Effect of antiandrogen flutamide on measures of hepatic regeneration in rats

Male rat liver undergoes a process of demasculinization during hepatic regeneration following partial hepatectomy. The possibility that antiandrogens might potentiate this demasculinization process and in so doing augment the hepatic regenerative response was investigated. Adult male Wistar rats were treated with the antiandrogen flutamide (2 mg/rat/day or 5 mg/rat/day subcutaneously) or vehicle for three days prior to and daily after a 70% partial hepatectomy. At various times after hepatectomy, the liver remnants were removed and weighed. Rates of DNA and polyamine synthesis were assessed by measuring thymidine kinase and ornithine decarboxylase activities, respectively. Hepatic estrogen receptor status and the activity of alcohol dehydrogenase, an androgen-sensitive protein, were measured. Prior to surgery, the administration of 5 mg/day flutamide reduced the hepatic cytosolic androgen receptor activity by 98% and hepatic cytosolic estrogen receptor content by 92% compared to that present in vehicle-treated controls. After hepatectomy, however, all differences in sex hormone receptor activity between the treatment groups were abolished. The rate of liver growth after partial hepatectomy in the three groups was identical. Moreover, hepatectomy-induced increases in ornithine decarboxylase activity and thymidine kinase activity were comparable. These data demonstrate that, although flutamide administration initially alters the sex hormone receptor status of the liver, these affects have no effect on the hepatic regenerative response following a partial hepatectomy.This work was supported by a grant from the NIAAA AA44205 and the Veterans Administration.     

Erythropoietin promotes hepatic regeneration after extended liver resection in rats

Background and Aim:  It has been proven in various animal studies that recombinant human erythropoietin (rHuEPO) protects renal, cardiac and neuronal, as well as hepatic, tissue from ischemia, and promotes regeneration of adult central nervous system neurons. To date, no data are available as to whether rHuEPO has the ability to stimulate liver regeneration after liver resection.
Methods:  Rats undergoing 70% or 90% hepatectomy received an intraportalvenous administration (i.p.) of rHuEPO prior to resection or a subcutaneous injection (s.c.) for 3 days postoperatively, control animals were treated with surgery and saline injection only. Regeneration capacity of remnant livers was studied over 7 days by histology and immunohistochemistry (Ki-67, proliferating cell nuclear antigen [PCNA]). Polymerase chain reaction was carried out to measure transforming growth factor β (TGF-β), hypoxia induced factor (HIF), signal transducing activator 3 and vascular endothelial growth factor.
Results:  Ten-day survival in rats undergoing 90% hepatectomy significantly increased in i.p.-pretreated animals. After 70% hepatectomy the mitotic index was significantly increased in both rHuEPO-treated groups. These data were confirmed by PCNA and Ki-67 expression, which was significantly increased in the treated groups 24 h and 2 days after liver resection. TGF-β and HIF mRNA both were upregulated in control animals 3 h after surgery.
Conclusion:  rHuEPO effectively increased liver regeneration in rats after 70% liver resection and enhanced survival after 90% hepatectomy. Thus, rHuEPO may increase the regenerative capacity after major hepatectomy.     

Effect of recombinant human growth hormone on age-related hepatocyte changes in old male and female Wistar rats

Aging induces changes in several organs, such as the liver, and this process might be due to damage caused by free radicals and inflammatory mediators. The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis shows a reduction with age, and this fact could be associated with some age-related changes. The aim of this study was to investigate the effect of GH administration on age-induced alterations in hepatocytes. Two and twenty two month-old male and female Wistar rats were used. Old rats were treated with human recombinant GH for 10 wk. At the end of the treatment, hepatocytes were isolated from the liver and cultured, and different parameters were measured in cells and medium. Plasma IGF-1 was also measured. Aging significantly decreased plasma IGF-1 in males. In females, plasma IGF-1 was also reduced, but not significantly. GH treatment restored plasma IGF-1 levels to values similar to young males. Aging was associated with a significant increase in lipid peroxidation (LPO), nitric oxide (NO), carbon monoxide (CO) and cyclic guanosyl-monophosphate (cGMP), as well as a reduction in adenosyl triphosphate (ATP) and phosphatidylcholine (PC) synthesis. GH administration partially prevented all these changes in males. In females, some of the parameters were significantly improved by GH (ATP, CO, cGMP), while others showed a tendency to improvement, although differences did not reach significance. In conclusion, GH administration could exert beneficial effects against age-related changes in hepatocytes, mainly in males.     

Effects of sildenafil citrate on hepatic function and regeneration in normal and alcohol-fed rats.

Sildenafil citrate is a potent inhibitor of specific phosphodiesterase-5, which mediates metabolism of intracellular second message -- cGMP. Sildenafil citrate has been widely used for erectile dysfunction in men. Moreover, it is known that men with liver diseases have higher rate of erectile dysfunction. Furthermore, it has been demonstrated that nitric oxide plays an important role in liver function and regeneration. The present study evaluates effects of sildenafil citrate on hepatic function and regeneration in normal and alcohol-fed rats. In normal rats sildenafil citrate has a trend to improve hepatic function after partial hepatectomy (PHx). Moreover, sildenafil citrate significantly reduces hepatic regenerative activity at the concentration of 5 mg/kg body weight. However, sildenafil had no effects on hepatic function and regeneration of alcohol-fed rats. In general, sildenafil citrate did not induce significant changes in hepatic function and regenerative activity after PHx in normal and alcohol-fed rats, except at concentration of 5 mg/kg sildenafil citrate significantly inhibit hepatic regeneration in normal rats.     

Mechanism of impairment on liver regeneration in elderly patients: Role of hepatic stellate cell function

Japan, along with most other countries in the world, is facing an increasingly aging population with a prolonged life expectancy. Concurrently, the need for medical intervention, including hepatectomy, has also increased for the elderly. Although surgical outcomes for older patients are reported to be comparable with those for younger patients, additional care in the selection of older patients for hepatectomy is considered necessary. Although the effect of aging on human liver regeneration is not fully understood, the regeneration of liver tissue after hepatectomy in elderly patients is shown to be generally worse than in younger patients and, to date, the mechanisms involved in the impairment of liver regeneration have not been fully clarified. Hepatic stellate cells (HSCs) are liver‐specific mesenchymal cells that play critical roles in liver physiology and fibrogenesis. Recent studies in liver regeneration have increasingly focused on HSCs rather than on hepatocytes, Kupffer cells, endothelial cells, or infiltrating immune cells and suggest that HSCs might play a critical role in liver regeneration. In this review, we summarize the mechanisms involved in the impairment of liver regeneration in elderly patients, especially focusing on HSCs. We also discuss how HSCs contribute to the impairment of liver regeneration.     

Effect of naked eukaryotic expression plasmid encoding rat augmenter of liver regeneration on acute hepatic injury and hepatic failure in rats

AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 μg/kg) was injected into the rats with acute hepatic injury intraven ously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl4 administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 μg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl4 after 4 h of CCl4 administration, respectively. Rats living over 96 h were considered as survivals. RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the most effective group after four times of injection of recombinant pcDNA3-ALR plasmid. The indexes of PCNA significantly increased in the recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The level of serum AST and ALT remarkably reduced in recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The results showed that the effect of 200 μg/kg recombinant pcDNA3-ALR plasmid in the rats with acute liver injury was stronger than that of 50 μg/kg pcDNA3-ALR DNA. The effect of intravenous injection of recombinant pcDNA3 ALR plasmid was better. After the rats with acute hepatic failure were treated with recombinant pcDNA3-ALR plasmid, the survival rate (40%) significantly increased in treatment groups compared to control group (15%, P<0.01). CONCLUSION: The ALR gene may play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and reducing level of AST and ALT in CCl4-intoxicated rats.     

Hepatic stimulator substance administration ameliorates liver regeneration in an animal model of fulminant hepatic failure and encephalopathy

Abstract: Aims/Background: Hepatic stimulator substance (HSS) is a liver‐specific growth factor implicated in hepatocellular proliferation and hepatoprotection in models of acute liver injury. In the present study, we examined the effect of exogenous HSS administration on liver proliferating capacity and survival outcome in an experimental animal model of fulminant hepatic failure (FHF) and encephalopathy, induced by repeated injections of thioacetamide (TAA) in rats. Methods: Fulminant hepatic failure was induced in adult male Wistar rats by three consecutive intraperitoneal injections of TAA (400 mg/kg of body weight), at 24 h time intervals. The animals received intraperitoneally either a saline solution or HSS (50 mg protein/kg of body weight), 2 h after the second and third TAA injections. The animals were killed at 6, 12 and 18 h post the last injection of TAA. Results: Levels of liver enzymes and urea in serum, blood ammonia values, liver histology, stage of hepatic encephalopathy and survival were statistically significantly improved in TAA‐intoxicated and HSS‐treated rats compared to TAA‐intoxicated and saline‐treated ones. Furthermore, HSS ameliorated liver regenerative indices – DNA biosynthesis, thymidine kinase activity and hepatocyte mitotic activity – in a statistically significant manner. Conclusions: Our data suggest the beneficial effect of HSS administration in this animal model of FHF and encephalopathy, supporting evidence for a possible use of HSS as supportive therapy, by increasing hepatocellular proliferation, in management of FHF.     

糖皮质激素联合生长激素对大鼠急性肝衰竭的保护作用及其机制初步探讨

目的研究糖皮质激素（氢化考的松琥珀酸钠,HCSS）联合生长激素（GH）对大鼠急性肝衰竭的影响.方法采用Wistar大鼠注射脂多糖（LPS）和D氨基半乳糖（D-GalN）制备急性肝衰竭模型,随机分为正常对照组、模型对照组、药物干预组及造模后4 h干预组,各组于末次给药6 h后处死大鼠,常规H-E染色观察大鼠肝脏病理变化,检测肝功能和血清TNF-α,IL-8,IL-6水平;分离培养大鼠库普否细胞（KC）,经LPS（10μg/ml）刺激,于刺激开始（0 h）和4 h后分别给药,并于0、2、4、8和24 h后收集上清,测定TNF-α.结果HCSS或HCSS联合GH预防给药可显著降低急性肝衰竭大鼠血清TBIL、ALT、AST和TNF-α、IL-8、IL-6水平,药物干预组较模型组肝细胞坏死有明显改善.LPS刺激2 h后,KC培养上清中TNF-α浓度开始上升并在4 h达高峰.在培养开始加用HCSS可明显降低TNF-α水平,而培养4 h后加用HCSS不能明显降低TNF-α水平.在培养开始加用GH可进一步升高TNF-α水平,而联合给予HCSS后可降低TNF-α水平.结论预防性给予大鼠药物干预可明显减轻肝损伤,而在造模4 h后使用HCSS则无明显效果.联合使用合理剂量的HCSS和GH仍能较好的减轻肝损伤.     

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.     

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.     

Effect of liver regeneration on malignant hepatic tumors

Liver regeneration after major surgery may activate occult micrometastases and facilitate tumor growth,leading to liver tumor recurrence.Molecular changes during liver regeneration can provide a microenvironment that stimulates intrahepatic tumor propagation through alterations in cellular signaling pathways,where activation and proliferation of mature hepatocytes,hepatic progenitor cells,non-parenchymal liver cells might favor both liver regeneration and tumor growth.This review highlights recent advances of tumor growth and development in the regenerating liver,possible mechanisms and clinical implications.     

Beneficial effects of Kampo medicine Inchin-ko-to on liver function and regeneration after hepatectomy in rats.

Aim: Inchin-ko-to (ICKT), Kampo medicine, is known to inhibit hepatocyte apoptosis as well as promote the secretion and excretion of bile. The aim of this study is to clarify the effects of ICKT on liver function and hepatic regeneration after massive hepatectomy in rats. Methods: Male Wistar rats received 2 g/kg ICKT from 3 days preoperatively and underwent 90% hepatectomy. Liver sections were stained using immunohistochemistry (hemeoxygenase-1 [HO-1], alpha-smooth muscle actin [SMA], and proliferating cell nuclear antigen [PCNA]). Results: The survival period was significantly prolonged, and the remnant liver/body weight ratio was significantly increased postoperatively in the ICKT group. The values of transaminase, total bile acid, and total bilirubin were significantly improved in the ICKT group. In the ICKT group, PCNA and HO-1 were strongly expressed early postoperatively, but the expression of alpha-SMA was weak. Conclusion: The preoperative administration of ICKT has been suggested to provide beneficial effects in promoting hepatic regeneration and preventing postoperative hepatic failure. The reduced activation of stellate cells may be involved in their mechanisms.     

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     

Effects of heparin on hepatic regeneration and function after partial hepatectomy in rats

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重组人肝再生增强因子对大鼠肝部分切除后肝再生的影响

目的观察原核表达的重组人肝再生增强因子（rhALR）对大鼠肝再生的影响。方法按Higgins方法进行大鼠34％肝切除。术后4-6h各实验组大鼠经腹腔注射rhALR剂量分别为50μg、100μg、200μg、400μg、800μg,对照组给生理盐水。术后30h杀鼠取肝,增殖核抗原（PCNA）免疫组化染色和HE染色,进行肝细胞核PCNA阳性细胞计数和有丝分裂计数。结果rhALR能促进肝部分切除后大鼠肝细胞的有丝分裂和细胞核PCNA的表达,并呈现一定的剂量依赖关系。结论rhALR能促进在鼠肝再生和肝细胞增殖。     

Effect of cyclosporine on liver regeneration after orthotopic reduced-size hepatic transplantation in the rat

These experiments were undertaken to study the effects of cyclosporine A (CsA) on liver regeneration after an isogeneic orthotopic reduced-size hepatic transplantation (RSHT) in rats. Male Wistar rats were treated with or without a daily injection of CsA beginning 24 hr before surgery and were subjected to a 68% partial hepatectomy. A isogeneic orthotopic reduced-size hepatic transplantation was performed in recipient rats pretreated with or without CsA. A daily injection of CsA was continued until the recipient rats were sacrificed. Animals were sacrificed at various time points (12, 24, 36, 48, and 72 hr) postoperatively. The incorporation of bromodeoxyuridine (BrdU) into the DNA of the remnant hepatocytes was evaluated by immunohistochemical staining with a monoclonal antibody against BrdU. CsA (10 mg/kg/day) significantly augmented BrdU incorporation into hepatocytes after hepatectomy. The maximum labeling index (LI) was observed at 24 hr after hepatectomy. In contrast, the maximum LI in the recipient rats not receiving CsA was seen at 36 hr after RSHT, and 10 mg/kg/day of CsA decreased the LI at 36 hr after RSHT. A lower dose of CsA (3 mg/kg/day), however, significantly increased the LI in the recipient rats (P<0.01), and it reached a peak at 24 hr after RSHT when compared to the transplant recipients not receiving CsA. The time course of the increase in the LI in the transplant recipient rats receiving 3 mg/kg/day of CsA was similar to that observed in the rats after hepatectomy. This dosage improved the delay in the reduced-size hepatic transplant LI reaching its peak. These findings suggest that after RSHT the liver graft is more sensitive to both hepatotrophic and hepatotoxic effects of CsA.This work was supported in part by Mika Fund.