Nano-DEN Solution Preparation and Experiment

The preparation of the nano-DEN solution containing 41.25 mg/mL diethylnitrosamine [12 (link)] used in this experiment was consistent with our previous report [23 (link)]. DEN was provided by Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Tween-80 was purchased from Amresco^® (Amresco, Solon, OH, USA). Lecithin from egg yolk and sesame oil were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure deionized water was used for all experiments. In addition, the control group and nano-DEN group were provided with regular feed, while the high-fat group and coexposure group were given obesity model series diets (DIO series Diets). The regular feed had a fat energy supply rate of 10%, whereas the HFD had a fat energy supply rate of 60%. Both the regular feed and high-fat feed were sourced from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). The composition of the HFD for mice (No. H10060) consisted of casein, dextrin, sucrose, cellulose, soybean oil, lard, various minerals, various vitamins, and choline, with fat accounting for 60% of the total energy intake, carbohydrates accounting for 20%, and protein accounting for 20%. The antibodies (Abcam Inc., Cambridge, UK) used in this study included antibodies against β-catenin (dilution of 1:200), HIF-1α (dilution of 1:200), p16 (dilution of 1:200), and COX-2 (dilution of 1:200).