Effect of N-n-butyl haloperidol iodide on ROS/JNK/Egr-1 signaling in H9c2 cells after hypoxia/reoxygenation

Abstract

Reactive oxygen species (ROS)-induced oxidative stress in cells is an important pathophysiological process during myocardial ischemia/reperfusion (I/R) injury, and the transcription factor Egr-1 is a master switch for various damage pathways during reperfusion injury. An in vitro model of myocardial I/R injury and H9c2 cardiomyoblast cells hypoxia/reoxygenation (H/R) was used to assess whether there is abnormal intracellular ROS/JNK/Egr-1 signaling. We also assessed whether N-n-butyl haloperidol (F2), which exerts protective effects during myocardial I/R injury, can modulate this pathway. H/R induced ROS generation, JNK activation, and increased the expression of Egr-1 protein in H9c2 cells. The ROS scavengers edaravone (EDA) and N-acetyl-L-cysteine (NAC) reduced ROS level, downregulated JNK activation, and Egr-1 expression in H9c2 cells after H/R. The JNK inhibitor SP600125 inhibited Egr-1 overexpression in H9c2 cells caused by H/R. F2 could downregulate H/R-induced ROS level, JNK activation, and Egr-1 expression in H9c2 cells in a dose-dependent manner. The ROS donor hypoxanthine-xanthine oxidase (XO/HX) and the JNK activator ANISO antagonized the effects of F2. Therefore, H/R activates ROS/Egr-1 signaling pathway in H9c2 cells, and JNK activation plays an important role in this pathway. F2 regulates H/R-induced ROS/JNK/Egr-1 signaling, which might be an important mechanism by which it antagonizes myocardial I/R injury.

Figure 1. ROS levels and Egr-1 protein expression in H9c2 cells with different durations of H/R, as assessed using flow cytometry and western blotting.

A. ROS levels during H/R; n = 6. B. Cropped blots show protein levels of Egr-1 and β-actin; n = 4. The bands were excised from the same gel. Data are expressed as the percentages of the control group. All values are expressed as mean ± S.E.M. ^*P < 0.05 vs. control; ^#P < 0.05 vs. the H:1 h/R:1 h group; ^†P < 0.05 vs. the H:2 h/R:1 h group.

Figure 2. Effects of different doses of ROS scavengers on ROS level and Egr-1 protein expression in H9c2 cells after H/R, as assessed using flow cytometry and western blotting.

A. ROS levels; n = 6. B. Cropped blots show protein levels of Egr-1 and β-actin; n = 3. The bands were excised from the same gel. Data are expressed as the percentages of the control or H/R groups. All values are expressed as mean ± S.E.M. ^*P < 0.05 vs. control; ^#P < 0.05 vs. H/R; ^†P < 0.05 vs. H/R + 2 × 10⁻⁶ M EDA; ^§P < 0.05 vs. H/R + 2 × 10⁻⁵ M EDA; ^&P < 0.05 vs. H/R + 5 × 10⁻⁴ M NAC; ^*P < 0.05 vs. H/R + 2 × 10⁻³ M NAC.

Figure 3. Effects of a ROS donor, ROS scavengers, and a JNK inhibitor on the levels of total and p-JNK expression in H9c2 cells, as assessed using western blotting.

A. Effects of a ROS donor; n = 6. B. Effects of ROS scavengers and a JNK inhibitor. n = 4. Cropped blots show protein levels of p-JNK, total JNK and β-actin. The bands were excised from different gels which were run under the same electrophoresis condition. Data are expressed as percentages of the control or H/R groups. All values are presented as mean ± S.E.M. ^*P < 0.05 vs. control; ^#P < 0.05 vs. H/R.

Figure 4. Effects of a JNK inhibitor on Egr-1 expression in H9c2 cells after H/R using western blotting. Cropped blots show protein levels of Egr-1 and β-actin.

The bands were excised from the same gel. Data are expressed as percentages of the H/R group. All values are presented as mean ± S.E.M; n = 4. ^*P < 0.05 vs. control; ^#P < 0.05 vs. H/R.

Figure 5. Effects of F₂ on ROS levels, JNK activation, and Egr-1 expression in H9c2 cells after H/R, as assessed using flow cytometry and western blotting.

A. ROS levels; n = 10. B. Cropped blots show total and p-JNK expressions; n = 6. The bands were excised from different gels which were run under the same electrophoresis condition. C. Cropped blots show Egr-1 and β-actin expressions; n = 6. The bands were excised from the same gel. Data are expressed as percentages of the levels of the control or H/R groups. All values are expressed as mean ± S.E.M. ^*P < 0.05 vs. control; ^#P < 0.05 vs. H/R; ^†P < 0.05 vs. H/R + 10⁻⁷ M F₂.

Figure 6. Influence of a ROS donor and JNK activator on the effects of F₂ on ROS level, JNK activation, and Egr-1 expression in H9c2 cells after H/R, as assessed using flow cytometry and western blotting.

A. ROS levels; n = 6. B. Cropped blots show total and p-JNK expressions; n = 3. The bands were excised from different gels which were run under the same electrophoresis condition. C. Cropped blots show Egr-1 and β-actin expressions; n = 3. The bands were excised from the same gel. Data are expressed as percentages of the levels of the control or H/R groups. All values are expressed as means ± S.E.M. ^*P < 0.05 vs. control; ^#P < 0.05 vs. H/R; ^†P < 0.05 vs. H/R + 10⁻⁶ M F₂.

Figure 7. Protocol of the experimental grouping and treatments.

A. Protocol used to investigate whether ROS /JNK/Egr-1 signaling occurs in H9c2 cells after H/R. B. Protocol used to investigate the effects of F₂ on ROS/JNK/Egr-1 signaling in H9c2 cells after H/R.