Role of ferroptosis in hypoxic preconditioning to reduce propofol neurotoxicity

Abstract

Background: An increasing number of studies have reported that neurotoxicity of propofol may cause long-term learning and cognitive dysfunction. Hypoxic preconditioning has been shown to have neuroprotective effects, reducing the neurotoxicity of propofol. Ferroptosis is a new form of death that is different from apoptosis, necrosis, autophagy and pyroptosis. However, it is unclear whether hypoxic preconditioning reduces propofol neurotoxicity associated with ferroptosis. Thus, we aimed to evaluate the effect of propofol on primary hippocampal neurons in vitro to investigate the neuroprotective mechanism of hypoxic preconditioning and the role of ferroptosis in the reduction of propofol neurotoxicity by hypoxic preconditioning. Methods: Primary hippocampal neurons were cultured for 8 days in vitro and pretreated with or without propofol, hypoxic preconditioning, agonists or inhibitors of ferroptosis. Cell counting kit-8, Calcein AM, Reactive oxygen species (ROS), Superoxide dismutase (SOD), Ferrous iron (Fe²⁺), Malondialdehyde (MDA) and Mitochondrial membrane potential assay kit with JC-1 (JC-1) assays were used to measure cell viability, Reactive oxygen species level, Superoxide dismutase content, Fe²⁺ level, MDA content, and mitochondrial membrane potential. Cell apoptosis was evaluated using flow cytometry analyses, and ferroptosis-related proteins were determined by Western blot analysis. Results: Propofol had neurotoxic effects that led to decreased hippocampal neuronal viability, reduced mitochondrial membrane potential, decreased SOD content, increased ROS level, increased Fe²⁺ level, increased MDA content, increased neuronal apoptosis, altered expression of ferroptosis-related proteins and activation of ferroptosis. However, hypoxic preconditioning reversed these effects, inhibited ferroptosis caused by propofol and reduced the neurotoxicity of propofol. Conclusion: The neurotoxicity of propofol in developing rats may be related to ferroptosis. Propofol may induce neurotoxicity by activating ferroptosis, while hypoxic preconditioning may reduce the neurotoxicity of propofol by inhibiting ferroptosis.

Keywords: ferroptosis; hippocampus; hypoxic preconditioning; neurotoxicity; propofol.

FIGURE 1

Experimental groups and treatments. In the first phase of the research, the cells in Group C and Group I were incubated with fresh maintenance medium and intralipid vehicle, respectively. The cells in Group P were incubated with 100 μM propofol for 3 h. Cells in Group H were treated with hypoxic preconditioning, and cells in Group HP were subjected to hypoxic preconditioning before incubation with propofol. In the second phase of the research, the cells in Group C, Group I, Group P, Group H and Group HP were processed as the first phase of the study. The cells in Group FP were treated with 2 μM ferrostatin-1 for 30 min before incubation with propofol. The cells in the HRP group were treated with 10 μM RSL3 for 6 h after hypoxic preconditioning and then incubated with 100 μM propofol for 3 h.

FIGURE 2

Changes in mitochondrial membrane potential, neuronal viability and ATP in primary hippocampal neurons exposed to propofol with or without hypoxic preconditioning. (A,B) Fluorescence images of JC-1 aggregates and JC-1 monomers indicating the mitochondrial membrane potential in each group (one-way ANOVA, *p < 0.05). (D,E) Cell viability and ATP content in each group (one-way ANOVA, *p < 0.05).

FIGURE 3

Changes in cell apoptosis in primary hippocampal neurons exposed to propofol with or without hypoxic preconditioning. (A,B) Cell apoptosis in each group (one-way ANOVA, *p < 0.05).

FIGURE 4

Changes in ROS in primary hippocampal neurons exposed to propofol with or without hypoxic preconditioning. (A,B) ROS content in each group (one-way ANOVA, *p < 0.05).

FIGURE 5

Protein expression levels of TFR1, FPN1, DMT1, SLC7A11 and GPX4 in primary hippocampal neurons exposed to propofol with or without hypoxic preconditioning. (A) TFR1, FPN1, DMT1, SLC7A11 and GPX4 protein expression was detected by Western blot analysis. (B–F) Quantitative analysis of the ratio of TFR1, FPN1, DMT1, SLC7A11 and GPX4 to β-actin (one-way ANOVA, *p < 0.05).

FIGURE 6

Cell viability in primary hippocampal neurons exposed to propofol with or without hypoxic preconditioning, inhibitor or agonist. (A,B) Calcein AM in each group (one-way ANOVA, *p < 0.05).

FIGURE 7

Changes in ROS in primary hippocampal neurons exposed to propofol with or without hypoxic preconditioning, inhibitor or agonist. (A,B) ROS content in each group (one-way ANOVA, *p < 0.05).

FIGURE 8

Changes in mitochondrial membrane potential, SOD level, Fe2+ level and MDA content in primary hippocampal neurons exposed to propofol with or without hypoxic preconditioning, inhibitor or agonist. (A,B) Fluorescence images of JC-1 aggregates and JC-1 monomers indicating the mitochondrial membrane potential in each group (one-way ANOVA, *p < 0.05). (D–F) The contents of SOD, Fe2+ and MDA in each group (one-way ANOVA, *p < 0.05).

FIGURE 9

Protein expression levels of TFR1, FPN1, DMT1, SLC7A11 and GPX4 in primary hippocampal neurons exposed to propofol with or without hypoxic preconditioning, inhibitor or agonist. (A) TFR1, FPN1, DMT1, SLC7A11 and GPX4 protein expression was detected by Western blot analysis. (B–F) Quantitative analysis of the ratio of TFR1, FPN1, DMT1, SLC7A11 and GPX4 to β-actin (one-way ANOVA, *p < 0.05).

FIGURE 10

The molecular mechanisms of ferroptosis.