Whole genome methylation combined with RNA-seq reveals the protective effects of Gualou-Xiebai herb pair in foam cells through DNA methylation mediated PI3K-AKT signaling pathway.
Whole genome methylation combined with RNA-seq reveals the protective effects of Gualou-Xiebai herb pair in foam cells through DNA methylation mediated PI3K-AKT signaling pathway.
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DOI：
10.3389/fimmu.2023.1054014
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发表时间：
2023
影响因子：
7.3
通讯作者：
Zhou, Qingbing
中科院分区：
文献类型：
--
作者：
Jia, Zijun;Mei, Jun;Zhang, Yan;Wang, Ya;Wang, Hongqin;Wang, Anlu;Xu, Fengqin;Zhou, Qingbing
DNA甲基化，包括异常的低甲基化和高甲基化，在动脉粥样硬化（AS）中起着重要作用，因此，针对AS中不平衡的甲基化是一种潜在的治疗策略。瓜蒌-泻白药对是治疗动脉粥样硬化相关性疾病的经典方剂。然而，GXHP对AS的作用及其机制尚不清楚。在这项研究中，CCK-8法被用来确定GXHP的无毒处理浓度。泡沫细胞的形成在AS的发生发展中起重要作用，因此，用ox-LDL处理RAW264.7细胞，建立泡沫细胞模型。采用气相色谱-质谱联用仪（GC-MS）测定总胆固醇（TC）和游离胆固醇（FC）的含量。采用酶联免疫吸附试验（ELISA）检测IL-1β、TNF-α、VCAM-1等炎症因子的表达。甲基捕获测序（MC-seq）和RNA-seq分别用于观察全基因组DNA甲基化和基因表达的变化。利用京都基因与基因组百科全书（KEGG）分析差异甲基化基因（DMG）和差异表达基因（DEG）。选择靶向信号通路并使用蛋白质印迹（WB）验证。结果表明，泡沫细胞内脂质和炎症因子明显增加。GXHP显著降低TC、FC和炎症因子的表达。MC-seq和RNA-seq结果表明，GXHP不仅能纠正ox-LDL诱导的泡沫细胞DNA高甲基化，还能纠正DNA低甲基化，从而恢复ox-LDL诱导的泡沫细胞DEG的异常。GXHP治疗可能靶向PI 3 K-Akt信号通路。GXHP可降低泡沫细胞中磷酸化（p）-PI 3 K和p-AKT的蛋白水平。我们的数据表明，GXHP通过抑制DNA甲基化介导的PI 3 K-AKT信号通路对AS具有保护作用，提示GXHP是一种新的基于甲基化的药物。
DNA methylation, including aberrant hypomethylation and hypermethylation, plays a significant role in atherosclerosis (AS); therefore, targeting the unbalanced methylation in AS is a potential treatment strategy. Gualou-xiebai herb pair (GXHP), a classic herb combination, have been used for the treatment of atherosclerotic-associated diseases in traditional Chinese medicine. However, the effects and underlying mechanism of GXHP on AS remain nebulous. In this study, the CCK-8 method was applied to determine the non-toxic treatment concentrations for GXHP. The formation of foam cells played a critical role in AS, so the foam cells model was established after RAW264.7 cells were treated with ox-LDL. The contents of total cholesterol (TC) and free cholesterol (FC) were determined by Gas chromatography-mass spectrometry (GC-MS). Enzyme-linked immunosorbent assay (ELISA) was used to check the expressions of inflammatory factors including IL-1β, TNF-α, and VCAM-1. Methyl-capture sequencing (MC-seq) and RNA-seq were applied to observe the changes in genome-wide DNA methylation and gene expression, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze differentially methylated genes (DMGs) and differentially expressed genes (DEGs). The targeted signaling pathway was selected and verified using western blotting (WB). The results showed that the lipids and inflammatory factors in foam cells significantly increased. GXHP significantly reduced the expression of TC, FC, and inflammatory factors. MC-seq and RNA-seq showed that GXHP not only corrected the aberrant DNA hypermethylation, but also DNA hypomethylation, thus restored the aberrant DEGs in foam cells induced by ox-LDL. GXHP treatment may target the PI3K-Akt signaling pathway. GXHP reduced the protein levels of phosphorylated(p)-PI3K and p-AKT in foam cells. Our data suggest that treatment with GXHP showed protective effects against AS through the inhibition of DNA methylation mediated PI3K-AKT signaling pathway, suggesting GXHP as a novel methylation-based agent.
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