*2.5. Experimental Design*

A pilot study was conducted with three di fferent doses of bee bread (0.5, 1.0, and 1.5 g/kg/day) (*n* = 3/group) administered for 6 weeks via oral gavage to determine the best dose of bee bread in HFD-induced obese rats. According to Reagen-Shaw et al. [23], the lowest dose, i.e., 0.5 g/kg, was calculated based on the body surface area normalization method, relative to the local human consumption of bee bread, which is 5 g/day. Bee bread at the dose of 0.5 g/kg/day was chosen as the best dose and used in the present study, as it reduced Lee obesity index, TC, and LDL levels in HFD-induced obese rats (unpublished observation).

Thirty-two male Sprague Dawley rats were randomly divided into four groups (*n* = 8/group); i.e., normal group (N, on normal rat chow pellet and distilled water), high-fat diet (HFD, on high-fat diet and distilled water), HFD + BB (on high-fat diet and bee bread at 0.5 g/kg/day), and HFD + O (on high-fat diet and orlistat at 10 mg/kg/day). Normal rat chow pellets and the HFD were given ad libitum. Distilled water, bee bread, and orlistat were administered to rats via oral gavage for 6 weeks. Body weight and food intake were measured every other day. At the end of experimental period, Lee obesity index was calculated using a previous method [24], and a value of less than 315 was considered as normal [25]. Animals were sacrificed after being anaesthetised with ketamine 90 mg/kg and xylazine 5 mg/kg. Blood was collected from posterior vena cava for serum biological markers. Thoracic aorta was immediately excised, rinsed in ice-cold phosphate bu ffer solution, and homogenized for assessment on the levels of oxidant-antioxidant markers and fatty acid synthase (FAS) activity. Section of aortic arch was analysed for the presence of atherosclerotic plaque. Adipose tissue was dissected out and stored in 10% formalin for histological study.

#### *2.6. Measurement of Lipid Profile and Atherogenic Index*

Total cholesterol was determined by Architect c total cholesterol kit (ARCHITECT c cholesterol kit, Abbott, IL, USA) using an enzymatic-colorimetric method, which produced the end product quinoneimine from hydrogen peroxide (coe fficient of variation, CV ≤ 3% and sensitivity 18.26 mmol/L). TG was determined using Architect c triglyceride kit (ARCHITECT c Triglyceride Reagent kit, Abbott, IL, USA), which hydrolysed lipase to free fatty acids and glycerol (CV ≤ 5% and sensitivity 16.05 mmol/L). Reaction changes for TC and TG were measured at 500 nm (ARCHITECT c System, Abbott, IL, USA). High-density lipoprotein was measured based on elimination of chylomicrons, LDL, and very-low density lipoprotein by cholesterol esterase, cholesterol oxidase, and catalase using Biosino Direct HDL-Cholesterol reagen<sup>t</sup> kit, Biosino Bio-Technology and Science Inc, Beijing, China (sensitivity up to 2.586 mmol/L). Absorbance value was determined at 600 nm (ARCHITECT c System, Abbott, IL, USA). LDL was determined by the formula described by Friedewald et al. [26]: LDL (mmol/L) = (TC − HDL − TG)/5. Atherogenic index was calculated using formula: AI = (TC − HDL-C)/HDL-C [27].

#### *2.7. Determination of Aortic Oxidant*/*Antioxidant Status Markers and Fatty Acid Synthase (FAS)*

Aortic tissue was homogenized using a tissue grinder (Tissue Grinder G50, Coyote Bioscience Co., Ltd., Beijing, China) in an ice-chilled 0.1 M phosphate bu ffer solution, pH 7.4 and centrifuged (Avanti J-HC, Beckman Coulter, IN, USA) at 4000 rpm for 15 min. Supernatant was collected and used to analyse oxidant-antioxidant markers and FAS using procedures described by respective experimental protocols. Aortic oxidised LDL (oxLDL) and MDA were determined by commercially available kits from Northwest (Vancouver, WA, USA) and Cloud-Clone (Houstan, TX, USA), respectively. Aortic antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were determined by commercially available kits from Bioassay (San Francisco, CA, USA). Level of FAS activity was determined using a commercially available kit (Cloud-Clone, Houstan, TX, USA).

#### *2.8. Assessment on the Presence of Atherosclerotic Plaque in Aortic Arch*

The aortic arch was transversely cut at about 2 mm from, where it emerged in 2 cm length. Clean aortas were fixed with 78% methanol followed by incubation with Oil Red O solution for 50–60 min. Aortas were washed twice with 78% methanol followed by phosphate bu ffer solution. En face images of aortic arches were visualized under a stereomicroscope (Olympus SZ, OLYMPUS, Tokyo, Japan) at ×20 magnification [28].

## *2.9. Histology of Adipose Tissue*

Adipose tissue was processed and embedded in para ffin. A sample block was cut into sections of 5 μm size and fixed on glass slides. After drying, all slides were stained with haematoxylin and eosin (H&E) and inspected under a light microscope (Leica DM750, LEICA, Wetzlar, Germany).
