| 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. Ethanol metabolized into acetaldehyde and acetic acid in the body and has a direct toxic effect on the liver. It can reduce the antioxidant capacity of the liver and cause lipid peroxidation, thus leads to the liver damage. In the process of ethanol oxidation, the production of Reduced form of nicotinamide adenine dinucleotide (NADH) is increased, which inhibit the tricarboxylic acid cycle, causing the disord of fatty metabolism and accumulation in the liver. AMP-activated protein kinase (AMPK) existing in the mitochondria, is a member of serine/threonine protein kinase family. It is a trimer made up ofα,βandγsubunits.αis the catalytic subunit,βandγare the regulatory subunit. The distribution of various subunits is different,α1 distributed widely,α2 is mainly in the liver, skeletal muscle and heart. The activity of AMPK is regulatde by the ratio of AMP/ATP. AMPK is regard as the cell's "metabolic receptors" or "fuel switching" which regulate the levels of ATP and AMP. When the ratio of AMP/ATP rising, AMPK is activated, which can enhance the liver, muscle and other organizations uptaking glucose, fatty oxidation and insulin sensitivity, and reduce the synthesis of glucose, cholesterol and triglyceride. Amount of AMPK expressed in the normal liver tissues, while the liver tissue damaged, the expression of AMPK reduced. Acetyl-CoA carboxylase(ACC) is the rate-limiting enzyme in fatty acid synthesis, AMPK can phosphorylated threonine on 79 points of ACC, and made it inactivation, so that activated fatty acid and then enter theβ-oxidation process. Sterol regulatory element binding protein (SREBP), a kind of membrane junction protein located in the endoplasmic reticulum and nuclear membrane, is involved in the major transcription factor genes of fat synthesis. Hydroxy-Methyglutary-CoA reductase(HMGR) is the rate-limiting enzyme of cholesterol synthesis, ACC, HMGR gene is the target genes of SREBP, and also is the important downstream Target molecules of AMPK, therefore, SREBP may be related to the role of AMPK that regulate fatty acids and cholesterol metabolism.AMPK can inhibit fatty synthesis and promote fatty oxidation and decomposition by reducing the activity of it's downstream kinases, such as ACC and SREBP. In this study, the model of rats with ALD was established by intergastric alcohol to detect the expression of AMPK-α2, ACC, SREBP-1c in the liver of rats by immunohistochemistry staining and AMPK, ACC, SREBPmRNA by reverse transcription poly-merase chain reaction (RT-PCR) and the content of AMPK protein by Western blotting, to explore the mechanism of AMPK-α2 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), 10, 9, 8 and 8 rats were sacrificed randomly at the end of 4th, 8th , 12th and 16th week, and the serum and liver samples were collected respectively. The contents of ALT, AST, CHE, TG, TC, LDL, VLDL, HDL in serum were examined. 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 stained by hematoxylin-eosin (HE) and Sirius red to observe the ordinary pathologic changes and liver fibrosis. Periodic Acid Schiff (PAS) staining to observe the glycogen changes of liver cytoplasm. Paraffin sections are also used for immunohistochemical staining to examine the expression of AMPK-α2, ACC and SREBP-1c. The rest tissues of liver were frozen quickly in liquid nitrogen, then in -80℃frig, to detect the expression of AMPK, ACC, SREBP mRNA by RT-PCR and detect the expression of AMPK protein by Western blotting.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 biochemical index of serum: With the consumption of ethanol increase, the level of TG, TC, LDL, VLDL in the serum increased while the HDL decreased gradually, compared with those of the normal control group (P<0.05 and P<0.01).3 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 PAS showed that a large number of purple granules in cytoplasm. With the process of ALD, the quantity of purple granules decreased gradually. And at 16th week, there were a little purple granules was observed in the hepatic tissue. 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.4 The expression of AMPK-α2, ACC and SREBP-1c in the liver of rats by immunohistochemistry staining: There was much more expression of AMPK-α2 in hepatic tissue of rats in normal control group. With the progress of ALD, the negative expression became more severe, and compared with those of the normal control group P<0.05. The main expression located in cytoplasm. There was little expression of ACC in hepatic tissue of rats in normal control group. With the progress of ALD, the positive expression became more severe, and compared with those of the normal control group P<0.05. The main expression located in cytoplasm. There was little expression of SREBP-1c in hepatic tissue of rats in normal control group. With the progress of ALD, the positive expression became more severe, and compared with those of the normal control group P<0.05. The main expression located in cytoplasm or nucleus.5 The expression of AMPK, ACC and SREBP mRNA in the liver of rats by RT-PCR: There were much more expression of AMPK in hepatic tissue of rats in normal control group. The expression of AMPK decreased gradually with the progress of ALD, and compared with those of the normal control group P<0.05. There were little expression of ACC in hepatic tissue of rats in normal control group. The expression of ACC increased gradually with the progress of ALD, and compared with those of the normal control group P<0.05. There were little expression of SREBP in hepatic tissue of rats in normal control group. The expression of SREBP increased gradually with the progress of ALD, and compared with those of the normal control group P<0.05.6 The expression of AMPK-α2 protein in the liver of rats by Western blotting: There were much more expression of AMPK in hepatic tissue of rats in normal control group. The expression of AMPK decreased gradually with the progress of ALD, and compared with those of the normal control group P<0.05.7 Pearson correlation analysis showed that the relative AMPK expression was negatively correlated with the level of ALT and AST (r=-0.702, P<0.01; r=-0.487, P<0.01), and positively correlated with the level of CHE (r=0.606, P<0.01).8 The relative AMPK expression was negatively correlated with the level of TG,TC,LDL and VLDL in the serum (r=-0.501,P<0.01; r=-0.619, P<0.01; r=-0.586, P<0.01; r=-0.388, P<0.05),and positively correlated with the level of HDL (r=0.563, P<0.01).9 The relative AMPK-α2 expression was negatively correlated with the expression of ACC and SREBP-1c of the hepatic tissue (r=-0.911, P<0.01; r=-0.907, 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 The disorders of lipid metabolism and fat accumulation were exist in the process of ALD.3 The expression of AMPK in hepatic tissue was decreased with the process of ALD, which inhibit the activation to ACC and SREBP, the downstreams of AMPK, and increased the expression of ACC and SREBP. It causes the increase of lipid synthesis and the reduction of oxidative decomposition, which would result in the fat accumulation. AMPK involved in the"firsr hit"of ALD pathogenesis and has a great effect on the liver injury of ALD.4 AMPK as a new pharmacological target, provides a new idea for the prevention and treatment of ALD.
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