An Experimental Study On The Regulation Of ATGL And TNF-a For NAFLD Rats By Fenofibrate And Simvastatin

BackgroundIn recent years, the incidence of Nonalcoholic fatty liver disease (NAFLD) is gradually increased with the changes of diet structure and lifestyle for human, the disease has become one of the most common chronic liver diseases. At present, the prevalence rate of the general population is10%-16%, the average global prevalence rate is about10.24%, and the rate has increased to57.5%-74%in obesity for human. The etiology of about42-90%of population with transaminase elevation, which is excluded from other etiology liver disease and without symptom, arise from NAFLD. NAFLD, which is characterized of bullous steatosis and lipid accumulation in liver cell, is a clinic histologic syndrome that is similar to alcoholic liver disease (ALD) in pathology but excessive drinking, including several consecutive pathological processes of simple steatosis, nonalcoholic steatohepatitis (NASH), hepatic fibrosis and hepatic cirrhosis. NAFLD, which is correlated with obesity, type2diabetes, hyperlipidemia, hyperinsulinemia and hypertension, is intimately associated with metabolism syndrome(MS).It has become a threaten human health.The incidence of NAFLD has been increasing rapidly, which became one of the most common chronic liver disease hazards to human health. However its etiopathogenesis is not clear. The "two-hit" theory supported by Day and James is currently recognized as the better Pathogenesis. The theory that insulin-resistance are "The First Hit" that make the fat deposits on the hepatocyte and lead to fatty liver."The Second Hit" involves oxidative stress (OS) and lipidperoxidation (LP). In1993, Tumor necrosis factor-a (TNF-a), which was identified firstly in the fat cells, was considered as a fat-adipokine secreted by fat cells. TNF-a, which is one of the most important adpokine leading to hepatocyte injure, may be involved in the progression of liver inflammation and fibrosis. There is a certain positive correlation between expression in part of fat tissue and levels of obesity. Recently, studies found that the adipokine, TNF-a, play an important role in inducing and aggravating IR.Besides acting on the signal transduction system for insulin to block the phosphorylation of insulin receptor substrate, that cause IR, TNF-a participates the process of hepatocyte injure caused by LP and inflammation with those cytokines of IL-1、IL-6induced by TNF-a, to provide matrixes for The "two-hit", and finally, that lead simple steatosis to be nonalcoholic steatohepatitis and hepatic cirrhosis.Fat accumulaotion in liver is the type characteristics of NAFLD. And triglyceride (TG) is the major lipid in the liver of patients with NAFLD. Liver fatty acid absorbed by liver mostly derives from decomposition of the fat stored in fat cells. However, when compared with normal people, TG source in patients with NAFLD only have60%from the free fatty acids (FFAs), while25%from it synthesized by the liver itself and15%from diet. Demonstrating that, fatty acids synthesized by the liver increase in patients with NAFLD and several pathways are involved in liver lipid accumulation. Among the plenty of evidences, increased peripheral source of fatty acids and DNL are the main factors of fat accumulation in liver of NAFLD. Excessive accumulations of triglyceride (TG) in liver is the prerequisite of NAFLD occurs,while the accumulation of triglyceride TG in liver is a results of imbalance of synthesis and transformation, suggesting that limitation process of decomposition metabolism of triglyceride TG may also exist in the liver lipid accumulation.For a long time, hormone sensitive lipase (HSL) is considered to be the rate-limiting enzyme of fat decomposition.However, with the emergence of HSL gene-knockout mice, researchers began to suppose that whether there was another adipose triglyceride lipase in the fat cells. In2004, Zimmermann and other researchers screened the gene and protein database in human and rats, in order to find the protein with homology to the known lipase. At last, they found the fat tissue triglyceride lipase ATGL, while Jenkins and Villena soon also found the same protein. By using transfection of COS-7cells, Zimmermann’s team proved that the ATGL diacylglycerol did not catalyze hydrolyzed cholesterol or retinol but triglyceride hydrolysis. ATGL is an important lipase which catalyzes the first part of hydrolysi, while HSL mainly hydrolyze diacylglycerol. Thereforce, both the ATGL and HSL are the speed limit enzymes of adipose decomposition reaction. So far, we learned that the ATGL was regulated by many factors. For example, Protein58(CGI-58), identified by Comparative genomics, which can specifically activate ATGL and worke together with ATGL in the fat cells, enhancing the activity of the triglyceride hydrolysis enzyme more than20times. The form process stimulated the fat decomposition, which demonstrated that the CGI-58plays an important role in ATGL enzyme activity. Peroxisome proliferative activated receptor-y (PPAR-y) is a member of PPAR family, which also consists of PPAR-a and PPAR-β. PPAR-γ can regulate fat metabolism in many ways, including free fatty acid oxidation, lipoprotein metabolism, fat cell differentiation, and can adjust the process such as intracellular fat metabolism related protein expression, including HSL, lipoprotein lipase (LPL), adiponectin, etc. In an in vitro experiment, PAR-y can promote the expression of ATGL in fat cells. And some experiments proved that the mice feeded with rosiglitazone, a PAR-y agonist, the mRNA and protein expression of ATGL and HSL were detected to increase significantly. A recent experiment detected that the expression of TNF alpha can significantly reduce ATGL mRNA level in the3T3-L1fat cells in a time and dose-dependent manner. And they also detected a similar effect of insulin compared with TNF-alpha on the regulation of ATGL gene expression. Studies have pointed out that the ATGL can expresse in the liver which can enhance the activity of triglyceride hydrolysis and promote the release of free fatty acid and oxidation. It also can improve liver lipid metabolism and reduced fatty liver. Thus, the inner link between triglyceride lipase ATGL and NAFLD is a hot topic among the researchers in recent years.At present, the drugs used in treatment of NAFLD mainly involved in insulin sensitization agent, antioxidant, liver cell protective agent, diet pills, lipid and so on. Hyperlipidemia is one of the bases in the development of NAFLD. Hence, there are more and more attention paid to the role of lipid drugs in the prevention and treatment of NAFLD. Thus, the gene regulation effect of lipid drug on ATGL may provide a new way for the treatment of NAFLD in the future.Part Ⅰ Analysis on the serum levels of ATGL in Rat models of NAFLD induced by High-fat dietObjective:To establish the rat model of NAFLD by high-fat diet, and then to investigate the changes of levels of serum adipose triglyceride lipase(ATGL) and tumor necrosis factor-α(TNF-α), as well as the relation between them during the developing process of NAFLD, which may further provide new insight into the pathogenesis of NAFLD.MethodsSeventy-eight male SD rats were randomized into2groups:normal control group (n=15, fed with normal diet,25g/rat/day) and NAFLD model group (n=63, fed with high-fat diet,25g/rat/day); Free of drinking and breeding continued for12weeks;2ml blood is collected from the inner canthus of rats after no eating and drinking to be isolated to serum. The serum level of TG, TC, ALT, AST and FBG were measured; The serum ATGL and TNF-a were assayed by ELISA quality kit strictly according to the manufacture’s instruction.5rats were randomly selected (3were from model group and2were from control group) and killed to undergo liver pathological examination after belly cavity injected10%Chloral hydrate (0.5ml/100g).Results 1. After continuing breeding12weeks using high-fat diet, the model rats were established, which had obesity and an increase in abdominal. Compared with the normal control group, the levels of serum TG、TC、ALT、AST、FBG of the NAFLD control group were shown to rise significantly(all p<0.05). The liver weight of model rats was increased, and lipid deposition was found in liver tissue, furthermore, the liver histology result displayed severe fatty degeneration with numerous inflammatory cells that belonged to the characteristic pathological changes of fatty liver. In pathological examination, the liver changes of model rats of were very similar with that of human obesity and/or diabetes as well as NASH which is dued by Hyperlipidemia;2. After12weeks, compared with the normal control group, the levels of serum TNF-a significantly raised, but the plasma activity dropped markedly(all p<0.05);3. During the developing process of NAFLD, the level of ATGL was highly negatively related to TNF-a(r=0.956, p<0.01).Conclusion1. The model of NAFLD rats can be successfully constructed by continuous breeding High-fat diet for12weeks;2. The level of ATGL was highly correlated with TNF-a in the NAFLD model rats, indicating that both ATGL and TNF-a might play important roles in the development of NAFLD.PartⅡ Effects of Simvastatin and Fenofibrate on serum level and expression of ATGL mRNA in Rat models of NAFLDObjective:To establish the rat model of NAFLD by high-fat diet, and then to investigate the effects of Simvastatine and Fenofibrate on the nonalcoholic fatty liver disease (NAFLD) and the changes of serum adipose triglyceride lipase (ATGL) as well as tumor necrosis factor-α (TNF-α) caused by these two drugs, to further improve the pathogenesis of NAFLD and explore the methods of treatment.Methods:Seventy-eight male SD rats were randomized into2groups:normal control group (n=15, fed with normal diet,25g/rat/day) and NAFLD model group (n=63, fed with high-fat diet,25g/rat/day); Free of drinking and breeding continued for12weeks; The level of serum TG, TC, ALT, AST were measured at the end of12weeks; Besides,5rats were randomly selected (3were from model group and2were from control group) and killed to undergo liver pathological examination; After the establishment of NAFLD rat model was confirmed the remaining60NAFLD rats were subdivided into4equal subgroups:NAFLD control group (fed with high-fat diet), Simvastatine-treated group (normal diet and sacrificed with Simvastatine4mg-kg-1·d-1), Fenofibrate-treated group (normal diet and sacrificed with Fenofibrate40mg-kg-1·d-1) and dietary treatment group (normal diet and sacrificed with normal saline); By the end of16th week, all rats anesthetized intraperitoneal by injecting10%Chloral hydrate(0.5ml/100g) were killed to isolate the samples of serum to measure the levels of TG、TC、ALT、AST、BUN、Scr and FBG; the liver index was counted; the ATGL and TNF-a of plasma were assayed by ELISA quality kit strictly according to the manufacture’s instruction; reverse transcription-polymerase chain reaction (RT-PCR) was performed to semi-quantitatively determine the liver mRNA expression of ATGL; and the pathologic changes of liver were recorded during experiment.Results1. After continued breeding16weeks using high-fat diet, it found the model rats had obesity and fat increase in abdominal, besides hyperlipidemia, liver enlargement, abnormal liver function all were appeared in model group; furthermore, the liver histology result displayed severe fatty degeneration with numerous inflammatory cells; Compared with the normal control group, the levels of serum TG、TC、ALT、 AST、BUN、Scr and TNF-a, liver index of the NAFLD control group were shown to increase significantly(all p<0.01), the gene expression and plasma activity of ATGL decreased markedly (both p<0.01);2. After drug treatment for four weeks, the levels of serum TG、TC、ALT、AST、BUN、Scr and liver index of two drug-treated groups became much lower than that of NAFLD control group (all p<0.05), moreover, the liver pathological results of two drug-treated groups both showed significant improvement with decreasing numerous inflammatory cells;3. After drug treatment for four weeks, compared with model control group, the levels of TNF-a of two drug-treated groups dropped markedly (all p<0.05), and the expression of ATGL mRNA had up-regulation(all p<0.01), the plasma activity of ATGL had been incresed (all p<0.01), but there was no difference between two drug-treated groups;4. The levels of serum TC、ALT of dietary treatment group without high-diet food for four weeks were lower significantly than that of model control group (all p<0.01), while still higher than that of two drug-treated groups; Although the liver pathological results showed that the lipid deposition had somewhat improvement, there were no difference on the level of serum TNF-a, gene expression and plasma activity of ATGL,compared with model control group;5. During the developing process of NAFLD, the level of ATGL was highly negatively correlated to TNF-a.Conclusion1. In rat model of NAFLD, the level of TNF-a increased significantly, but the expression of ATGL mRNA in liver and the plasma activity of ATGL dropped markedly, indicating that TNF-a could be weaken the plasma activity of ATGL by down-regulating the expression of ATGL mRNA, causing the accumulation of TG in liver in in the development of NAFLD;2. When the expression of ATGL mRNA is down-regulated, it may be followed by the poorer activity of plasma ATGL;3. Simvastatine and Fenofibrate both can treat rats NAFLD effectively, and in the two drug-treated groups, the expression of ATGL mRNA had up-regulation, the plasma activity of ATGL had been increased as well.