| Objective: “Non-alcoholic Fatty Liver Disease” which was coined by Ludwing in 1980 early is a clinical syndrome characterized by the excess fat accumulation in the liver and hepatic cellular degeneration even in the absence of alcohol consumption. Defined as a genetic-environmental-metabolic stress-related disease, NAFLD encompasses a spectrum of disease from simple steatosis to nonalcoholic steatohepatitis(NASH), which can progress to cirrhosis and hepatocellular carcinoma. NAFLD affects 10% to 24% of the general population in various countries and the prevalence has even been up to 75 percent in obese people. In United States it translates to approximately 30.1 million obese people affected with steatosis and 8.6 million with steatohepatitis.There are increasing evidences that NAFLD represent the hepatic component of a metabolic syndrome characterized by obesity, hyperinsulinemia, peripheral insulin resistance, diabetes, hypertriglyceridemia, and hypertension.In recent years, with the improvement of living standard and sedentary lifestyle, especially the change of the diet structure and decrease in physical activity, the NAFLD patients increase year by year, and their ages are to be younger. So far, NAFLD has become a global public health problem.The mechanisms of NAFLD have not been completely eluicated and new targetsfor preventing and curing NAFLD need to be further researched.The fatty change of liver involves excess fat accumulation, which primarily is triglyceride, the hallmaker of NAFLD. Lipid is stored in hepatic lipid droplets, mainly in the form of triglyceride. Lipid droplets are spherical intracellular organells, in the center of neutral lipid storage surrounded by a phospholipid monolayer and coated by many special proteins. Lipid droplets play a central role in lipid homeostasis by mediating the transient storage of fatty acids in the form of triglyceride.The accumulation of excess lipid droplets in Non-Adipose Tissue, for example, liver, coronary artery, islet leads to metabolic dieases such as fatty liver disease, coronary heart diease,type2 diabetes.LD-associated proteins that are involved in lipid metabolism and transport, intracellular trafficking, signaling and cytoskeletal organization.The best-characterized are members of the PAT family,with similar squenches and locating in lipid droplets.Prior researchs have suggested the expression of PAT family proteins in the adipose tissue, steroidogenic cells, mammary gland tissue, in alcohol related liver diease.Some studies haved demonstrated PAT family proteins are associated with NAFLD. PAT family proteins include: perilipin1(perilipin), perilipin2(ADRP), perilipin3(TIP47), perilipin4(S3-12), perilipin5(OXPAT).Recently, People have done some studies about the relations of perilipin and obesity, diabetes, insulin resistance, atherosclerosis and tumor, but the research is less abou the relation of perilipin, metabolism of lipid droplets and non-alcoholic fatty liver disease.Therefore, using the model of non-alcoholic fatty liver disease induced with high-fat diet and intervened with pioglitazone to further clarify the mechanism of NAFLD, this will be a potential drug target for prevention and curing of NAFLD.Methods:1Selected 24 male SD(Sprague Dawley) rats weighing 180±20g;the rats were randomly divided into three groups after fed with normal diet for a week:normal controlled group(NC group), n=8, high-fat diet group(HF group, n=8), drug prevention group(DP group n=8);2 Normal controlled group rats were fed with normal diet for 8 weeks; high-fat diet group rats were fed with high-fat diet(82.5% common food, 2% cholesterol, 10% lard, 5% egg yolk powder, 0.5% sodium tauroglycocholate) and given the same Sodium Chloride by intragastric administration intervention for 8 Weeks; drug prevention group rats were fed with high-fat die and given pioglitazone(4 mg/kg.d) by intragastric administration for 8 Weeks. Weighted and recorded the body weight every week, all rats were killed at the end of the eighth week.3 Completely remove the liver tissue and observe the general condition,weigh liver wet weight and count liver index; take the serum samples of rats, automatic biochemical analyzer for the detection of serum biochemical indexes:serum triglyceride(TG), cholesterol(TC), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol(LDL-C), alanine aminotransferase(ALT), aspartate aminotransferase(AST); copper reagent chromogenic method for the testing of the level of serum FFA and FFA of liver tissue; hematoxylin and eosin(HE) staining for observing the change of liver pathology by light microscopy; the expression of perilipin in the liver tissue was detected by immunohistochemical method; the level of perilipin was tested by western-blot.Results: 1 Changes of body weight,liver wet weight and liver index in each group rats 1.1 WeightThe weight of Each group rats has no significant difference at the end of the fist week.At the end of the eighth week, compared with NC group(411.54±35.16),HFgroup weight(455.62±30.95) and DP group(448.23±28.01) weight was higher(P<0.05); Compared with HF group, DP group weight was slightly lower, but they had no significant difference(P =0644>0.05). 1.2 Liver wet weightCompared with NC group(11.51±2.08), the liver wet weight of HF group(23.65±3.13) and DP group(20.30±3.16) were significantly higher(P <0.05).Compared with HF group, the liver wet weight of DP group was significantly lower(P <0.05). 1.3 Liver indexCompared with NC group(2.76±0.36), the liver index of HF group(5.20±0.63)and DP group(4.47±0.60) were significantly higher(P<0.05); Compared with HF group, the liver index of DP group was significantly lower(P <0.05). 2 Comparison of serum biochemical indexes 2.1 Serum triglyceride(TG), cholesterol(TC)The level of TG in HF group rats(0.81±0.25)was sharply higher than NC group(0.43±0.30)(P<0.05), DP group TG(0.45±0.34)was sharply lower than HF group(P<0.05), was slightly higher than NC group, but did not have statistical difference(P=0.89>0.05);Compared with NC group(1.64±0.31), HF group TC(2.65±0.50) was sharply increased(P<0.05), DP group TC(2.17±0.53)was significantly lower than HF group(P<0.05), while it was still higher than NC group(P<0.05). 2.2 Serum HDL-C, LDL-CCompared with NC group(0.99±0.27), HDL-C of HF group(0.71±0.24) was significantly decreased(P<0.05), DP group(0.66±0.22)was lower than HF group, but did not have a difference between them(P=0.692>0.05).Compared with NC group LDL-C(0.97±0.25), HF group(1.66±0.40) was significantly increased(P<0.05). Compared with HF group, DP group LDL-C(1.09±0.39) was significantly declined(P<0.05). DP group was higher than NC group, while DP group did not have a difference with NC group(P =0.520>0.05). 2.3 Serum FFAThe serum FFA of HF group(731.7±49.59) was sharply higher than NC group FFA(641.05±50.94)(P<0.05), DP group(655.58±48.07) was significantly decreased, compared with HF group(P<0.05), slightly higher than NC group,but DP group did not have a difference with NC group(P =0.564>0.05). 2.4 Serum ALT and ASTThe ALT of HF group(70.02±19.45) was significantly higher than NC group(23.33±7.26)(P<0.05), but DP group was obviously decreased,compared with HF group(P<0.05); Compared with NC group AST(99.59±7.10), HF group(182.02±17.91) was significantly increased(P<0.05) and DP group(144.33±22.34) was lower than HF group(P<0.05). 3 Comparison of FFA in liver tissueCompared with NC group(84.10±9.21), FFA of HF group(223.37±21.28) and DP group(137.23±28.92) were obviously increased(P<0.05); Compared with HF group, FFA of DP group was significantly decreased(P<0.05). 4 The general and pathological changes of liver10In the NC group rats, liver was smooth and shiny with reddish-brown appearance.HE staining did not have fatty liver cells and inflammatory infiltration in the NC group rats;The liver of the HF group rats was not shiny and smooth, with yellow appearance and greasy feeling.Small and large lipid droplets were in liver cells of the HF group rats, lobules of liver and liver portal canal had inflammatory cells infiltration and mixed cell necrosis in HE staining;The fatty change of liver cells in the DP group had a significant improvement with the HF group. 5 Comparison of Immunohistochemistry of Perilipin in liver tissue in each group ratsThe positive cells of expressing perilipin were brown-yellow.Perilipin of NC group rats was on the cell membrance of liver.Perilipin of HF group and DP group was on the cell membrance of liver and larger lipid droplets. 6 Comparison of expression of perilipin protein in each group ratsThe expression of perilipin protein in HF group(0.84±0.05) was sharply higher than NC group(0.55±0.06)(P<0.05); DP group(0.61±0.06) was significantly lower than HF group(P<0.05), and slightly higher than NC group, but there was no obvious difference between DP group and NC group(p =0.137>0.05).Conclusions:1 High-fat diet can successfully establish the model of NAFLD.2 The expression of perilipin in the HF group was increased, and serum TG, FFA was also high, which suggested perilipin was related with the disorder of lipid metabolism. Perilipin was mainly on larger lipid droplets and the cell membrance of liver, which clarified perilipin had a relationship with the maturity and size of lipid droplets. Decreasing the expression of perilipin could prevent the progress of NAFLD.3 Pioglitazone decreased the level of serum TG and FFA, so it improved the disorder of lipid metabolism. Decreasing of expression of perilipin protein could decrease the amount of lipid droplets in the liver, make lipid droplets smaller and improve hepatic steatosis and inflammation. |
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