Epimedin C: A promising neuroprotective agent that can participate in mediating the JNK/Nrf2/HO-1 signaling pathway to prevent neurodegenerative diseases.

Epimedium can be used to treat neurodegenerative diseases. Flavonol glycosides are the major bioactive compounds within Epimedium extract, including icariin and Epimedin C, which is found at the highest concentration among all flavonol glycosides. The present study aimed to explore the potential pharmacological mechanisms by which Epimedin C prevents neurodegenerative diseases. The present study first identified the active ingredients in Epimedium by performing ultra-high performance liquid chromatography-quadrupole-Exactive Orbitrap high resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS). Subsequently, its potential mechanism in preventing neurodegenerative diseases was explored by combining the identification results with network pharmacological analysis (using Alzheimer's disease as an example). Subsequently, the optimal concentration of Epimedin C for the intervention of PC12 cells was screened using Cell Counting Kit-8 (CCK-8). PC12 cells were divided into the following groups: Normal control, H(2)O(2) (150 µM for 4 h), or 24 h pretreatment with either 17β-estradiol (1 nM) or Epimedin C (1, 5 and 10 µM) followed by exposure to H(2)O(2) (150 µM for 4 h). Lactate dehydrogenase was measured to detect the cytotoxicity of each group under different intervention methods, and malondialdehyde and reactive oxygen species assay kits were employed to detect the oxidative stress (OS) of each group of cells. Subsequently, the apoptosis levels of each group were evaluated by flow cytometry and TUNEL staining. Transmission electron microscopy and JC-1 were adopted to evaluate the mitochondrial function of cells. Subsequently, according to network pharmacological analysis, western blotting was performed to detect the protein levels of JNK, phosphorylated (p)-JNK, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), Bcl-2 and Bax in cells. Finally, to further verify whether Epimedin C mediates the JNK pathway, the JNK agonist anisomycin was added to the PC12 cells after H(2)O(2) intervention and after H(2)O(2) combined with Epimedin C intervention. The differences in protein levels in PC12 cells under different intervention methods were then compared. The CCK-8 results showed that cells treated with Epimedin C at concentrations of 1, 5 and 10 µM had improved cell survival rates compared with other concentrations. When compared with the control group, the H(2)O(2)-induced group displayed severe OS damage and a significantly increased incidence of apoptosis. By contrast, after intervention with Epimedin C, the OS damage in PC12 cells was markedly inhibited and mitochondrial apoptosis was evidently decreased. Among the concentrations, 10 µM Epimedin C had a more notable effect. Through UHPLC-Q-Exactive Orbitrap HRMS plus network pharmacological analysis, 108 shared targets (with Alzheimer's disease) were enriched, and the top 20 core genes included BCL2, APP and JUN. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that the common targets were closely related to apoptosis. The results of the western blot double validation experiment also confirmed that the JNK pathway was significantly activated in PC12 cells exposed to H(2)O(2), and that Epimedin C can inhibit JNK phosphorylation. Notably, western blotting results showed that compared with in the H(2)O(2) group, after intervention with Epimedin C, p-JNK was significantly downregulated, Nrf2 and HO-1 were significantly upregulated, BAX was significantly downregulated and Bcl-2 was upregulated. In conclusion Epimedin C may improve OS in PC12 cells, exert neuroprotective effects and reduce apoptosis by inhibiting JNK phosphorylation and activating Nrf2/HO-1. Epimedin C may thus be considered a potential candidate neuroprotective agent for preventing neurodegenerative diseases.