Lignans from Eucommia ulmoides Oliver leaves exhibit neuroprotective effects via activation of the PI3K/Akt/GSK-3β/Nrf2 signaling pathways in HO-treated PC-12 cells.

Background: 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.

Purpose: This study aimed to evaluate the neuroprotective effects of lignans in E. ulmoides leaves and to explore the underlying mechanism.

Methods: 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.

Results: 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.

Conclusion: 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.