Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, China.
Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, China.
J Adv Res. 2020 Sep 9;29:83-94. doi: 10.1016/j.jare.2020.09.001. eCollection 2021 Mar.
Inflammation is a key factor in myocardial ischemia/reperfusion (MI/R) injury. Targeting leucocyte-mediated inflammation is an important strategy for MI/R therapy. Iminostilbene (ISB), a simple dibenzoazepine small molecule compound, has a strong anti-neurodegenerative effect. However, no study has shown the cardioprotective effect of ISB.
This study aimed to investigate the role of ISB against MI/R injury and identify its molecular target.
To verify the cardiac protection of ISB and , we performed rat MI/R surgery and subjected inflammatory modeling of macrophages. In terms of molecular mechanisms, we designed and synthesized a small molecular probe of ISB and employed it on the click chemistry-activity-based protein profiling technique to fish for ISB targets in macrophages. To identify the target, we applied the competitive inhibition assay, surface-plasmon resonance (SPR), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay.
, ISB showed robust anti-myocardial injury activity by improving cardiac function, reducing myocardial infarction, and inhibiting macrophage-mediated inflammation. , ISB strongly inhibited the transcription and the expression levels of inflammatory cytokines in macrophages. The pyruvate kinase isozyme type M2 (PKM2) was identified as the potential target of ISB through proteomic analysis and the competitive assay was performed for specific binding verification. Further thermodynamic and kinetic experiments showed that ISB was bound to PKM2 in a dose-dependent manner. Moreover, in terms of the biological function of ISB on PKM2, ISB reduced the expression of PKM2, thereby reducing the expression of HIF1α and the phosphorylation of STAT3.
This study for the first time demonstrated that ISB targeted PKM2 to reduce macrophage inflammation thereby significantly alleviating MI/R injury.
炎症是心肌缺血/再灌注（MI/R）损伤的关键因素。针对白细胞介介导的炎症是 MI/R 治疗的重要策略。亚氨基苯并二氮杂（ISB），一种简单的二苯并氮杂小分子化合物，具有很强的抗神经退行性作用。然而，没有研究表明 ISB 具有心脏保护作用。
本研究旨在探讨 ISB 对抗 MI/R 损伤的作用及其分子靶点。
为了验证 ISB 的心脏保护作用，我们进行了大鼠 MI/R 手术，并对巨噬细胞进行了炎症模型构建。在分子机制方面，我们设计并合成了 ISB 的小分子探针，并将其应用于点击化学-活性基蛋白谱技术，以在巨噬细胞中寻找 ISB 的靶标。为了鉴定靶标，我们应用了竞争抑制试验、表面等离子体共振（SPR）、细胞热转移试验（CETSA）和药物亲和响应靶标稳定性（DARTS）试验。
ISB 通过改善心脏功能、减少心肌梗死和抑制巨噬细胞介导的炎症，显示出强大的抗心肌损伤活性。ISB 强烈抑制了巨噬细胞中炎症细胞因子的转录和表达水平。通过蛋白质组学分析和竞争性试验进行特定结合验证，鉴定出丙酮酸激酶同工酶 M2（PKM2）是 ISB 的潜在靶标。进一步的热力学和动力学实验表明，ISB 与 PKM2 呈剂量依赖性结合。此外，就 ISB 对 PKM2 的生物学功能而言，ISB 降低了 PKM2 的表达，从而降低了 HIF1α的表达和 STAT3 的磷酸化。
本研究首次表明，ISB 通过靶向 PKM2 减少巨噬细胞炎症，从而显著减轻 MI/R 损伤。
