黄连解毒汤阻断Neu-1/TLR4信号通路改善心肌缺血损伤机制研究

According to Traditional Chinese Medicine Theory, the heat-toxin is an important initial pathogenic factor to coronary heart diseases. The therapy rule of clearing heat-toxin is important in the treatment of coronary heart diseases. Clinical observation showed that Huang-Lian-Jie-Du Decoction, a traditional Chinese herbal prescription, can be used to alleviate the symptoms of angina pectoris. The mechanism by which Huang-Lian-Jie-Du Decoction protects cardiac injury, however, remains largely unknown. Our previous study observed that Huang-Lian-Jie-Du Decoction could protect the rats from myocardial ischemic injury by inhibiting the TLR4-NF-κB signaling pathway. Coptisine, a major ingredient from Huang-Lian-Jie-Du Decoction, significantly inhibited the activity of neuraminidase-1 (Neu-1). The SPR and coimmunoprecipitation results showed that Neu-1 could directly interact with TLR4. As a result, this project proposes a hypothesis that: Huang-Lian-Jie-Du Decoction alleviates the myocardial ischemic injury by blocking the interaction between Neu-1 and TLR4. The major contents of this project include: The active components from the Huang-Lian-Jie-Du Decoction bounding to Neu-1 will be screened by target molecule affinity mass spectrometry. The underlying mechanisms of how Neu-1 regulates TLR4 will be elucidated. The mechanisms by which Huang-Lian-Jie-Du Decoction regulates the Neu-1/TLR4 signaling pathway to inhibit myocardial ischemia will be investigated. This project will not only provide theoretical basis for the clinical application of Huang-Lian-Jie-Du Decoction in coronary heart disease, but also confirm that Neu-1 is a new therapeutic strategy for coronary heart disease.

瘀热壅毒，毒损心营是引起冠心病胸痹的重要病机，“清热解毒法”为胸痹心痛的中医重要治疗方法，临床研究表明清热解毒药—黄连解毒汤能改善冠心病心绞痛症状，但机制尚不明确。课题组前期研究发现：黄连解毒汤可通过抑制TLR4-NF-κB信号通路改善缺血导致的大鼠心肌损伤；其中黄连碱等显著抑制神经氨酸酶(Neu-1)活力；SPR及免疫共沉淀实验显示Neu-1与TLR4之间存在蛋白-蛋白相互作用。基于此，本项目提出科学假说：黄连解毒汤中活性成分通过靶向Neu-1，阻断Neu-1与TLR4相互作用，改善心肌缺血损伤。本课题拟通过靶分子亲和-质谱联用技术发现靶向抑制Neu-1的黄连解毒汤活性成分（群）；研究Neu-1调控TLR4的作用机制，阐明黄连解毒汤抑制Neu-1/TLR4信号通路改善心肌缺血损伤的作用机理。研究成果将为黄连解毒汤的临床应用提供依据，对于以Neu-1为干预靶点的冠心病治疗具有推动作用。

“清热解毒法”是胸痹心痛的中医重要治疗方法之一，黄连解毒汤是中药经典名方，近年来临床研究发现，黄连解毒汤具有明显的心肌保护作用，但有效成分不清楚，作用机制不明确。.本项目利用左前降支结扎和主动脉弓结扎小鼠模型，明确黄连解毒汤对小鼠的心肌保护作用；基于小鼠血清和心肌组织代谢组学分析，明确黄连解毒汤可从整体水平调节脂质代谢紊乱，纠偏损伤心肌异常代谢。机制研究发现黄连解毒汤显著抑制神经氨酸酶NEU1（neuramnidase1）；利用临床样本、多种心肌损伤动物模型、心肌特异性敲除鼠，系统阐明了NEU1是心肌损伤的启动因子，是黄连解毒汤发挥心肌保护作用的主要靶标分子。通过高分辨质谱技术解析了黄连解毒汤的主要化学成分；利用二维涡流、同源模建、化学发光等多种手段，明确黄连解毒汤中小檗碱、黄芩苷等活成成分，对人源NEU1具有较强亲和力，是靶向抑制NEU1的化学成分。利用分子生物学手段，明确NEU1与TLR4直接结合，并且NEU1结合TLR4后激活下游炎症反应；黄连解毒汤中活性成分通过抑制NEU1，改善NEU1/TLR4介导的IL-6, TNF-α, IL-1β等促炎因子表达。研究成果以通讯作者发表在European Heart Journal（2021，IF=29.98），Journal of Ethnopharmacology（2021，IF=4.36），the American Journal of Chinese Medicine（2021，IF=4.66）；授权美国发明专利1项，授权中国发明专利2项。.该研究为揭示黄连解毒汤通过清热解毒治疗心血管病的科学内涵提供了实验基础和理论支持，同时研究成果将为黄连解毒汤的临床应用提供依据，对于以NEU1为干预靶点的心血管病治疗具有推动作用。