Analysis of serum metabolic profile

Body weight and food intake of animals were recorded weekly. Intraperitoneal glucose tolerance test (IPGTT), with injection of 20% glucose at a dose of 2g/kg, was administered with tail vein blood at week 14 with One Touch Ultra meter (Lifescan; Johnson & Johnson, USA) at 0, 15, 30, 60 and 120 min. For the measurement of the blood lipids profile, blood was collected from orbital sinus of the animals which already fasting for 8 hours and anesthetized by isoflurane. We centrifuge the blood samples at 1500g 4^oC for 10min to obtain the serums and store at -80oC. The measurement of the serum levels of TG, TC, LDL, HDL, VLDL are operated on BECKMAN AU-5800 automatic biochemical analyzer with Olympus original reagent, calibration and quality control solution(Olympus, Japan). Serum TG levels were measured by Glycerophosphate oxidase - peroxidase (GPO-PAP) method. Serum TC levels were determined by the enzymatic method, HDL levels were determined by chemical modification method. LDL and VLDL levels were determined by selective lysis enzymatic method.

And the serum soluble adhesion molecules were determined by enzyme-linked immune-sorbent assay (R&D Systems, UK). In addition, aortas and other tissues were collected at the end of the study and quickly frozen in liquid nitrogen and then stored at −80°C for later analysis.

Quantification of atherosclerotic lesion area and measurements of atherosclerotic plaques histological composition

The aortas after opening and fixing in 10% formalin for 36h were stained with Sudan IV to detect the atherosclerotic lesion area and then photographed by a digital camera connected to a dissection microscope. The amount of atherosclerosis lesion was evaluated as the ratio of the atherosclerotic lesion area to the whole aorta area by Image-Pro Plus 6.0.

The aorta fixed in 10% formalin after 24h was paraffin embedded and cross-sectioned to analyze its histological composition. Masson’s trichrome stain kit (Maiwei, Xiamen, China) was used to assess the content of collagen fibers in atherosclerotic plaques and immune-histochemistry incubated with goat anti-macrophage-2 antigen (MAC-2) mouse macrophage or anti-alpha-smooth muscle cell (α-SMA) specific actin polyclonal antibody respectively (Bioss, Beijing, China) to qualify the contents of vascular smooth muscle cells and macrophage cells. All cross-sections were analyzed under an upright microscope (Nikon, Tokyo, Japan) .The amounts of macrophage and smooth muscle cells as well as the collagen fibers were quantified by image-processing software (Image Pro-plus 6.0).