Propofol Protects Against H2O2-Induced Oxidative Injury in Differentiated PC12 Cells via Inhibition of Ca(2+)-Dependent NADPH Oxidase

Abstract

Propofol (2,6-diisopropylphenol) is a widely used general anesthetic with anti-oxidant activities. This study aims to investigate protective capacity of propofol against hydrogen peroxide (H2O2)-induced oxidative injury in neural cells and whether the anti-oxidative effects of propofol occur through a mechanism involving the modulation of NADPH oxidase (NOX) in a manner of calcium-dependent. The rat differentiated PC12 cell was subjected to H2O2 exposure for 24 h to mimic a neuronal in vitro model of oxidative injury. Our data demonstrated that pretreatment of PC12 cells with propofol significantly reversed the H2O2-induced decrease in cell viability, prevented H2O2-induced morphological changes, and reduced the ratio of apoptotic cells. We further found that propofol attenuated the accumulation of malondialdehyde (biomarker of oxidative stress), counteracted the overexpression of NOX core subunit gp91(phox) (NOX2) as well as the NOX activity following H2O2 exposure in PC12 cells. In addition, blocking of L-type Ca(2+) channels with nimodipine reduced H2O2-induced overexpression of NOX2 and caspase-3 activation in PC12 cells. Moreover, NOX inhibitor apocynin alone or plus propofol neither induces a significant downregulation of NOX activity nor increases cell viability compared with propofol alone in the PC12 cells exposed to H2O2. These results demonstrate that the protective effects of propofol against oxidative injury in PC12 cells are mediated, at least in part, through inhibition of Ca(2+)-dependent NADPH oxidase.

Keywords: Hydrogen peroxide; NADPH oxidase; Oxidative injury; PC12 cells; Propofol.

Fig. 1

Effects of propofol and H₂O₂ on cell viability. The viability of PC12 cells was detected by MTT assay. Cells were incubated for 24 h in different concentrations of H₂O₂ alone (a). Cells were exposed to (250 μM) H₂O₂ for time-course study (b). Cells were incubated for 24 h in different concentrations of propofol alone (c). Cells were pre-incubated with different concentrations of propofol for 6 h and then co-treated with 250 μM H₂O₂ for additional 24 h (d). The results are expressed as mean ± SD (n = 5). ^# P < 0.05, ^## P < 0.01 versus untreated control cells; *P < 0.05, **P < 0.01 versus H₂O₂-treated cells

Fig. 2

Effects of propofol on H₂O₂-induced oxidative injury in PC12 cells. Cell morphology was observed with inverted phase contrast microscope, scale bars represent 100 μM (A); Cell apoptosis was evaluated by apoptotic nuclei staining with Hoechst 33258 dye. The apoptotic cells are indicated by arrows, scale bars represent 50 μM (B); Percentage of apoptotic cells per total number of PC12 cells in each group (C). Control cells cultured in drug-free medium; Propofol cells exposed to 100 μM propofol for 24 h; H ₂ O ₂ cells exposed to 250 μM H₂O₂ for 24 h; H ₂ O ₂ + Propofol cells were pretreated with 100 μM propofol for 6 h, and then co-incubated in 250 μM H₂O₂ for an additional 24 h. The results are expressed as mean ± SD (n = 5). ^## P < 0.01 versus untreated control cells; **P < 0.01 versus H₂O₂-treated cells

Fig. 3

Effects of propofol on H₂O₂-induced elevation of intracellular MDA levels. MDA contents were detected by a Lipid Peroxidation MDA Assay Kit. The cells were divided into four groups, Control cells cultured in drug-free medium; Propofol cells exposed to 100 μM propofol for 24 h; H ₂ O ₂ cells exposed to 250 μM H₂O₂ for 24 h; H ₂ O ₂ + Propofol cells were pretreated with 100 μM propofol for 6 h and then incubated in 250 μM H₂O₂ for an additional 24 h. Data are expressed as micromoles of MDA per milligram of protein The results are expressed as mean ± SD (n = 5). ^## P < 0.01 versus untreated control cells; **P < 0.01 versus H₂O₂-treated cells

Fig. 4

Effects of propofol on NOX2 protein expression and NOX activity. a NOX2 protein expression was detected by Western blotting. GAPDH served as a loading control. The upper panel is a representative experiment. The lower panel shows densitometric data; b NOX activity was detected by lucigenin-enhanced chemiluminiscence with Colorimetric assay kit. Data are presented as percentage of control-treated values. The results are expressed as mean ± SD (n = 5). ^## P < 0.01 versus untreated control cells; **P < 0.01 versus H₂O₂-treated cells

Fig. 5

Effects of Ca²⁺ influx in NOX2 expression and caspase-3 activation. The cells were divided into four groups. Control cells cultured in drug-free medium; H ₂ O ₂ cells exposed to 250 μM H₂O₂ for 24 h; Propofol + H ₂ O ₂ cells exposed to 100 μM propofol for 6 h, and then co-incubated in 250 μM H₂O₂ for an additional 24 h; Nimodipine + H ₂ O ₂ cells exposed to 10 μM Nimodipine for 6 h, and then co-incubated in 250 μM H₂O₂ for an additional 24 h. NOX2 (a) and cleaved caspase 3 (b) expression were detected by Western blotting. GAPDH served as a loading control. The upper panel is a representative experiment. The lower panel shows densitometric data. The results are expressed as mean ± SD (n = 5). ^## P < 0.01 versus untreated control cells; **P < 0.01 versus H₂O₂-treated cells

Fig. 6

Effects of NOX inhibitor apocynin on NOX activity and cell viability following H₂O₂ exposure. The cells were divided into five groups. Control cells cultured in drug-free medium; H ₂ O ₂ cells exposed to 250 μM H₂O₂ for 24 h; Propofol + H ₂ O ₂ cells exposed to 100 μM propofol for 6 h, and then co-incubated in 250 μM H₂O₂ for an additional 24 h; Apocynin + Propofol + H ₂ O ₂ cells exposed to 100 μM propofol plus 50 μM apocynin for 6 h, and then co-incubated in 250 μM H₂O₂ for an additional 24 h; Apocynin + H ₂ O ₂ cells exposed to 50 μM apocynin for 6 h, and then co-incubated in 250 μM H₂O₂ for an additional 24 h. a NOX activity was detected by lucigenin-enhanced chemiluminiscence with Colorimetric assay kit; b cell viability was detected by MTT assay. Data are presented as percentage of control-treated values. The results are expressed as mean ± SD (n = 3). ^## P < 0.01 versus untreated control cells; **P < 0.01 versus H₂O₂-treated cells. n.s. no significance