摘要
目的观察栀子苷对ox-LDL诱导的RAW264.7来源泡沫细胞DNA甲基化的作用。方法以ox-LDL诱导RAW264.7细胞形成泡沫细胞模型;CCK-8法检测不同浓度栀子苷对泡沫细胞的增殖抑制情况;将细胞分为4组,分别为空白对照组(无处理)、模型组(80μg/mL ox-LDL处理)、栀子苷小剂量组(80μg/mL ox-LDL+30μg/mL栀子苷)、栀子苷大剂量组(80μg/mL ox-LDL+100μg/mL栀子苷),处理24 h后收集细胞,总胆固醇试剂盒检测栀子苷降脂效应;同时提取DNA,进行甲基化免疫共沉淀联合测序(Medip-seq)检测,并对差异甲基化基因进行GO与Pathway分析。结果红油O染色结果提示:以80μg/mL ox-LDL处理的RAW264.7细胞泡沫化最为明显;栀子苷对RAW264.7来源泡沫细胞无细胞毒作用;大剂量栀子苷(100μg/mL)能够降低细胞总胆固醇(P=0.04)。与空白组比较,模型组共有大量异常差异基因甲基化(包括异常的基因高甲基化与基因低甲基化)。涉及多个AS相关功能与通路,如蛋白激酶C活性,MAPK信号通路、PPAR信号通路、Wnt信号通路等;小剂量与大剂量栀子苷(30μg/mL与100μg/mL)均能改变泡沫细胞中异常的基因甲基化,涉及蛋白激酶C功能、Wnt信号通路、MAPK信号通路、PPAR信号通路等。栀子苷不仅能去甲基化,同时还能促甲基化。结论栀子苷可能通过对ox-LDL诱导的泡沫细胞异常基因甲基化进行双向调控实现抗AS效应。
Objective To observe the effect of geniposide on the DNA methylation in foamed cells derived from RAW264.7 cell line induced by ox-LDL. Methods ox-LDL was used to induce RAW264.7 cells to form the foam cells;CCK-8 method was used to detect the cell proliferation of foam cells treated by different concentrations of geniposide;Cholesterol test kit was applied for the changing of total cholesterol in cells after the treatment of geniposide. Then cells were divided into four groups: blank control group(no treatment), model group(treated only by 80 μg/mL ox-LDL), low-dose geniposide group(treated by 80 μg/mL ox-LDL and 30 μg/mL geniposide), high-dose gardenoside group(treated by 80 μg/mL ox-LDL and 100 μg/mL geniposide). After treatment of different concentrations of geniposide for 24 h, the foam cells were collected and Cholesterol test kit was applied to test the changing of total cholesterol in cells. Then DNA was extracted for methylation immunoprecipitation sequencing(Medip-seq). GO and Pathway databases were used to analyze the data from Medip-seq. Results The oil red O staining showed that RAW264.7 cells treated by 80 μg/mL ox-LDL turned out to be foam cells well. CCK-8 showed that geniposide had no cytotoxic effects on the RAW264.7 derived foam cells;total cholesterol in foam cells decreased after treatment of high-dose gardenoside(P=0.04). Medip-seq showed that compared with blank control group, there were a large number of abnormal methylated genes in model group(including abnormal hypermethlated genes and hypomethylated genes). These abnormal methylated genes were involved in multiple AS related functions and pathways, such as the activity of protein kinase C, MAPK signaling pathways, PPAR signal Pathway, Wnt signaling pathways, etc. 30 μg/mL and 100 μg/mL geniposide could change the methylation level of genes in foams cells. These altered methylatd genes were involved in the protein kinase C function, Wnt signaling Pathway, MAPK signaling Pathway, PPAR signaling Pathway and so on. At the same time,
作者
周庆兵
吴立旗
徐凤芹
ZHOU Qing-bing;WU Li-qi;XU Feng-qin(Institute of Geriatric Medicine ,Xiyuan Hospital ,China Academy of Chinese Medical Sciences,Beijing 100091)
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2019年第7期853-858,共6页
Chinese Journal of Integrated Traditional and Western Medicine
基金
国家自然科学基金资助项目(No.81603490,No.81473529)
中国博士后科学基金资助项目(No.2016m590187)
