Curcumin pretreatment and post-treatment both improve the antioxidative ability of neurons with oxygen-glucose deprivation

Abstract

Recent studies have shown that induced expression of endogenous antioxidative enzymes thr-ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway may be a neuroprotective strategy. In this study, rat cerebral cortical neurons cultured in vitro were pretreated with 10 μM curcumin or post-treated with 5 μM curcumin, respectively before or after being subjected to oxygen-glucose deprivation and reoxygenation for 24 hours. Both pretreatment and post-treatment resulted in a significant decrease of cell injury as indicated by propidium iodide/Hoechst 33258 staining, a prominent increase of Nrf2 protein expression as indicated by western blot analysis, and a remarkable increase of protein expression and enzyme activity in whole cell lysates of thioredoxin before ischemia, after ischemia, and after reoxygenation. In addition, post-treatment with curcumin inhibited early DNA/RNA oxidation as indicated by immunocytochemistry and increased nuclear Nrf2 protein by inducing nuclear accumulation of Nrf2. These findings suggest that curcumin activates the expression of thioredoxin, an antioxidant protein in the Nrf2 pathway, and protects neurons from death caused by oxygen-glucose deprivation in an in vitro model of ischemia/reperfusion. We speculate that pharmacologic stimulation of antioxidant gene expression may be a promising approach to neuroprotection after cerebral ischemia.

Keywords: NSFC grant; brain injury; cortical neurons; curcumin; ischemia/reperfusion injury; nerve regeneration; neural regeneration; oxidative stress; oxygen-glucose deprivation; post-treatment; pretreatment; primary cell culture.

Figure 1

Curcumin protects neurons against oxygen-glucose deprivation-induced injury. Curcumin pretreatment protects neurons against cell injury. (A) Rat cortical neuron cultures pretreated with curcumin (10 μM) or vehicle (dimethyl sulfoxide 0.01%) for 1 hour and reoxygenation for 24 hours. Control cells were incubated for 24 hours with either curcumin or vehicle in neurobasal medium without serum. (B) Rat cortical neuron cultures were exposed to oxygen-glucose deprivation in a deoxygenated, glucose-free ionic shift solution for 1 hour. Curcumin (5 μM) was added to the culture at the beginning of reoxygenation and cultures were maintained for 24 hours. After 24 hours of reoxygenation, the injury ratio of cortical neurons was determined by propidium iodide/Hoechst staining. Data are expressed as the mean ± SEM (n = 4). Data were analyzed using one-way analysis of variance and the Student Newman-Keuls post-hoc test. *P < 0.01, vs. control group; #P < 0.05, vs. OGD/R + vehicle group. OGD/R: Oxygen-glucose deprivation/reoxygenation.

Figure 2

Immunoreactivity of 8-hydroxydeoxyguanosine (8-OHdG) in cortical neurons after 24 hours of reoxygenation (immunocytochemical staining). Curcumin post-treatment inhibits early DNA/RNA oxidation after transient oxygen-glucose deprivation. (A) DNA/RNA oxidation was detected by immunocytochemical staining for 8-OHdG, a common marker for the evaluation of oxidative DNA damage. 8-OHdG is shown in green and nuclei were counterstained blue with 4,6-diamidino-2-phenylindole. Arrows show 8-OHdG and Hoechst double-positive cells. (B) Relative fluorescence intensities were obtained as described in the Methods and are expressed as the mean ± SEM (n = 4). Data were analyzed using one-way analysis of variance and the Student Newman-Keuls post-hoc test. #P < 0.05, vs. OGD/R + vehicle group. Scale bar: 100 μm. OGD/R: Oxygen-glucose deprivation/ reoxygenation.

Figure 3

Curcumin increases Nrf2 protein expression in rat cortical neurons (western blot analysis). (A) Cortical neurons were pretreated for 24 hours with curcumin or vehicle followed by OGD/R, or maintained in neurobasal medium without serum (control). (B) Cortical neurons were post-treated for 24 hours with 5 μM curcumin after a 1 hour period of OGD. After 24 hours, the nuclear fractions were isolated as described in the Methods. The immunoblots were probed with anti-Nrf2 antibody then stripped and re-probed for histone H3 as loading control (n = 4). The values were square transformed and the data were analyzed using one-way analysis of variance and the Student-Newman-Keuls post-hoc test. #P < 0.05, vs. OGD/R + vehicle group. Data were expressed as the mean ± SEM. OGD/R: Oxygen-glucose deprivation/reoxygenation; Nrf2: nuclear factor erythroid 2-related factor 2.

Figure 4

Curcumin increases the nuclear translocation of Nrf2 in cortical neurons (immunocytochemical staining, fluorescence microscope). Cortical neurons cultured on chamber slides were exposed to 5 μM curcumin after a 1 hour period of OGD. After 24 hours of reoxygenation, neurons were fixed in 4% paraformaldehyde, permeabilized, and immunostained for active Nrf2 [cyanine-3 (Cy3); red]. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). A merge of Cy3 and DAPI is shown in the third panel. The images presented here are representative of multiple fields from three independent experiments. Arrows show the curcumin-induced translocation of Nrf2 to the nucleus, cells expressing Nrf2 in the cytoplasm, but not in the nucleus. After the neurons were treated with curcumin for 24 hours, a 1 hour exposure to OGD caused increased Cy3 signaling for Nrf2 in the nucleus. This overlapped with DAPI staining. Scale bar: 100 μm. OGD/R: Oxygen-glucose deprivation/reoxygenation; Nrf2: nuclear factor erythroid 2-related factor 2.

Figure 5

Curcumin increases Trx expression and Trx enzyme activity in rat cortical neurons. (A) Rat cortical neurons were pretreated for 24 hours with curcumin, then exposed to OGD (1 hour) and reoxygenation (24 hours). The immunoblots were probed with anti-Trx antibody, and then stripped and re-probed for β-actin as a loading control (n = 4). Controls represent the following: control-before OGD, neurons maintained in neurobasal medium without serum; control-end of OGD, neurons maintained 1 hour in serum-free neurobasal medium; control-24 hours reoxygenation, neurons maintained 24 hours in serum-free neurobasal medium. The ratio of densitometry values for Trx and β-actin was analyzed. *P < 0.05, vs. control group; #P < 0.01, vs. vehicle group. (B) The Trx enzyme activity in total cell lysates of neurons pretreated with curcumin was determined at the same time points as those used for measuring the Trx total protein level. The results are expressed as the mean ± SEM (n = 4). #P < 0.01, vs. control group; *P < 0.01, vs. vehicle group. (C) Curcumin post-treatment increases the protein espression level of Trx after 24 hours of REOX. The ratio of densitometry values of Trx and β-actin was analyzed (n = 4). #P < 0.01, vs. control group; *P < 0.01, vs. vehicle group. (D) Curcumin post-treatment increases Trx enzyme activity after 24 hours of REOX. The results are expressed as the mean ± SEM (n = 4). The values were square transformed and the data were analyzed using one-way analysis of variance and the Student-Newman-Keuls test. #P < 0.01, vs. control group; *P < 0.01, vs. vehicle group. OGD: Oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reoxygenation; Trx: thioredoxin; min: minute.