Protective effects of parecoxib on rat primary astrocytes from oxidative stress induced by hydrogen peroxide

Abstract

Objective: To investigate the protective effects of parecoxib from oxidative stress induced by hydrogen peroxide (H2O2) in rat astrocytes in vitro.

Methods: All experiments included 4 groups: (1) negative control (NC) group, without any treatment; (2) H2O2 treatment group, 100 μmol/L H2O2 treatment for 24 h; (3) and (4) parecoxib pretreatment groups, 80 and 160 μmol/L parecoxib treatment for 24 h, respectively, and then treated with 100 μmol/L H2O2. Several indices were investigated, and the expressions of Bax, Bcl-2, and brain-derived neurotrophic factor (BDNF) were quantified.

Results: Compared to the NC group, exposure to H2O2 resulted in significant morphological changes, which could be reversed by pretreatment of parecoxib. In addition, H2O2 treatment led to loss of viability (P=0.026) and increased intracellular reactive oxygen species (ROS) levels (P<0.001), and induced apoptosis (P<0.01) in the primary astrocytes relative to the NC group. However, in the parecoxib pretreatment groups, all the above changes reversed significantly (P<0.05) as compared to the H2O2 treatment group, and were nearly unchanged when compared to the NC group. Mechanical investigation showed that dysregulated Bax, Bcl-2, and BDNF could be implicated in these changes.

Conclusions: Our results indicated that parecoxib provided a protective effect from oxidative stress induced by exposure to H2O2.

Keywords: Bax; Bcl-2; Brain-derived neurotrophic factor (BDNF); Hydrogen peroxide; Parecoxib; Primary astrocyte.

Fig. 1

Parecoxib resisted morphological changes of the primary astrocytes due to exposure to H₂O₂ (a) NC group; (b) H₂O₂ treatment group (100 μmol/L); (c) Parecoxib pretreatment group (80 μmol/L); (d) Parecoxib pretreatment group (160 μmol/L)

Fig. 2

H₂O₂ exposure led to loss of viability in the primary astrocytes, which was reversed by parecoxib Data shown were representative of three independent experiments and error bars represent mean±SEM. ^* P<0.05 vs. H₂O₂ treatment group

Fig. 3

Protection of parecoxib from the increase of the intracellular ROS levels of the primary astrocytes due to exposure to H₂O₂ Intracellular peroxides of the primary astrocytes were indicated by fluorescence intensity in the NC group (a), H₂O₂ (100 μmol/L) treatment group (b), parecoxib (80 μmol/L) pretreatment group (c), and parecoxib (160 μmol/L) pretreatment group (d). (e) Mean intracellular fluorescence intensity of the 10⁴ cells. Data shown were representative of three independent experiments and the error bars represent the mean±SEM. ^** P<0.01, ^*** P<0.001 vs. H₂O₂ treatment group

Fig. 4

Parecoxib resisted apoptosis of the primary astrocytes induced by exposure to H₂O₂ Cell apoptosis was indicated through flow cytometry using propidium iodide (PI) staining in the NC group (a), H₂O₂ (100 μmol/L) treatment group (b), parecoxib (80 μmol/L) pretreatment group (c), and parecoxib (160 μmol/L) pretreatment group (d). (e) Ratio of apoptotic cells in four groups. Data shown were representative of three independent experiments and the error bars represent the mean±SEM. ^** P<0.01 vs. H₂O₂ treatment group

Fig. 5

Exposure to H₂O₂ resulted in dysregulated Bax and Bcl-2 expressions, which were reversed using parecoxib (a, b) Gel electrophoresis images of the RT-PCR products of Bax and Bcl-2 in four groups: (1) NC group; (2) H₂O₂ (100 μmol/L) treatment group; (3) parecoxib (80 μmol/L) pretreatment group; and (4) parecoxib (160 μmol/L) pretreatment group. β-Actin was used as an internal control. The RT-PCR product lengths of Bax, Bcl-2, and β-actin were 377, 450, and 285 bp, respectively. (c) Gray intensity of the gel electrophoresis images of the RT-PCR products of Bax and Bcl-2 in four groups. Data shown were representative of three independent experiments and the error bars represent the mean±SEM. ^* P<0.05 vs. H₂O₂ treatment group

Fig. 6

Exposure to H₂O₂ resulted in dysregulated BDNF expression, which was reversed by parecoxib Data shown were representative of three independent experiments and the error bars represent the mean±SEM. ^* P<0.05 vs. H₂O₂ treatment group. β-actin was used an internal control