加味六味地黄汤对巨首相关蛋白-1基因的影响
Effect of Modified Liuwei Dihuang Decoction on Cytosine Hydroxymethylation of Dickkopf-Related Protein 1 Gene in BMSCs under High Glucose Stress
刘明明 1陈翀 2许海燕 2杨伏猛 1吕南宁 1李洪叶1
作者信息
- 1. 徐州医科大学连云港临床学院(江苏 连云港,222003)
- 2. 徐州医科大学
- 折叠
摘要
目的:探讨加味六味地黄汤在骨髓间充质干细胞(BMSCs)高糖应激下巨首相关蛋白-1(DKK1)基因胞嘧啶羟甲基化的作用机制.方法:将雄性SD大鼠随机分为空白组(等量生理盐水)和加味六味地黄汤干预组(24.255 g/kg)灌胃干预,制备含药血清.加味六味地黄汤含药血清干预骨髓间充质干细胞48 h,通过qRT-PCR和ELISA测定高糖应激下DKK1基因及蛋白的表达;用双荧光素酶报告基因检测高糖应激下DKK1基因启动子活性;5-hmC和5-mC分析加味六味地黄汤含药血清对高糖应激下DKK1基因启动子区的CpG甲基化和羟甲基化水平及Abct和Pgk1基因启动子区的羟甲基化水平;通过TETs活性及qRT-PCR测定加味六味地黄汤含药血清对TETs活性及TETs基因表达水平的影响.结果:与0％含药血清相比,20％加味六味地黄汤含药血清可以显著抑制高糖状态下DKK1 mRNA表达水平,差异有统计学意义(P＜0.01);同时显著减少培养上清中DKK1的浓度,差异有统计学意义(P＜0.01);抑制了高糖诱导的DKK1启动子活性,差异有统计学意义(P＜0.01).5-hmC和5-mC分析结果显示20％加味六味地黄汤含药血清降低了 DKK1启动子区羟甲基化水平,差异有统计学意义(P＜0.05);同时提高了该区域甲基化水平,差异有统计学意义(P＜0.01);加味六味地黄汤含药血清对TETs的活性没有显著影响,差异无统计学意义(P＞0.05),提高了 TET2基因表达水平,差异有统计学意义(P＜0.05).结论:加味六味地黄汤选择性抑制DKK1基因胞嘧啶羟甲基化,减轻高糖应激诱发的小鼠骨髓间充质干细胞DKK1表达.
Abstract
Objective:To investigate the effect of modified Liuwei Dihuang decoction on cytosine hydroxymethylation of dickkopf-related protein 1(DKK1)gene in bone marrow mesenchymal stem cells(BMSCs)under high glucose stress.Methods:Male SD rats were randomly divided into blank group(equivalent normal saline)and intervention group treated with a modified Liuwei Dihuang decoction(24.255 g/kg)by gavage to prepare medicated serum.BMSCs were treated with 20％serum containing modified Liuwei Dihuang decoction for 48 h,and the relative protein and gene expression levels of DKK1 were analyzed by qRT-PCR and ELISA tests under high-glucose environments.Dual lucifer-ase reporter gene assay was employed to evaluate the the DKK1 promoter activity.The 5-hmC and 5-mC was used to analyze the methylation level of the DKK1 gene and hydroxy-methylation level of CpG island of the DKK1,Abct and Pgk1 genes under high glucose stress.TETs activity and qRT-PCR were used to detected the TET(ten-eleven translocation)ac-tivity of TETs and expression level of TETs.Results:Compared with 0％modified Liuwei Dihuang decoction containing serum,20％modified Liuwei Dihuang decoction containing serum significantly inhibited the expression level of DKK1 mR-NA under high glucose conditions(P＜0.01),while significantly reduced the concentration of DKK1(P＜0.01).Addi-tionally,high-glucose-induced DKK1 promoter activity overexpression was attenuated(P＜0.01).The results of 5-hmC and 5-mC analysis showed that 20％modified Liuwei Dihuang decoction containing serum reduced the hydroxymethylation level in the DKK1 promoter region(P＜0.05),while increased the methylation level in this region(P＜0.01).The 20％modified Liuwei Dihuang decoction containing serum had no significant effect on the activity of TETs(P＞0.05).Howev-er,it can increase the expression level of TET2 gene(P＜0.05).Conclusion:Modified Liuwei Dihuang decoction selectively inhibits cytosine hydroxymethylation of DKK1 gene and alleviates the expression of DKK1 in high-glucose-induced BMSCs.
关键词
骨髓间充质干细胞/加味六味地黄汤/巨首相关蛋白-1/胞嘧啶羟甲基化/糖尿病性骨质疏松症Key words
bone marrow mesenchymal stem cells/modified Liuwei Dihuang decoction/dickkopf-related protein 1/cytosine hydroxymethylation/diabetic osteoporosis引用本文复制引用
基金项目
江苏省卫生健康委医学科研项目(H2023049)
连云港市第六期"521高层次人才培养工程"科研项目(LYG06521202159)
连云港市中医药科技发展计划(重点项目)(ZD202210)
连云港市"十四五"花果山科教强医工程(连卫科教[2021]1号)
出版年
2024
国家科技期刊平台
NSTL
万方数据