基于RNA-seq技术的何首乌对衰老小鼠脑组织转录组学影响的研究

Study on the effect of Polygonum multiflorum on the transcriptomics of brain tissue in aging mice based on RNA-seq technology

目的通过分析D-半乳糖导致的衰老小鼠脑组织以及经何首乌提取液治疗后的衰老小鼠脑组织的转录组测序结果,筛选出差异表达的基因,探索何首乌提取液对延缓脑衰老的潜在作用靶点。方法将30只C 57 BL/6小鼠随机分为空白对照组(CC)、D-Gal衰老组(MC)、何首乌提取液治疗组(MM)。MC组和MM组小鼠腹腔注射D-半乳糖,连续60 d。而后,MM组灌胃何首乌提取液,连续60 d。断头处死小鼠后取脑组织进行转录组测序(Transcriptome sequencing,RNA-Seq),通过对测序结果进行基因本体论(Gene Ontology,GO)功能注释分析和基因组百科全书(Kyoto Encyclopedia of Genes and Gnomes,KEGG)途径富集分析等,寻找何首乌提取液作用的可能靶点,并通过实时荧光定量PCR(qPCR)进行验证。结果MC组与CC组相比,筛选出明显差异表达基因331个,其中110个基因上调,221个基因下调。MM组与MC组相比,筛选出明显差异表达基因225个,其中122个基因上调,103个基因下调。同时变化的基因103个,多富集在细胞组分中的膜组成中,主要参与调节心律失常性右室心肌病通路及细胞外基质受体相互作用通路。其中Tnfrsf4在MC组下调,在MM组上调;Xkr4、Gprin3、Plcxd3在MC组均上调,在MM组均下调。RT-qPCR结果与其一致。结论何首乌可能通过上调Tnfrsf4的表达,下调Xkr4、Gprin3、Plcxd3的表达实现其抗衰老的作用。

Objective To analyze the transcriptome sequencing results of brain tissue of aging mice induced by D-galactose and brain tissue of aging mice treated with Polygonum multiflorum extract and screen out the differentially expressed genes and further explore the target of Polygonum multiflorum extract on delaying brain aging.Methods c57BL/6 mice were randomly divided into control group(CC),D-Gal aging group(MC)and Polygonum multiflorum extract treatment group(MM).Mice in MC group and MM group were injected intraperitoneally with D-galactose for 60 consecutive days.Then,the MM group was given the extract of Polygonum multiflorum for 60 consecutive days.After the mice were killed by decapitation,the brain tissues were taken for transcriptome sequencing(Transcriptome Sequencing,RNA-Seq).The sequencing results were analyzed by Gene Ontology(GO)functional annotation analysis and the Kyoto Encyclopedia of Genes and Gnomes(KEGG)pathway enrichment analysis,etc,to find possible targets for the effect of Polygonum multiflorum extract.Finally,the target gene was verified via real-time fluorescence quantitative PCR(qPCR)to verify.Results Compared with CC group,a total of 331 differentially expressed genes were screened out,of which 110 genes were up-regulated and 221 genes were down-regulated.Compared with MC group,a total of 225 differentially expressed genes were screened out,among which 122 genes were up-regulated and 103 genes were down-regulated.Among them,a total of 103 genes changed simultaneously,most of which were enriched in the membrane of the cellular component,and mainly involved in the arrhythmogenic right ventricular cardiomyopathy and ECM-receptor interaction.For example,Tnfrsf4 was down-regulated in the MC group and up-regulated in the MM group.Xkr4,Gprin3,and Plcxd3 were all up-regulated in MC group and down-regulated in MM group.Conclusion results were shown in RT-qPCR results.Conclusion Polygonum multiflorum Thunb might increase the expression of T nfrsf4 and decrease the expression of Xkr4,Gprin3 and Plcxd3 to achieve its anti-aging effect.