坐骨神经损伤大鼠背根神经节基因表达与机械痛阈的相关性

Correlation between gene expression in dorsal root ganglion and mechanical pain threshold in rats with sciatic nerve injury

目的:基于生物信息学方法观察大鼠坐骨神经损伤(SNI)模型构建后1、4、7及14 d背根神经节(DRG)中核心差异分子的表达改变并分析与机械性痛阈的相关性。方法:下载GEO数据库中已构建的SNI模型大鼠的DRG基因数据集(GSE30165),应用生物信息学方法筛选DRG中的差异表达基因,将SNI不同时间点的差异基因进行共交集处理后通过STRING数据库分析蛋白质互作(PPI)网络的中心节点蛋白。使用Cytoscape软件对中心节点分子进行最大集团中心度(MCC)、边缘渗出组件(EPC)、度数(degree)、发散性(radiality)及紧密度(closeness)等拓扑分析法评分,并共交集出关键节点分子,分析每个关键节点分子不同时间点的表达量与机械性痛阈的相关性。结果:数据质控和标准化后,PCA结果显示组间区分较好,火山图结果显示,与对照组相比,SNI后1、4、7及14 d分别有2166、2590、3557及6369个差异基因被筛选,其中上调基因分别为1493、1524、1962及3277个,下调基因分别为673、1066、1595及3092个。SNI后1、4、7及14 d的上调和下调基因进行共交集处理,获取SNI后不同时间点的共同上调基因188个,共同下调基因98个。GO富集结果显示上调基因主要参与炎症反应、细胞凋亡及细胞分化过程,富集于胞浆及细胞间隙,介导蛋白质同源二聚体的活化、细胞因子以及激素活力。KEGG富集结果显示上调基因主要涉及MAPK信号通路、神经活性配体-受体的相互作用以及细胞因子-细胞因子受体相互作用等。MCC、EPC、Degree、Radiality及Closeness算法评分结果显示,白细胞介素-6(IL-6)、转录激活因子3(ATF3)、碱性成纤维细胞生长因子(FGF2)、基质金属蛋白酶3(MMP3)、Janus激酶2(JAK2)及胱天蛋白酶3(CASP3)共6个基因为所获得的核心差异基因。行为学结果显示,与对照组相比,SNI后1 d(P<0.01)、4 d(P<0.001)、7 d(P<0.001)及14 d(P<0.001)大鼠的机械性疼痛阈值显著降低,差异具有统计学意义。相关性分析结果显示ATF3(P=0.021)、FGF2(P=0.005)、MMP3(P=0.002)、JAK2(P=0.013)及CASP3(P=0.006)的mRNA水平与机械性疼痛阈值存在显著相关性,未见IL-6与机械性疼痛阈值存在显著相关性(P=0.133)。结论:ATF3、FGF2、MMP3、JAK2及CASP3在SNI诱发的神经病理性疼痛的早期和慢性期阶段均发挥着关键的作用,并且可能与影响细胞因子及经典MAPK信号通路密切相关,深入的分子机制探索仍需进一步的实验验证。

Objective:To observe the expression changes of core differential molecules in dorsal root ganglion(DRG)at 1,4,7 and 14 after sciatic nerve injury(SNI)model was established in rats by bioinformatics method and to analyze the correlation with mechanical pain threshold.Methods:The DRG gene data set(GSE30165)of SNI model rats constructed in GEO database was downloaded.The differentially expressed genes in DRG were screened by bioinformatics methods.The differentially expressed genes at different time points of SNI were intersected and the central node proteins of protein interaction(PPI)network were analyzed by STRING database.The maximal clique centrality(MCC),edge percolated component(EPC),degree,radiality and closeness algorithms were used to score the central node molecules by Cytoscape software,and the key node molecules were intersected to analyze the correlation between the expression of each key node molecule at different time points and the mechanical pain threshold.Results:After data quality control and standardization,PCA results showed a good distinction between groups.Volcano plot results showed that compared with the control group,2166,2590,3557 and 6369 differentially expressed genes were screened at 1,4,7 and 14 d after SNI,respectively.The up-regulated genes were 1493,1524,1962 and 3277,and the down-regulated genes were 673,1066,1595 and 3092,respectively.The up-regulated and down-regulated genes at 1,4,7 and 14 d after SNI were co-intersected to obtain 188 common up-regulated genes and 98 common down-regulated genes at different time points after SNI.GO enrichment results showed that up-regulated genes were mainly involved in inflammatory response,apoptosis and cell differentiation,enriched in cytoplasm and intercellular space,and mediated the activation of protein homodimers,cytokines and hormone activity.KEGG enrichment results showed that the up-regulated genes were mainly involved in MAPK signaling pathway,neuroactive ligand-receptor interaction and cytokine-cytokine receptor interaction.The results of MCC,EPC,Degree,Radiality and Closeness algorithms showed that Interleukin-6(IL-6),activator of transcription 3(ATF3),beta-fibroblast growth factor(FGF2),matrix metalloproteinase 3(MMP3),Janus kinase 2(JAK2)and caspase 3(CASP3)were the core differential genes.Behavioral results showed that compared with the control group,the mechanical pain threshold of rats at 1 d(P<0.01),4 d(P<0.001),7 d(P<0.001)and 14 d(P<0.001)after SNI was significantly reduced,and the difference was statistically significant.Correlation analysis showed that ATF3(P=0.021),FGF2(P=0.005),MMP3(P=0.002),JAK2(P=0.013)and CASP3(P=0.006)mRNA level were significantly correlated with mechanical pain threshold.There was no significant correlation between IL-6 and mechanical pain threshold(P=0.133).Conclusion:ATF3,FGF2,MMP3,JAK2 and CASP3 play a key role in the early and chronic stages of SNI-induced neuropathic pain and may be closely related to the influence of cytokines and classical MAPK signaling pathways.Further molecular mechanism exploration still needs further experimental verification.