| PurposeNAFLD (Non-alcoholic fatty liver disease) is a common liver disease, which refers to a clinical syndrome with the steatosis of parenchymal liver cells and lipid storage in liver without excess alcohol intake history. NAFLD has become a disease difficult to treat and has epidemiologic characters universally found around the world. It is a chronic liver disease which draws more and more attention. In recent years, many reports on NAFLD have been released. However, its pathogenesis has remained unclear. At present, the two-hits theory is regarded as a uniform pathogenesis. The first hit is insulin resistance (IR) and the second oxidative stress. The generation and increase of ROS initiates lipid peroxidation, cell factors and Fas ligands, inducing inflammation and fibrosis as a result. Besides, oxygen deficiency, endotoxin, iron, hepatotoxic drugs, or enhanced CYP2E1 expression with PPAR-α may become an additional factor to enhance the hit. Fatty acid plays a critical role in the development of non-alcoholic liver disease. The storage of fatty acid is not only a character of NAFLD but also an important factor inducing IR (insulin resistant), lipid peroxidation, changes of energy metabolism, hepatic cell damage and inflammatoryreaction.The abnormal metabolism of fatty acid is throughout the course of the development of non-alcoholic fatty liver disease. The experiment herein studies the changes of fatty acid composition in the liver tissues of rats with NAFLD. The purpose of the study is to learn various fatty acid compositions and changes in the model liver tissues of rats with NAFLD, and the relation between the changes and other factors so that discusses the role of fatty acid metabolism in the development of NAFLD.Materials and methods1. Source of animals for the experiment: 46 male SD rats; clean grade; 80-100g; provided by Zhejiang Provincial Experimental Animal Center.2. Establishment of the animal model:The male SD rats were randomly divided into several groups according to their weight. The normal comparison group was fed basic feedstuff. The model group was fed high-fat feedstuff, i.e., adding 2% high cholesterol, 7% lard, 10% yolk powder and 0.5% bile salt in the basic feedstuff (80.5%).2-month group:The normal comparison group (normal 2-month) was fed basic feedstuff and performed intragastric administration with saline, and put to death at the end of the eighth week.The model group (high-fat 2-month) was fed high-fat feedstuff and performed intragastric administration with saline, and put to death at the end of the eighth week.4-month group:The normal comparison group (normal 4-month) was fed basic feedstuff; performed intragastric administration with saline after the 8th week, and put to death at the end of the 16th week.The model group (high-fat 2-month) was fed high-fat feedstuff; performedintragastric administration with saline after the 8th week, and put to death at the end of the 16th week.3. Blood test methodUse Hitachi 7600 Automatic Bio-Test to determine ALT, AST, ALP, CHE, TBA, TG, TC, HDL, LDL, VLDL and FBS values.4. Pathological sectionCut a small liver tissue at 5mm from the edge of the biggest hepatic lobes and put it into formalin, paraffin imbedded, sliced, HE stained, and observed it under the light microscope. Refer to the standards proposed by Dixon JB in 2004 for assessing the changes of fat and the inflammatory activity: steatosis <5%, 0 score; <25%, 1 score; <50%, 2 scores; <75%, 3 scores; >75%, 4 scores; no hepatic lobular inflammation, 0 score; sporadic inflammation in zone 3, 1 score; mild inflammation in zone 3, 2 scores; apparent inflammation in zone 3, 3 scores; severe inflammation in zone 3, 4 scores; degree of portal inflammation: none, 0 score; mild, 1 score; medium, 2 scores; severe, 3 scores; extent of portal inflammation: none, 0 score; <25%, 1 score; <50%, 2 scores; <75%, 3 scores; >75%, 4 scores.5. Determination of energy metabolism substance: Use HPLC to measure the hepatocyte energy metabolism substance. Chromatogram condition: reversed-Phase Partition Chromatography (Lichrospher 5-C18, 250mm×4.6mm×5μm), mobile phase 0.1mol/l diammonium phosphate (PH=6.25), flow rate 0.8ml/min, wavelength 254nm, injection volume 10μl. Preparation for standard curve: Prepared ATP, ADP, AMP samples of different volume, detected with above condition, calculated their respective peak area ,worked out the linear regression and got the linear calibration equation of the standard curve. Worked out the peak area of each sample , calculated the different ATP, ADP, AMP content of each sample according to the linear calibration equation of the standard curve, and calculated EC(energy charge).6. Determination of fatty acid: Use gas chromatographic detection. For pre-treatmentof the samples, adopted classical Folch method. Added C17:0 internal standard and mixed them well. Treatment of derivative: The extracted lipids were methylesterized by 14% BF3- methanol/ ether. Conditions of chromatograph: use FFAP (30m*0.53mm*0.5μm) and PEG-20m (30m*0.32mm*0.5μm) for glass capillary columns; Nitrogen (99.999%) as carrier gas; sample introduction: 1μl; column inlet pressure: 50Kpa; split rate: 1/50; make-up gas 40ml/min; temperature of sample introduction: 240°C; temperature of detector: 260°C; column programmed temperature: initial column temperature 40°C kept for 1 minute; programmed temperature 6°C/min rose to 170°C and kept for 0 minute; then 5°C/min to 240°C and kept for 5 minutes. Preparation for standard curve:Prepared six standard unsaturated fatty acid samples and six saturated fatty acid samples of different concentrations. Added internal standard solution (C17:0) and calculated their respective peak area and internal standard ratio. Worked out the linear regression of the internal standard ratio and the fatty acid concentration and got the linear calibration equation of the standard curve.Worked out the peak area of each fatty acid sample with chromatographic work station (N2000). Calculated the different fatty acid content of each sample according to the linear calibration equation of the standard curve, and calculated U/S (unsaturated fatty acid content/saturated fatty acid content). Results 1. General indicatorsWeighted the body weight and the wet liver weight of all rats and calculated liver indices (liver index= wet liver weight/body weight ×100%). The experiment result showed that the body weight of rats from the 2-month groups had no significant difference. The wet liver weight and liver index of the model group (high-fat 2-month) had significant increases (P＜0.05) compared to the comparisongroup (normal 2-month). The body weight of rats from 4-month groups had no significant difference either. The wet liver weight and liver index of the model group (high-fat 4-month) had significant increases (P＜0.05) compared to the comparison group (normal 4-month). Meanwhile, it was found that the body weight of the normal 4-month group was significantly higher than that of the normal 2-month group (P＜0.05) while the figure of the high-fat 4-month group significantly higher than that of the high-fat 2-month group (P＜0.05).2. Serological results: The blood ALT, AST, ALP, TC, LDL, and VLDL of the high-fat 2-month group were all significantly higher (P＜.05) than that of the normal 2-month group while TG and HDL decreased significantly (P＜0.05). The ALT, AST, ALP, CHE, TBA, TC, LDL, VLDL and FBS of the high-fat 4-month group were all significantly increased (P＜0.05) than that of the normal 4-month group. TG lowered significantly (P＜0.05). The ALP of the normal 4-month group was lower (P＜0.05) than that of the normal 2-month group while HDL (P＜0.05) higher than the normal 2-month group. Other indicators had no significant difference (P>0.05). The TBA, TG, HDL and FBS of the high-fat 4-month were higher (P＜ 0.05) than that of the high-fat 2-month group. Meanwhile, ALT and AST also rose to some degree. However, the difference was not significant (P>0.05).3. Pathological detectionMorphological observation of liver: The livers of the normal group rats had a smooth surface, fresh dark-red color, no node and were soft. The livers of the model group rats were enlarged, had tight membrane, yellowish color and an oily cut surface. Besides, with the modeling time extending, the appearance difference of the livers of the two groups became bigger. Observing the HE stained rat liver tissues of the normal groups under the light microscope, no fatty liver appearance was found. All sections of the model groups at the 8th week showed steatosis, which was a diffused steatosis. The relative sizes of the hepatic cell nucleus were uneven. Sinuscells were active. The lobular and portal areas showed inflammatory cell infiltration, much more than that of the normal group. All model rats showed severe steatosis and considerable inflammatory cell infiltration at the 16 week, which was most noticeable in lobule. Scored the samples according to the standards proposed by Dixon JB in 2004: Comparing the high-fat 2-month group with the normal 2-month group, noticeable steatosis, ballooning degeneration and lobular inflammation (P＜0.05) were found. The total inflammation scores increased significantly (P<0.05) but inflammation in portal area was inapparent. Comparing high-fat 4-month with the normal 4-month, there was noticeable steatosis and Mallory body formed while ballooning degeneration, lobular inflammation (P＜0.05) and portal inflammation (.P<0.05) became more serious and total inflammation scores increased significantly (P<0.05). Comparing the normal 4-month group with the normal 2-month group, the pathological scores showed no difference (P>0.05). However, the lobular inflammation of the high-fat 4-month group was more noticeable (P<0.05) than the high-fat 2-month group.4. Energy metabolism substance detectionComparing the high-fat 2-month group with the normal 2-month group, the contents of ATP, ADP, AMP in liver tissue showed no difference (P>0.05). Comparing high-fat 4-month with the normal 4-month, the contents of ATP, ADP, AMP decreased significantly(P<0.05). Comparing the normal 4-month group with the normal 2-month group, ADP and AMP increased (P＜0.05). Comparing the normal 4-month group with the normal 2-month group, the contents of ATP, ADP, AMP showed no difference (P>0.05). The EC showed no difference with all groups(P>0.05).5. Fatty acid CompositionComparing the high-fat 2-month group with the normal 2-month group, the contents of C12:0, C14:0, C15:0, C16:0, C18:0, C18:2, C18:3, C20:0, C22:0, andC23:0 increased significantly (P＜0.05) and U/S reduced (P＜0.05). Comparing the high-fat 4-month group with the normal 4-month group, the contents of C12:0, C14:0, C15:0, C16:0, C18:0, C18:2, C18:3, C20:0, C20:4, C22:0, and C23:0 increased significantly (P＜0.05). Comparing the normal 4-month group with the normal 2-month group, C14:0, C15:0, and C20:0 increased (P＜0.05) while U/S decreased (P＜0.05). The C15:0 and C20:4 of high-fat 4-month group increased compared with the high-fat 2-month group (P＜0.05). 6. Correlation analysisALT, AST had a negative correlation with U/S; ALT had a positive correlation with C16:0, C18:0, C18:2, and C20:0; ALP had a positive correlation with C12:0, C14:0, C16:0, C18:0, C18:2, C18:3, C20:0, C22.0 and C23:0; TBA had a correlation with C15:0, C16:0, C18:0, C18:2, C20:0, and C23:0; TC, LDL, VLDL all had a negative correlation with U/S; TG had a positive correlation with C24:0; LDL had a positive correlation with C18:0, C18:2, and C20.0; FBS had a positive correlation with C12:0, C14:0, C15:0, C16:0, C18:0, C18:2, C18:3, C20:0, C20:4, C22:0, and C23:0.Liver steatosis, ballooning degeneration, lobular inflammation and total inflammation scores had positive correlations with C12:0, C13:0, C14:0, C15:0, C16:0, C18:0, C18:2, C18:3, C20:0, and C22:0; ballooning degeneration and lobular inflammation were also positively correlated to C23:0; the degree of portal inflammation was positively correlated to C13:0; the extent of portal inflammation was negatively correlative to C20:4; steatosis, ballooning degeneration, portal inflammation, lobular inflammation, and total inflammation scores were negatively correlated with U/S.The contents of ATP had negative correlations with C18:0, C18:2, C18:3, C20.0; The contents of AMP had negative correlations with C12:0, C18:3.Conclusions:1. The contents of ATP, ADP, AMP in the liver tissue of the rats with NAFLD decreased significantly. It indicates that the hepatocyte mitochondria had damaged and fatty acid β-oxidation is abnormal.2. Both saturated fatty acid and unsaturated fatty acid in the liver tissue of the rats with NAFLD increased significantly while the U/S ratio decreased significantly. In addition, BSL and liver inflammation have a positive correlation with both saturated fatty acid and unsaturated fatty acid but a negative correlation with U/S. It indicates that saturated and unsaturated fatty acids accumulate in the liver tissue of NAFLD rats. Both saturated fatty acid and unsaturated fatty acid have a pushing effect in the development of NAFLD.3. The contents of ATP and ADP in the liver tissue of the rats with NAFLD had negative correlations with C18:0, C18:2, C18:3, et al. The result indicates that the accumulation of fatty acids in the liver tissue maybe have considerable connection with the energy metabolism.
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