## 3 Results

Table 1: Fasting blood concentrations of biochemical parameters in ApoE/- mice fed with a CD or HFD, without or with NR at the end of the study 1. Figure 1: Weekly food and energy intakes and final body weights in ApoE/- mice fed with a CD or HFD, without or with NR at the end of the study. (**A**) Weekly food intake; (**B**) Weekly energy intake; (**C**) Final body weight at the end of the study. Data were presented as mean \(\pm\) SEM. A statistical analysis was performed using a two-way analysis of variance (ANOVA) or a Scheirer-Ray-Hare test with the main effects of diet and NR and diet*NR interaction, depending on whether the data were under normal distribution. Means with different letters (a or b) indicated a significant difference from each other. CD (control), n = 10; CD + NR, n = 11; HFD, n = 10; HFD + NR, n = 10. CD, control diet; HFD, high-fat and high-cholesterol diet; + NR, a diet plus oral gavage with 0.3 g/kg BW/day of NR. Effect of Supplementation with NR on Gene Expressions in Cholesterol Metabolism Pathituays in CD- or HFD-Fed ApoE-/ Mice  
The hepatic mRNA expression of HMGCR, which is involved in cholesterol synthesis, was higher in the CD + NR and HFD + NR groups than the CD and HFD groups, respectively (_P-NR_ = 0.0323, _P-diet_ = 0.0123) (Figure 3A). The hepatic mRNA expression of PCSK9, which was involved in cholesterol uptake, was lower in the HFD + NR group than the HFD group (_P-diet_*NR_ = 0.0002) (Figure 3C). Compared to the CD and HFD groups, the hepatic mRNA expression of CYP7A1, which is involved in bile acid synthesis, was higher in the CD + NR and HFD + NR groups (_P-NR_ = 0.0010) (Figure 3D). The intestinal mRNA expression of ABCA1, which is involved in cholesterol efflux, was higher in the HFD + NR than the HFD group (_P-diet_*NR_ = 0.0134, _P-NR_ = 0.0407) (Figure 3D). There were no significant differences in the mRNA expression of other genes that are involved in cholesterol synthesis (hepatic SREBP-2), absorption (intestinal LXR-\(\alpha\) and NPC1L1), uptake (hepatic LXR-\(\alpha\), IDOL and LDLR), or efflux (hepatic FXR and intestinal ABCG5 and ABCG8) pathways among four groups (Figure 3A-D). Effect of Supplementation with NR on Gut Microbial Composition and Diversity in CD- or HFD-Fed ApoE-/ Mice  
A total of 6,611,437 raw 16S rRNA reads from fecal samples of 41 mice were obtained in this study. After quality control filtering of 79,941-231,415 raw reads from each sample, we obtained 71,081-207,951 16S rRNA reads per sample for further analysis. Based on the Venn diagrams, both the CD and CD + NR groups had similar numbers of total ASVs, while there were 169 more ASVs in the HFD + NR group than in the HFD group. The number of group-specific ASVs was highest in the HFD + NR group, which was 27.8% of the total ASVs in this group, followed by 18.7%, 22.7% and 16.2% in the CD, CD + NR, and HFD groups, respectively (Figure 4A). There were no significant differences in the richness indexes of \(\alpha\)-diversity among the four groups, including Observe, Chao 1 and ACE (Figure 4B). In contrast, the evenness indexes of \(\alpha\)-diversity, including Shannon, Simpson and J, were significantly decreased in the HFD and HFD + NR groups compared with the CD and CD + NR groups (all _P-diet_ < 0.05) (Figure 4C). The \(\beta\)-diversity of gut microbiota was similar among groups, and no distinct separation among the four groups was observed from the principal component analysis based on ASVs abundance (Figure 4D,E). An  
Figure 2: Histopathological changes of the entire aorta in ApoE-/- mice fed a CD or HFD, without or with NR at the end of the study. (**A**) Oil Red O staining (\(\times 1\) magnification); (**B**) Percentage of plaque area in aorta. Data were presented as mean \(\pm\) SEM. A statistical analysis was performed using a two-way analysis of variance (ANOVA) with the main effects of diet and NR and diet*NR interaction. Means with different letters (a or b) indicated a significant difference from each other. CD (control), n = 10; CD + NR, n = 11; HFD, n = 10; HFD + NR, n = 10.