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

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。

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 andg ene expression levels of DKK1 were analyzed by qRT-PCR and ELISA tests under high-glucose environments.Dual luciferase 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 DKKl gene and hydroxymethylation 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)activity 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 mRNA under high glucose conditions(P<0.01),while significantly reduced the concentration of DKK1(P<0.01).Additionally,high-glucose-induced DKK1 promoter activity overexpression was attenuated(P<o.O1).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.O5).However,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.