Studies On Expression Of Uncoupling Protein 2 And Its Correlations With Proliferation And Function Of Hepatocytes In Regenerating Liver In Rats

Objectives: The liver has a great regeneration capacity following 70% partial hepatectomy. The regeneration of residual liver lobes starts in a few hours and lasts 5 to 7 days. The residual lobes enlarge to make up for the mass of the removed lobes. Liver regeneration is a complex process with many facets still unresolved. Uncoupling protein 2(UCP2) is a recently identified inner mitochondrial membrane anion carrier. The tissue distribution of UCP2 is more diverse in rodents. UCP2 catalyses proton leak when they are activated and protons can return to the matrix in a process that bypasses ATP synthase. It was suggested that the purpose of the uncoupling protein 2 might be to accurately decrease mitochondrial membrane potential and consequently prevent the excess reactive oxygen species (ROS) production. UCP2 are thought to curtail the release of oxygen free radical precursors from the electron transport chain by partially uncoupling ATP synthesis from oxygen consumption. Therefore, UCP2 may play a role in regulating mitochondrial production of ROS and energy metabolism. Hpatocytes of the adult rat liver do not express UCP2, but it might be induced to produce in hepatocytes such as after partial hepatectomy, and its function is not clear yet.In this study, we investigate the expression of uncoupling protein 2, and explore correlations with proliferation and function of the regenerating liver in hepatectomied rats.Methods: According to the method of Higgins and Anderson, two-thirds partial hepatectomy was performed on male Wistar rats(180±20g). The rats were divided randomly into two groups (n=5): sham hepatectomy(SH), partial hepatectomy(PH), PH groups included 24h,48h,72h and 96h after partial hepatectomy. The sham operation was done with the same procedures aforementioned except for liver resection. Micewere sacrificed at different time points(24,48,72 or 96 hours) and the liver remnants were removed, weighed, and either frozen at -70℃for studies. After centrifuging the blood serum were frozen at -4℃for studies. The liver tissues 0.5g was weighed for the biochemical assessment. Fresh mitochondria were used for the measurement of mitochondrial membrane potential and western blot analysis of UCP2. The mitochondrial membrane potential was measured in the presence or absence of 0.4 mM GDP. Paraffin slides were used for histological assessment and immunohistochemistry.Result:1. Liver remnant growth and the changes of histological in rats regenerating liver in rats.The growth of liver remnants was monitored up to 4 days after surgery. The liver weight/body weight ratio was decreased at 24 hours after partial hepatectomy (P < 0.05, compared with SH group). Afterward, the ratios went up. It approached nearest the normal at 96 hours. HE staining showed that the nuclear of hepatocytes were normal in SH group. The proliferation of cells has increased rapidly in the regenerating liver at 24 and 48 hours after PH with abundant nucleus division. The center veins were dilative and hepatocytes arranged loosely around it. Whereas the rearrangement of cells arranged was cordlike in the regenerating liver at 96 hours after PH, representing that the regenerating liver was telophase.2. Measurements of serum ALT, AST, albumin levels of liver tissues during liver regeneration in rats.The content of albumin markedly reduced at 24 hours after PH (P < 0.05, compared with SH group). Afterward, the content of albumin was increased. It reached the level of SH group at 96 hours after PH. The levels of AST and ALT were increased after PH. AST and ALT were increased 3 times and 6 times at 24h after PH comparing with SH group respectively (P < 0.05). Subsequently, it reduced and restored at 96 hours after PH.3. Changes of MDA of liver tissues in regenerating liver in rats.The contents of MDA were high in the regenerating liver after PH and reached peak value with 7 times at 24 hours after PH relative to SH group (P < 0.05). Then it decreased at 96 hours but still higher than SH group.4. Expression of UCP2 in regenerating liver in rats.The UCP2 expression was analyzed by immunnohistochemistry assessment. The hepatocytes in the liver of SH group have few UCP2 expression. UCP2 positivehepatocytes markedly increased at 24 hours after PH, and the UCP2 positive hepatocytes were seldom at 96 hours after PH. Western blot analysis was corresponded with Immunnohistochemistry analysis. The result showed that the expression of UCP2 was obviously increased at 24 hours after PH, and then decreased afterward. The expression of UCP2 at 48 hours is higher than SH group. It approached nearest the SH at 96 hours.5. The expression of proliferating cell nuclear antigen (PCNA) during liver regeneration in rats.The PCNA expression was analyzed by immunnohistochemistry method. The PCNA showed few expression in hepatocytes in SH group, but PCNA positive hepatocytes markedly increased at 24 hours after PH. At 48 hours the expression declined, and the positive hepatocytes were seldom at 96 hours after PH.6. Changes of mitochondrial membrane potential (MMP) and hepatic adenosine triphosphate(ATP) during liver regeneration in rats.Mitochondrial membrane potentials (MMP) were higher in the regenerating liver at the different time points after PH than the SH group. MMP was 211.19mv at 24h after PH (P < 0.001, compared with SH group), and 187.1mv at 48 hours after PH (P < 0.01, compared with SH group). It decreased but still higher than SH group at 96h. After the action of GDP, MMP markedly decline from 211.19mv to 181.70mv at 24 hours after PH. Hepatic ATP levels was the lowest during liver regeneration at 24 hours after PH (P < 0.001, compared with SH group), and then increased afterward. ATP level was higher than SH group at 96 hours (P < 0.001).7. Correlation of UCP2 expression, proliferation of cells, MMP during liver regeneration in ratsAccording to the correlation analysis, UCP2 expression was significant correlation with MMP (P < 0.001, r=0.813) and PCNA expression (P < 0.001, r=0.946). There was a statistically significant correlation between PCNA expression and MMP (P < 0.001, r=0.843).Conclusion:1. The decrease of liver function is due to partial hepatectomy (PH) and returns to the normality following the liver regeneration in rats.2. UCP2 expression is increased significantly in regenerating liver at 24h after PH, and decreases with the liver regeneration.3. There are a statistically significant correlation among UCP2 expression andPCNA expression and MMP.4. Increased UCP2 expression in the regenerating liver could limit the production of ROS by appropriately decreasing MMP.