Study On UCP2 Inhibited By Genipin In The Mechanism Of Regenerating Liver Against CCl4 In Rats

Objectives:Uncoupling protein 2(UCP2)is a member of the anion carrier family localized in the inner mitochondrial membrane. It could dissipate the proton gradient across the inner membrane of cell mitochondria because of the uncoupling effect and decrease the ROS production. Our previous research revealed that expression of UCP2 in regenerating liver in rats increase dramaticly, it suggested that UCP2 might be involved in the self-protective effect of the regenerating liver. Through preparing the model of regenerating liver of rates, our experiment investigated the role of UCP2 in the regenerating liver after CCl4 treatment by using genipin which is a specific inhibitor of UCP2.Methods: Healthy male SD rats were divided randomly into six groups: sham-operated (SO), partial hepatectomy (PH), sham operated treated with CCl4 (SO+CCl4), partial hepatectomy treated with CCl4 (PH+CCl4), partial hepatectomy treated with genipin (PH+G) and partial hepatectomy treated with genipin and CCl4 (PH+G+CCl4). Two-thirds partial hepatectomy was performed under anesthesia with ethyl ether according to the method of Higgins and Anderson. Rates that performed partial hepatectomy administed by genipin which is specific inhibitor of UCP2 at 84 and 95 hours after partial hepatectomy, intraperitoneal injection with genipin 20mg of body weight add 0.5ml physiological saline. Then rats received intraperitoneal injection with CCl4 which was used as the hepatic toxin. Observed the morphological change by using hematoxylin-eosin (HE) staining. Detected the level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of rats. Detected the content of oxidative stress index such as malondialdehyde (MDA) and superoxide dismutase (SOD) in liver tissues. Immunohistochemistry analysis was used to detect the expression of UCP2 protein in liver tissues.Result:1. The HE staining showed that in the SO group, hepatocytes were normal and hepatic cords aligned compactly. In the PH group, the regenerating liver hepatocytes showed a little swelling, nuclear division and the structure of hepatic cords could be seen clearly. In the SO+CCl4 group, sham-operated liver induced by CCl4 poisoning showed disordered arrangement on hepatic cords, cells suffer from inflammation or necrosis increased, developed ballooning and steatosis. In the PH+CCl4 group, most hepatic cells of the regenerating liver after CCl4 treatment showed normal appearance,hyperplasy well, only a focal region could be seen infiltration by inflammatory cells without obviously necrosis. In the PH+G group, regenerating liver hepatocytes after genipin treatment showed a little swelling, nuclear division and the structure of hepatic cords still regularly. In the PH+G+CCl4 group, most hepatic cells of the partial hepatectomy treated with genipin and CCl4 showed normal appearance, only a little inflammatory and necrosis cells could be seen.2. The ALT and AST level in serum at 96h after partial hepatectomy had no significant differences between partial hepatectomy and sham-operated groups. The ALT and AST levels in the groups which undergoed CCl4 poisoning increased significantly, the ALT and AST levels in SO+CCl4 group was more than that of PH+ CCl4 group(P<0.001). There was no significant differences in the ALT and AST level in serum between PH+G group and PH group(P>0.05). Compared with the PH+G group, the ALT and AST level in serum were increased obviously in PH+G+CCl4 group (P<0.001). Treatment with genipin could increase the ALT and AST level in groups that undergo partial hepatectomy then treated with CCl4 (P<0.05).3. At 96h after partial hepatectomy, there was no significant difference in the content of MDA and SOD between partial hepatectomy and sham-operated groups(P>0.05). The content of MDA and SOD in the groups that CCl4 poisoning changed significantly, the MDA content increased(P < 0.01)and SOD content decreased dramaticly(P<0.05). Treatment with genipin could not change the content of MDA and SOD in the liver of partial hepatectomy group(P>0.05). Compared with the PH+G group, the content of MDA were increased (P<0.001)and the content of SOD were decreased obviously in PH+G+CCl4 group (P<0.01). Treatment with genipin could increase the content of MDA (P<0.05) and decrease the content of SOD in groups that undergo partial hepatectomy then treated with CCl4 (P<0.05). 4. Immunohistochemistry staining indicated that there was no UCP2 positive hepatocytes in the sham-operated liver and abundant brown positive UCP2 granules were detected in the cytoplasm of hepatocytes in the regenerating liver. The brown positive granules in the sham-operated liver and the regenerating liver after CCl4 treatment were more abundant than livers without CCl4 treatment respectively, there was almost filled with UCP2 in the cytoplasm of hepatocytes. There was a little UCP2 protein expression in PH+G group. The expression of UCP2 in the PH+G+CCl4 group was less than that of PH+CCl4 group and more than SO+CCl4 group.Conclusions:1. UCP2 could elevate the ability against damage by CCl4 in regenerating liver by limiting the oxidative stress.2. Genipin could degrade the ability against damage by CCl4 in regenerating liver by specific inhibiting of UCP2.