基于HSP70和IKK双靶标调控的黄姜抗脑缺血再灌注损伤药效物质及机制研究

It is a crucial strategy to treat the ischemic cerebrovascular disease in clinic to suppress the cerebral ischemia reperfusion (CIR). The HSP70 expression and the NF-κB signal pathway activation mediated by IKK kinase are closed related with inflammatory disequilibrium of CIR. It is the research hotspot at present to discover new bioactive ingredients, which are used to alleviate the inflammatory reaction of CIR through regulating and controlling the key targets, from Traditional Chinese Medicine (TCM) with higher efficiency and lower toxicity in the treatment with CIR. We have pioneered to the study of total steroid saponins in treating CIR, which have been used to cure cardiovascular disease in clinic, from Dioscorea zingiberensis C.H. Wright (D. zingiberensis) and obvious pharmacological effect has displayed based on the TCM theory of treating heart and brain with common theory and methods. According to the available basis, this research project is firstly to separate a series of single steroid saponins from D. zingiberensis by preparative HPLC. Then, on account of HSP70 expression and NF-κB signal pathway regulated by upstream IKK, these compounds against CIR are in vitro screened on PC12 cell line subjected to oxygen-glucose deprivation/reperfusion (OGD/RP). The target active ingredients potentially treated CIR are finally confirmed in vivo from D. zingiberensis after validating on the CIR rats induced by middle cerebral artery occlusion (MCAO). The current research will not only elaborate the substance basis and underlying mechanisms of curing CIR of D. zingiberensis, but also supply the theoretical basis of developing this TCM into a new drug for the treatment of CIR.

抑制脑缺血再灌注损伤（Cerebral ischemia reperfusion，CIR）是临床上治疗缺血性脑血管病的重要策略。HSP70及IKK介导的NF-κB通路激活与CIR的炎症平衡失调密切相关。从中药中发现高效低毒的活性成分，作用于关键靶点减轻CIR炎症反应是当前治疗CIR的研究热点。黄姜中甾体总皂苷在临床上用于治疗心血管疾病，基于中医“心脑同治”理论，我们开创性地将其应用于抗CIR的研究中，且疗效明显。本项目拟在此基础上采用HPLC制备系列甾体皂苷单体成分；通过OGD/RP建立PC12细胞CIR模型，基于对HSP70和IKK/NF-κB通路上游IKK的调控作用，体外筛选抗CIR单体成分；采用MCAO建立大鼠CIR模型，根据上述双靶标作用特点通过体内验证最终确定黄姜抗CIR的目标活性成分。本研究不仅阐明黄姜治疗CIR的物质基础及机制，也为将黄姜开发成为治疗CIR的新药提供理论依据。

脑缺血再灌注损伤（Cerebral ischemia reperfusion，CIR）是临床上治疗缺血性脑血管疾病的次级并发症。炎症异常是诱发CIR的关键病因，从中药中发现高效低毒、副作用小的有效部位、组分群或单体化合物，用于调控炎症因子平衡失调，抑制CIR的病程，是当前研发治疗CIR药物的热点。黄姜又名盾叶薯蓣，是薯蓣科薯蓣属多年生草本植物，为陕西秦巴山区特色药材。由其根茎提取物研发而成的“盾叶冠心宁”是治疗冠心病和心绞痛等心血管疾病的药物。基于中医“心脑同治”理论，首次将其用于治疗CIR。虽然疗效显著，但抗CIR的药效物质基础及作用机制尚不清楚。. 采用乙醇溶液提取和大孔吸附树脂除杂等过程从黄姜根茎中富集甾体总皂苷，通过色谱法对其进行分离，用波谱学法鉴定化学结构和表征理化参数。按照OGD/RP法致SH-SY5Y和PC12损伤，模拟CIR临床病症，体外考察抗CIR活性，明确黄姜甾体皂苷药效成分。对大鼠施以线栓法致MACO诱导CIR模型，体内验证前述成分的药效。构建HSP70-siRNA和IKK-cDNA，研究黄姜目标成分通过HSP70和IKK调控NF-κB介导的炎症反应，最终发挥抗CIR的作用机制。. 通过色谱法从黄姜中鉴定了47个甾体皂苷成分，其中有9个新化合物。经细胞体外抗炎和抗CIR活性筛选、大鼠体内药效验证发现：不同类型甾体苷元及其上糖配基少者化合物，尤其是薯蓣皂素及其系列皂苷的活性显著。明确的药效成分致使因CIR引起的高表达HSP70升高、高表达IKK降低，进而恢复NF-κB介导的异常炎症反应。采用蛋白质组学和代谢组学从薯蓣皂素干预治疗的大鼠CIR脑组织中共鉴定了5043个蛋白和1495个代谢物，其中有418个差异蛋白和51个差异代谢物。通过GO和KEGG生信学分析，发现了HSP70和NF-κB上游调控的蛋白HIKESHI和STAT2，表明薯蓣皂素抗CIR的药效作用与其抗炎作用机制相关。. 上述研究成果已有5篇论文发表在本领域相关的SCI源期刊上，并以访问学者的身份赴英国帝国理工学院学习，开展为期一年的合作研究。. 本项目的完成明确了黄姜抗CIR的药效物质基础及作用机制，为从黄姜中发现临床上用于治疗CIR的先导化合物奠定了基础，并拓展了陕西道地药材的适用领域。

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