Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine, Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China, International Institute for Translational Chinese Medicine, School of Pharmaceutical Sciences, Guangzhou Univ Chinese Med, Guangzhou, Guangdong, 510006, China.
School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian, 361102, China.
Theranostics. 2021 Jan 1;11(2):861-877. doi: 10.7150/thno.48436. eCollection 2021.
Kelch ECH-associating protein 1 (Keap1) is a crucial chaperonin for E3 ubiquitin ligases. Modification of the key reactive cysteine residues in Keap1 affects the interaction between Keap1 and its substrate nuclear factor erythroid 2-related factor 2 (Nrf2), subsequently regulating oxidative stress and NLPR3 inflammasome activation, which are important factors for myocardial ischemia-reperfusion injury (MI/RI). Pubescenoside A (PBA), an active compound from has antithrombotic and anti-inflammatory effects. However, the effect of PBA on MI/RI is still unknown. In the present study, we aimed to determine whether PBA can protect the heart against MI/RI and clarify the direct target and the underlying mechanism of PBA. The left anterior descending artery (LAD) ligation-induced MI/RI mice model or oxygen and glucose deprivation/reperfusion (OGD/R) were used to evaluate the cardioprotective effect of PBA. Pull-down assays, co-immunoprecipitation (Co-IP) assays, LC/MS/MS, isothermal calorimetry (ITC) experiments and covalent docking were used to identify the target of PBA. PBA protected cardiomyocytes against OGD/R and LAD-induced MI/RI . PBA suppressed NLRP3 inflammation activation and induced the Nrf2 signaling pathway. Interestingly, PBA targeted Keap1 by selectively covalently binding to conserved cysteine residues, cysteine 77 (Cys77) in the BTB domain and cysteine 434 (Cys434) in the Kelch domain of Keap1, subsequently inhibiting ubiquitination of Nrf2 and activating antioxidant enzymes. Additionally, the cysteines of Keap1 has different degree of activation by PBA as follows: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which further elucidates the cysteine sensitivity of Keap1. Our results indicated that PBA might be a new Nrf2 activator that covalently binds to two critical domains of Keap1, and shows cardioprotective activities against ischemia-reperfusion injury.
Kelch ECH 相关蛋白 1（Keap1）是 E3 泛素连接酶的关键伴侣蛋白。Keap1 中关键反应性半胱氨酸残基的修饰会影响 Keap1 与其底物核因子红细胞 2 相关因子 2（Nrf2）之间的相互作用，进而调节氧化应激和 NLPR3 炎性体激活，这是心肌缺血再灌注损伤（MI/RI）的重要因素。淫羊藿苷 A（PBA）是一种来自淫羊藿的活性化合物，具有抗血栓和抗炎作用。然而，PBA 对 MI/RI 的作用尚不清楚。在本研究中，我们旨在确定 PBA 是否可以保护心脏免受 MI/RI 的影响，并阐明 PBA 的直接靶标和潜在机制。使用左前降支（LAD）结扎诱导的 MI/RI 小鼠模型或氧葡萄糖剥夺/再灌注（OGD/R）来评估 PBA 的心脏保护作用。下拉实验、共免疫沉淀（Co-IP）实验、LC/MS/MS、等温滴定量热法（ITC）实验和共价对接用于鉴定 PBA 的靶标。PBA 可保护心肌细胞免受 OGD/R 和 LAD 诱导的 MI/RI。PBA 抑制 NLRP3 炎症激活并诱导 Nrf2 信号通路。有趣的是，PBA 通过选择性共价结合到 Keap1 的保守半胱氨酸残基，BTB 结构域中的半胱氨酸 77（Cys77）和 Kelch 结构域中的半胱氨酸 434（Cys434）来靶向 Keap1，从而抑制 Nrf2 的泛素化并激活抗氧化酶。此外，PBA 对半胱氨酸的激活程度不同：Cys77>Cys434>Cys23>Cys38>Cys226>Cys273，这进一步阐明了 Keap1 的半胱氨酸敏感性。我们的结果表明，PBA 可能是一种新的 Nrf2 激活剂，它可共价结合到 Keap1 的两个关键结构域，并对缺血再灌注损伤表现出心脏保护活性。
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