The Energy Metabolism Of Non-alcoholic Fatty Liver Disease And Its Varieties Exposed To The Extract Of Compound Embryonic Bovine Liver

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a liver disease correlated with the heredity-environment-metabolize stress. In pathology, NAFLD includes non-alcoholic fatty liver (NAFL)), and its following production that is non-alcoholic sleatohepatitis (NASH) and fatty liver cirrhosis (FLC) and so on.In order to explain the pathogenesis of NAFLD, Day brought forward the "twice strike theory" in 1998: the first strike is insulin resistance (IR), which can make hepatocyte fat accumulating by promoting degradation of the peripheral fat and hyperinsulinemia, and made hepatocyte become more sensitive to damaging factors. The second strike is the oxygen stress, lipid overoxidation, overexpression of mitochondrial uncoupling protein (the regulative gene of TNF-α), and the activation of cytokine and Fas ligand, all of these can induce inflammation and fibrosis.The "twice strike theory" emphasize the effect of mitochondrial, oxygen stress, lipid overoxidation and cell defence changes and there interaction. This indeed can promote the study of the relatived molecule biology, find gene which has relation with NAFLD hereditary sensitivity, and exploit the new strategy for clinic prevention and cure. However, the credibility biochemistry symbol is short for oxygen stress and lipid overoxidation, so in the clinic, in order to offer reasonable and effective therapy, the histology examination is often used for the patients who suffer from inflammation and fibrosis. Although the basic conception has been brought, this theory is still in its beginning, and the theory system is not integrated, so we need further study.NAFLD is the typical metabolism disease. The abnormity of lipid metabolism exists during the wholeprocess. Normally the internal free fatty acid circulates between liver and peripheral fatty tissue. There is no lipid accumulated in the hepatocyte. But high fat diet for a long time (cause the body ingests too much FFA) and IR (fatty tissue degradation, release more FFA,) will cause too much FFA enter the hepatocyte. FFA is a zwitterion with high cell toxicity, it can cause the functional abnormal of the hepatocyte mitochondrion, the P-oxidation disorder of the fatty acids, and the changes of hepatocyte energy stable state. We can estimate the capability of the mitochondrion for storing ATP and transforming ADP, AMP to ATP by measuring the content of the energy substance of the liver tissue.This research is to establish the trophism fatty liver rat model, and intervene with ANFATE, then measure the content of the ATP, ADP and AMP in hepatic tissue by using the HPLC, calculate the change of the energy charge (EC) and compare them with biochemistry index and pathological morphology, so that to further explain the pathogenesis and observe the effect.ObjectivesThe objective of this research is to explore the liver energy metabolism in NAFLC rat and the changes with drug-intervention, illuminate the pathogenesis further, and provide methods for exploring anti-fatty liver drugs and observing the clinical effect.The aim of this research is to establish the trophism fatty liver rat model, and intervene with ANFATE, then measure the content of the ATP, ADP and AMP in hepatic tissue by using the HPLC and calculate the change of the energy charge (EC).MethodsChoose 60 SD rats (S, weight 150-160g), and then divides them randomly after encoding. So the 5 groups are normal control group, high-fatty model group, ANFATE treated group with dosage of high, middle and low. The high-fatty feed formula is basic feedstuff (80.5%) +2%high cholesterin+7%lard+10%yolk powder+0.5%bile salt. After the model is succeed, giving the ANFATE (large middle and small dosage are 40,24, 15mg/kg) one time each day from the end of the 8th week in oral dosing. Record the weight of animal every week and adjusted the dosage according to the weight. This work is done continuously until the end of the 16th week. Feed condition: temperature is 25°C±rC, relative humidity is 55%±5%, giving sufficient water, and feed freely.After the administration, we took blood from the fermoral vein, measured the index of TG, TC, AST and ALT. Observed the general state of liver. After that, completely resected and weighed the liver, conserved one part of them in the fluid nitrogen, solidified another part them with formalin, embedded with paraffin,sliced up, staining with HE, and observed under optical microscope.We use HPLC to measure the hepatocyte energy metabolism substance. Chromatogram condition: reversed-Phase Partition Chromatography (Lichrospher 5-C18, 250mmx4.6mmx5um), mobile phase O.lmol/1 diammonium phosphate (PH=6.25), flow rate 0.8ml/min, wavelength 254nm, injection volume 10|j.l. The results for ATP, ADP, AMP were shown with ng/mg..ResultsThe surface of the liver in the normal group is slippery, ruddy, knotless and soft. But in the high-fatty model group, the volume of the liver increased, envelope becomes tensioned, also its color changed to khaki, and the profile is so oil. Under the light microscope, the hepatocyte of the normal control group (8 and 16 weeks) is normally arranged, the hepatic lobule is regulated, and no fatty drop is observed in the cytoplast. Also there is no inflammatory infiltration in the portal area and lobules. For the High-fatty model group at the end of the 8th week, the cytoplast of the hepatocyte is taken up by the large and small vacuoles which extrude the nucleus to the side of the hepatocyte. In the end of the 16th week, the large vacuoles in the hepatocyte cytoplast fuse together and became different size of fatty vesicle, and large mount of ? inflammation infiltration appearance in the lobule. These pathology changes show that in the 8* week, we have successfully established the non-alcoholic fatty liver disease (NASH) model. The disease is aggravated along with the time, which shows that this model can be used to study the pathogenesis and the effectiveness of the drug.The general morphologic change of every ANFATE treated group is between the normal and high-fatty model group. The volume of the liver increased, envelope becomes tensioned, the color become khaki, and soft. The degree of every treated group is lower than the model group. The difference between high, middle dosage is small. Many small fatty drops exist in the cytoplast, but have' not extrude the nuclear. The inflammation infiltration also can be seen in it. In the low dosage group, the large vacuoles around the nuclear fuse and became different size of fatty vesicle, and the inflammation infiltration also can be seen.The average weight of the animal has no obvious differences when grouping. At the 16th week, the high-fatty model group has no difference with the normal group, but the liver wet weight and liver index are all increased, the difference has statistic value (P<0.05);the average weight of the high dosage group is lower than the high-fatty model group (P<0.05). Both the liver wet weight and liver index (liver green weight/weight X100%) of the treated group are all increased compared with normal group (PO.05);The liver wet weight of every treated group are all decreased compared with the high-fatty model group (PO.05).The liver index of the high and middle treated group is decreased (PO.05).Compared the index of liver function of the high-fatty model group with the normal group, both the ALT and AST are increase (PO.01). ALT and AST are obviously increased in every ANFATE treated group compared with the normal group (P<0.05), but has no statistic value compared with the high-fatty model group (P>0.05).Comparing the blood fat index of the high-fatty model group with the normal group, TC increased, TG decreased (PO.01). Comparing every ANFATE treated group with normal group, all TC increased, TG decreased (PO.05);there are no statistic value of the TG and TC between treated groups and high-fatty model group (P>0.05). No statistic value between each two treated group (P>0.05).Compared with the normal group, in the high-fatty model group, the hepatic energy metabolism substance ATP, ADP, AMP is all decrease, the hepatocyte storage of EC is also decrease (P <0.05).In the ANFATE treated group the energy metabolism index (ATP, ADP, AMP and EC) are all decrease than the normal group (P< 0.05), but these index are all increase than the high-fatty model group, all these have statistics value (P< 0.05) except AMP. These is no statistics value of index between each high, middle, low group (P>0.05).ConclusionThe result of the changes of energy metabolism of the NAFLD rat can be showed as the reduced synthesize of ATP and the reduced storage of cell EC. These indicated the hepatocyte mitochondria damage and the abnormal of fatty acid p-oxidation and the glucose utilization.This research shows that the ANFATE treating is effective for the changes of liver energy metabolism. Combining with the index of pathology and serology, the cell energy metabolism index ATP, ADP and EC is more sensitive and stable for evaluating the effect ANFATE.Due to its high sensitivity and repeatability, HPLC can be used to measure the hepatocyte energy metabolism substance. Because ATP is easily depredated, we must keep the samples at low temperature during material collection and pre-treating.