Integration of Metabolomics and Transcriptomics Reveals HDL-Driven Biliary Reverse Cholesterol Transport Involved in the Antiatherogenic Effect of San-wei-tan-xiang Capsule

Biliary reverse cholesterol transport (RCT) plays a crucial role in cholesterol clearance and regulation of atherogenesis. San-wei-tan-xiang capsule (SWTX), a traditional Chinese medicine, has shown potential in inhibiting atherogenesis by increasing high-density lipoprotein (HDL) cholesterol levels and promoting macrophage-mediated cholesterol efflux. However, the specific role of HDL-driven cholesterol metabolism in the anti-atherogenic effects of SWTX remains unclear. In this study, liquid chromatography coupled with tandem mass spectrometry was used to analyze the circulating metabolic profile, and RNA sequencing was performed on liver samples from ApoE−/− mice fed a cholesterol-enriched diet. We found that SWTX treatment induced significantly differential expression of metabolites and genes involved in cholesterol and lipid metabolism, as well as bile secretion pathways, which are critical for HDL-driven biliary RCT. Furthermore, alterations in L-carnitine and choline metabolism induced by SWTX treatment was involved in the atheroprotective effects of SWTX. Notably, SWTX treatment led to a significant increase in the expression of cholesterol 7α-hydroxylase (CYP7A1), a key enzyme involved in bile acid synthesis during atherogenesis. Additionally, the expression of CYP7A1 and CYP7A1-mediated bile acid secretion were enhanced by the addition of choline in hepatic cells, suggesting that SWTX-induced elevation of choline metabolic products may contribute to the upregulation of CYP7A1 and CYP7A1-mediated biliary RCT. Overall, SWTX demonstrated its ability to attenuate atherosclerotic plaque formation, which can be attributed to alterations in carnitine and choline metabolism, as well as the modulation of CYP7A1-mediated HDL-driven biliary RCT. The SWTX used in this study was obtained from Kaimeng Pharmaceutical Group Co., Ltd. (Hohhot, China) and had the following lot numbers: 20051401.1571, 20051901.1814, and 20051401.1555. The quality control of SWTX was performed according to the standards outlined in the Chinese Pharmacopoeia. The chemical fingerprint analysis of SWTX was conducted as described in our previous report (Ye et al., 2023). For administration, SWTX particles were dissolved in normal saline (NS) using ultrasonication for 40 minutes to prepare a suspension for treatment. ApoE−/− male mice with a C57BL/6J background (Gempharmatech, Guanzhou, China) at 8 weeks of age were used in this study. The mice were housed in specific pathogen-free facilities maintained at a temperature of 22-23°C with a 12-hour light:12-hour dark circadian cycle. They were fed a Paigen atherogenic diet (PAD, Dyets, Wuxi, China) containing 12.5 g/kg cholesterol and 5 g/kg sodium cholic acid for a duration of 8 weeks. The mice were randomly divided into two groups: the model group, which received NS administered by gavage, and the SWTX group, which received a dosage of 3.5 g/kg/d administered by gavage.