目的 研究凉血通瘀方对高血压大鼠急性脑出血模型脑组织miRNA表达的影响，对差异表达的miRNA靶基因进行分析，探索凉血通瘀方可能的药效机制。方法 将自发性高血压大鼠随机分成对照组（B）和实验组（C）。适应性饲养一周后，C组灌胃凉血通瘀方，B组灌胃等体积生理盐水，连续5天，每天1次。构建脑出血模型后收集脑组织，借助全转录组测序技术获得miRNA表达量，与miRBase数据库比对获取已知miRNA，使用miRDeep2预测新miRNA。差异分析软件为DESeq2，筛选阈值为|log₂FC| ≥1 并且P <0.05。对显著差异表达的miRNA进行靶基因预测，对靶基因进行GO功能、KEGG通路富集和PPI网络分析。结果 实验组和对照组对比，共发现21个显著差异表达的miRNA，上调有9个，下调有12个，共预测得到1243个有统计学意义的靶基因。GO富集分析发现，生物过程中突触囊泡分泌的调节、神经递质分泌的调节和神经递质运输的调节占前三位，神经元投射终点、全膜、质膜区域和细胞投射则是主要的细胞成分。分子功能分别为小GTPase绑定、底物特异性跨膜转运蛋白活性和离子跨膜转运体活性。通路分析结果显示，靶基因在癌证通路、pI3K-Akt信号通路、人类乳头瘤病毒感染、神经活性配体-受体相互作用和MAPK通路等分布广泛。采用STRING网站和Cytoscape软件，根据MCC算法筛选出ADRA2C、CASR、CCL28、CCR1、DRD2、GNAT3、GRM2、DYNC1LI1、GABBR1、GNAI1等核心靶基因。结论 凉血通瘀方对脑出血急性期鼠脑组织内miRNA的表达有重要影响；显著差异表达miRNAs可能通过靶向核心基因调控凉血通瘀方干预急性脑出血的病理过程及预后。

Objective To study the effect of Liangxue Tongyu Prescription (LTP) on the expression of miRNA in brain tissue of hypertensive rats with acute cerebral hemorrhage, analyze the differentially expressed miRNA target genes, and explore the possible mechanism of LTP.Methods Spontaneously hypertensive rats were randomly divided into control group (B) and experimental group (C). After one week of adaptive feeding, group C was intragastrically administered with Liangxue Tongyu Prescription and group B was intragastrically administered with an equal volume of normal saline once a day for 5 consecutive days. Brain tissues were collected after the intracerebral hemorrhage model was constructed. miRNA expression was obtained with the help of whole transcriptome sequencing technology. Known miRNAs were obtained by alignment with the miRBase database, and new miRNAs were predicted using miRDeep2. The difference analysis software was DESeq2 with a screening threshold of | log2FC |≥1 (P<0.05). Target gene prediction was performed for significantly differentially expressed miRNAs, and GO function, KEGG pathway enrichment analysis and PPI Network Analysis were performed for target genes.Results A total of 21 significantly differentially expressed miRNAs were found in control group and experimental group, 9 were up-regulated and 12 were down-regulated, and a total of 1243 statistically significant target genes were predicted. GO enrichment analysis revealed that the regulation of synaptic vesicle secretion, regulation of neurotransmitter secretion and regulation of neurotransmitter transport accounted for the top three in biological processes, and neuronal projection membrane, whole membrane, plasma membrane region and cell projection part were the main cellular components. The molecular functions are small GTPase binding, substrate-specific transmembrane transporter activity and ion transmembrane transporter activity. The results of pathway analysis showed that the target genes were widely distributed in the pathway in cancer, p13K-Akt signaling pathway, human papillomavirus infection, neuroactive ligand-receptor interaction and MAPK pathway. Finally, with the help of STRING websites and Maximal Clique Centrality (MCC) of Cytoscape software, ADRA2C, CASR, CCL28, CCR1, DRD2, GNAT3, GRM2, DYNC1LI1, GABBR1, GNAI3 and other core target genes were screened out.Conclusion Liangxue Tongyu Prescription can induce the differential expression of miRNAs in the brain tissue of rats with acute intracerebral hemorrhage. Significantly differential expression of miRNAs regulates the intervention of LTP in the pathological process and prognosis of acute cerebral hemorrhage by targeting core genes.

国家自然科学基金委员会面上项目（81373512）：急性脑出血瘀热病机单元的动态测量及其生物标志物的研究，负责人：李国春。