| Abstract:Objective:Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome, which is characterized mainly by macrovesicular hepatic steatosis in the absence of significant alcohol ingestion and excluding other liver diseases. With the improvement of lifestyle and changes of living habit and dietary choices, obesity and diabetes are epidemic, subsequently increasing the risk for developing NAFLD.But the pathogenesis of NAFLD remains poorly understood yet. Recent research indicates hepatocyte apoptosis are abnormally overexpressed in NAFLD. It can lead to the disruption of membrane potentia(△Ψm),make the cytochrome-c(cyt-c) and apoptosis-inducing factor(AIF) to be released into the cytosol that the mitochondrial permeability transition pore(MPTP),which is the centre of the communication between inner and outer message for the mitochondrial,is in the openning state.This decides the mitochondrial's function educing and induces the apoptosis.Our research intends to establish the rat model of NAFLD by the improved high-fat diet fed method, continuously time to observe the openning of MPTP, hepatocyte apoptosis and the change of the hepatic fat in NAFLD,and investigates the association between MPTP and NAFLD at last. That is helpful to reveal the pathogenesis of NAFLD and to provide a strong theoretical basis for searching for drugs that inhibit the development of hepatocyte apoptosis, block the progress of NAFLD. Methods:Forty healthy adult male Spague-Dawlay (SD) rats were divided into two groups randomly, normal control group (C group) 20 rats, the model group (F group) 20 rats. Each group included 4,8,12,16 week team, five in each team. The normal diet group was fed with the foundation feed (provided by Luzhou Medical college animal experimental center), the model group was fed with high-fat diet(preparation of modified high fat diet:2% cholesterol,0.5% sodium cholate, 0.2% PTU,5% sucrose,10% lard,82.3% foundation feed), two group drank water freely. Five rats of both control group and model group were killed at the time of 4th,8th,12th,16th weekends. Blood through abdominal aorta injection was collected and the rat's blood lipid and serum aminopherase were determinated in each group in order to observe the progress of the change of the hepatic fat in NAFLD model.Then the liver was taken out rapidly to take it's wet weight, the liver index was calculated as follows:liver wet weight/body mass×100%. The liver tissue samples were taken to preparate paraffin sections. The liver tissue pathological changes were observed through the hematoxylin-eosin (HE) staining under light microscope and the degree of liver fibrosis was detected. The hepatocellular apoptosis index(AI) was measured through TUNEL; the expression of cyt-c in the hepatocellular cytoplasm, which was dectected by enzyme linked immunosorbent assay(ELISA), and the mitochondrial absorption at 540nm(A540nm), which is dectected by ultraviolet spectrophotometry, are regarded as indexes of the openning of MPTP. Measuring data was demonstrated with mean±standard deviation (x±S).With t test, the groups were compared with variance analysis. If there was homogeneity of variance, one-way ANOVA analysis was used,and if there was heterogeneity of variance H test was used. The difference between the model group and the control group in the serum, pathology, extent of apoptosis and openning of MPTP was compared. Count data was used chi-square test; the difference of P<0.05 has statistical significance. Results:All 40 rats were entered into the final analysis. (1)With the mode time expanding, the liver index, serum lipid and transaminase levels of the model group rat were significantly increased; fatty change in liver tissue and the degree of inflammation gradually increased;the fibrosis performance of a small number in the later period. (2)With time gradually increasing, hepatocyte in the model group rat appeared severe fatty degeneration after 4,8,12,16 weeks later; hepatocyte around the central vein was most obvious. The degree of fatty change in liver became serious as the time extending, significant higher than the control group(P<0.05). The model group rats'liver could be seen with varying degrees of lobular inflammation,especially in vessel district department. The model group rat's liver had more or less degree of inflammation in folial or in vessel department,especially in vessel district department, and degeneration or focal necrosis, partial necrosis and even integration into films. At 4th,8th,12th,16th the model rat's liver inflammation activity score were significant higher than the control group (P<0.01). HE staining showed that hepatic fibrosis could not be seen at 4th,8th week model rats'liver, but perisinusoidal fibrosis of liver cells could be seen in a small number at 12th,16th week model group rats'(1/5,2/5). (3)At at 4th,8th,12th,16th the F group rat's AI, which was as follows:3.26±1.03, 5.37±0.97,8.46±1.43,9.51±2.21, were increasing. However, the C group rat's AI, which was follows:2.43±0.96.2.48±0.91,2.23±1.00,2.38±0.93, were almost same(P>0.005). The difference of AI between C and F group was obvious(P<0.01). The degree of apoptosis of experimental rat hepatocyte was positively correlated with the degree of lipid changes in liver tissue, the degree of inflammation and the degree of fibrosis (r=0.675, P<0.01; r=0.607, P<0.01; r=0.560, P<0.05). (4)The cyt-c's concentration in the hepatocellular cytoplasm, detected by ELISA, were advancing at the F group rat. The value at 4th,8th,12th, 16th weekend was 0.33±0.02(mmol/L),0.35±0.02,0.41±0.01,0.59±0.12. There was not obvious difference between the 4-week team and the 8-week team(P>0.05), but it was different compared the 8-week team with the 12-week team(P<0.01) or the 12-week team with the 16-week team(P<0.05). The value at 4th,8th,12th,16th weekend of C group rat was 0.25+0.04(mmol/L),0.26±0.07,0.25±0.03,0.25±0.02 and there was no difference between each team (P>0.05).It was obvously different compared F group with C group(P<0.01). The concentration of cyt-c in hepatocellular cytoplasm at F group rat was positively correlated with the degree of lipid changes in liver tissue, the degree of inflammation and the hepatocellular apoptosis(r=0.603, P<0.01; r=0.531, P<0.05; r=0.674, P<0.01). (5)The A540nm at 4th,8th,12th,16th weeks of F group rat, which was as follows:0.71±0.02,0.56±0.03,0.33±0.02,0.21±0.01, was decreasing. There was obvous difference between each team(P<0.01). The A54onm at 4th,8th,12th,16th week of C group rat, which was as follows:0.85±0.03,0.82±0.05,0.83±0.08,0.83±0.05, was decreasing. There was not obvous difference between each team(P>0.05). It was obvously different compared F group with C group(P<0.01). The A540nm at F group rat was negatively correlated with the degree of lipid changes in liver tissue, the degree of inflammation and the hepatocellular apoptosis(r=-0.690, P<0.01; r=-0.617, P <0.01; r=-0.601, P<0.01). Conclusion:1. We could use improved high-fat diet to establish the rat model of non-alcoholic fatty liver, which could short the time of the construction, reduce experimental cost and basically simulate the occurrence and progression of non-alcoholic fatty liver in human beings.It is an ideal modeling way.2. In rat model of non-alcoholic fatty liver disease, the degree of hepatocyte apoptosis is closely related to the degree of liver injury.There is more severe fatty change, that there will be more hepatocyte apoptosis in inflammation liver tissue. Pathological hepatocyte apoptosis promots the progress of the disease NAFLD.3. The degree of hepatic tissue's pathological change is positively correlated with the concentration of the cyt-c in hepatocellular cytoplasm, but is negatively correlated with the A540nm, the same to say, is positicely cprrelated with the openning of MPTP in the development of NAFLD.This indicates the openning of MPTP is increasing by the progressing of NAFLD.
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