基于多维谱效网络差示捕获红花和黄芪中调控DAPK1-NR2B信号通路的药效物质

Cerebral ischemia–reperfusion injury (CI/RI) is critical for the treatment of stroke. Our previous studies have shown that astragalus and safflower from Fangbu Yang Huan Wu Tang, a classic formula from the “benefiting qi for activating blood circulation in Medical Classics”, can significantly inhibit extrasynaptic NR2B phosphorylation, reduce neurotoxicity and dramatically alleviate CI/RI. Because of the variety of effective substance, the complexity of CI/RI and the particularity of blood-brain barriers, it was difficult to elucidate the true efficacy substances and compatibility mechanisms with astragalus and safflower. The effective substance basis of Safflower, Dan Hong injection and Safflower-Frankincense have been successfully revealed through the Differential efficacy of serum chromatographic analysis (DESCA). In the present study, we firstly proposed the multidimensional differential spectrum-effect network base on the DESCA. The efficacy indicator including the symptomatic body, cell and DAPK1-of NR2B signaling pathway were used. We will conduct multidimensional comparisons between spectrums from the “Indicators” and in vitro substances in the extract-serum-cerebrospinal fluid to investigate the efficacy substances. Followed, the efficacy substances and compatibility mechanisms will be investigated by the methods of specific substance knockout, reverse virtual, component compatibility. to the study will provide new strategies and approaches for discovering effective substances form Chinese medicine against cerebral injury.

脑缺血/再灌注损伤(cerebral ischemia–reperfusion injury,CI/RI)已成为影响脑卒中治疗的关键。我们研究表明，补阳还五汤（益气活血名方）中红花和黄芪可抑制DAPK1-NR2B信号通路，降低兴奋性神经毒性，有效改善CI/RI。但由于药效物质的多样性、脑损伤的复杂性和血脑屏障的特殊性，使两者脑保护药效物质和配伍机制至今不明。本课题组前期依托“药效差示血清色谱法”已成功揭示红花、丹红注射液和乳香/没药中改善血液流变学的药效物质，据此进一步提出“多维谱效网络差示捕获”新策略，即拟以对证机体、细胞和信号通路（DAPK1-NR2B）靶分子药效改变为导示，差示比对药效物质在提取物-血清-脑脊液的“多维谱效网络”中移行变化，再通过特异性敲除、反向虚拟验证和组分配伍方法，阐明红花和黄芪中脑保护的真正药效物质和配伍机制，以期为发现中药脑保护药效物质提供新策略和方法。

本课题组在构建寒凝气滞型CI/RI大鼠模型的基础上，观察凝血四项、血液流变学、神经功能评分、脑梗死面积等指标评价，明确黄芪-红花配伍组疗效优红花组和黄芪组，疗效与补阳还五汤组相当。在体外实验中通过细胞增殖、钙离子检测报告、流式细胞凋亡、JC-1检测、细胞ROS检测和氧化应激指标NO、GSH、MDA、SOD进行考察。筛选确定了黄芪-红花伍用配比和剂量，结果5:1-1倍剂量组最优。课题组在明确其改善CI/RI后1小时为最大药效时间点、5小时为最小药效时间点基础上，利用“差示色谱技术”，发现模型大鼠血液中的主要成分是羟基红花黄色素A（HSYA）、黄芪甲苷（AS-IV）和毛蕊异黄酮葡萄糖苷（CG），同时进行大鼠PK研究，相关内容发表在Biomedical Chromatography上。.为进一步验证捕获有效单体是否为真正药效物质，课题组首先应用神经元细胞对三种单体进行体外验证，结果表明：HSYA+CG+AS-IV组对神经元细胞增殖和游离钙离子浓度的影响优于单用和两两配伍组；通过流式细胞检测、JC-1检测发现三药组对神经元细胞凋亡的影响均优于单用和两两配伍组；ROS检测及对NO、GSH、MDA、SOD含量变化发现对神经元细胞氧化应激的影响仍是三药合用组疗效最优；体外NR2B/ DAPK1的机制研究也表明，三个有效单体均可以激活NR2B/ DAPK1通路，提高Bcl-2，降低Caspase-3表达，减轻细胞凋亡程度，发挥神经保护作用，且三药合用组优于单药和两药组。体内药效验证实验中，却发现HSYA+AS-IV组在改善MCAO/R大鼠上述整体四个指标优于单药、其他两两组合和三药组；脑组织机制研究也发现，HSYA、CG和AS-IV均可激活NR2B/DAPK1的通路，同样HSYA+AS-IV组最优，推测体内可能还通过其他通路发挥作用。为此，对红花和黄芪及其单体在代谢组学、蛋白组学、血管新生和自噬等方面进行外延性研究和探讨。以上内容分别发表在 Journal of Neuroinflammation.、Rejuvenation Res.、Mol Med Rep.、现代生物医学进展和中国医药导报等杂志上。.总之，课题组人员分工明确，严格按照年度计划执行，达到预期实验目的。在完成期间，培养博士研究生2名，硕士2名，累计发表论文6篇，其中SCI 4篇，中文核心2篇；获发明专利1项。