Resveratrol protects C6 astrocyte cell line against hydrogen peroxide-induced oxidative stress through heme oxygenase 1

Abstract

Resveratrol, a polyphenol presents in grapes and wine, displays antioxidant and anti-inflammatory properties and cytoprotective effect in brain pathologies associated to oxidative stress and neurodegeneration. In previous work, we demonstrated that resveratrol exerts neuroglial modulation, improving glial functions, mainly related to glutamate metabolism. Astrocytes are a major class of glial cells and regulate neurotransmitter systems, synaptic processing, energy metabolism and defense against oxidative stress. This study sought to determine the protective effect of resveratrol against hydrogen peroxide (H2O2)-induced cytotoxicity in C6 astrocyte cell line, an astrocytic lineage, on neurochemical parameters and their cellular and biochemical mechanisms. H2O2 exposure increased oxidative-nitrosative stress, iNOS expression, cytokine proinflammatory release (TNFα levels) and mitochondrial membrane potential dysfunction and decreased antioxidant defenses, such as SOD, CAT and creatine kinase activity. Resveratrol strongly prevented C6 cells from H2O2-induced toxicity by modulating glial, oxidative and inflammatory responses. Resveratrol per se increased heme oxygenase 1 (HO1) expression and extracellular GSH content. In addition, HO1 signaling pathway is involved in the protective effect of resveratrol against H2O2-induced oxidative damage in astroglial cells. Taken together, these results show that resveratrol represents an important mechanism for protection of glial cells against oxidative stress.

Figure 1. Resveratrol decreased nitrite production.

Cells were pre-treated for 1 h with 100 µM resveratrol (RSV) followed by the addition of 1 mM H₂O₂ for 0.5 h. Nitrite production was measured as described in the Experimental procedures section. Data represent mean ± S.E.M of three independent experimental determinations performed in triplicate, analyzed statistically by two-way ANOVA followed by the Tukey’s test. (a) indicates significant differences from control (P<0.05). (b) indicates significant differences from H₂O₂ (P<0.05). (c) indicates significant differences from L-NAME (P<0.05).

Figure 2. Effects of resveratrol on iNOS expression.

Cells were pre-treated for 1 h with 100 µM resveratrol (RSV) followed by the addition of 1 mM H₂O₂ for 0.5 h. Western blot for iNOS was performed as described in the Experimental procedures section. Data are expressed as percentage of control value (assumed to be 100%) and represent the optical density (OD) of mean ± S.E.M of three independent experimental determinations performed in triplicate, analyzed statistically by two-way ANOVA followed by the Tukey’s test. (a) indicates significant differences from control (P<0.01). (b) indicates significant differences from H₂O₂ (P<0.01).

Figure 3. Resveratrol increased HO1 expression.

Cells were pre-treated for 1 h with 100 µM resveratrol (RSV) followed by the addition of 1 mM H₂O₂ for 0.5 h. Western blot for HO1 was performed as described in the Experimental procedures section. Data are expressed as percentage of control value (assumed to be 100%) and represent the optical density (OD) of mean ± S.E.M of three independent experimental determinations performed in triplicate, analyzed statistically by two-way ANOVA followed by the Tukey’s test. (a) indicates significant differences from control (P<0.01). (b) indicates significant differences from H₂O₂ (P<0.01).

Figure 4. Effects of resveratrol on intracellular ROS production.

Cells were pre-treated for 1 h with 100 µM resveratrol (RSV) followed by the addition of 1 mM H₂O₂ for 0.5 h. Cells were also pre-incubated for 0.5 h with ZnPP IX (10 µM), a HO1 inhibitor, before the pre-treatment with resveratrol. Intracellular ROS production was measured as described in the Experimental procedures section. Data are expressed as percentage of control value and represent mean ± S.E.M of three independent experimental determinations performed in triplicate, analyzed statistically by two-way ANOVA followed by the Tukey’s test. (a) indicates significant differences from control (P<0.05). (b) indicates significant differences from H₂O₂ (P<0.05) and (c) indicates significant differences from ZnPP IX inhibitor (P<0.05).

Figure 5. Effects of resveratrol on TAR levels.

Cells were pre-treated for 1 h with 100 µM resveratrol (RSV). After pre-treatment, cells were exposed to 1 mM H₂O₂ for 0.5 h. TAR levels were measured as described in the Experimental procedures section. The basal TAR levels, assumed to be 100%, are indicated by the line. Each value is the mean ± S.E.M of three independent experimental determinations performed in triplicate, analyzed statistically by two-way ANOVA followed by the Tukey’s test. (a) indicates significant differences from control (P<0.001). (b) indicates significant differences from H₂O₂ (P<0.001).

Figure 6. Resveratrol prevented mitochondrial dysfunction.

Cells were pre-treated for 1 h with 100 µM resveratrol (RSV). After pre-treatment, cells were exposed to 1 mM H₂O₂ for 0.5 h. Mitochondrial membrane potential was measured as described in the Experimental procedures section. The control conditions, assumed to be 100%, are indicated by the line. Each value [the ratio of 590 nm (red fluorescent J-aggregates)/540 nm (green monomeric)] is the mean ± S.E.M of three independent experimental determinations performed in triplicate, analyzed statistically by two-way ANOVA followed by the Tukey’s test. (a) indicates significant differences from control (P<0.05). (b) indicates significant differences from H₂O₂ (P<0.05).

Figure 7. Effects of resveratrol on CK activity.

Cells were pre-treated for 1 h with 100 µM resveratrol (RSV). After pre-treatment, cells were exposed to 1 mM H₂O₂ for 0.5 h. CK activity was measured as described in the Experimental procedures section. Data represent mean ± S.E.M of three independent experimental determinations performed in triplicate, analyzed statistically by two-way ANOVA followed by the Tukey’s test. (a) indicates significant differences from control (P<0.05). (b) indicates significant differences from H₂O₂ (P<0.05).

Figure 8. Effects of resveratrol on SOD, CAT and GPx activity.

Cells were pre-treated for 1 h in the presence of 100 µM resveratrol (RSV) followed by the addition of 1 mM H₂O₂ for 0.5 h. SOD (A), CAT (B) and GPx (C) activities were measured as described in the Experimental procedures section. All data represent mean ± S.E.M. of three independent experimental determinations performed in triplicate, analyzed statistically by two-way ANOVA followed by the Tukey’s test. (a) indicates significant differences from control (P<0.05). (b) indicates significant differences from H₂O₂ (P<0.05).

Figure 9. Schematic illustration of the mechanism underlying the protective effect of resveratrol against H₂O₂-induced oxidative stress in C6 astrocyte cell line.

H₂O₂ exposure induces increase in ROS/RNS levels, which are prevented by resveratrol (RSV). Oxidative-nitrosative stress induces impairment in mitochondrial membrane potential (referred in the figure as MMP), SOD, CAT, GPx, CK, extracellular GSH (referred in the figure as eGSH) and TNFα. RSV enhances HO1 expression, followed by decreased iNOS expression and consequently NO levels attenuation. Thus, resveratrol is able to confer protection against H₂O₂-induced oxidative stress in C6 astrocyte cell line, probably, via HO1 activation, antioxidant/scavenger activity and/or anti-inflammatory effect.