补阳还五汤对脑缺血大鼠海马组织circRNA-miRNA-mRNA转录网络的影响

Effect of Buyang Huanwu Decoction on circRNA-miRNA-mRNA transcriptional network in hippocampus of rats with cerebral ischemia

目的研究补阳还五汤对脑缺血大鼠海马组织环状RNA(circular RNA,circRNA)-微小RNA(microRNA,miRNA)-mRNA转录网络的影响,探讨其治疗脑缺血的作用机制。方法运用超高效液相色谱-四极杆-飞行时间质谱联用仪(UPLCQ-TOF/MS)分析补阳还五汤化学成分;SD大鼠随机分为对照组、模型组、补阳还五汤(5 g/kg)组及丁苯酞组(54 mg/kg)组,除对照组外,其余各组采用大脑中动脉栓塞法复制脑缺血模型,给予药物干预7 d后,采用神经行为学评分、苏木素-伊红(HE)染色法及免疫组化法评估补阳还五汤疗效,采用竞争性内源RNA(competing endogenous RNA,ceRNA)芯片筛选差异表达的circRNAs及mRNAs,通过基因本体(gene ontology,GO)功能及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析差异基因参与的主要生物学过程,采用qRT-PCR法验证基因芯片结果,最后构建circRNA-miRNA-mRNA转录网络。结果补阳还五汤中共鉴定出黄芪甲苷Ⅳ、芒柄花素、阿魏酸及芍药内酯苷等21个成分。大鼠脑缺血后出现神经功能障碍及海马神经细胞损伤,补阳还五汤能部分逆转上述损伤并提高海马齿状回区增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)及巢蛋白(Nestin)蛋白表达。ceRNA芯片筛选出大鼠脑缺血后有27个差异表达的circRNAs和767个差异表达的mRNAs,补阳还五汤治疗后有70个差异表达的circRNAs和692个差异表达的mRNAs。构建了由7个circRNAs、9个miRNAs及15个mRNAs组成的三元转录网络。生物信息学分析显示这些靶点可能通过自噬、血管内皮生长因子(vascular endothelial growth factor,VEGF)信号通路、磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase,PI3K)-蛋白激酶B(protein kinase B,Akt)信号通路等发挥作用。结论补阳还五汤可能通过影响脑缺血大鼠海马组织的circRNA-miRNA-mRNA转录网络促进海马神经前体细胞增殖,诱导神经再生,涉及多条通路及多个生物学过程。

Objective To reveal the effects of Buyang Huanwu Decoction(补阳还五汤,BHD)on circular RNA(circRNA)-microRNA(miRNA)-mRNA transcriptional network in hippocampal of rats with cerebral ischemia,explore its mechanism for treatment of cerebral ischemia.Methods UPLC-Q-TOF/MS was used to analyze the chemical composition of BHD.SD rats were randomly divided into control group,model group,BHD(5 g/kg)group and butylphthalide(54 mg/kg)group.The middle cerebral artery embolization method was used to replicate cerebral ischemia model in all groups except control group.After 7 d of intervention,neurobehavioral scores,HE staining and immunohistochemistry were used to evaluate the efficacy of BHD,competing endogenous RNA(ceRNA)microarray was used to screen circRNAs and mRNAs differentially expressed.Main biological processes involved in differential genes was analyzed by gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichmen;qRT-PCR was used to validate the gene microarray results.Finally,circRNA-miRNA-mRNA transcriptional network was constructed.Results A total of 21 compounds including astragalosideⅣ,dormononetin,ferulic acid and paeonilactone glycosides were identified in BHD.Rats developed neurological dysfunction and hippocampal neuronal cell damage after cerebral ischemia,and BHD could partially reverse the above injury,increase the expression of proliferating cell nuclear antigen(PCNA)and Nestin in hippocampal dentate gyrus.The ceRNA microarray identified 27 differentially expressed circRNAs and 767 differentially expressed m RNAs after cerebral ischemia in rats,and 70 differentially expressed circRNAs and 692 differentially expressed mRNAs after BHD treatment.A ternary transcriptional network consisting of seven circRNAs,nine miRNAs and 15 mRNAs was constructed.Bioinformatics analysis showed that these targets may play a therapeutic role through autophagy,vascular endothelial growth factor(VEGF)signaling pathway and phosphatidylinositol 3-kinase(PI3 K)-protein kinase B(Akt)signaling pathway.Conclusion BHD may promote the proliferation of hippocampal neural progenitor cells and induce nerve regeneration by affecting circRNA-miRNA-mRNA transcriptional network in the hippocampus of cerebral ischemia rats,involving multiple pathways and multiple biological processes.