Expression And Significance Of UCP2 In Alcoholic Liver Disease

Objective: Alcoholic liver disease (ALD) is a liver impairment caused by alcohol abuse. Histopathological features of ALD include adiposis hepatica, steatohepatitis, followed by fibrosis and cirrhosis. Now the etiopathogenesis of ALD remains obscure. In recent years, surveys presents with the hypothesis of"two hits"which is characteristic by center of oxidative stress and lipid peroxidation to account for the mechanism of alcoholic induced liver injury. The"firsr hit"causes lipid deposition in the hepatocytes which leads to adiposis hepatica. The"second hit"involves oxidative stress and lipid peroxidation. Endotoxin and proinflammatory cytokines are two inseparated main parts to take part in this process. They can activate Kupffer cells, and the activated Kupffer cells produce oxygen free radicals (OFR) and cytokines to recruit neutrophilic leukocytes and lymphocytes'infiltration which results in inflammation to take part in the etiopathogenesis of ALD. Uncoupling proteins (UCPs), possessing uncoupling activity, are the carrier proteins in the inner membrane of mitochondria. UCPs comprise five subtypes, including UCP1, UCP2, UCP3, UCP4 and UCP5. UCP2 expresses in many tissues. But, in the normal hepatic tissue, it can be found only in Kupffer cells. UCP2 does not express in the hepatocytes or has a little expression. UCP2 can accommodate transmembrane transport of protons, it is associated with energy expenditure and lipid metabolism. It can take part in the process of oxidative stress and lipid peroxidation in the pathogenesis of ALD. In this study, the model of rats with ALD was established by intergastric alcohol to detect the expression of UCP2 in the liver of rats by immunohistochemistry staining and reverse transcription poly -merase chain reaction (RT-PCR), to explore the mechanism of UCP2 in ALD, and to provide a potential way of the prevention and treatment of ALD.Methods: 50 male depuratory grade Wistar rats, weighting 200±20g, were acclimatized for 7 days and then 10 rats were randomly assigned to the normal control group. Others were to develop the rats model of ALD by intragastric alcohol of increasing concentration gradually (30%-60%, 5-9g·kg-1·d-1), 8, 8, 8 and 9 rats were sacrificed randomly at the end of 4th, 8th , 12th and 16th week, and the serum, liver samples and liver homogenate (10%) were collected respectively. The contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and cholinesterase (CHE) in serum were examined by OLYMPUS AU-600 automatic biochemical analyzer and the concentrations of triglyceride (TG), oxygen free radical (OFR), malondialdehyde (MDA) and superoxide dismutase (SOD) in the liver homogenate were also measured by biochemical chromatometry. The level of IL-1β, IL-6 and TNF-αin the serum of rats were detected by the mean of enzyme-linked immunosorbent assay (ELISA). Some of the hepatic tissue were made of frozen sections and stained by SudanⅣand to observe the liver steatosis. Some part of hepatic tissue were fixed with 4% neutral formalin and embedded in paraffinum, and tissue secions of 5μm were cut and stained by hematoxylin-eosin (HE), Sirius red and DPAS to observe the ordinary pathologic changes, liver fibrosis and the change of Kupffer cell. Paraffin sections are also used for immunohistochemical staining to examine the expression of UCP2. The rest tissues of liver were frozen quickly in liquid nitrogen, then in -80℃frig , to detect the expression of UCP2 mRNA by RT-PCR.Result: 1 The change of biochemical index of serum: With the consumption of ethanol increase, the level of ALT and AST in the serum increased while the CHE decreased gradually, compared with those of the normal control group P<0.05 and P<0.01.2 The change of contents of IL-1β, IL-6 and TNF-αin the serum: With the consumption of ethanol increase, the contents of IL-1β, IL-6 and TNF-αin the serum increased gradually, compared with those of the normal control group P<0.05 and P<0.01.3 The examination of oxidative stress index of hepatic tissue homogenate: With the consumption of ethanol increase, the contents of TG, OFR and MDA in the hepatic tissue homogenate increased while the SOD decreased gradually, compared with those of the normal control group P<0.05 and P<0.01. 4 Histopathological changes of hepatic tissue: At the 4th week, the rats developed mild steatosis in pericentral to midzonal regions of hepatic lobules. With the progress of ALD, hepatocytes steatosis, necrosis and inflammation in liver lobules as well as fibroplasias became aggravated. At the 16th week, diffuse microvesicular adipose degeneration, fibrosis in liver sinus and fibrosis septa in the portal area were observed in hepatic tissue. The frozen sections stained by SudanⅣshowed that nonsteatosis was observed in the hepatic tissue of normal control group. At 4th week, there were a little lipid droplets in cytoplasm. With the process of ALD, the quantity of lipid droplets increased gradually, and at 16th week, a large quantity of lipid droplets distributed in hepatocytes. The frozen sections stained by sirius red showed that at 4th week, the fibrosis of the hepatic tissues were not obvious. At 12th week, fibrosis in liver sinus and fibrosis septa in the portal area were observed in hepatic tissue. And at 16th week, the fibrosis septa were obvious.5 The change of Kupffer cells: A few DPAS positive Kupffer cells were existed in the fibrosis septa in the portal area of hepatic tissue of the rats in normal group. After 4 weeks of intragastric alcohol, there were an increase number of DPAS positive Kupffer cells inⅠzone of hepatic lobules of severe. After 16 weeks, there were diffused distribution of DPAS positibe Kupffer cells in hepatic lobules. Furthermore, those cells became hypertrophia and rich in cytoplasm.6 The expression of UCP2 protein in the liver of rats: There was only a little expression of UCP2 in hepatic tissue of rats in normal control group. With the progress of ALD, the positive expression became more severe, and the main expression located in cytoplasm.7 The expression of UCP2 mRNA in the liver of rats: There were only a little expression of UCP2 in hepatic tissue of rats in normal control group. The expression of UCP2 increased gradually with the progress of ALD, and compared with those of the normal control group P<0.05.8 Pearson correlation analysis showed that the relative UCP2 expression was positively correlated with the level of ALT and AST (r=0.38, P<0.05; r=0.63, P<0.01), and negatively correlate- ed with the level of CHE (r=0.43, P<0.01).9 The relative UCP2 expression was positively correlated with the level of IL-1β, IL-6 and TNF-αin the serum (r=0.85, P<0.01; r=0.75, P<0.01; r=0.70, P<0.01).10 The relative UCP2 expression was positively correlated with the contents of TG, OFR and MDA of the hepatic tissue homogenate (r=0.75, P<0.01; r=0.74, P<0.01; r=0.73, P<0.01), and negatively correlated with the contents of SOD (r=-0.55, P<0.01).Conclusion: 1 The model of rats with ALD can be established successfully by intragastric alcohol of increasing concentration gradually. The pathological changes, such as steatosis, inflammation and fibrosis, can reflect human ALD.2 Oxidative stress plays an important role in the process of ALD, it takes part in the etiopathogenesis of ALD.3 Activation and increase in the number of Kupffer cells present in the hepatic tissue of rats with ALD. Activated Kupffer cells take part in the process of inflammation reaction and oxidative stress, and play an important role in ALD.4 The expression of UCP2 in hepatic tissue was increased with the process of ALD. And the high expression of UCP2 was positively correlated with the level of proinflammatory cytokin- es in the serum, and it has a great effect on the inflammatory injury of ALD. The high expression of UCP2 was positively correlated with the level of oxidative stress,and it participates in oxidative stress and lipid peroxidation, it plays an important role in the etiopathogenesis of ALD.