An aliquot (50 g) of sample proteins were denatured in sample buffer containing SDS and -mercaptoethanol, separated on a 420% gradient SDS-PAGE gel, and electroblotted onto nitrocellulose membranes

An aliquot (50 g) of sample proteins were denatured in sample buffer containing SDS and -mercaptoethanol, separated on a 420% gradient SDS-PAGE gel, and electroblotted onto nitrocellulose membranes. potential natural cholesterol decreasing agent, operating through mechanisms unique from statins. == Intro == Hypercholesterolemia is a contributing element to atherosclerosis and consequent cardiovascular and cerebrovascular disease. Clinically, statins efficiently lower plasma cholesterol by inhibiting HMG-CoA reductase activity[1]. However, some individuals under statin treatment can not tolerate statins well or do not reach the low-density lipoprotein-cholesterol (LDL-C) goal recommended by the US National Institutes of Health guidelines[2]. Therefore, it is desirable to develop natural drugs that have cholesterol-lowering effect NAV-2729 comparable to statins, but could be tolerated well from the individuals. Astragalus polysaccharides (APS), an draw out of Radix Astragali, is one of the main efficacious principles. APS-I and APS-II are well known to NAV-2729 become the major structural components of APS. APS-I (molecular weight = 1,699,100 Da) consists of arabinose and glucose inside a molar percentage of 13.45, while APS-II (molecular weight = 1,197,600 Da) consists of rhamnose, arabinose and glucose inside a molar ratio of 16.2517.86[3]. In China, APS has been extensively used to treat viral illness[4],[5], acute myocarditis[6], glomerulonephritis[7], diabetes[8], tumor[9], KRT13 antibody and many other illnesses, with no harmful record in medical center. Previous reports indicated that some NAV-2729 dietary soluble polysaccharides lower plasma cholesterol via reduction of intestinal cholesterol absorption[10],[11],[12]or interference with the enterohepatic blood circulation of bile acids[13],[14]. However, the cholesterol-lowering effect of APS has not been well analyzed and underlying mechanisms behind are still elusive. Hence, we are here to determine how APS regulates plasma cholesterol and the cholesterol metabolic pathways in hyperlipidemia hamsters. == Materials and Methods == == Animals and diet programs == Twenty-four male Golden Syrian hamsters weighing 130140 g (Animal experimental center, Guangdong, China) were housed one per cage having a 12 h light: dark cycle and NAV-2729 moisture 5060%. The hamsters were fed a regular rodent cholesterol-free chow, with free access to food and water. After two weeks of adaptation, animals were weighed and randomly divided into 3 organizations (n = 8/group): APS group, Simvastatin group and Control group. The food of all the hamsters was switched to a high-fat diet for a period of 3 months. The diet contained 10% lard, 10% yoke powder, 1% cholesterol and 79% standard chow blend (Animal experimental center, Guangdong, China). Diets were prepared once for the 1st two weeks and on a weekly basis thereafter. For the next 3 months, based on the high-fat diet, animals in APS group and Simvastatin group were given orally APS at 0.25 g/kg/d and simvastatin at 10 mg/kg/d, respectively. Control animals received an equal volume of vehicle (0.9% saline). The purity of APS was >95% (Dabang animal Pharmaceutical Co, Ltd. Inner Mongolia, China). Simvastatin tablets were from Merck & Co., Inc. USA. The study protocol was authorized by the ethics committee of the 1st Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). Blood (0.5 mL) was collected from your tail vein into a heparinized capillary tube at the end of month 0,1.5, 3, 4.5, and 6 after overnight fasting. Plasma was harvested for plasma lipids and plasma liver enzyme measurements. At the end of month 0, 3, 6, fecal samples collected over 4 days for each animal were freeze- dried, weighed, and floor to powder for bile acid and natural sterol analysis. A week prior to sacrifice, the hamsters were gavaged with 2 Ci [3H]–sitosterol and 1 Ci [14C]-cholesterol, and the feces were collected for three days for fractional cholesterol absorption measurement. One hour before sacrifice, 40 mCi [3H] NAV-2729 water was injected intraperitoneally for.