风轮菜中双重调控NF-κB和Nrf2的新颖混源萜及其抗心肌缺血再灌注损伤作用研究

Myocardial ischenfical reperfusion injury (MIRI) is medical puzzle and is attracting more attentions from researchers all over the world. However, long-term use of anti-MIRI drugs can lead to drug-resistant. In addition, little progress has been made on clinical treatment in recent years. Traditional Chinese medicine has been proved to be of multi-targets effects and lower side effects getting increasing attention. Our previous studies found that Clinopodium chinense extracts showed significantly protective effect against myocardial ischenfical reperfusion injury. Further chemical studies led to the isolation of six novel favonoid-triterpene saponin meroterpenoids with unprecedented structures from the aerial parts of C. chinense. All compounds exhibited significantly protective effects on H9c2 cardiomyocyte may via dual regulation NF-κB and Nrf2 signaling pathway. On the basis of previous work, favonoid-triterpene saponin meroterpenoids will be rapidly screened, isolated and elucidated. Meanwhile, H9c2 cardiomyocyte model will be established to evaluate activity of the isolated compounds, RT-PCR and western-blot will be applied to test the NF-κB inhibition and Nrf2 activation effects of the active ingredients. Then, the structure-activity relationship of against MIRI in vitro will be analyzed in the favonoid-triterpene saponin meroterpenoids. The protective effect against MIRI will be verified on rat model after accumulation of the selected meroterpenoids with better protective effects and clear mechanism. In this way, our findings will establish solid scientific foundation for the development of new protective effect against myocardial ischenfical reperfusion injury drug of C. chinense.

心肌缺血再灌注损伤（MIRI）是当今医学研究热点和难点，长时间连续使用某一类药物易导致耐药，近年来临床治疗方法却没有明显进步，中药因其毒副作用小及多靶点治疗越来越受到人们的关注。前期研究我们发现风轮菜具有显著的抗MIRI作用，且其有效部位中含新颖的黄酮皂苷混源萜类成分，该骨架类型首次在自然界中发现，此类成分可能通过NF-κB和Nrf2双重调节作用而显示出显著的心肌保护活性。本项目拟在前期工作的基础上，对风轮菜黄酮皂苷混源萜类成分快速识别后，实现目标化合物的快速分离与鉴定；采用H9c2心肌细胞进行体外活性评价；运用RT-PCR、WB检测活性成分对细胞内NF-κB抑制和Nrf2激活作用；进而深入剖析黄酮皂苷混源萜类成分的体外抗MIRI构效关系；优选活性较强、作用机制基本清晰的成分，经足量制备后，利用大鼠模型验证其抗MIRI药效；为开发风轮菜中黄酮皂苷混源萜类成分作为抗MIRI药物提供科学依据。

本课题采用超高效液相色谱四级杆串联飞行时间质谱联用技术，对风轮菜黄酮皂苷混源萜类成分进行快速识别和指认，结合多种现代色谱联用技术、光谱和波谱方法共分离鉴定了66个化合物，其中新化合物12个，包括7个新的黄酮皂苷混源萜类成分；采用缺氧复氧心肌细胞损伤为模型对黄酮皂苷混源萜类成分进行活性筛选，发现黄酮皂苷C-11的构型为R，C-2′的构型为S时心肌保护活性最好，本课题基本阐明活性先导化合物通过抑制 NF-кB和激活 Nrf2 活性抗 MIRI 作用的分子机制，离体动物实验发现抗clinoposide G具有抗MIRI作用。另外本课题发现tournefolic acid B具有显著改善糖尿病心肌病的作用；以人乳腺癌耐药细胞（MCF-7/ADR细胞）为模型，对部分化合物进行了抗肿瘤筛选，发现didymin具有协同增加阿奇霉素的抗肿瘤的药理活性。本课题已发表相关学术论文5篇，其中包括SCI论文4篇，中文核心期刊论文1篇；申请国家发明专利2项；培养毕业硕士研究生2名和在读研究生1名，本科生6名，其中1人获得校优秀毕业论文。