| Objective and backgroundNonalcoholic fatty liver disease (NAFLD) is a chronic liver disease with a wide spectrum of liver damage ranging from simple steatosis to steatohepatitis (NASH) to advanced fibrosis and cirrhosis. NASH has been implicated as a major risk factor for cryptogenic cirrhosis. NAFLD is a clinical syndrome with lesion of steatosis and fat storage in hepatic lobules but without alcohol abuse. Obesity, type2 (non-insulin-dependent) diabetes mellitus, and hyperlipidemia which comprise the metabolic syndrome, are coexisting conditions frequently associated with NAFLD. Global epidemiological survey shows that the prevalence of NAFLD varied between 17~33%, and it has exceeded those of virus hepatitis and alcoholic liver disease (ALD), becoming a medical and social problem with common concern. Recently, Fan Jian-gao and colleagues have shown that the prevalence of NAFLD is 17.4% among 3175 adults in Shanghai. The pathogenesis of NAFLD might revolve the accumulation of free fatty acids (FFA) and triglycerides (TG) within the liver, lipid peroxidation and insulin resistance (IR).Sterol regulatory element-binding proteins (SREBPs) play a crucial role in regulation the transcription and expression of lipid synthetic enzymes, which areassociated with the lipogenesis of cholesterol (Chol), FA, and TG The mammalian genome encodes three SREBP isoforms, designated SREBP-la, -lc, and -2. SREBP-lc plays a crucial role in inducing the expression of lipid synthetic enzymes in the liver. It associatd with the over accumulatin of TG and FFA in the liver which lead to lipotoxity, and enhanced IR. SREBP-lc increases FFA levels within hepatocytes lead to up-regulated of cytochrome P450 enzyme (CYP) 4A10, CYP4A14 and CYP2E1 genes which enhance FFA to generate reactive oxygen species(ROS) and lipid peroxidases(LPO). These factors may play a role in the development of hepatic inflammation, and result in NASH/NAFLD.Methods30 Male Sprague-Dawley rats weighing 250 to 300g, from Experimental Animal Center of Zhejiang Provice, were housed at the temperature of 23 to 25 centigrade and fed on a standard food with free access to drinking water. The animals were randomized into three groups. One was a control group (group I) (n=10) with natural history; The second group (group II) was hyperlipidemic diet group (n=10), feed on 78% common food plus 2% cholesterol and 10% lard, rats were sacrificed after one month; The third group (groupIII) was also hyperlipidemic diet group (n=10) as well as the second, but rats were sacrificed after six months. At one to six months, the rats were killed after weighing. Blood were got from femoral vein for laboratory investigations. Liver specimens from each animal were prepared with 10% formalin for paraffin section, and some fresh specimen was frozen in liquid nitrogen and stored in-80℃ for RT-PCR.Total RNA was isolated from approximately 200mg of frozen liver specimens according to the manufacturer's recommendation (TRIzol reagent, GIBOCOBRL). RT was carried out using a Revert Aid M-MLV Reverse Trancriptase (MBI Fermentas). Resulting cDNA were amplified by using primers that recognize SREBP-lc, CYP4A10,CYP4A14 and CYP2E1 mRNA for SREBP-lc, CYP4A10,CYP4A14 and CYP2E1. Primers recognize GAPDH were used with the same cDNA. Primers recognizing SREBP-lc mRNA were 5'- CTG CTG CTG ACA GCT GTA AA -3'(sense) and 5'- AGC GCT TCT CAA TGG CAT TG -3' (antisense); Primers recognizing CYP4A10 mRNA were 5'- CGT CTA CAG ATT GCT AGC TC -3' (sense) and 5'- GCA CAC TTC ATG ACA GTG TC -3' (antisense); Primers recognizing CYP4A14 mRNA were 5'- CTT GAT GAC ACT GGA CAC TG -3' (sense) and 5'- ACT CCA TCT GTG TGC TCA TG -3' (antisense); Primers recognizing CYP2E1 mRNA were 5'- TCA AGG AGG TGC TGC TGA AC -3' (sense) and 5'- ATG GCT TCC AGG TAG GTA TC -3' (antisense);The primers for GAPDH were 5'- CAT GGT CTA CAT GTT CCA GT -3' (sense) and 5'- GGC TAA GCA GTT GGT GTT GC-3' (antisense). The primers were purchased from Sangon Biotechnology Co, Ltd. PCR were performed with 2 ul of cDNA in a 20 ul of buffer containing 10mM Tri... |
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