Edaravone protects against oxygen-glucose-serum deprivation/restoration-induced apoptosis in spinal cord astrocytes by inhibiting integrated stress response

Abstract

We previously found that oxygen-glucose-serum deprivation/restoration (OGSD/R) induces apoptosis of spinal cord astrocytes, possibly via caspase-12 and the integrated stress response, which involves protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2-alpha (eIF2α) and activating transcription factor 4 (ATF4). We hypothesized that edaravone, a low molecular weight, lipophilic free radical scavenger, would reduce OGSD/R-induced apoptosis of spinal cord astrocytes. To test this, we established primary cultures of rat astrocytes, and exposed them to 8 hours/6 hours of OGSD/R with or without edaravone (0.1, 1, 10, 100 μM) treatment. We found that 100 μM of edaravone significantly suppressed astrocyte apoptosis and inhibited the release of reactive oxygen species. It also inhibited the activation of caspase-12 and caspase-3, and reduced the expression of homologous CCAAT/enhancer binding protein, phosphorylated (p)-PERK, p-eIF2α, and ATF4. These results point to a new use of an established drug in the prevention of OGSD/R-mediated spinal cord astrocyte apoptosis via the integrated stress response.

Keywords: CCAAT/enhancer binding protein homologous protein; PERK; activating transcription factor 4; apoptosis; astrocytes; caspase-12; caspase-3; eIF2α; edaravone; integrated stress response; nerve regeneration; neural regeneration; reactive oxygen species.

Figure 1

Edaravone (Eda) reduces oxygen-glucose-serum deprivation/restoration (OGSD/R)-induced apoptosis of spinal cord astrocytes. (A) Apoptotic cells with condensed or fragmented nuclei were detected after OGSD/R (Hoechst 33342 staining; original magnification, × 200). (B) Quantification of apoptotic cells. Eda (0.1, 1, 10, 100 μM) suppressed OGSD/R-induced apoptosis. (C) Cell viability (Cell Counting Kit-8 assay). Data are normalized to control (Cont; no treatment). All data are presented as the mean ± SD (n = 30; one-way analysis of variance followed by Student-Newman-Keuls test). **P < 0.01, vs. Cont; #P < 0.05, ##P < 0.01, vs. OGSD/R. h: Hours.

Figure 2

Edaravone (Eda) reduces oxygen-glucose-serum deprivation/restoration (OGSD/R)-induced release of reactive oxygen species (ROS) in spinal cord astrocytes. (A) Representative images of ROS immunofluorescence (staining with fluorescent dye H₂DCF-DA; × 200). (B) Quantification of ROS production. OGSD/R induced ROS production. Pre-treatment with Eda (100 μM) suppressed OGSD/R-induced ROS production. OGSD/R led to an increase of ROS release. Data are presented as the mean ± SD (n = 34; one-way analysis of variance followed by the Student-Newman-Keuls test). **P < 0.01, vs. control (Cont; no treatment); ##P < 0.01, vs. OGSD/R. h: Hours.

Figure 3

Edaravone (Eda) inhibits the caspase-3 signaling pathway after oxygen-glucose-serum deprivation/restoration (OGSD/R). (A) Western blots of cleaved caspase-3. (B) Quantification of cleaved caspase-3 protein expression. Eda (100 μM) inhibited OGSD/R-induced activation of caspase-3 from spinal cord astrocytes. Data are presented as the mean ± SD (n = 6; one-way analysis of variance followed by the Student-Newman-Keuls test). *P < 0.05, **P < 0.01, vs. control (Cont; no treatment); #P < 0.05, vs. OGSD/R group.

Figure 4

Edaravone (Eda) reduces oxygen-glucose-serum deprivation/restoration (OGSD/R)-induced increase in caspase-12 protein expression. (A) Changes in immunoreactivity and cellular distribution of caspase-12 in control (Cont), OGSD/R and OGSD/R + Eda (100 μM) groups. There was little caspase-12 protein expression in the Cont group (no treatment). Cytoplasm staining of caspase-12 was increased in the OGSD/R group. Green, glial fibrillary acidic protein (GFAP); red, caspase-12; blue, 4′,6-diamidino-2-phenylindole (DAPI); merged images are shown on the right. Eda inhibited OGSD/R-induced activation of caspase-12 from astrocytes (immunofluorescence staining, × 200). (B) Western blots of cleaved caspase-12 protein expression. (C) Quantification of caspase-12 protein expression. Eda inhibited OGSD/R-induced activation of caspase-12 from spinal cord astrocytes. Data are presented as the mean ± SD (n = 6; one-way analysis of variance followed by Student-Newman-Keuls test). **P < 0.01, vs. Cont; ##P < 0.01, vs. OGSD/R.

Figure 5

Effects of edaravone (Eda) on the CCAAT/enhancer binding protein homologous protein (CHOP) signaling pathway after oxygen-glucose-serum deprivation/restoration (OGSD/R). (A) Western blots of CHOP protein expression. (B) Quantification of CHOP protein expression (relative optical density of blots). Eda (100 μM) suppressed OGSD/R-induced CHOP expression in astrocytes. Data are presented as the mean ± SD (n = 6; one-way analysis of variance followed by Student-Newman-Keuls test). *P < 0.05, **P < 0.01, vs. control (Cont; no treatment); ##P < 0.01, vs. OGSD/R.

Figure 6

Effects of edaravone (Eda) on the PERK/eIF2α/ATF4 signaling pathway after oxygen-glucose-serum deprivation/restoration (OGSD/R). (A) Western blots of PERK, p-PERK, eIF2α, p-eIF2α, and ATF4 protein expression. (B–D) Quantification of p-PERK and PERK (B), p-eIF2α and eIF2α (C) and ATF4 (D) protein expression (relative optical density of bands). Eda (100 μM) suppressed activation of the PERK/eIF2α/ATF4 pathway in spinal cord astrocytes during OGSD/R. Data are presented as the mean ± SD (n = 6; one-way analysis of variance followed by the Student-Newman-Keuls tests). *P < 0.05, **P < 0.01, vs. control (Cont; no treatment); ##P < 0.01, vs. OGSD/R.