Curcumin inhibits oxidative stress and autophagy in C17.2 neural stem cell through ERK1/2 signaling pathways

Abstract

Objectives: This study investigates curcumin's neuroprotective role and its potential in promoting neurogenesis in progenitor cells within the brain. Notably, curcumin's antioxidant properties have been implicated in Alzheimer's disease treatment. However, the association between curcumin's antioxidative effects and its impact on neural stem cells (NSCs) remains to be elucidated.

Methods: C17.2 neural stem cells were utilized as a model to simulate oxidative stress, induced by hydrogen peroxide (H₂O₂). We quantified the levels of superoxide dismutase (SOD), malondialdehyde (MDA), and intracellular reactive oxygen species (ROS), alongside the gene expression of SOD1 and SOD2, to assess intracellular oxidative stress. Additionally, Western blot analysis was conducted to measure the expressions of LC3-II, Beclin-1, and phosphorylated ERK (p-ERK), thereby evaluating autophagy and ERK signaling pathway activation.

Results: Treatment with curcumin resulted in a reduction of MDA and ROS levels, suggesting a protective effect on NSCs against oxidative damage induced by H₂O₂. Furthermore, a decrease in the relative expressions of LC3-II, Beclin-1, and p-ERK was observed post-curcumin treatment.

Conclusions: The findings suggest that curcumin may confer protection against oxidative stress by attenuating autophagy and deactivating the ERK1/2 signaling pathways, which could contribute to therapeutic strategies for Alzheimer's disease.

Keywords: autophagy; curcumin; oxidative stress.

FIGURE 1

Viability of C17.2 neural stem cells treated with curcumin (Cur), H₂O₂ or Cur combined with H₂O₂. (A–D) The viability of C17.2 neural stem cells was measured via CCK‐8 after 6,12, 24, 48 h of intervention with Cur (5, 10, 15, 20 μM), H₂O₂ (400, 600, 800, 1000 μM) and Cur + H₂O₂ (Cur: 10 μM; H₂O₂: 600 μM for 6 h). The results were mean ± SD of three independent examination. All groups were compared with Control, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Levels of SOD and MDA after 6,12, 24, 48 h treatment with H₂O₂ or H₂O₂ combined with Cur. (A, B) The levels of SOD and MDA in the supernatant of cells treated with H₂O₂ or H₂O₂ combined with Cur for indicated time sets were determined by ELISA. (C, D) The relative expression levels of SOD1 and SOD2 of cells treated with H₂O₂ or H₂O₂ combined with Cur for indicated time sets were detected by real‐time PCR calculated against the GAPDH. The results were mean ± SD of three independent examination. *p < 0.05, **p < 0.01, *p < 0.001.

FIGURE 3

Role of autophagy on H₂O₂‐treated cells suppressed by cur. The H₂O₂ induced autophagy reversed by Cur was detected by western blot (LC3‐2 and Beclin‐1) (A), quantified in (B, C), visualized in tf‐LC3 fluorescence images (D) and TEM images (E). The results were mean ± SD of three independent examination. *p < 0.05, **p < 0.01, *p < 0.001.

FIGURE 4

Effects of Cur on H₂O₂‐induced ROS in C17.2 neural stem cells. The geomean fluorescence intensity in positive cells and the percentages of positive cells of C17.2 cells were analyzed by flow cytometry (A‐F), quantified in (G‐H); ROS within cells labeled by (D, E, H) were visualized in fluorescence images (J–O) and quantified in (I). The results were mean  ± SD of three independent examination. *p < 0.05, **p < 0.01, *p < 0.001.

FIGURE 5

Effect of Cur on ERK1/2 signaling in C17.2 neural stem cells. (A) Western blot was used to evaluate the expression of p‐ERK in U0126‐treated C17.2 cells, calculated against the t‐ERK (B). (C) Effect of Cur on H₂O₂ induced activation of MAPK/ERK1/2 pathway in C17.2 neural stem cells, calculated against the t‐ERK (D). The results were mean ± SD of three independent examination. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Cur alleviates autophagy through ERK1/2 pathway. (A) Decreased expression of LC3‐II and Beclin‐1 upon down‐regulated autophagy was detected by western blot (A), quantified in (B, C). The results were mean ± SD of three independent examination. *p < 0.05, **p < 0.01, *p < 0.001.