Key Laboratory of Bioresource Research and Development of Liaoning Province, College of Life and Health Sciences, Northeastern University, Shenyang, China; National Frontiers Science Center for Industrial Intelligence and Systems Optimization, Key Laboratory of Data Analytics and Optimization for Smart Industry, Ministry of Education, Northeastern University, Shenyang, China.
Key Laboratory of Bioresource Research and Development of Liaoning Province, College of Life and Health Sciences, Northeastern University, Shenyang, China.
Phytomedicine. 2023 May;113:154729. doi: 10.1016/j.phymed.2023.154729. Epub 2023 Feb 26.
Ischemic stroke (IS) is considered as a serious cerebral vascular disease. Ferroptosis is a novel type of regulated cell death (RCD), that closely related to the occurrence and progress of IS. Loureirin C, a type of dihydrochalcone compound derived from the Chinese Dragon's blood (CDB). The effective components extracted from CDB have shown neuroprotective effects in ischemia reperfusion models. However, the role of Loureirin C in mice after IS is not well understood. Thus, it is worth to identify the effect and mechanism of Loureirin C on IS.
The present research aims to prove the existence of ferroptosis in IS and explore whether Loureirin C can inhibit ferroptosis by regulating nuclear factor E2 related factor 2 (Nrf2) pathway in mice and exert neuroprotective effects on IS models.
Middle cerebral artery occlusion and reperfusion (MCAO/R) model was established to evaluate the occurrence of ferroptosis and the potential Loureirin C brain-protective effect in vivo. The analysis of free iron, glutamate content, reactive oxygen species (ROS) and lipid peroxidation levels, along with transmission electron microscope (TEM) was applied to prove the existence of ferroptosis. The function of Loureirin C on Nrf2 nuclear translocation was verified by immunofluorescence staining. In vitro, primary neurons and SH-SY5Y cells were processed with Loureirin C after oxygen and glucose deprivation-reperfusion (OGD/R). ELISA kits, western blotting, co-immunoprecipitation (Co-IP) analysis, immunofluorescence, and quantitative real-time PCR were devoted to proving the neuroprotective effects of Loureirin C on IS via regulating ferroptosis and Nrf2 pathways.
The results showed that Loureirin C not only dramatically alleviated brain injury and inhibited neurons ferroptosis in mice after MCAO/R, but also dose-dependently reduce ROS accumulation in ferroptosis after OGD/R. Further, Loureirin C inhibits ferroptosis by activating Nrf2 pathway, and promoting nuclear translocation of Nrf2. Besides, Loureirin C increases heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1) and glutathione peroxidase 4 (GPX4) content after IS. Intriguingly, the anti-ferroptosis effect of Loureirin C is weakened by Nrf2 knockdown.
Our discoveries first revealed that the inhibitory action of Loureirin C on ferroptosis may greatly depend on its adjusting effect on the Nrf2 pathway, suggesting that Loureirin C could act as a novel anti-ferroptosis candidate and play a therapeutic role in IS. These novel discoveries on the role of Loureirin C on IS models reveal an innovative method that may contribute to neuroprotection for the prevention of IS.
缺血性脑卒中（IS）被认为是一种严重的脑血管疾病。铁死亡是一种新型的调控细胞死亡（RCD），与 IS 的发生和进展密切相关。Loureirin C 是一种从中国龙血树（CDB）中提取的二氢查尔酮化合物。从 CDB 中提取的有效成分在缺血再灌注模型中显示出神经保护作用。然而，Loureirin C 对 IS 后小鼠的作用尚不清楚。因此，有必要确定 Loureirin C 对 IS 的作用及其机制。
本研究旨在证明 IS 中存在铁死亡，并探讨 Loureirin C 是否可以通过调节核因子 E2 相关因子 2（Nrf2）通路来抑制铁死亡，并对 IS 模型发挥神经保护作用。
采用大脑中动脉闭塞再灌注（MCAO/R）模型，在体内评估铁死亡的发生和 Loureirin C 潜在的脑保护作用。通过检测游离铁、谷氨酸含量、活性氧（ROS）和脂质过氧化水平，以及透射电镜（TEM）来证明铁死亡的存在。通过免疫荧光染色验证 Loureirin C 对 Nrf2 核易位的作用。体外，用 Loureirin C 处理氧葡萄糖剥夺再灌注（OGD/R）后的原代神经元和 SH-SY5Y 细胞。通过 ELISA 试剂盒、Western blot、免疫共沉淀（Co-IP）分析、免疫荧光和实时定量 PCR 来证明 Loureirin C 通过调节铁死亡和 Nrf2 通路对 IS 的神经保护作用。
结果表明，Loureirin C 不仅能显著减轻 MCAO/R 后小鼠的脑损伤和抑制神经元铁死亡，还能剂量依赖性地减少 OGD/R 后铁死亡中 ROS 的积累。此外，Loureirin C 通过激活 Nrf2 通路，促进 Nrf2 的核易位，抑制铁死亡。此外，Loureirin C 增加 IS 后血红素加氧酶 1（HO-1）、醌氧化还原酶 1（NQO1）和谷胱甘肽过氧化物酶 4（GPX4）的含量。有趣的是，Nrf2 敲低削弱了 Loureirin C 的抗铁死亡作用。
本研究首次揭示，Loureirin C 对铁死亡的抑制作用可能在很大程度上取决于其对 Nrf2 通路的调节作用，提示 Loureirin C 可能作为一种新型的抗铁死亡候选药物，在 IS 中发挥治疗作用。这些关于 Loureirin C 对 IS 模型作用的新发现揭示了一种创新的方法，可能有助于神经保护，预防 IS 的发生。
