Study On Dynamic Change And UCP2 Of Protection Against Carbon Tetrachloride Poisoning In Regenerating Liver In Rats

Objectives:The liver has the potent capacity to regenerate. It is a compensated regeneration of liver to damage or lost liver cell mass. During liver regeneration after 70% partial hepatectomy (PH), quiescent hepatocytes enter the cell cycle after several hours and finish after 7¹0 days. Regenerating liver has an important physiological characteristic that is the capacity of potent self-protection against damage. It is known that regenerating liver possess the protection against many hepatic toxins such as carbon tetrachloride (CCl₄). But the self-protection against damage of regenerating liver is unclear. If we can observe the physiological and biochemical indicators of different time points in regenerating liver, the mechanism of self-protection against damage will be more understood. The well known of the regenerating liver would help in understanding the hepatic physiology and the mechanism of liver regeneration, giving an experimental gist for clinical liver disease, partial hepatectomy and transplantation. Therefore, our experiment investigated the change of protection against carbon tetrachloride damaging in different time points regenerating liver.Uncoupling protein 2 is a new member of the anion carrier family localized in the inner mitochondrial membrane. It distributes ubiquitously in tissues of the rodent. The exact physiological function of UCP2 is unclear, but it has been established that UCP2 can decreases the ROS production. Recent studies have shown that UCP2 has the protective effect on many cells through the function of controlling the production of superoxide and other downstream reactive oxygen species. CCl₄ is the hepatic toxin commonly used. CCl₄ is metabolized with P-450 enzyme of microsome in hepatocytes and increased reactive oxygen species ( ROS ) production. ROS cause serious hepatocytes damage by eliciting deleterious effects primarily by oxidizing intracellular components, which is involved in many liver damage induced by hepatic toxins. It is a key point for the liver guarding against hepatic poisoning to limit ROS production andto eliminate the already generated ones. We thus hypothesized that UCP2 might exert its function in the protection against damage of regenerating liver. UCP2 is the anion channel in the inner mitochondrial membrane, and it dissipates the proton gradient across the inner membrane, uncouples oxidative phosphorylation and reduces intracellular ATP levels. So up-regulation of UCP2 may compromise cellular ATP levels and worsen liver damage. Therefore, our experiment investigated the changes of mitochondrial ROS production and the hepatic ATP levels in the regenerating liver after CCl₄ treatment to explore how UCP2 is involved in the protection against damage of the regenerating liver and the action of UCP2 in regulating ROS and ATP.Methods: We prefer CCl₄ as the hepatic toxin. Healthy male Wistar rats were divided randomly into four groups: control, sham operation treated with CCl₄ (SO/CCl₄), partial hepatectomy (PH) and partial hepatectomy treated with CCl₄ (PH/CCl₄). Two-thirds partial hepatectomy was performed under anesthesia with ethyl ether according to the method of Higgins and Anderson. The sham operation treated with CCl₄ group was done with the same procedures aforementioned except for liver resection. In SO/CCl₄ and PH/CCl₄ groups after surgery, rats received a single administration (5ml/kg of body weight) of either 50% CCl₄ (1:1, CCl₄: corn oil) intragastrically. Partial hepatectomy group received corn oil alone intragastrically. At 24h, 48h, 72h, 96h after surgery, the rats were sacrificed. The blood samples were collected for assay of serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Liver tissues were collected, and homogenization of the tissues was prepared for the determination of the levels of malondialdehyde (MDA) and adenosine triphosphate (ATP). Fresh mitochondria were prepared for mitochondrial membrane potential (MMP) and western blot analysis of UCP2. Quantified analysis of western blot was performed later. Paraffin slides were used for histological assessment and immunohistochemistry analysis of UCP2 and proliferating cell nuclear antigen (PCNA).Result:1. Changes of Histology in Different Time Points of Liver Regeneration after CCl₄ IntoxicationThe HE staining showed in control liver hepatocytes were normal and hepatic cords aligned compactly. The sham operation liver treated with CCl₄ developed severe steatohepatitis with hepatocyte ballooning, and necrosis in the portal areas; the structure of the hepatic lobule was destroyed, the hepatic cords disappeared. The 24hregenerating liver after partial hepatectomy treated with CCl₄ (PH24h/CCl₄) developed considerable and severe steatohepatitis with hepatocyte ballooning, inflammation, bleeding and necrosis; the structure of the hepatic lobule was severe destroyed, and the hepatic cords disappeared. Along with continual regeneration of liver, steatohepatitis, inflammation, bleeding and necrosis decreased gradually. The structure of the hepatic lobule recovered gradually, the hepatic cords appeared. When the 96h regenerating liver after partial hepatectomy treated with CCl₄ (PH96h/CCl₄), the structure of the hepatic lobule was destroyed slightly, the hepatic cords was remained. It has slight steatohepatitis and has no necrosis.2. Changes of Serum ALT, AST Levels in Different Time Points of Liver Regeneration after CCl₄ IntoxicationAfter CCl₄ intoxication, the levels of serum ALT and AST at PH24h/CCl₄ increased significantly compared with the SO24h/CCl₄ group (P < 0.001). Along with continual regeneration of liver after partial hepatectomy, the ALT and AST levels decreased gradually. To PH96h/CCl₄, the ALT and AST levels were obviously lower than the SO96h/CCl₄ group (P < 0.001), and also lower than that of PH24h/CCl₄ (P < 0.001).3. Changes of MDA of Liver Tissues in Different Time Points of Liver Regeneration after CCl₄ IntoxicationAfter CCl₄ intoxication, MDA contents in hepatic tissues at PH24h/CCl₄ increased significantly compared with SO24h/CCl₄ group (P < 0.001). Along with continual regeneration of liver, MDA contents decreased. When PH96h/CCl₄, the elevation of MDA induced by CCl₄ poisoning was significantly suppressed compared with SO96h/CCl₄ group (P < 0.01) and the PH24h/CCl₄ group (P < 0.001).4. Changes of Hepatic Adenosine Triphosphate Levels in Different Time Points of Liver Regeneration after CCl₄ IntoxicationAfter CCl₄ Intoxication, hepatic ATP level of 24h regenerating liver was the lower, it has a significant change compared with the SO24h/CCl₄ group (P < 0.001).And then ATP level increased gradually .To 96h ,the ATP level increased markedly relative to the SO96h/CCl₄ group(P < 0.001),and much higher than the PH24h/CCl₄ group (P < 0.001).5. Changes of Mitochondrial Membrane Potential of hepatocyte in Different Time Points of Liver Regeneration after CCl₄ IntoxicationAfter CCl₄ poisoning the MMP of 24h was much higher than the SO24h/CCl₄group (P < 0.01). The MMP of 96h was higher than the SO96h/CCl₄ group (P < 0.001) too, and higher than the PH24h/CCl₄ group (P < 0.01).6. Expression of UCP2 in Different Time Points of Liver Regeneration after CCl₄ IntoxicationImmunohistochemistry staining indicated that there is no UCP2 positive hepatocytes in the control liver, and abundant brown positive UCP2 granules were detected in the cytoplasm of hepatocytes in the SO/CCl₄ group and different time points of regenerating liver. In PH24h/CCl₄, the brown positive granules were most abundant. In the 96h group brown positive granules were more abundant than the SO/CCl₄ group.Furthermore, Western blot analysis showed that the average relative photodensity was obviously increased in the 24h regenerating liver after CCl₄ poisoning compared with the SO24h/CCl₄ group(P<0.001). The average relative photodensity of 96h regenerating liver after CCl₄ poisoning was higher than the SO96h/CCl₄ group (P<0.001). But the average relative photodensity of 96h was much lower than that of PH24h/CCl₄ group (P < 0.001).7. Expression of PCNA in Different Time Points of Liver Regeneration after CCl₄ IntoxicationImmunohistochemistry staining indicated that positive cells with PCNA antibodies were very few in the control liver. However, they were found to be increased at 24h and 48h regenerating liver after CCl₄ poisoning, and expression of PCNA reached peak. Then expression of PCNA decreased gradually. To the 96h, expression of PCNA approached the control level.Conclusions: CCl₄ could induce hepatocellular damage through the increase of ROS. Regenerating liver possessed the weakest self-protection against damage at 24 hours after PH. By 96 hours after PH, the capacity of self-protection against was the strongest. At 24 hours after PH, ATP in regenerating liver was lower. Along with continual regeneration, ATP increased. ATP was important for self-protection against CCl₄ in regenerating liver. UCP2 expression in regenerating liver and damaged regenerating liver increased, it indicated that UCP2 had a close correlation with proliferation of hepatocytes and protection against damage in regenerating liver.