Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China.
College of Biology Pharmacy & Food Engineering, Shangluo University, Shangluo, Shaanxi 726000, People's Republic of China.
Phytomedicine. 2022 Jul;101:154124. doi: 10.1016/j.phymed.2022.154124. Epub 2022 Apr 19.
Neuronal apoptosis and oxidative stress have the most crucial influence on neurodegenerative diseases, including Parkinson's disease. Rat adrenal pheochromocytoma cells (PC-12) induced by HO are one of the primary in vitro models of Parkinson's disease (PD) . Previous studies have found that E ulmoides leaf extract exerts good neuroprotective activity and has the potential to treat neurodegenerative diseases. However, the molecular pathways involved in the neuroprotective effects of its primary leaf component, lignans, have not yet been well elucidated yet.
This study aimed to evaluate the neuroprotective effects of lignans in E. ulmoides leaves and to explore the underlying mechanism.
Cell viability was measured using the CCK-8 assay. Apoptosis was assessed by calcein/PI staining. The release levels of ROS and LDH were assessed using a commercial assay kit. The enzyme activities of SOD and GPx were measured using kits. The establishment of the compound-target-pathway-disease network was performed using a database and computer software. Antioxidant proteins (HO-1, NQO-1, and Cat) and related regulatory proteins (Nrf2, GSK-3β, p-GSK 3β (Ser 9), Akt, p-Akt (Tyr326), PI3K) were detected by western blotting. Apoptosis in the zebrafish head was assessed using acridine orange (AO) staining.
In the present study, 12 lignans were isolated and characterized from E. ulmoides leaves, including a new compound, (-)-7‑epi-pinoresinol mr1 (1). Compounds 1-12 exerted neuroprotective effects in HO-treated PC-12 cells by increasing cell viability, improving the enzyme activity of SOD and GPx, and reducing levels of ROS and LDH. Compared to the positive control group (25 μM hesperetin), cell viability in response to 25 μM compound 1 (78.0 ± 0.8%) was highest, but its relative percent LDH release (20.1 ± 2.5%) was the lowest; 25 μM compound 4 resulted in the lowest ROS release levels (101.7 ± 2.6%) and highest SOD enzyme activity (35.9 ± 4.2 U/mg), and the GPx enzyme activity of 25 μM compound 1 was strongest (197.6 ± 0.6 U/mg). Next, the potential targets (PI3K, GSK-3β) of the test compounds' antioxidant activity were identified using pharmacological network analysis. Using DAVID software for pharmacological network analysis, potential targets (PI3K, GSK-3β, and SOD2) of 12 lignans were identified. Based on the initial screening results, biological experiments confirmed that diepoxylignans 1, 2, and 4 exerted significant neuroprotection by regulating the PI3K/AKT/GSK-3β/Nrf2 signaling pathways, increasing protein expression of HO-1, NQO-1, and CAT, and enhancing the antioxidant enzyme activity of SOD and GPx.
Our experiments first propose that the diepoxylignans from E. ulmoides leaves exert neuroprotective effects via activation of the PI3K/Akt/GSK-3β/Nrf2 signaling pathway. These findings further indicate that lignans could be the primary components of E. ulmoides Oliver as agents for the prevention and treatment of neurodegenerative diseases. Collectively, Eucommia ulmoides leaves with important research value may be a potential candidate for traditional Chinese medicine for treating oxidative stress-related neurodegenerative diseases.
神经元凋亡和氧化应激对包括帕金森病在内的神经退行性疾病有最关键的影响。 由 HO 诱导的大鼠肾上腺嗜铬细胞瘤细胞 (PC-12) 是帕金森病 (PD) 的主要体外模型之一。 先前的研究发现，榆树叶片提取物具有良好的神经保护活性，并有潜力治疗神经退行性疾病。 然而，其主要叶成分木脂素发挥神经保护作用的分子途径尚未得到很好的阐明。
本研究旨在评估榆树叶片木脂素的神经保护作用，并探讨其潜在机制。
使用 CCK-8 测定法测定细胞活力。 通过钙黄绿素/PI 染色评估细胞凋亡。 使用商业测定试剂盒评估 ROS 和 LDH 的释放水平。 使用试剂盒测定 SOD 和 GPx 的酶活性。 使用数据库和计算机软件建立化合物-靶标-通路-疾病网络。 通过 western blot 检测抗氧化蛋白（HO-1、NQO-1 和 Cat）和相关调节蛋白（Nrf2、GSK-3β、p-GSK 3β (Ser 9)、Akt、p-Akt (Tyr326)、PI3K）。 通过吖啶橙 (AO) 染色评估斑马鱼头部的细胞凋亡。
在本研究中，从榆树叶片中分离并鉴定了 12 种木脂素，包括一种新化合物 (-)-7-epi-pinoresinol mr1 (1)。 化合物 1-12 通过增加细胞活力、改善 SOD 和 GPx 的酶活性以及降低 ROS 和 LDH 水平，对 HO 处理的 PC-12 细胞发挥神经保护作用。 与阳性对照组 (25 μM 橙皮苷) 相比，25 μM 化合物 1 的细胞活力最高 (78.0±0.8%)，但相对 LDH 释放率 (20.1±2.5%) 最低； 25 μM 化合物 4 导致 ROS 释放水平最低 (101.7±2.6%) 和 SOD 酶活性最高 (35.9±4.2 U/mg)，25 μM 化合物 1 的 GPx 酶活性最强 (197.6±0.6 U/mg)。 接下来，使用药理学网络分析鉴定了测试化合物抗氧化活性的潜在靶标 (PI3K、GSK-3β)。 使用 DAVID 软件进行药理学网络分析，鉴定了 12 种木脂素的潜在靶标 (PI3K、GSK-3β 和 SOD2)。 根据初步筛选结果，生物实验证实二环氧木脂素 1、2 和 4 通过调节 PI3K/AKT/GSK-3β/Nrf2 信号通路发挥显著的神经保护作用，增加 HO-1、NQO-1 和 CAT 的蛋白表达，并增强 SOD 和 GPx 的抗氧化酶活性。
我们的实验首次提出，榆树叶片中的二环氧木脂素通过激活 PI3K/Akt/GSK-3β/Nrf2 信号通路发挥神经保护作用。 这些发现进一步表明木脂素可能是榆树作为预防和治疗神经退行性疾病的中药的主要成分。 总之，具有重要研究价值的榆树叶片可能是治疗氧化应激相关神经退行性疾病的传统中药的潜在候选药物。
