaluated. METHODS: Bilateral adrenalectomies were performed on ether-anesthetized rats 3 days before PH. Corticosterone in sesame oil was injected subcutaneously to adrenalectomized rats. ODC mRNA, ODC protein and enzyme activity were detected by in situ hybridization, Western blot and high performance liquid chromatography (HPLC), respectively. RESULTS: The ODC mRNA levels, protein accumulation and enzyme activity were lower in the intact liver compared to the regenerating liver. After PH, mRNA levels were remarkably enhanced in all groups and peaked at 5 h post-PH, and presented a persistent increase only in adrenalectomy rats during the regeneration process. Corticosterone treatment resulted in a dose-dependent decrease in ODC mRNA content after 5 h post-PH. ODC protein accumulation in adrenalectomy rats was higher than that in sham-adrenalectomy rats, but it decreased in corticosterone-treated (10 mg/kg) rats until 24 h post-PH, with a strong decline seen in 40 mg/kg corticosterone-treated rats. ODC activity was rapidly promoted, and the highest levels were observed at 6 h after PH in all groups. After corticosterone treatment, the activities declined significantly at 6 h post-PH, with the lowest value found in the 40 mg/kg group. CONCLUSIONS: Corticosterone treatment results in dose-dependent decreases in ODC mRNA and enzyme protein both in the intact liver and the regenerating liver. The change in ODC activity is partially related to alterations of ODC mRNA and protein accumulation.     

Effect of granulocyte-macrophage colony-stimulating factor on hepatic regeneration after 70% hepatectomy in normal and cirrhotic rats

BackgroundPost-hepatectomy liver insufficiency is one of the most serious postoperative problems and its prevention is important after major hepatic resection, especially in the cirrhotic liver. Some growth factors and cytokines appear to play important roles in liver regeneration. In the present study we have investigated the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on hepatic regeneration after 70% partial hepatectomy (PH) in cirrhotic and non-cirrhotic rats.MethodsA rat model of liver cirrhosis was prepared using thioacetamide (TAA) (a dose of 20 mg/100 g body w, intra-peritoneally) on three days a week for 12 weeks. Adult male rats were divided into four groups:Group 1 (n=10) no cirrhosis and no GM-CSF; Group 2 (n=10) no cirrhosis and GM-CSF; Group 3 (n=10) cirrhosis and no GM-CSF; and Group 4 (n=10) cirrhosis and GM-CSF. All the rats underwent a 70% hepatectomy, and GM-CSF was administrated immediately after operation in Groups 2 and 4. On postoperative days 2 and 7, fresh samples from the remnant liver were obtained to evaluate its regenerative capacity.The liver regenerative process was estimated by DNA synthesis, using flow cytometry.ResultsProliferation index (PI) of hepatocytes at 48 h was higher in Group 4 rats than Group 3 rats (p<0.05). On postoperative day 7, PI was elevated in Group 3 rats compared with Group 4 rats, but this difference was not statistically significant. In non-cirrhotic rats given GM-CSF, PI was increased compared with Group 1 rats at day 2 (p<0.05), but not at day 7.ConclusionsThe findings suggest that the proliferative capacity of liver cells is impaired and delayed after 70% PH in cirrhotic rat liver. GM-CSF administration might enhance the liver PI in both normal and TAA-induced cirrhotic rats.     

Effect of Acute Ethanol Exposure on Hepatic Stimulator Substance (HSS) Levels During Liver Regeneration: Protective Function of HSS

Ethanol administration in rats induces liver damage and suppression of liver regeneration. To further understand the underlying mechanism, we investigated the effects of ethanol on hepatic stimulator substance (HSS) levels during liver regeneration caused by partial hepatectomy. The hepatotrophic action of HSS to ethanol-treated partially hepatectomized rats was also examined. Rats received repetitive ethanol or saline doses beginning 1 hr prior to 70% partial hepatectomy (PH), and the animals were killed at 16, 24, 32, 40, 48, and 60 hr after PH. Our results showed that ethanol inhibited hepatic regenerative capacity and prolonged liver regenerative process. HSS biological activity in ethanol-administered rats peaked at 48 hr after PH, in contrast to saline-treated ones where activity peaked at 24 hr. Additionally, exogenous HSS administration to ethanol-treated partially hepatectomized rats increased liver proliferating capacity and suppressed the elevation of serum ALT activity. These results showed that ethanol modifies the time course of HSS biological activity during the regenerating process. The observed suppression of HSS activity at 24 hr after PH was in relation with a reduction of DNA synthesis. Exogenous administration of HSS to ethanol-treated partially hepatectomized rats restored DNA synthesis and ameliorated serum AST levels, indicating that HSS could be used in the treatment of ethanol-induced hepatic failures.     

Effects of Bicyclol on Liver Regeneration After Partial Hepatectomy in Rats

Bicyclol is a synthetic antihepatitis drug with antioxidative property. The present study was performed to investigate the effect of bicyclol on liver regeneration after partial hepatectomy in rats. Bicyclol (300 mg/kg) was given to rats subjected to 70% hepatectomy three times before operation. At 6, 24, and 48 h after resection, samples were collected for the measurement of serum alanine aminotransferase (ALT), total bilirubin (TBil), hepatic glycogen, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH). Moreover, liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling, proliferation index, and histopathological examination were evaluated at 48 h after hepatectomy. As a result, bicyclol significantly increased regeneration rate, mitotic index (MI), PCNA labeling index, and proliferation index in PH rats. Additionally, bicyclol remarkably inhibited the elevation of serum ALT and TBil levels, alleviated the formation of liver MDA, restored impaired antioxidant SOD and GSH, increased hepatic glycogen content, and also attenuated hepatic vacuolar degeneration. These results suggested that bicyclol had a beneficial effect on liver regenerative capacity of the remnant liver tissue after hepatectomy, probably due to its antioxidative property.     

Evaluation of safety for hepatectomy in a novel mouse model with nonalcoholic-steatohepatitis

AIM To investigate whether the liver resection volume in a newly developed nonalcoholic steatohepatitis(NASH) model influences surgical outcome.METHODS For establishment of a NASH model, mice were fed a high-fat diet for 4 wk, administered CCl_4 for the last 2 wk, and administered T0901317 for the last 5 d. We divided these mice into two groups: A 30% partial hepatectomy(PH) of NASH liver group and a 70% PH of NASH liver group. In addition, a 70% PH of normal liver group served as the control. Each group was evaluated for survival rate, regeneration, apoptosis, necrosis and DNA expression after PH.RESULTS In the 70% PH of NASH group, the survival rate was significantly decreased compared with that in the control and 30% PH of NASH groups(P 0.01). 10 of 32 mice in the NASH 70% PH group died within 48 h after PH. Serum aspartate aminotransferase(AST) levels and total bilirubin(T-Bil) in the NASH 70% PH group were significantly higher than the levels in the other two groups(AST: P 0.05, T-Bil: P 0.01). In both PH of NASH groups, signaling proteins involved in regeneration were expressed at lower levels than those in the control group(P 0.01). The 70% PH of NASH group also exhibited a lower number of Ki-67-positive cells and higher rates of apoptosis and necrosis than the NASH 30% PH group(P 0.01). In addition, DNA microarray assays showed differences in gene expression associated with cell cycle arrest and apoptosis.CONCLUSION The function of the residual liver is impaired in fatty liver compared to normal liver. A larger residual volume is required to maintain liver functions in mice with NASH.     

Effects of octreotide on liver regeneration and tumour growth in the regenerating liver

The ability of the liver to regenerate following resection is remarkable. However, there is evidence to suggest that tumour growth within the regenerating liver is significantly increased. As octreotide (a synthetic analogue of somatostatin) inhibits the growth and development of hepatic tumour in rats, we have investigated its effects on liver regeneration, liver blood flow, hepatic reticuloendothelial system activity and tumour growth in the rat following partial hepatectomy (PH). Octreotide significantly inhibited liver regeneration in the rat 1 and 2 weeks following PH when compared with controls (regeneration index: 1.0 and 1.14 cf. 1.14 and 1.4, respectively). There was no significant difference in hepatic arterial or portal venous blood flow following PH in control or octreotide-treated rats. However, portal pressure was significantly reduced in octreotide-treated rats. Hepatic reticuloendothelial system activity was significantly increased in octreotide-treated rats compared with control animals 1 and 2 weeks after hepatectomy (uptake of radiolabelled technetium-99m albumin colloid: 2.2 and 3.9 cf. 1.6 and 1.9). The growth of both HSN (fibrosarcoma) and K12-Tr (colonic adenocarcinoma) cells in the regenerating liver was significantly decreased by octreotide treatment compared with controls (median percentage hepatic replacement: HSN control 71.3%, Octreotide 8.4%, K12-Tr Control 38.3%, Octreotide 4.5%). The results of the present study demonstrate that octreotide inhibits both liver regeneration and tumour growth following PH, possibly via a similar mechanism.     

Enhancement of Liver Regeneration by the Association of Hyptis pectinata with Laser Therapy

Since new molecules that normally would accelerate regeneration can also be potentialized by light, the use of new substances combined with laser therapy seems to be a natural type of experiment. Therefore, the purpose of this study was to assess the effects of Hyptis pectinata leaves on liver regeneration after partial hepatectomy (PH) associated with laser therapy. Twenty-four rats were divided into four groups—PH(control), PHL (laser therapy), PH200 (200 mg/kg of Hyptis pectinata), and PHL200 (200 mg/kg of the plant and laser)—which were submitted to 67% hepatectomy. Laser treatment consisted of focusing the light on the remaining liver after hepatectomy. The data analyzed were serum levels of aminotransferases, liver regeneration, and mitochondrial function. Group PH200 showed a statistically significant decrease in AST levels, and PHL200 disclosed an augmentation in ALT levels. The liver regeneration index was significantly increased in group PHL200. Concerning liver mitochondrial respiratory assay, groups PH200 and PHL200 showed lower state 3 levels than groups PH and PHL. Group PHL showed an increase in state 4 levels and a reduction in membrane potential and RCR. The present study shows that the association of the aqueous extract of Hyptis pectinata leaves at 200 mg/kg with intraoperative laser therapy can stimulate liver regeneration and cause a reduction in liver mitochondrial respiratory function without altering its phosphorylative activity.     

Liver regeneration is impaired by FK778 in partially hepatectomized rats, while supplemental uridine restores both liver growth and hepatocyte proliferation

Aim:  The impact of mandatory immunosuppression on liver regeneration after segmental liver transplantation is of clinical importance. FK778, a novel immunosuppressant, inhibits pyrimidine biosynthesis and prevents rejection after organ transplantation in a dose-dependent manner. We investigated the effect of FK778 at a highly effective dose on liver regeneration in a small animal model.
Methods:  Inbred Lewis rats were subjected to 70% partial hepatectomy (PH) and treated with saline ( n  = 28), uridine ( n  = 16), FK778 alone ( n  = 28) or in combination with uridine ( n  = 16). FK778 was given intravenously daily at a dose of 25 mg/kg bodyweight (bw) and uridine was given daily intraperitoneally at a dose of 250 mg/kg bw. Liver bodyweight ratio (LBR), hepatocyte proliferation index (PI), blood chemistry and morphological analysis were incorporated. PI was determined by Ki-67 immunostaining. De Ritis ratio was calculated to assess the extent of liver damage.
Results:  In FK778-treated animals PI was decreased at 24 h and 72 h and LBR was lower at 48 h and 72 h ( P  < 0.05) after the PH. In addition, morphological analysis showed confluent central lobular necrosis at 72 h in four of seven animals. Uridine supplementation restored PI, LBR and the de Ritis ratio in FK778-treated animals and no confluent necroses were observed.
Conclusion:  FK778 is antihepatotrophic as well as antiproliferative during rat liver regeneration. Both liver growth and hepatocyte proliferation are completely restored by supplementation with uridine. In addition, supplemental uridine markedly reduces the severity of morphological abnormalities consistent with FK778 toxicity.     

熊脱氧胆酸促进肝脏部分切除后肝细胞再生

目的 探讨熊脱氧胆酸（ｕｒｓｏｄｅｏｘｙｃｈｏｌｉｃ ａｃｉｄ，ＵＤＣＡ）对胆道梗阻肝脏部分切除（ＰＨ）后肝细胞再生的影响。方法Ｗｉｓｔａｒ大鼠随机分为正常７０％肝部分切除组（Ｎ－ＰＨ）、胆道梗阻２周７０％ＰＨ组（ＢＤＯ－ＰＨ）、ＢＤＯ—ＰＨ ＵＤＣＡ治疗组及ＢＤＯ—ＰＨ生理盐水治疗组。观察肝组织学改变，检测７０％ＰＨ后肝细胞ＢｒｄＵ标记、肝内肝细胞生长因子（ＨＧＦ）及其受体Ｍｅｔ ｍＲＮＡ表达。结果 ＵＤＣＡ治疗能促进胆道梗阻后肝功能好转并减轻肝组织学病变；ＵＤＣＡ治疗组大鼠７０％ＰＨ后肝内ＨＧＦ／Ｍｅｔ ｍＲＮＡ高峰表达值均高于ＢＤＯ—ＰＨ组（Ｐ ＜ ０．０５），肝细胞 ＢｒｄＵ高峰标记指数（５９．３９±１０．８２）％高于 ＢＤＯ—ＰＨ组肝细胞 ＢｒｄＵ高峰标记指数（３６．２２±８．３７％（ｔ＝４．１４９，Ｐ＜０．０１），而与Ｎ－ＰＮ组肝细胞ＢｒｄＵ高峰标记指数（６８．６４±１１．２６％）％相比差异无显著性（ｔ＝１．４５１，Ｐ＞０．０５）。结论 ＵＤＣＡ通过缓解胆道梗阻后肝组织损害并上调７０％ＰＨ后肝内ＨＧＦ／Ｍｅｔ ｍＲＮＡ表达，从而促进胆道梗阻肝脏部分切除后肝细胞再生。     

Role of the oestrogen receptor in liver regeneration in the male rat

Partial hepatectomy in male rats results in raised serum oestrogen levels, nuclear binding of oestrogen receptor (ER) and feminization of certain aspects of hepatic metabolism. It has been proposed that these changes may have an important role in liver regeneration. The present study was performed to ascertain the effects of the oestrogen agonist diethylstilbestrol (DES), 2 mg/kg, and the oestrogen antagonist tamoxifen (TAM), 2 mg/kg, on liver regeneration induced by partial hepatectomy in the male rat. Regenerative activity was determined by incorporation of [³H]-thymidine into hepatic DNA as well as by measurement of liver remnant weight. Following partial hepatectomy, there was a trend towards an increase in liver remnant weight at 24 h in rats treated with DES (DES, 5.95 ± 1.52 g; vehicle, 4.87 ± 0.66 g; P= 0.06) though by 48 h no effect was found. Tamoxifen treatment did not significantly affect liver weight at 24 h but by 48 h there was a highly significant reduction in liver remnant weight (TAM, 5.41 ± 0.85 g; vehicle, 7.31 ± 1.43 g; P < 0.001). Neither DES nor TAM treatment influenced liver regeneration as determined by [³H]-thymidine incorporation into hepatic DNA. We conclude that pharmacologic manipulation of oestrogens does not influence the initiation of the regenerative process but that oestrogen may facilitate later phases of hepatic growth.     

Vascular endothelial growth factor does not improve liver regeneration and survival after 90% subtotal liver resection

Aim: Vascular endothelial growth factor (VEGF) has been shown to stimulate liver regeneration after 70% partial hepatectomy (PH). It is unclear, however, whether exogenous administration of VEGF can also be used to improve liver regeneration and survival after 90% subtotal liver resection. The aim of this study was to determine the effect of exogenous and endogenous VEGF after 90% subtotal hepatectomy (SH). Methods: Rats were subjected to 90% SH and treated with VEGF, anti-VEGF or NaCl. Postoperatively (3 h - 5 days) liver body weight ratio (LBR), hepatocyte proliferation and biochemical markers were assessed. ELISA was performed to measure protein levels for VEGF. Gene expression was determined by customized cDNA arrays and quantitative RT-PCR. Results: Administration of VEGF did not enhance LBR or hepatic proliferation, or reduce the serum parameters. VEGF levels were the highest in VEGF-treated animals. The overall survival after 90% SH reached 78% in VEGF-treated animals, but did not differ significantly from that of anti-VEGF or NaCl-treated animals (74% and 75%, respectively). Gene expression analysis showed a modulation of anti-apoptotic and cell cycle control genes that was independent of VEGF. Conclusions: In contrast to PH, liver regeneration and survival after SH cannot be modulated by VEGF. This indicates that the relevant mechanisms that stimulate liver regeneration after hepatectomy at least partially depend upon the extent of liver resection.     

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.     

Alteration of bile acid metabolism in two-thirds hepatectomized rat

Bile acid metabolism after two-thirds partial hepatectomy (PH) in rat was studied. Bile acid kinetics (i.e. synthesis rate and pool size) were determined by wash out method combined with gas liquid chromatography, and serum bile acids by gas liquid chromatography-mass spectrometry. Serum bile acid concentration was highest on the third day after PH, as the liver regeneration progressed but it gradually decreased with increasing cholic acid (CA)/chenodeoxycholic acid (CDCA), reflecting impaired hepatic uptake of bile acids and/or cholestasis during the early post-hepatectomy period. Predominance of CA in bile acid synthesis, pool, and biliary secretion was also found. On the third day after PH, liver weight recovered to 66% of the control value, but enhancement of bile acid synthesis was not observed. Consequently, pool size remained at 50% of control. On the seventh day, synthesis of bile acid, especially of CA, was enhanced and pool size and liver weight returned to 68 and 72% of the respective control values. Bile acid synthesis was returned to the control value on the fourteenth day with concomitant restoration of liver weight and bile acid pool size. These changes in bile acid kinetics parallel the events during hepatic regeneration after PH.     

Type beta transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat.

Type beta transforming growth factor (TGF-beta), a factor produced by many cell types, is a potent inhibitor of hepatocyte DNA synthesis in vitro. To determine whether TGF-beta can influence hepatocyte proliferation in vivo, its effects were examined on the regenerative response of liver to partial hepatectomy (PH) in the rat. Porcine platelet-derived TGF-beta 1 (0.5 micrograms), administered intravenously at the time of PH and 11 hr later, reduced the fraction of hepatocytes engaged in DNA synthesis 22 hr after PH by 67% and inhibited the rate of hepatic [3H]thymidine incorporation by 50%. TGF-beta 2 produced a similar effect. A single dose of 0.5 micrograms of TGF-beta 1 given 11 hr after PH reduced liver [3H]thymidine incorporation by 32%; 4.5 micrograms of TGF-beta 1 or TGF-beta 2 inhibited DNA synthesis by 88% and the labeling index by 86%. Although sensitive to TGF-beta administered 11 hr after PH, late in the G1 phase of the cell cycle, a single dose of 0.5 micrograms given at the time of PH did not significantly influence DNA synthesis 22 hr after PH. The inhibitory effects of TGF-beta were transient; rats treated with two 0.5-microgram doses of TGF-beta at 0 and 11 hr had completely restored their original liver DNA mass 8 days after PH. Administration of 0.5 microgram of either TGF-beta 1 or TGF-beta 2 every 12 hr for 5 days failed to suppress the recovery of hepatic DNA mass. However, the nuclear labeling index of the TGF-beta-treated animals was significantly higher than that of the controls. There was no evidence of cytotoxicity from TGF-beta, as determined by liver histology and plasma concentrations of glucose, insulin-like growth factor I, and two hepatic enzymes. Thus, TGF-beta 1 and TGF-beta 2 reversibly inhibit the proliferative response of liver to PH and may be important in the modulation of normal liver growth and repair.