The relevant kits were purchased from SunBio Biomedical Technology (Beijing, China). the inflammatory cytokines tumor necrosis factor- (TNF-), IL-6 and metalloproteinase-9 (MMP-9), the blood lipids triglyceride (TG), total cholesterol (TC), LDL cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured, and the severity of plaque lesions was also evaluated. Our results showed that the saline group, the rosuvastatin group and the low-dose demethylzeylasteral group had significantly Rabbit Polyclonal to YOD1 lower activated T lymphocyte parameters CD3+, CD4+, CD8+ and CD4+/CD8+ (P 0.05), and significantly higher levels of sIL-2R, immunoglobulins IgG, IgA and IgM, complements C3 and C4, anti-ox-LDL antibody, TNF-, IL-6 and MMP-9 CHAPS (P 0.01) when compared with the high-dose demethylzeylasteral group. Moreover, TG, TC, LDL-C contents were found significantly lower and their CHAPS HDL-C contents were significantly higher in high-dose demethylzeylasteral group (P 0.01) as compared to the other three groups. Furthermore, Sudan staining and haematoxylin and eosin staining of the thoracic aorta showed that, after 30-day treatment, the high-dose demethylzeylasteral group had the smoothest intima and the lightest plaque lesions among the four groups. Based on these results, we concluded that AS is a systemic immune-mediated chronic inflammatory disease and the relatively high dose of demethylzeylasteral used in the treatment of atherosclerotic rabbits could significantly alleviate AS. This implies that demethylzeylasteral may be considered as a suitable drug for anti-immunization therapy. would reduce or delay AS has not been reported. Therefore, the main focus of this study was to explore the anti-atherosclerotic effect of demethylzeylasteral. Materials and methods Animals For this study, 60 male New Zealand white rabbits (weight, 2.0C2.5 kg) were housed in the SPF level animal facility with one rabbit per cage. Feed and water was given to the animals, and litter and cages were CHAPS sterilized before use. The indoor temperature was kept at 20C22C, and the animals had each day 12-h light and 12-h dark. Animal feed and demethylzeylasteral intervention The rabbits were given 150 g high-fat diet (1% cholesterol, 5% CHAPS lard and 15% egg yolk powder) daily for 90 days. On day 61, the animals were randomly divided into the saline group, the rosuvastatin group, the low-dose demethylzeylasteral group, and the high-dose demethylzeylasteral group, each consisting of 15 rabbits. The rabbits from each group received saline, 0.5 mg/kg/day rosuvastatin (National Medicine Permit no. J20090092), 10 mg/kg/day demethylzeylasteral (provided by the Department of Pharmacy, Zhongshan Hospital of Fudan University, Shanghai, China) and 40 mg/kg/day demethylzeylasteral, respectively by intragastrical administration. The treatment was continued for 30 days. Collection of blood samples Venous blood samples were collected before the high-fat feeding, and before and after the interventions with saline, rosuvastatin and demethylzeylasteral. Ethics approval for animal experiments was from the Animal Ethics Committee of Shanghai University of Medicine and Health Sciences. Collection of pathology specimen On day 91, the rabbits were sacrified by air injection after they were given anesthesia by intraperitoneal injection of ketamine (35 mg/kg) and diazepam (5 mg/kg). Then the abdominal cavities of the animals of each group were opened and the epicardial adipose tissues were peeled from the aortic root to the level of the iliac artery. After that, the aortas were stored in 4% paraformaldehyde (PBS, pH 7.4). Then, a 0.5-cm fragment starting from the descending aorta was separated and sliced into 4 m pathology sections for immunohistochemical [hematoxylin and eosin (H&E)] staining. The rest of the aorta was opened longitudinally and stained with Sudan III. Measurements of cellular immune indexes The CD3+ T lymphocytes and the subsets CD4+, CD8+, and CD4+/CD8+ were measured by direct immunofluorescence CHAPS labeling before and after the treatment. An assay (ELISA) was used to measure the soluble interleukin-2 receptor (sIL-2R) with the sIL-2R kit (Meixuan Biotechnology Co., Ltd., Shanghai, China) by following the manufacturer’s operating manual. Determination of humoral immune indexes The rate nephelometry was used to determine the serum level of IgG, IgA, IgM, C3 and C4. Measurement of autoimmune index The anti-oxidized LDL (ox-LDL) antibody was measured by using ELISA with the anti-rabbit ox-LDL antibody ELISA kit (Nanjing SenBeijia Biotechnology Co., Ltd., Nanjing, China) following the instruction of manufacturer’s manual. Determination of inflammatory cytokines To measure the serum tumor necrosis factor- (TNF-), IL-6, metalloproteinase-9 (MMP-9) contents, radioimmunoassay was used. The relevant kits were purchased from SunBio Biomedical Technology (Beijing, China). The operations were conducted according to the instructions given on the manufacturers’ manual. Determination of blood lipid levels The contents of blood triglyceride (TG), high-density lipoprotein cholesterol.