Table_2_Acteoside From Ligustrum robustum (Roxb.) Blume Ameliorates Lipid Metabolism and Synthesis in a HepG2 Cell Model of Lipid Accumulation.docx

We aimed to ascertain the mechanism underlying the effects of acteoside (ACT) from Ligustrum robustum (Roxb.) Blume (Oleaceae) on lipid metabolism and synthesis. ACT, a water-soluble phenylpropanoid glycoside, is the most abundant and major active component of L. robustum; the leaves of L. robustum, known as kudingcha (bitter tea), have long been used in China as an herbal tea for weight loss. Recently, based on previous studies, our team reached a preliminary conclusion that phenylpropanoid glycosides from L. robustum most likely contribute substantially to reducing lipid levels, but the mechanism remains unclear. Here, we conducted an in silico screen of currently known phenylethanoid glycosides from L. robustum and attempted to explore the hypolipidemic mechanism of ACT, the representative component of phenylethanoid glycosides in L. robustum, using RNA-seq technology, quantitative real-time PCR (qPCR) and Western blotting. First, the screening results for six compounds were docked with 15 human protein targets, and 3 of 15 protein targets were related to cardiovascular diseases. Based on previous experimental data and docking results, we selected ACT, which exerted positive effects, for further study. We generated a lipid accumulation model using HepG2 cells treated with a high concentration of oleic acid and then extracted RNA from cells treated for 24 h with 50 μmol/L ACT. Subsequently, we performed a transcriptomic analysis of the RNA-seq results, which revealed a large number of differentially expressed genes. Finally, we randomly selected some genes and proteins for further validation using qPCR and Western blotting; the results agreed with the RNA-seq data and confirmed their reliability. In conclusion, our experiments proved that ACT from L. robustum alters lipid metabolism and synthesis by regulating the expression of multiple genes, including Scarb1, Scarb2, Srebf1, Dhcr7, Acat2, Hmgcr, Fdft1, and Lss, which are involved several pathways, such as the glycolytic, AMPK, and fatty acid degradation pathways.