## 4 Discussion  
In the current study, supplementation with NR resulted in lower fasting blood glucose concentrations in ApoE\({}^{\
icefrac{{7}}{{-}}}\) mice fed with HFD, with no significant effect on lipid and lipoprotein profiles. NR supplementation resulted in higher mRNA expressions of intestinal ABCA1 and hepatic CYP7A1, and the lower mRNA expression of hepatic PCSK9, concurrent with the lower relative abundance of phylum _Actinobacteria_ and genera _Coriobacteriaceae_UCG-002_, _Bacteroides_ and _Lactococcus_ in ApoE\({}^{\
icefrac{{7}}{{-}}}\) mice fed with HFD. The relative abundance of _Coriobacteriaceae_UCG-002_ was positively correlated with the hepatic mRNA expression of PCSK9 and inversely correlated with intestinal mRNA expression of ABCA1, and the relative abundance of _Lactococcus_ was inversely correlated with the hepatic mRNA expression of CYP7A1. These findings collectively suggested that NR  
Figure 5: (**A**) Correlations between the relative abundance of gut microbiota (Top 16 genera) and blood biochemical parameters, Oil Red O staining of aorta, gene expressions in cholesterol metabolism and concentrations of SCFAs in ApoE\({}^{\
icefrac{{7}}{{-}}}\) mice fed with a CD and HFD, without or with NR at the end of the study. (**B**) Changes of corresponding parameters after different diet and NR interventions. (**C**) Changes in the relative abundance of gut microbiota after different diet and NR interventions. Pearson or Spearman correlation tests were used depending on the normality of the data (N = 41). The data were presented as Pearson or Spearman correlation coefficients. * \(P<0.05\), ** \(P<0.01\), *** \(P<0.001\), and *** \(P<0.001\) indicate statistically significant correlations. 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 with 0.3 g/kg BW/day supplementation of natto and red yeast. \(\uparrow\) represented the parameters in the CD + NR group or the HFD + NR group that were significantly higher than those in the CD group or HFD group, respectively. \(\downarrow\) represented the parameters in the CD + NR group or the HFD + NR group were significantly lower than those in CD group or HFD group, respectively. The color blue or green in the merged grid that represented the parameters in both HFD groups were significantly lower or higher than those in both CD groups. The color green in a single grid that represented the parameters in the HFD group were significantly higher than the CD group. supplementation may regulate gene expressions in cholesterol metabolism via changes in the gut microbiota in ApoE/- mice fed with HFD. Previous studies have reported the cholesterol-lowering effect of supplementation with natto or RYR alone in both human subjects and experimental animals [10; 11; 12; 13; 16; 17; 18; 19; 20]. However, in the current study, combined supplementation with NR at a dose of 0.3 g/kg BW/day for 12 weeks did not significantly change fasting plasma concentrations of TC, LDL-C, HDL-C or non-HDL-C. The reason for the discordant results may be attributed, in part, to the lower dose of NR used in the current study in comparison to the doses used in previous studies, including natto ranging from 1.0-2.0 g/kg BW/day [33; 34; 35] and red yeast rice ranging from 0.3-1.0 g/kg BW/day [19; 36; 37]. Of note, on the basis of body surface area, the dose of NR in the current study equaled 1.4 g for an adult with a standard body weight of 50 kg, which was equivalent to the recommended dose of commercially available NR supplements. Future studies are required to examine the dose-response of NR supplementation on lipid and lipoprotein profiles in order to find the optimal dose for the cholesterol-lowering effect. Previous studies have reported the ability of RYR or monacolin K to downregulate the expression of HMGCR [19] or inhibit HMG-CoA reductase [6]. Inconsistent with these findings, we found that supplementation with NR resulted in higher mRNA expression of HMGCR in ApoE/- fed with either CD or HFD.