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.

诸多研究表明，包括异常低甲基化与高甲基化在内的DNA甲基化（DNA methylation）在动脉粥样硬化（atherosclerosis, AS）的发生发展中发挥着重要作用。因此，针对动脉粥样硬化患者体内失衡的甲基化状态进行干预，有望成为该病治疗的全新策略。瓜蒌薤白药对（Gualou-xiebai herb pair, GXHP）作为经典的中药配伍组合，在传统中医药中用于治疗动脉粥样硬化性疾病已有两千余年的历史。然而，瓜蒌薤白药对在动脉粥样硬化中的治疗作用及其潜在分子机制仍有待阐明。本研究首先采用CCK-8法（Cell Counting Kit-8）确定瓜蒌薤白药对的有效干预浓度；采用氧化低密度脂蛋白（oxidized low-density lipoprotein, ox-LDL）诱导RAW264.7细胞构建泡沫细胞模型；分别采用气相色谱-质谱联用法（gas chromatography-mass spectrometry, GC-MS）与酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）检测细胞内总胆固醇、游离胆固醇以及炎症因子的含量。此外，本研究通过甲基化捕获测序（methyl-capture sequencing）与RNA测序（RNA sequencing, RNA-seq），分别检测细胞全基因组DNA甲基化水平与基因表达谱的变化；采用京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）对差异甲基化基因与差异表达基因进行富集分析，筛选得到核心调控通路后，通过蛋白质免疫印迹法（Western blotting, WB）对其进行验证。实验结果显示，氧化低密度脂蛋白诱导的泡沫细胞内脂质与炎症因子水平显著升高；瓜蒌薤白药对可显著降低上述脂质与炎症因子的水平。甲基化捕获测序与RNA测序结果表明，瓜蒌薤白药对不仅可纠正异常升高的DNA甲基化水平，还可修复异常降低的DNA甲基化状态，进而逆转氧化低密度脂蛋白诱导泡沫细胞产生的差异表达基因异常表达。京都基因与基因组百科全书富集分析结果显示，PI3K-Akt信号通路可能是瓜蒌薤白药对发挥治疗作用的核心通路；蛋白质免疫印迹实验进一步证实，瓜蒌薤白药对可显著降低泡沫细胞内磷酸化PI3K（p-PI3K）与磷酸化AKT（p-AKT）的蛋白表达水平。体外实验结果表明，瓜蒌薤白药对可通过抑制DNA甲基化介导的PI3K-Akt信号通路，发挥抗动脉粥样硬化的保护作用，提示瓜蒌薤白药对有望成为基于DNA甲基化调控的新型治疗药物。