丹参素异丙酯通过p38/NLRP3焦亡通路保护心脏缺血再灌注损伤研究

Ischemia/reperfusion (I/R) in acute myocardial infarction has become a major clinic problem, so the development of novel drugs is urgently needed. In previous work, we had developed Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxylpropanoate (IDHP), a new chemical molecule developed in the metabolites of Compound Salvia Miltiorrhiza Prescription. The preliminary results indicated that IDHP could protect heart from I/R injury, and such protection was associated with p38, NLRP3 and GSDMD. Thus, we propose a hypothesis that IDHP protects myocardial I/R injury heart through p38/NLRP3 pathway. To investigate the protective function of IDHP in myocardial I/R, IDHP will be supplemented with different doses and time points. Meanwhile, the effect of IDHP on hypoxia/reoxygenation (H/R) injury in neonatal rat cardiac myocytes (NRCMs) will be detected. Next, we will detect that IDHP protects myocytes through NLRP3 induced by I/R and H/R in vivo and in vitro. With the overexpression of NLRP3A350V which constitutively actives NRLP3 by cardiac specific AAV，the change in protection with IDHP will be detected in myocardial I/R models. At the same time, the protection of IDHP on H/R injury of NRCMs will be studied using adenovirus to overexpress NLRP3A350V. Furthermore, the heart and NRCMs will be overxpressed p38α, the protection of IDHP on I/R injury and H/R injury will be detected. While the NLRP3 inflammasome activation and pyroptosis activation will be detected to clarify IDHP protects myocardial injury via p38/NLRP3 pathway. The project will reveal the role and molecular mechanism of IDHP in protection of myocardial I/R injury. It provides evidence for clinical translational research of IDHP and a new choice for myocardial I/R treatment.

缺血再灌注（I/R）损伤是急性心肌梗死血流再通后影响患者预后的瓶颈问题，亟待开发新的治疗药物。丹参素异丙酯（IDHP）为项目组自主研发的1类化学新药候选化合物，预实验结果显示IDHP对心脏I/R有保护作用，机制与p38、NLRP3、GSDMD有相关性。因此，本项目假设IDHP通过p38/NLRP3焦亡通路保护心脏I/R损伤。本项目在前期工作基础上，进一步利用不同给药剂量和时间点，在细胞水平和整体动物水平明确IDHP对心脏I/R损伤的保护作用；应用AAV和腺病毒在心脏和心肌细胞过表达NLRP3 A350V持续激活突变体，明确IDHP通过NLRP3焦亡信号保护心脏I/R损伤的作用；进一步过表达p38，明确IDHP通过p38/NLRP3焦亡通路保护心脏I/R损伤的机制。为IDHP临床转化提供实验依据；为基于p38/NLRP3焦亡通路的新药设计与创制，为心脏I/R治疗与预后提供新的支撑与思路。

缺血再灌注（I/R）损伤是急性心肌梗死血流再通后影响患者预后的瓶颈问题，亟待开发新的治疗药物。丹参素异丙酯（IDHP）为项目组自主研发的1类化学新药候选化合物，GLP急毒评价表明按化学药品最大给药量2000mg/kg给药后，实验动物无生理、生化、病理等方面异常，显示IDHP安全。团队成功创建了IDHP原料药合成生物学+化学合成工艺，构建了独立的原料药及滴丸（片）制剂的中试生产、包装线，实现了10公斤级原料药和制剂的生产能力，产品达到了国家新药注册要求，为IDHP的药理药效学实验进行提供了保障。项目在优化了心脏缺血/再灌注手术，建立成年小鼠心肌细胞缺氧/复氧模型的基础上，IDHP可改善I/R诱导的心脏早期及晚期损伤，通过抑制GSDMD介导的细胞焦亡以实现改善损伤作用。项目工作为IDHP临床转化提供实验依据, 为心脏I/R治疗与预后提供新的支撑与思路。