Integration of Metabolomics, Transcriptomics, and 16s rRNA Sequencing Reveals the Mechanism of Morus alba L. (Sangzhi) Alkaloids (SZ-A) in Improving Cholesterol Metabolism in Diabetic Rats

Mulberry (Morus alba L.) is a plant widely used in the agricultural, food and pharmaceutical fields. Sangzhi alkaloids (SZ-A) are a series of alkaloids extracted from mulberry branches that are approved in China for treating type 2 diabetes mellitus (T2DM). T2DM is always accompanied by hypercholesterolemia, which increases the risk of macrovascular complications. However, the mechanism by which SZ-A influences cholesterol metabolism remains to be elucidated. In this study, SZ-A was orally administered to Zucker diabetic fatty rats at doses of 100 and 200 mg/kg once daily for 9 weeks. Cholesterol and bile acid levels in the blood and feces were determined using biochemical assays and targeted metabolomics, and the gut microbial profile was analyzed using 16s rRNA sequencing. Transcriptomic analysis and quantitative real-time polymerase chain reaction were used to explore the underlying genes involved in cholesterol metabolism. The results showed that repeated treatment with 200 mg/kg SZ-A significantly decreased the total cholesterol and low-density lipoprotein cholesterol levels in the blood and increased the fecal content of two conjugated bile acids, taurodeoxycholic acid (TDCA) and taurolithocholic acid (TLCA). The abundances of the beneficial bacteria Akkermansiaceae, Tannerellaceae, and Rikenellaceae were also increased, while the abundance of bile salt hydrolase-producing bacteria was modulated; notably, the abundances of Bacteroidaceae and Bifidobacteriaceae were increased and those of Clostridiaceae and Lactobacillaceae were decreased. Decreases in Clostridiaceae and Lactobacillaceae were negatively correlated with increases in fecal TLCA and TDCA, respectively, and increases in Akkermansiaceae, Tannerellaceae, and Rikenellaceae were positively correlated with increases in TLCA. SZ-A also significantly lowered the expression of aldo-keto reductase 1b7 and its upstream gene farnesoid X receptor (FXR) and increased the expression of cholesterol 7α-hydroxylase and small heterodimer partner. Taken together, repeated treatment with SZ-A ameliorated hypercholesterolemia partly by regulating bile acids production and excretion. Additionally, the gut microbiota and hepatic FXR signaling played important roles in this process.