N-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway

Abstract

Endothelium dysfunction induced by reactive oxygen species (ROS) is an important initial event at the onset of myocardial ischemia/reperfusion in which the Egr-1 transcription factor often serves as a master switch for various damage pathways following reperfusion injury. We hypothesized that an intracellular ROS/MAPK/Egr-1 signaling pathway is activated in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). ROS generation, by either H/R or the ROS donor xanthine oxidase-hypoxanthine (XO/HX) activated all three MAPKs (ERK1/2, JNK, p38), and induced Egr-1 expression and Egr-1 DNA-binding activity in CMECs, whereas ROS scavengers (EDA and NAC) had the opposite effect following H/R. Inhibitors of all three MAPKs individually inhibited induction of Egr-1 expression by H/R in CMECs. Moreover, N-n-butyl haloperidol (F₂), previously shown to protect cardiomyocytes subjected to I/R, dose-dependently downregulated H/R-induced ROS generation, MAPK activation, and Egr-1 expression and activity in CMECs, whereas XO/HX and MAPK activators (EGF, anisomycin) antagonized the effects of F₂. Inhibition of the ROS/MAPK/Egr-1 signaling pathway, by either F₂, NAC, or inhibition of MAPK, increased CMEC viability and the GSH/GSSG ratio, and decreased Egr-1 nuclear translocation. These results show that the ROS/MAPK/Egr-1 signaling pathway mediates H/R injury in CMECs, and F₂ blocks this pathway to protect against H/R injury and further alleviate myocardial I/R injury.

Keywords: Egr-1; N-n-butyl haloperidol; cardiac microvascular endothelial cell; hypoxia/reoxygenation; mitogen-activated protein kinase; reactive oxygen species.

Figure 1

Protocol for experimental grouping and treatments. (A) Protocol used to investigate whether the ROS/MAPK/Egr-1 signaling pathway occurs in CMECs after H/R. (B) Protocol used to investigate the protective effects of F₂ on CMEC H/R injury through regulating ROS/MAPK/Egr-1 signaling.

Figure 2

ROS level and Egr-1 protein expression in CMECs following different durations of hypoxia, and a 1-h reperfusion, as assessed using flow cytometry and western blotting. (A) ROS levels during H/R; n = 3. (B) Protein levels of Egr-1 and β-actin; n = 3. Quantitative 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 H1/R1 group; ^†P < 0.05 vs. the H2/R1 group; ^‡P < 0.05 vs. the H3/R1 group.

Figure 3

Effects of different doses of a ROS donor on ROS level, and Egr-1 gene and protein expression in CMECs. (A) Flow cytometry was performed to determine ROS levels; n = 4. (B) Egr-1 protein expression was detected by western blot; n = 3. (C) RT-PCR was performed to determine Egr-1 mRNA levels; n = 3. Quantitative 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 1 mU/ml XO + 1.2 × 10⁻⁴ M HX group; ^†P < 0.05 vs. the 3 mU/ml XO + 3.6 × 10⁻⁴ M HX group.

Figure 4

Effects of different doses of ROS scavengers on ROS level, and Egr-1 gene and protein expression in CMECs after H/R, as assessed by flow cytometry, western blot and RT-PCR. (A) Effect of NAC on ROS levels in H/R CMECs; n = 3. (B) Effect of EDA on ROS levels in H/R CMECs; n = 3. (C) Effects of EDA and NAC on protein levels of Egr-1 and β-actin; n = 3. (D) Effect of EDA on Egr-1 mRNA levels; n = 3. Quantitative data are expressed as the percentages of 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 × 10⁻⁵ M NAC or H/R + 5 × 10⁻⁵ M EDA; ^‡P < 0.05 vs. H/R + 1 × 1 0⁻⁴ M NAC or H/R + 1 × 10⁻⁴ M EDA.

Figure 5

Effects of MAPKs (ERK1/2, JNK, and p38) on ROS/Egr-1 signaling in CMECs subjected to H/R, as detected by western blotting. (A) Effects of XO/HX and NAC on ERK1/2; n = 3. (B) Effects of XO/HX and NAC on JNK; n = 4. (C) Effects of XO/HX and NAC on p38; n = 3. (D) Effects of MAPK activators and inhibitors on expression of Egr-1 protein. n = 3. Quantitative densitometric 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 6

Effects of F₂ on ROS level, MAPK (ERK1/2, JNK, and p38) activation, and Egr-1 gene and protein expression in CMECs after H/R, as assessed using flow cytometry, western blotting and RT-PCR. (A) ROS levels; n = 3. (B) Total ERK1/2 and p-ERK1/2 protein expression; n = 4. (C) Total JNK and p-JNK protein expression; n = 3. (D) Total p38 and p-p38 protein expression; n = 3. (E) Egr-1 and β-actin protein expression; n = 3. (F) Egr-1 mRNA and GAPDH mRNA expression; n = 3. Quantitative data are expressed as percentages of the levels of 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; ^‡P < 0.05 vs. H/R + 10⁻⁶ M F₂.

Figure 7

Effect of F₂ on DNA binding activity of Egr-1. EMSA was performed with a biotin-labeled consensus Egr-1 oligonucleotide probe and CMEC nuclear extracts (6 μg/lane of nuclear protein). Competitor samples were incubated with an extra 50- or 200-fold non-biotin-labeled probe.

Figure 8

Influence of ROS donor and MAPK activators on the effects of F₂ on ROS level, MAPK activation, and Egr-1 expression in CMECs after H/R, as determined using flow cytometry and western blot. (A) ROS levels; n = 3. (B) Total ERK1/2 and p-ERK1/2 protein expression; n = 3. (C) Total JNK and p-JNK protein expression; n = 3. (D) Total p38 and p-p38 protein expression; n = 3. (E) Egr-1 and β-actin protein expression; n = 3. Quantitative data are expressed as percentages of the levels of the 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 + F₂.

Figure 9

Effects of signaling pathway inhibitors (NAC, U0126, SP600125, and SB203580) and F₂ on cell viability, MDA levels and the GSH/GSSG ratio of CMECs after H/R. (A) Cell viability; n = 4. (B) Level of MDA; n = 6. (C) GSH/GSSG ratio; n = 5. All values are expressed as means ± S.E.M. ^*P < 0.05 vs. control; ^#P < 0.05 vs. H/R.