Dimethylnitrosamine-Induced Liver Cirrhosis and Expression of Hepatocyte Growth Factor (HGF), its Receptor c-Met, and the Transforming Growth Factor (TGF)-beta1 in Sprague-Dawley Rats

BACKGROUND: Liver fibrosis and cirrhosis are the ultimate histologic consequences of chronic liver damage. Efforts have been made to study the mechanisms of cirrhosis and to discover effective therapeutic strategies. However, to date, no animal model reproduces the disease in man. The purpose of this work is to establish a model of DMN-induced liver cirrhosis for treatment of liver cirrhosis, to understand the basic characteristics of DMN-induced liver cirrhosis, and to confirm the expression of HGF, its receptor c-Met, and TGF-beta1 in Sprague-Dawley rats. METHODS: Five-week-old male Sprague-Dawley rats (n=56) were used for this study. Liver cirrhosis was induced in the rats by using DMN (1 ml/kg body weight, i.p.) given 3 consecutive days a week for 6 weeks. Changes in the portal vein pressure were measured by a venous catheter during the duration of the DMN-treatment. The levels of serum albumin, bilirubin, and ammonia were determined in a clinical laboratory by routive methods. Pieces of the median lobe were cut and fixed in 10% buffered neutral formalin, embedded in paraffin, and stained by hematoxylin-eosin (H&E) & masson-trichrome (M&T). Changes in the extracellular matrix were measured by image analysis and hydroxyproline content. Immunohistochemical staining of alpa-smooth muscle actin was performed to confirm the activation ofhepatic stellate cells. Northern blot analyses were performed to confirm the expression of HGF and TGF-beta1 and western blotting was performed c-Met, HGF receptor. RESULTS: Pressures in the portal vein were significantly increased during the DMN-treatment time (p<0.05). Biochemical parameters were significantly correlated with the progression of liver cirrhosis. H&E staining of 4-week DMN-treated rats demonstrated fibrous tissue bridging between the periportal and the pericentral areas with gradual widening of fibrous bands. Both the extracellular matrix measured by image analysis of the M&T staining and the hydroxyproline content rose continuously throughout the 6 weeks of DMN treatment. alpa-smooth muscle actin was observed in the stellate cells of DMN-treated rats. The northern blot analyses showed that the expression of HGF mRNA decreased with the progression of DMN-induced liver cirrhosis but that of TGF-beta1 mRNA did not. The western blot analyses showed that the expression of the c-Met receptor protein increased continuously, but the expression of HGF mRNA a decreased. CONCLUSION:The model of cirrhosis induced by chronic, discontinuous treatment with a low dose of DMN in rats was simple and predictable and displayed many of the features of human cirrhosis. The decrease in the expression of HGF mRNA may be responsible for the reduced hepatocyte regeneration in liver cirrhosis. The expression of the c-Met protein was related with the decreased expression of HGF. The exact significance of TGF-beta1 was not determined in this study.

Biodistribution of Intrasplenically Transplanted Hepatocytes in the Rat

Liver failure still constitutes a major cause of morbidity and death in patients with liver disease. Various potential treatments have been discussed to reduce liver failure-induced deaths. Recently, interest in hepatocyte transplantation(HT) has markedly increased, and HT has been tried for the treatment of liver failure. Both experimental and clinical data indicate that HT may be beneficial both for the support of an acutely devastated liver and for the correction of genetic disorders resulting in states of metabolic deficiency. For this purpose, the location of the HT has been suggested as a critical point. Therefore, we tried to study the biodistribution of hepatocytes in acute and chronic liver failure model in rats at 2 hours and 24 hours after the injection of 125I-labeled hepatocytes into the spleen. A ninety percent partial hepatectomy model and a dimethylnitrosamine-induced liver-cirrhosis model were used as the acute and chronic liver-failure models respectively. An SV-40 T-transfected immotilized cell line named L2A2 was transplanted intrasplenically. The biodistribution of the transplanted hepatocytes was similar in both the acute and the chronic liver-failure groups. In both groups, the biodistribution studies at 2 hours and 24 hours after intrasplenic transplantation demonstrated that the hepatocytes were localized predominantly in the spleen. However, intrasplenic retention of the transplanted hepatocytes progressively decreased with time. We conclude that methods for preventing the progressive loss of intrasplenic transplanted hepatocytes should be investigated.

Influence of Bile Duct Ligation on 54 Liver Regeneration in Rats

N/AWe evaluate the regenerating capacity of rat liver according to the severity and duration of the obstructive jaundice. Also we evaluate the regenerating capacity of hepatectomized rat liver according to the duration of the obstructive jaundice. The 10 week-old Sprague-Dawley rats were used. Common bile duct ligation and sequential partial hepatectomies were done. The regenerating capacity was measured by tritiated thymidine incorporation into rat liver DNA. The rate of incorporation of thymidine into DNA in the jaundiced livers increased significantly compared with that in the sham-operated liver on day 3.In jaundiced rats, the regenerating capacity after partial epatectomy decreased markedly without a return toward normal on day 3 and kept low thereafter. The duration of obstructive jaundice may be a key factor in the regeneration of rat liver after partial hepatectomy.

Changes in the Fxpression of DNA Synthesis According to Time Hepatocyte Growth Factor, Transforming Growth Factor-beta1 mRNA and c-met mRNA in Regenerating Rat Liver after Partial Hepatectomy during 24 Hours

The capacity of the liver to regenerate after a reduction in liver mass induced by surgical removal is quite remarkable. The most widely studied example of this phenomenon is accompanied by many investigators but the exact mechanism is not clear. Over the past decade, it has been demonstrated that growth factors play a major role in liver regeneration. Several growth factors such as epidermal growth factor, transforming growth factor(TGF), acidic fibroblast growth factor and hepatocyte growth factor(HGF) are involved in liver regeneration. Recently, HGF has aroused increasing interest due to its powerful mitogenic effect and multiple functions on various cell types. The receptor for HGF has recently been characterized as the product of the protooncogene c-met. We have examined the changes of synthesis of DNA and the expression of HGF mRNA and c-met mRNA that be involved in liver regeneration, after partial hepatectomy in the rat. Also we have examined the expression of TGF- mRNA that might be involved in inhibition of epithelial cell regeneration and differentiation of hepatocyte. Proliferative index was determined to use in vivo thymidine uptake. Nothern blot analysis used to assay HGF mRNA an TGF- mRNA. RNase protection assay used to assay c-met mRNA. The thymidine uptake of the remnant liver was abruptly increased after 18 hours, was maintained 24 hours. The nothern blot analysis showed that the expression of HGF mRNA and TGF- mRNA increased continuously within 24 hours. The RNase protection assay showed that the expression of c-met mRNA decreased continuously during one day after partial hepatectomy. The data presented here suggests that HGF is involved in liver regeneration and continuous decrease of c-met mRNA is phenomenon of receptor down-regulation. The increase of TGF- mRNA, well known as inhibitor of cell regeneration, suggests that TGF- is control mechanism of liver regeneration.