| Objectives: Liver is one of the most important organs of the human body. Clinically the ischemia / reperfusion, trauma and tumor can lead to liver damage. How to prevent liver from injurying and to improve liver function, is the clinical problem solved. A full-grown animal's liver on the condition of normal physiology seldom undergo any cell division or proliferation, but regenerating liver possesses a potent self-protection and regeneration against hepatic toxins or hepatectomy. The well understanding of hepatic physiology and the gradually recognition of mechanism of regenerating liver's self-protection, giving a bright future in the cure of hepatic disease. During liver regeneration after 2/3 partial hepatectomy (PH), the quiescent hepatocytes enter a stage of rapid cell proliferation. Regenerating liver at different time points possesses the corresponding self-protection against hepatic toxins such as galactosamine (GalN), lipopolysaccharide (LPS) and so on, and the self-protection of regenerating liver after PH 96 hr shows the strongest. Though GalN or LPS induces hepatic damage through different mechanisms, they both generate reactive oxygen species (ROS) in the hepatocytes. ROS cause serious hepatocytes damage by eliciting pathogenic effects primarily by oxidizing intracellular components, which is involved in many liver damage induced by hepatic pathogenic factor. It is a key point for the liver guarding against hepatic damage to limit ROS production and to eliminate the already generated ones.Uncoupling protein 2 (UCP2) is a new member of the anion carrier family localized in the inner mitochondrial membrane. It distributes ubiquitously in tissues of the human being and the rodent. At present, it is not quite clear about the exact physiological function of UCP2, but it has been proved that UCP2 activity decreases the production of ROS, and reduces the cell damage caused by oxidation. At the same time the UCP2 shows a great increase in the hepatocytes of regenerating liver. We thus hypothesized that UCP2 might exert its function on the regenerating liver's self-protection. Therefore, our experiment tries to use the UCP2 specificity inhibitor genipin to restrain the function of UCP2, cell mitochondrial membrane potential and intracellular total ROS was evaluated by fluorospectrophotometry and the cell values was evaluated by MTT, and investigates the changes of UCP2 content and the biochemical indicator in the restrained regenerating liver after the joint damage caused by GalN and LPS to explore how UCP2 is involved in the self-protection of the regenerating liver.Methods: We chose healthy male Kunming mice which were divided randomly into 6 groups as our experiment sample: sham-operated (SO), sham operated treated with GalN and LPS (SO + GalN/LPS), partial hepatectomy (PH), partial hepatectomy treated with GalN and LPS (PH + GalN/LPS), partial hepatectomy treated with genipin, GalN and LPS (PH + Genipin + GalN/LPS) and partial hepatectomy with genipin (PH + Genipin).2/3 partial hepatectomy was performed under anesthesia with 10% chloral hydrate intraperitoneal injection according to the method of Higgins and Anderson. The sham operation was done with the same procedures aforementioned except for liver resection. 12 hr before damage and 1 hr before damage the PH + Genipin + GalN/LPS and PH + Genipin received a single administration of genipin (20mg/kg of body weight) intraperitoneal injection respectively. 96 hr after surgery SO + GalN/LPS, PH + GalN/LPS and PH + Genipin + GalN/LPS received a single administration of GalN (400mg/kg of body weight) plus LPS (10mg/kg of body weight) intraperitoneal injection. The rest SO and PH just received normal saline intraperitoneal injection with the same dosage.At the 24th hr after administration of the chemical, the animals were sacrificed after 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 superoxide dismutase (SOD). Paraffin slides were used for histological assessment.Chang Liver normal hepatic cell strain and Chang liver cell strain overexpressing UCP2 are used as the experimental subjects. They were cultivated in thermostatic incubator with PRMI-1640 culture solution containing 10% fetal bovine serum under the conditions of 5% CO2 and 37℃. Cells will be used for the experiment when they are in good conditions after the second or the third generation. Genipin is applied as UCP2 inhibitor. All cells were divided into four groups: control, 25μM genipin group, 50μM genipin group and 100μM genipin group. The Culture solution was changed regularly in control while different concentrations of genipin were administratered in other groups for 40 minutes. Cell mitochondrial membrane potential and intracellular total ROS was evaluated by fluorospectrophotometry. Chang Liver normal hepatic cell strain and Chang liver cell strain overexpressing UCP2 were divided into five groups: control, 0.5μM GalN/10mg/L LPS group, 1μM GalN/10mg/L LPS group, 1.5μM GalN/10mg/L LPS group, 2μM GalN/10mg/L LPS group for 12 hours and were evaluated the cell values by MTT .Result:1.Changes of animal mortalityThe mortality of SO + GalN/LPS group was the highest, while the mortality of PH + GalN/LPS group was the lowest and the resistance to injury was the strongest. The mortality of PH + GalN/LPS group was significantly decreased compared with SO + GalN/LPS group (P <0.05), however, there was not significant difference of the mortality between PH + Genipin + GalN/LPS group and SO + GalN/LPS group.2.Changes of Histological DamageThe HE staining showed that the sham-operated liver treated with GalN and LPS suffered more damage with the structure of the hepatic lobule destroyed, hepatocytes inflammation and necrosis in the portal areas than the sham-operated liver did which acted normal. The regenerating liver acted normal too, as expected, while the regenerating liver after GalN and LPS treatment lessened destruction of the structure of hepatic lobule with the symptom of hepatic cords still able to be identified, a few inflammatory portal areas found and only mild hydropic degeneration with few focal necrosis. The regenerating liver after GalN and LPS mingled with genipin treatment suffered seriously with the structure of the hepatic lobule destroyed, hepatocytes inflammation and necrosis in the portal areas, while the regenerating liver only treated with genipin acted normal.3.Changes of Serum ALT and AST LevelsThe ALT and AST levels in the SO + GalN/LPS group increased significantly in 24 hr. The ALT levels: compared with SO, the SO + GalN/LPS increased 60 times (P<0.01); compared with PH, the PH + GalN/LPS increased 29 times (P<0.01); compared with PH + GalN/LPS, the PH + Genipin + GalN/LPS increased 1.5 times (P<0.01); The AST levels: compared with SO, the SO + GalN/LPS increased 24 times (P<0.01); compared with PH, the PH + GalN/LPS increased 11 times (P<0.01); compared with PH + GalN/LPS, the PH + Genipin + GalN/LPS increased 1.7 times (P<0.01). So we get the conclusion that the serum ALT and AST levels went down obviously in the regenerating liver after GalN and LPS treatment compared with SO + GalN/LPS (P<0.01) and PH + Genipin + GalN/LPS (P<0.01).4.Changes of MDA and SOD in Liver TissuesThe MDA and SOD contents in hepatic tissues of regenerating liver group did not make obviously difference from the sham-operated one. However, compared with the sham-operated liver, the MDA contents in the sham-operated liver after GalN and LPS treatment made a great increase (P<0.01), whereas the SOD contents made a great decrease (P<0.05). It is the same trend for the regenerating liver after GalN and LPS treatment compared with the regenerating liver, but a low level (P<0.05). Compared with the regenerating liver after GalN and LPS treatment, the MDA contents in the regenerating liver after GalN and LPS mingled with genipin made a significant increase, whereas the SOD contents remained little changed.5.Changes of membrane potential and intracellular total ROSCell mitochondrial membrane potential and intracellular total ROS were significantly increased with dose-dependent manner after administratering genipin for 40 minutes, compared with control (P<0.001).6.Changes of cell survival rateCell values of Chang liver cell strain overexpressing UCP2 were significantly increased after administratering GalN and LPS for 12 hours, compared with control (P<0.001).Conclusions:1.Regenerating liver has a strong anti-injury ability. The ability of the regenerating liver against injury significantly decreased after administratering genipin. The results suggest that UCP2 is an important factor to improve the ability of the regenerating liver against GalN/LPS.2.UCP2 may reduce the levels of cell mitochondrial membrane potential and intracellular total ROS. It maybe one of the mechanisms of Liver cells against GalN / LPS. |
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