Study On Apoptosis Related Genes And Other Factors In The Rat Model Of Alcoholic Liver Disease

Objective:To provide the experimental and theoretical basis for the pathogenesis of ALD by studying the expression and significance of apoptosis of hepatocyte and its closely related factors in the rat model of alcoholic liver disease (ALD) established by intragastric administration of alcohol.Methods:(1) Establishment of animal model of ALD and grouping:Wistar rats were randomly divided into two groups-the model group and control group.The rat model of ALD was established by intragastric administration of 40%alcohol with 8 g / kg / d for 12 weeks,and the control group was administrated with equivalent volume of physiological saline.After killing the rats at 8 and 12 weeks,the blood was taken from left ve(?)tricle and was centrifugated,conserved to measure the liver function. Part of the liver tissue was fixed with 10%neutral formalin to do the examination of histopathology,apoptosis and immunohistochemistry,and with 2.5%glutaraldehyde to do the electron microscopy(EM),and was kept at -70℃with liquid nitrogen to do the PCR,respectively.(2) Pathological changes of the liver were observed by HE staining and EM,apoptosis of hepatocytes was detected by the TUNEL method, content of the alanine amino-transferase(ALT) and aspartate aminotransferase(AST) was detected by the automatic biochemical analyzer,expression of the cystein-dependent aspartate-specefic pro gramed-3(Caspase-3),B-cell leukemia-lymphoma -2(Bcl-2),nuclear factor-κB(NF-κB) and tumor necrosis factor-α(TNF-α) was observed by immunohistochemistry(SABC),content of the malondialdehyde (MDA) and activity of superoxide dismutase(SOD) in the liver was determined by the thiobarbituric acid(TBA) and xanthine oxidase enzyme,level of serum TNF-α was detected by the radioimmunoassay(RIA),and expression of the cytochrome P4502E1(CYP2E1) was analyzed by the PCR,respectively.Results:(1) Values of serum ALT(116.12±14.30 vs 43.56±7.89 IU/L) and AST(248.83±20.25 vs 84.67±12.67 IU/L) in the model group were increased significantly as compared with those in the control group,the ratio of ALT and AST was larger than 2,and the difference was statistically significant(P＜0.05).(2) It was showed in liver tissue sections stained by HE staining under light microscope and EM in the model group as compared with those in the control group that obvious swelling of hepatocytes,different size of fat vacuoles in cytoplasm,the Councillman body, point and focus necrosis of hepatocytes with the infiltration of inflammatory cells,and mild hyperplasia of collagen fibers,mitochondrial swelling,unclear and vacuolar degeneration of crista mitochondriales,prosperity of endoplasmic reticulum, apoptosis of hepatocytes and endothelial cells of hepatic sinusoid occurred.(3) Apoptotic hepatocytes were mainly in and/or around the point,focus and piecemeal necrosis in liver tissue,and the apoptotic index of hepatocytes was significantly higher in the model group(6.2±1.7%) than that in the control group(1.7±0.8%, P＜0.05) and increased along with the time extension of making model of ALD.(4) The Caspase-3,Bcl-2 and NF-κB positive cells were mainly distributed around the central venous and necrotic foci of hepatocytes,and the intensity of gene expression of Caspase-3,Bcl-2,NF-κB was significantly higher in the model group than that in the control group(P＜0.05),and the expression between Bcl-2 and NF-κB was positively correlated(r=0.576,P＜0.01).(5) The content of serum MDA(15.72±2.06 vs 41.53±7.43 nmol/ml) was significantly increased and activity of SOD (636.82±138.60 vs 353.12±61.02 nmol/ml) was obviously decreased in the model group as compared with that in the control group,and the difference was statistically significant(P＜0.05),and there was negative correlation between MDA and SOD(r=-0.5818,P＜0.05).(6) Apoptotic index of hepatocytes was positively correlated with serum MDA,and negatively correlated with serum SOD(r_MDA=-0.6437,r_SOD=-0.5115,P＜0.05).(7) The content of serum TNF-α(745.6±174.8 ng/L) in the model group was obviously increased as compared with that in the control group(1236.4±283.5 ng/L),and there was significant difference between the two groups(P＜0.05),and was positively correlated with the apoptotic index of hepatocytes(r=0.8358,P＜0.05).(8) The content of serum TNF-αwas positively correlated with the MDA,but negatively correlated with the SOD(r_MDA=-0.4654, r_SOD=-0.3817,P＜0.05).(9) The expression of TNF-αwas higher in the model group than that in the control group(P＜0.05),and increased along with the time extension of making model of ALD.(10) The expression of TNF-αhad positive correlation with the Caspase-3 and NF-κB in liver tissue(r=0.648,r=0.678,P＜0.01),respectively.(11) The expression of CYP2E1 genotype B(c1/c2) and genotype C(c2/c2) in the liver tissue was significantly increased in the model group than that in the control group(P＜0.05),and the decreased gene frequency of c1(53.4%) and increased gene frequency of c2(46.7%) had statistically significant difference(P＜0.05).Conclusion:(1) The Caspase-3,Bcl-2 and NF-κB genes participate in the apoptosis of hepatocytes in development and progress of ALD of rats,and NF-κB as a transcription factor for inhibiting apoptosis plays a role in regulating the expression of a downstream anti-apoptosis gene of Bcl-2.(2) The TNF-αand injuries of lipid peroxidation play an important role in the apoptotic process of hepatocytes in ALD, and TNF-αparticipates in the development of ALD through its receptor-mediated activation of Caspase-3.(3) The NF-κB activated by TNF-αparticipate concomitantly in the apoptosis of hepatocytes of ALD.(4) The restriction fragment length polymorphisms(RFLPs) of CYP2E1 genetic PstI and RsaI participate in the apoptosis of hepatocytes of ALD,in which the c2 gene plays a decisive action in the apoptosis of hepatocytes.