何首乌提取液对衰老小鼠脑组织DNA甲基化和基因转录的影响

Effects of Polygonum multiflorum extract on DNA methylation and gene transcription in brain tissue of aging mice

目的分析何首乌提取液(Polygonum multiflorum extract,PME)对D-半乳糖致衰老C57BL/6小鼠脑组织DNA甲基化和转录组水平的影响,探讨何首乌延缓衰老的作用机制。方法 50只C57BL/6小鼠分为5组:正常对照组(CC)、衰老组(MC)、PME高剂量组(MH)、中剂量组(MM)及低剂量组(ML);除CC组外,其余4组用D-半乳糖诱导建立衰老模型,60 d后MH组、MM组及ML组分别以1、0.6、0.3 g·mL-1·kg-1的PME灌胃60 d,CC组及MC组灌胃等体积生理盐水。各组处理好后,麻醉小鼠并摘除眼球取血,通过检测血清中丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性判断衰老模型建立及何首乌延缓小鼠衰老的作用;采用简化甲基化测序(reduced representation bisulfite sequencing,RRBS)和RNA-seq测序方法检测各组小鼠脑组织DNA甲基化水平和转录水平,并利用生物信息学方法分析各组样本间差异性甲基化区域(differentially methylated region,DMR)以及差异表达基因,对DMR位于启动子区的相关基因进行GO聚类及KEGG通路分析,并通过实时荧光定量PCR验证部分基因的表达。结果与CC组比较,MC组中SOD、GSH-Px活性明显下降(P<0.05),MDA含量明显升高(P<0.05);PME作用后,衰老小鼠中抗氧化酶活性及MDA含量均明显缓解,以MM组较为明显(P<0.05)。在DNA甲基化测序中发现,CC组与MC组间及MC组与MM组间均发生甲基化变化的DMR位于启动子区的相关基因有52个,其中有7个基因的甲基化水平与转录组结果呈负相关,包括与衰老相关的Synpo和Mapkapk5;13个基因的甲基化水平与转录组呈正相关,包括与衰老相关的Tcf7和Rab15;且这4个基因mRNA水平均与转录组测序结果一致。结论何首乌提取液可能通过调控衰老小鼠中Synpo、Mapkapk5、Tcf7和Rab15基因甲基化水平,进而调节其转录水平,发挥延缓小鼠衰老的作用。

Objective To determine the effect of Polygonum multiflorum extract(PME) on DNA methylation and transcriptome levels in the brain tissue of D-galactose-induced senescence C57 BL/6 mice, and explore its mechanism of delaying aging. Methods Fifty C57 BL/6 mice were randomly divided into 5 groups, that is, normal control group(CC), aging group(MC), high-, medium-and low-dosed PME groups(MH, MM and ML). Except the CC group, the mice of the other 4 groups were induced to establish an aging model by D-galactose. Sixty days later, the mice of the MH, MM and ML groups were given intragastrical infusion with PME at 1, 0.6 and 0.3 g·mL-1·kg-1 for 60 d, and those of CC group and the MC group received same volume of normal saline. All the mice were anesthetized, and the eyeballs were removed to collect blood samples for malondialdehyde(MDA) content, and superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) activities to determine the establishment of aging model and the effect of Polygonum multiflorum in delaying aging in mice. The DNA methylation level and transcription level in brain tissue of each group of mice were detected by reduced representation bisulfite sequencing(RRBS) and RNA-seq sequencing. And bioinformatics analysis was used to analyze the differentially methylated region(DMR) and differentially expressed genes in the brain samples of each group. The related genes in the promoter region of DMR were analyzed by GO clustering and KEGG pathway analysis. Real-time qPCR was used to verify the expression of some genes. Results Compared with the CC group, the activities of SOD and GSH-Px were decreased obviously(P<0.05), and the content of MDA was increased significantly in the MC group(P<0.05). After PME treatment, the activities of antioxidant enzymes and MDA content in aging mice were distinctly relieved, especially in the MM group(P<0.05). In DNA methylation sequencing, there were 52 related genes in the promoter region of DMR with methylation changes between the CC group and the MC group, and between the MC group and the MM group. Among these genes, the methylation levels of 7 genes were negatively correlated with transcriptome results, including Synpo and Mapkapk5(associated with senescence), while the methylation levels of 13 genes were positively correlated with the transcriptome, including Tcf7 and Rab15(associated with senescence). The mRNA levels of these 4 genes were consistent with the transcriptome sequencing results. Conclusion PME exerts a delaying senescence effect probably by regulating the methylation of Synpo, Mapkapk5, Tcf7 and Rab15 genes, and thereby regulating their transcription levels in senescent mice.