| Objective: D-3-Hydroxyacyl-CoA dehydratase /D-3- Hydroxyacyl-CoA dehydrogenase (D-bifunctional protein, DBP), located in mammalian peroxisomes and involved in peroxisomal beta-oxidation, was first found and named by Jiang LL et al in 1996,which catalyzes the dehydration and dehydrogenation of D-3-hydroxyacyl-CoA.The synthesis of bile acids is associated with peroxisomal beta-oxidation. In the classical pathway for the disposal of cholesterol in mammals, Cholesterol 7α-hydroxylase(CYP7A) is the first and rate-limiting enzyme that catalyzes the conversion of cholesterol to bile acids. In peroxisomes,conversion of the enoyl-forms of intermediates tri- and dihydroxycholestanoyl-CoA (THC-CoA or DHC-CoA) in bile acid formation to oxo-forms via D-3-hydroxyacyl-CoA is catalyzed by DBP but not L-bifunctional protein. In DBP deficiency patients, the serum unmature 27-carbon-bile acids increased remarkably. The same phenomenon was observed in DBP knock-out mice. From these evidences ,we can draw a conclusion that DBP takes part in theβ-oxidation reactions of cholesterol side chains and it is a necessary enzyme for bile acid synthesis. But, in vivo, whether D-bifunctional protein is a key enzyme in the regulation of bile acids synthesis, whether the mRNA expression and enzyme activity change with the amount of bile acids synthesis are unclear.Recently, many works have been done on the factors which regulating the synthesis and secretion of bile acids. These may be the potential ways to prevent and treat the hypercholesterolemia and related diseases in the future. So the research on relationships between D-bifunctional protein and bile acid synthesis in vivo is a valuable work.The target of our experiment is to find whether there are some relationships between mRNA expression and activity of DBP and the synthesis of bile acids, and to investigate whether D-bifunctional protein regulates the bile acid synthesis.For this purpose, we design our experiment in two directions. One is to observe the changes of expression and activity of hepatic DBP of rats fed high cholesterol or cholestyramine which are used to induce bile acid synthesis. On the other hand, we want to know how the rat bile acids synthesis change after the quantity and activity of liver DBP was induced by Diethylhexyl phthalate(DEHP), one of the peroxisome proliferators.Methods1 Animals and dietsForty male Wistar rats were divided randomly into four groups:①control group(Control),fed common rodent diet②cholestyramine group(CHY),fed 2% cholestyramine diet③cholesterol group(CH),fed 2% cholesterol plus 10%corn oil diet④inducer group(DEHP),fed 2% DEHP diet. Rats were given free access to food and water for 2 wk. After overnight fasting, rats were killed by releasing blood from carotid artery. Blood were selected for serum total cholesterol assay. Liver samples were cut rapidly, and a portion of liver was throw into liquid Nitrogen and stored at -70℃ for the extraction of RNA. In paralleled, 5g of liver were quickly homogenized to isolate crude peroxisome for measurement of DBP activity.2 Isolation of liver crude peroxisome5 g of liver were quickly homogenized in 20ml of an ice-cold buffer(Tris-HCL 10mM,sucrose 250mM, EDTA 1mM,ethanol 0.1%, PMSF 0.5mM,Benzamidin 1mM, PH7.4).The homogenate was first centrifuged at 3000g (10min,4℃);the resulting supernatant kept in ice-water, the pellets were homogenized again in 20ml buffer .The supernatant of two times were put together adding volume to 50ml and then centrifuged at 7000g(5min,4℃).The resulting supernatant was then centrifuged at 11000g(20min,4℃). The pellets were resuspended in 15ml of the buffer. The centrifugation procedure was repeated and the resulting pellets resuspended in 1ml of the buffer. The peroxisomal preparation was stored at -20℃ until measurement of DBP activity.3 Serum total cholesterol assaySerum total cholesterol was measured by Auto-biochemical-analyst using enzymatic procedure. 4 DBP activity assayThe activity of D-3-hydoxyacyl-CoA d...
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