## 2 Materials and Methods  
### Animals and Experimental Diets  
The animal study was conducted according to established guidelines and protocols approved by the Sun Yat-sen University Animal Care and Use Committee (approval no. SYSU-IACUC-2021-000228). Because there are extremely limited published data on the effects of supplementation with both natto and RYR on circulating cholesterol concentrations, gene expressions in cholesterol metabolism and changes in gut microbiota, we based our sample size estimation on a previously established method called \"resource equation\" [26]. According to this method, the difference between the total number of animals and the total number of groups should lie between 10 and 20. Based on our study design, the total number of animals for four groups was between 14 (n = 4 per group) to 24 (n = 6 per group), and n = 10-11 per group was used in the current experiment. A total of 41 male ApoE-/- mice of 6-7 weeks of age were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and were housed in ventilated cages on 12 h light/dark cycle with access to food and water ad libitum in the animal facility of Sun Yat-sen University (Guangzhou, China). After acclimation to the normal chow diet for 7 days, mice were randomly allocated to four diet groups according to a 2 \(\times\) 2 factorial design: (1) control diet (CD, MD12014, 12% kcal from fat, n = 10); (2) CD plus oral gavage with 0.3 g/kg BW/day solution made from natto red yeast rice (NR) Capsules (CD + NR, n = 11); (3) high-fat and high-cholesterol diet (HFD, MD12015, 41% kcal from fat, 0.15% cholesterol, n = 10), and (4) HFD plus oral gavage with 0.3 g/kg BW/day NR (HFD + NR, n = 10). Diets were purchased from Medison Biomedical Co., Ltd. (Yangzhou, Jiangsu, China) and NR capsules were purchased from Weihai Nanbowan Biotechnology Co., Ltd. (Weihai, Shandong, China). The main components of NR were natto and red yeast rice, and the content of monacolin K (lovastatin) was 0.2 g in 100 g of NR. The NR powders isolated from capsules were dissolved 3:100 (\(w/v\)) with a sterile phosphate-buffered saline solution (Corning, Corning, NY, USA), and an NR suspension or sterile phosphate-buffered saline was administered once daily (100 \(\mu\)L/10 g BW) by oral gavage. The food intakes and body weights were monitored and recorded weekly. At the end of the 12-week treatment, mice were fasted for 14 h and were then anesthetized and euthanized. Fasting blood samples were collected in anticoagulation tubes, and plasma samples were isolated following centrifugation at 10,000\(\times\)\(g\) for 10 min at 4 \({}^{\circ}\)C. The liver and intestine samples and entire hearts with the aorta were carefully harvested and rinsed. Liver samples were dissected into smaller pieces and snap-frozen in liquid nitrogen and stored at -80 \({}^{\circ}\)C for subsequent analysis. After removal of the intestinal contents, the entire intestine was rinsed with ice-cold 1 \(\times\) phosphate-buffered saline (pH 7.4) (Servicebio, Wuhan, Hubei, China), cut longitudinally and spread out on a glass plate on top of ice. The opened intestines were gently scraped with glass microscope slides, and intestinal mucosa was collected and stored at \(-\)80 \({}^{\circ}\)C for the subsequent analysis with a Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). The aorta was isolated from the pulmonary trunk and the base of the heart, and the heart and aorta were preserved in 4% paraformaldehyde for no less than 48 h for further histologic measurement.