基于甲基化捕获联合转录组测序探讨栀子-川芎药对抗动脉粥样硬化的作用机制

Antiatherosclerotic Mechanism of Fructus Gardeniae-Chuanxiong Rhizoma Based on MC-seq and RNA-seq

目的:观察栀子-川芎药对对氧化型低密度脂蛋白(ox-LDL)诱导的RAW264.7泡沫细胞DNA甲基化和转录组表达的作用。方法:细胞毒性试验(CCK-8)法检测不同浓度栀子-川芎冻干粉对RAW 264.7细胞的增殖抑制情况;以ox-LDL处理RAW 264.7细胞48 h形成泡沫细胞,将细胞分为栀子-川芎冻干粉组(80 mg/L ox-LDL、0.3 g/L栀子-川芎冻干粉干预)、模型组(80 mg/L ox-LDL处理)和空白对照组(未处理);气相、色谱质谱法(GC-MS)和酶联免疫吸附法(ELISA)分别检测各组总胆固醇(TC)、游离胆固醇(FC)以及炎性因子[肿瘤坏死因子α(TNF-α)、白介素6(IL-6)和细胞间黏附分子1(ICAM-1)]的表达;采用DNA甲基化捕获测序(MC-seq)和转录组测序(RNA-seq)方法检测各组细胞全基因组DNA甲基化和转录组基因表达水平,使用DSS工具分析各组样本间的差异甲基化位点(DMS)和差异表达基因(DEGs),并利用生物信息学方法对差异基因进行京都基因与基因组百科全书(KEGG)和基因本体(GO)富集分析。结果:CCK-8检测发现,栀子-川芎冻干粉对RAW264.7细胞具有一定的细胞毒效应;与空白对照组相比,模型组TC、FC水平以及炎性因子TNF-α、IL-6和ICAM-1的表达明显升高(P<0.01);与模型组相比,栀子-川芎冻干粉组TC、FC水平以及TNF-α、IL-6、ICAM-1的表达明显降低(P<0.01)。MC-Seq检测发现,在启动子区域,模型组与空白对照组相比,共有8421个DMSs,分别对应5061个低甲基化和3405个高甲基化基因位点。栀子-川芎冻干粉组与模型组比较,共有8395个DMSs,分别有4257个高甲基化基因位点和4138个低甲基化基因位点。RNA-seq检测方面,经两两比较取交集(栀子-川芎冻干粉组与模型组比较,模型组与空白对照组比较)显示栀子-川芎冻干粉共逆转了119个DEGs,其中下调39个,上调80个。对MC-Seq与RNA-seq数据进行联合分析发现,与模型组相比,栀子-川芎冻干粉组有29个低甲基化高表达基因,包括YAM1、硫酯酶超家族成员4(THEM4)、神经型一氧化氮合酶(NOS1)等;33个高甲化低表达基因,包括血小板源性生长因子受体α多肽(PDGFRα)、纤维连接蛋白1(FN1)及Ⅰ型胶原蛋白α2(COL1A2)等。KEGG富集分析显示上述基因涉及Focal adhesion、PI3K-Akt和AGE-RAGE等信号通路;GO富集分析显示涉及白细胞介素-1产生的调节和上皮细胞增殖的正向调节等生物学过程。结论:体外实验表明,栀子-川芎冻干粉具有明显的抗动脉粥样硬化效应,其机制可能与调控心血管相关基因的甲基化水平进而影响下游基因的表达有关。

Objective:To observe the effect of Fructus Gardeniae-Chuanxiong Rhizoma on DNA methylation and RNA expression in foamed cells derived from RAW264.7 cell line induced by oxidized low-density lipoprotein(ox-LDL).Methods:The cell proliferation of Raw 264.7 cells treated by different concentrations of Fructus Gardeniae-Chuanxiong Rhizoma was detected by CCK-8 method.Raw 264.7 cells were treated with ox-LDL for 48 hours to form foam cells.The cells were divided into three groups:Fructus Gardeniae-Chuanxiong Rhizoma group(80 mg/L ox-LDL and 0.3 g/L Fructus Gardeniae-Chuanxiong Rhizoma lyophilized powder),model group(80 mg/L ox-LDL),and blank control group(no treatment).Gas chromatography-mass spectrometry(GC-MS)and enzyme-linked immunosorbent assay(ELISA)were used to detect the levels of intracellular total cholesterol(TC),free cholesterol(FC),and inflammatory factors including tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and intercellular adhesion molecule-1(ICAM-1)were used to detect the changes of genome-wide DNA methylation and gene expressions in cells of each group respectively.Differentially methylated sites(DMSs)and expressed genes(DEGs)were analyzed by dispersion shrinkage for sequencing data(DSS)tool.In addition,Kyoto Encyclopedia of Genes and Genomes(KEGG)and gene ontology(GO)enrichment analysis were performed using bioinformatics methods.Results:CCK-8 assay showed that Fructus Gardeniae-Chuanxiong Rhizoma showed a cytotoxic effect on RAW264.7 cells.Compared with blank control group,the expressions of TC,FC,and inflammatory factors(TNF-α,IL-6,and ICAM-1)in model group were significantly increased(P<0.01).Compared with model group,TC and FC levels and expressions of TNF-α,IL-6,and ICAM-1 in Fructus Gardeniae-Chuanxiong Rhizoma group were significantly decreased(P<0.01).Methylation capture sequencing(MC-seq)detection showed that compared with blank control group,there were a total of 8421 DMSs in the promoter region,corresponding to 5061 hypomethylated and 3405 hypermethylated genes respectively in model group.Compared with model group,there were 8395 DMSs in Fructus Gardeniae-Chuanxiong Rhizoma group,with 4257 hypermethylated and 4138 hypomethylated genes,respectively.In terms of RNA sequencing(RNA-seq),the intersection of pairwise comparisons(Fructus Gardeniae-Chuanxiong Rhizoma group vs model group,model group vs blank control group)showed that Fructus Gardeniae-Chuanxiong Rhizoma reversed a total of 119 DEGs.Specifically,39 DEGs that were up-regulated by ox-LDL were down-regulated by Fructus Gardeniae-Chuanxiong Rhizoma treatment,and 80 DEGs that were down-regulated were up-regulated by Fructus Gardeniae-Chuanxiong Rhizoma treatment.The correlation analysis of MC-seq and RNA-seq data showed that compared with model group,there were 29 hypomethylated genes,whose downstream RNA expression was up-regulated and 33 hypermethylated genes,whose downstream RNA expression was dow-n-regulated in Fructus Gardeniae-Chuanxiong Rhizoma group.KEGG enrichment analysis indicated that these genes were involved in signaling pathways such as focal adhesion,phosphatidylinositol 3-hydroxykinase(PI3K)-protein kinase(Akt),and advanced glycation end products(AGEs)-receptor for advanced glycation end products(RAGE).GO enrichment analysis revealed biological processes involved in cell adhesion,regulation of interleukin-1 production,and positive regulation of epithelial cell proliferation.Conclusion:In vitro experiments showed that Fructus Gardeniae-Chuanxiong Rhizoma shows a significant antiatherosclerotic effect.The mechanism may be related to regulating the methylation level of cardiovascule-related genes and then affecting the expression of downstream genes.