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

  • Authors:
    • Chao Cong
    • Xuan-Ling Li
    • Guang-Yao Lin
    • Lian-Wei Xu
  • View Affiliations / Copyright

    Affiliations: Department of Gynecology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, P.R. China
    Copyright: © Cong et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 4.0].
  • Article Number: 3
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    Published online on: October 21, 2025
       https://doi.org/10.3892/etm.2025.12998
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Abstract

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, H2O2 (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 H2O2 (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 H2O2 intervention and after H2O2 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 H2O2‑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 H2O2, and that Epimedin C can inhibit JNK phosphorylation. Notably, western blotting results showed that compared with in the H2O2 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.
View Figures

Figure 1

Total ion chromatograms in (A)
negative and (B) positive ion modes of Epimedium identified
by ultra-high performance liquid chromatography-quadrupole-Exactive
Orbitrap high resolution mass spectrometry.

Figure 2

Venn diagram. Intersection targets of
Epimedium and Alzheimer's disease.

Figure 3

Epimedin C improves PC12 cell
viability and protects against oxidative damage (A) Molecular
structure of Epimedin C. (B) Comparison of cell viability of PC12
cells treated with different concentrations of Epimedin C. (C) Cell
viability of PC12 cells in response to different intervention
measures. Comparison of (D) LDH release, (E) MDA contents and (F)
ROS levels in PC12 cells among the different treatment groups. Data
are presented as the mean ± SD from six replicates.
##P<0.01 vs. control group; *P<0.05,
**P<0.01 vs. H2O2 group.
17β-E2, 17β-estradiol; LDH, lactate dehydrogenase; MDA,
malondialdehyde.

Figure 4

Flow cytometric analysis of apoptosis
and MMP changes in PC12 cells (A) Apoptosis rate of each group of
cells was detected by flow cytometry; cell populations stained with
Annexin V-FITC and PI (quadrants 2 and 3) indicated apoptotic
cells. (B) Summary of comparisons of apoptosis between groups. (C)
JC-1 staining: Determination of MMP in each group of cells using
flow cytometry. (D) MMP was determined by red/green fluorescence
ratio. Data are presented as the mean ± SD from three replicates.
##P<0.01 vs. control group; **P<0.01
vs. H2O2 group. 17β-E2,
17β-estradiol; MMP, mitochondrial membrane potential.

Figure 5

Comparison of cell apoptosis,
detected by TUNEL staining, among the different groups. (A)
Representative immunofluorescence images of TUNEL (red) and DAPI
(blue) staining, and merged images. Scale bar, 50 µm (n=5). (B)
Summary of the fluorescent area for each treatment group. All
results are presented as the mean ± SD from five replicates.
##P<0.01 vs. control group; **P<0.01
vs. H2O2 group. 17β-E2,
17β-estradiol.

Figure 6

Transmission electron microscopy.
Comparison of the ultrastructure of PC12 cells in different groups.
Red arrows indicate mitochondria. 17β-E2,
17β-estradiol.

Figure 7

PPI network and functional enrichment
analysis. (A) Network diagram of ‘drug disease target’. (B) PPI
common target gene network. (C) Kyoto Encyclopedia of Genes and
Genomes analysis. Gene Ontology analysis: (D) BP terms, (E) MF
terms and (F) CC terms. PPI, protein-protein interaction; BP,
biological processes; MF, molecular functions; CC, cellular
components.

Figure 8

Effect of Epimedin C on the
JNK/Nrf2/HO-1 signaling pathway and related protein levels detected
by western blot analysis. (A) Western blot analysis of PC12 cells
in each group (n=3). Semi-quantitative analysis of (B) Nrf2/GAPDH;
(C) p-JNK/JNK; (D) HO-1/GAPDH; (E) Bcl-2/Bax. (F) Western blot
analysis of PC12 cells in each group after JNK agonist (5 µM Ani)
intervention (n=3). Semi-quantitative analysis of (G) Nrf2/GAPDH;
(H) p-JNK/JNK; (I) HO-1/GAPDH. All results are presented as the
mean ± SD from three replicates. &P<0.05;
#P<0.05, ##P<0.01 vs. control group;
*P<0.05, **P<0.01 vs.
H2O2 group; $P<0.05,
$$P<0.01 as indicated. 17β-E2,
17β-estradiol; Ani, anisomycin; HO-1, heme oxygenase-1; Nrf2,
nuclear factor erythroid 2-related factor 2; p-,
phosphorylated.
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Copy and paste a formatted citation
Spandidos Publications style
Cong C, Li X, Lin G and Xu L: Epimedin C: A promising neuroprotective agent that can participate in mediating the JNK/Nrf2/HO‑1 signaling pathway to prevent neurodegenerative diseases. Exp Ther Med 31: 3, 2026.
APA
Cong, C., Li, X., Lin, G., & Xu, L. (2026). Epimedin C: A promising neuroprotective agent that can participate in mediating the JNK/Nrf2/HO‑1 signaling pathway to prevent neurodegenerative diseases. Experimental and Therapeutic Medicine, 31, 3. https://doi.org/10.3892/etm.2025.12998
MLA
Cong, C., Li, X., Lin, G., Xu, L."Epimedin C: A promising neuroprotective agent that can participate in mediating the JNK/Nrf2/HO‑1 signaling pathway to prevent neurodegenerative diseases". Experimental and Therapeutic Medicine 31.1 (2026): 3.
Chicago
Cong, C., Li, X., Lin, G., Xu, L."Epimedin C: A promising neuroprotective agent that can participate in mediating the JNK/Nrf2/HO‑1 signaling pathway to prevent neurodegenerative diseases". Experimental and Therapeutic Medicine 31, no. 1 (2026): 3. https://doi.org/10.3892/etm.2025.12998
Copy and paste a formatted citation
x
Spandidos Publications style
Cong C, Li X, Lin G and Xu L: Epimedin C: A promising neuroprotective agent that can participate in mediating the JNK/Nrf2/HO‑1 signaling pathway to prevent neurodegenerative diseases. Exp Ther Med 31: 3, 2026.
APA
Cong, C., Li, X., Lin, G., & Xu, L. (2026). Epimedin C: A promising neuroprotective agent that can participate in mediating the JNK/Nrf2/HO‑1 signaling pathway to prevent neurodegenerative diseases. Experimental and Therapeutic Medicine, 31, 3. https://doi.org/10.3892/etm.2025.12998
MLA
Cong, C., Li, X., Lin, G., Xu, L."Epimedin C: A promising neuroprotective agent that can participate in mediating the JNK/Nrf2/HO‑1 signaling pathway to prevent neurodegenerative diseases". Experimental and Therapeutic Medicine 31.1 (2026): 3.
Chicago
Cong, C., Li, X., Lin, G., Xu, L."Epimedin C: A promising neuroprotective agent that can participate in mediating the JNK/Nrf2/HO‑1 signaling pathway to prevent neurodegenerative diseases". Experimental and Therapeutic Medicine 31, no. 1 (2026): 3. https://doi.org/10.3892/etm.2025.12998
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