Agilent methyl-capture sequencing of Gualou-xiebai herb pair in foam cells

Numerous studies have revealed that DNA methylation including aberrant hypomethylation and hypermethylation plays a significant role in atherosclerosis. Thus, targeting the unbalanced methylation in AS could be a novel treatment strategy for AS patients. Gualou-xiebai herb pair, a classic herb combination, have been used for the treatment of atherosclerotic diseases for over 2000 years in traditional Chinese medicine. However, the effects and underlying mechanism of GXHP on AS remain unclear. In this study, the CCK-8 method was applied to determine the treatment concentrations for Gualou-xiebai herb pair. The foam cells model was established after RAW264.7 cells were treated with ox-LDL. Gas chromatography-mass spectrometry and enzyme-linked immunosorbent assay was used to determine the contents of total cholesterol, free cholesterol and inflammatory factors. Furthermore, methyl-capture sequencing and RNA sequencing were applied to observe the changes of genome-wide DNA methylation and gene expressions in cells. Kyoto Encyclopedia of Genes and Genomes were performed to analyze differentially methylated genes and differentially expressed genes. The targeted signaling pathway was chosen and then verified by Western blotting. The results showed that the lipids and inflammatory factors in foam cells increased significantly. Gualou-xiebai herb pair could significantly reduce the expression of total cholesterol, free cholesterol and inflammatory factors. methyl-capture sequencing and RNA sequencing showed that Gualou-xiebai herb pair not only corrected the aberrant DNA hypermethylation, but also DNA hypomethylation, thus reversed the aberrant DEGs in foam cells induced by ox-LDL. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the PI3K-Akt signaling pathway maybe the target for Gualou-xiebai herb pair treatment. Western blotting confirmed that Gualou-xiebai herb pair reduced the protein levels of phosphorylated(p)-PI3K and p-AKT in foam cells. The vitro data suggested that treatment with Gualou-xiebai herb pair showed the protective effects on AS through the inhibition of DNA methylation mediated PI3K-AKT signaling pathway, indicating that Gualou-xiebai herb pair may be a novel methylation-based agent.