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Objective: Alcoholic liver disease (ALD) is a toxic liver disease caused by alcohol abuse. Histopathological features of ALD include mild alcoholic injury, alcoholic fatty liver, alco- holic hepatitis, alcoholic hepatic fibrosis and cirrhosis. The pathogenesis of ALD which remains obscure as yet is very complicated. Endoplasmic reticulum (ER) stress can induce hepatocyte apoptosis which may play an important role in the pathogenesis of ALD.ER stress is a subcellular pathological state characterized by Physi- ological dysfunction of endoplasmic reticulum include calcium homeostasis disruption, the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum lumen from all causes, it is a necessary step which can recover the correct conformation of protein for further processing. ER stress is a self-protecting mechanism of the body, but if the response is too long or too strong, too serious injured hepatocyte may lead to apoptosis.Previous stduies have shown that ER stress can be induced by Hyperhomocysteinemia (Hhcy) in ALD, and the role of Hhcy may be more important. Cystathionine beta synthase (CBS), the pyridoxal phosphate- dependent enzymes, mainly synthesized in the liver.It is the key enzyme in the metabolic process of homocysteine (Hcy). The decrease of activity of CBS may be the important reason of HHcy. Glucose-regulated protein-78 (GRP78) is the largest chaperonin in the endoplasmic and is a sign molecules of endoplasmic reticulum stress response. GRP78 is strongly expressed under ER stress to recover the correct conformation of protein by combining with unfolded protein. The pathway of ER stress- mediated hepatic apoptosis includes a series of enzymatic reactions and signal transductions, in which calpain 2 and caspase-12, the cysteine proteinases in hepatocytes, play an important role. Methods: 50 male 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 establish the rats model of ALD by intragastric alcohol of increasing concentration gradually (30%-60%, 5-9g·kg-1·d-1), 9, 9, 8 and 8 rats were sacrificed randomly after 12 hours fasting at the end of 4th, 8th , 12th and 16th week, and the serum and liver samples were collected respectively. The serum levels of ALT, AST, CHE, TG, TC, LDL, VLDL, HDL, tHcy were examined by chemical methods and the activity of CBS in liver tissue was measured by chromatometry. Some of the hepatic tissue were made of frozen sections and stained by SudanⅣto observe the liver steatosis. Some part of hepatic tissue were made of Paraffin sections and stained by hematoxylin-eosin (HE),Sirius red and periodic acid Schiff reaction(PAS)to observe the ordinary pathologic changes,liver fibrosis and glycogen in the cytoplasm of hepatocytes. The mRNA expressions of GRP-78, calpain 2, and caspase-12 were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). GRP-78, calpain 2 protein expressions and the precursor protein expressions of caspase-12 (procaspase-12) were assessed by immunohisto- chemistry. Hepatocyte apoptosis was determined by TdT-mediated dUTP nick end labeling (TUNEL) assay.Result:1 The change of biochemical index of serum: As the duration of ethanol administration extended, the level of ALT, 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: As the duration of ethanol administration extended, 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:In the normal control group, hepaticcords radiate out from central veins of hepatic lobules. As the duration of ethanol administration extended, the hepatocyte swelling, steatosis, apoptosis and necrosis in liver lobules can be found.At the 16th week, diffuse microvesicular adipose degeneration, fibrosis in liver sinus and fibrosis septa in the portal area were observed in hepatic tissue, while the apoptotic body and necrotic focus were increased. The frozen sections stained by SudanⅣshowed that nonsteatosis was observed in the hepatic tissue of the normal control group. With the process of ALD, the quantity of lipid droplets increased gradually, and at the 16th week, a large quantity of lipid droplets distributed in hepatocytes. The Paraffin sections stained by PAS showed that a number of purple guanules in Cytoplasm.With the process of ALD, the quantity of purple guanules decreased gradually. The Paraffin sections stained by sirius red showed that at the 4th week, the fibrosis of the hepatic tissues were not obvious. As the duration of ethanol administration extended, fibrosis septa appeared gradually in hepatic tissue. At the 16th weeks of model group, the fibrosis septa were obvious.4 The change of tHcy in the serum: As the duration of ethanol administration extended, the serum level of tHcy increased gradually , compared with those of the normal control group P<0.01.5 The activity of CBS in the liver: As the duration of ethanol administration extended, the activity of CBS decreased gradually, compared with the normal control group P<0.05 and P<0.01 .6 The expressions of GRP-78, calpain 2 and procaspase-12 in the liver by immunohistochemistry staining: There were only a little expressions of GRP–78 and calpain 2 in hepatic tissue of rats in normal control group. which were increased gradually with the progress of ALD, and the main expression located in cytoplasm. Abundant expression of procaspase-12 can be observed in normal control group. With the consumption of ethanol increase, the expression of procaspase-12 decreased gradually , and the main expression located in cytoplasm. Compared with the normal control group P<0.01.7 The mRNA expressions of GRP-78, calpain 2 and caspase-12 in the liver by RT-PCR: There were only a little mRNA expressions of GRP-78, calpain 2 and caspase-12 in hepatic tissue in normal control group, which were increased gradually with the progress of ALD, and compared with the normal control group P<0.01.8 The change of hepatocyte apoptosis by TUNEL: There were few apoptotic cells in hepatic tissue of rats in normal control group, with the consumption of ethanol increase, the hepatocyte apoptosis index (AI) exhibited an increasing trend, compared with the normal control group (P<0.01).9 The relative CBS expression of the hepatic tissue was negatively correlated with the serum level of tHcy (r=-0.632,P<0.01).10 The relative tHcy expression of the hepatic tissue was positively correlated with the expression of GRP-78 of the hepatic tissue (r=0.617, P<0.01).11 The hepatocyte apoptosis index was positively correlated with the mRNA expressions of calpain 2 and caspase-12 of the hepatic tissue. (r=0.959 and r=0.887,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 In our experimental ALD model, decreased CBS activity results in Hhcy that further induces ER stress and activates calpain 2 and caspase-12. These changes may provoke hepatic apoptosis which may play an important role in the pathogenesis of ALD.
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