益气活血法调控脑出血后lncRNA-miRNA-mRNA三元转录网络促进脑组织修复的机制研究

Based on our previous NSFC projects, and our finding that qi-tonifying and stasis-eliminating therapy mediates lncRNA-mRNA co-expression of transcriptional networks in intracerebral hemorrhage (ICH), this program aims to target lncRNA-miRNA-mRNA ternary transcriptional network to explore the tissue repair-promoting mechanism of qi-tonifying and stasis-eliminating therapy following ICH. The qi-tonifying and stasis-eliminating prescription-Buyang Huangwu Decoction (BYHWD), qi-tonifying drugs of BYHWD, and stasis-eliminating drugs of BYHWD are used as tools, transcriptome technology is adopted to seize transcriptional networks in brain tissue repair after the qi-tonifying drug huangqi (knock out/knock in) treatment; pivotal lncRNA-miRNA-mRNA pathways are targeted by Bioinformatics technology; dual luciferase report gene, gene silencing and overexpression combination with cytobiology and molecular biology technology are used to investigate the ceRNA mechanism in the ternary transcriptional network, and to confirm the effect of qi-tonifying and stasis-eliminating therapy on the promotion of ICH tissue repair. This study will reveal the vital ternary transcriptional network which implicated in the ICH tissue repair by qi-tonifying and stasis-eliminating therapy, further explain the therapeutic cooperation rule. Our work will provide novel evidence for ICH treated with qi-tonifying and stasis-eliminating therapy, and explore new ideas for therapeutic cooperation.

在以往NSFC项目工作基础上，结合前期发现：益气活血调控脑出血（ICH）组织lncRNA-mRNA的共表达转录网络。以此为线索，本项目拟靶向lncRNA-miRNA-mRNA三元转录网络进一步探索益气活血法促进ICH组织修复的机制。内容：以益气活血经典方-补阳还五汤及方中的益气药、活血药为工具，采用转录组学技术，捕获敲入/敲出益气药黄芪干预ICH后组织修复的差异三元转录网络；借助生物信息学技术锚定网络中关键的lncRNA-miRNA-mRNA通路；运用双荧光素报告基因和基因沉默/过表达等技术，结合细胞生物学、分子生物学等技术，明确上述三元转录通路的ceRNA机制，验证其在益气活血法促进ICH组织修复中的作用。本研究可望揭示益气活血法促进ICH组织修复的关键三元转录网络，诠释益气活血法“气血共济”治疗ICH的治法协同规律，为益气活血法治疗ICH研究提供新的证据，为治法协同科学性探索新的思路。

在以往NSFC项目工作基础上，结合前期发现：补阳还五汤（BYHWD）调控脑出血（ICH）组织lncRNA-mRNA的共表达转录网络。以此为线索，本项目靶向lncRNA-miRNA-mRNA三元转录网络进一步探索BYHWD促进ICH组织修复的机制。内容：构建了自体血小鼠模型，通过错足试验、转角实验和改良神经功能评分进行评价补阳还五汤对小鼠神经功能的改善作用；通过形态学染色等方法评价补阳还五汤对脑出血小鼠的神经系统修复作用。进行了lncRNA-miRNA-mRNA测序，ICH 7d共检测到570个mRNA和313个lncRNA差异表达，其中差异表达的mRNA主要参与炎症反应、血管新生、神经退行性改变及神经保护等多种ICH相关的重要生理病理过程。构建了lncRNA-mRNA共表达网络和ceRNA网络，发现了38个lncRNA-mRNA共表达对和3个核心子集。筛选到5个BYHWD的效应miRNA，在动物水平验证了miR-2137、miR-3547-3p、miR-1224-5p、miR-7a-2-3p的表达变化。通过网络药理学联合转录组学分析策略共筛选出5个关键靶蛋白，分别是Ccl12，Abcb1b，Tlr4，Birc5和Ctsb，其中ctsb的mRNA表达变化最显著，进一步研究证实其与细胞自噬相关蛋白变化趋势相符。ICH 14d后，BYHWD显著改善ICH小鼠的神经凋亡，主要通过调节933404O12Rik/miR-185-5p/Sh2b3 ceRNA网络，并介导ERK1/2信号通路，缓解神经细胞凋亡。BYHWD介导GPR17蛋白的表达，通过抑制GPR17调控少突胶质细胞的分化和成熟，促进ICH小鼠髓鞘修复，其主要通过miR-760-3p抑制GPR17发挥作用。另外，我们绘制了ICH小鼠脑组织的时空代谢图谱，首次揭示了ICH后脑组织代谢的动态变化，有助于发现ICH潜在的疾病标志物及治疗靶标。本研究为补阳还五汤治疗ICH研究提供新的证据，为治法协同科学性探索新的思路。