Lipoxin a4 preconditioning and postconditioning protect myocardial ischemia/reperfusion injury in rats

Abstract

This study aims to investigate the pre- and postconditioning effects of lipoxin A4 (LXA4) on myocardial damage caused by ischemia/reperfusion (I/R) injury. Seventy-two rats were divided into 6 groups: sham groups (C1 and C2), I/R groups (I/R1 and I/R2), and I/R plus LXA4 preconditioning and postconditioning groups (LX1 and LX2). The serum levels of IL-1 β , IL-6, IL-8, IL-10, TNF- α , and cardiac troponin I (cTnI) were measured. The content and the activity of Na(+)-K(+)-ATPase as well as the superoxide dismutase (SOD), and malondialdehyde (MDA) levels were determined. Along with the examination of myocardium ultrastructure and ventricular arrhythmia scores (VAS), connexin 43 (Cx43) expression were also detected. Lower levels of IL-1 β , IL-6, IL-8, TNF- α , cTnI, MDA content, and VAS and higher levels of IL-10, SOD activity, Na(+)-K(+)-ATPase content and activity, and Cx43 expression appeared in LX groups than I/R groups. Besides, H&E staining, TEM examination as well as analysis of gene, and protein confirmed that LXA4 preconditioning was more effective than postconditioning in preventing arrhythmogenesis via the upregulation of Cx43. That is, LXA4 postconditioning had better protective effect on Na(+)-K(+)-ATPase and myocardial ultrastructure.

Figure 1

Comparison of concentrations of serum IL-1β, IL-6, IL-8, IL-10; TNF-α;, and cTnI at T₁ and T₂ among all groups (n = 12 for each group). At time point T₁, blood was collected immediately before thoracotomy. At time point T₂, blood was collected right after the I/R procedure was over. (a) IL-1β; (b) IL-6; (c) IL-8; (d) IL-10; (e) TNF-α; (f) cTnI. *P < 0.05 for comparisons of I/R₁ and LX₁ groups with C₁ group, P < 0.05 for comparisons of I/R₂ and LX₂ with C₂ group; ^# P < 0.05 for comparisons of LX₁ group with I/R₁ group, P < 0.05 for comparisons of LX₂ group with I/R₂ group; ^Δ P < 0.05 for comparisons of LX₂ group with LX₁ group.

Figure 2

Comparison of cardiac MDA content and SOD and Na⁺-K⁺-ATPase activities at T₂ among all groups (n = 12 for each group). At time point T₂, myocardial tissue was collected right after the I/R procedure was over and kept frozen in liquid nitrogen. MDA content and SOD and Na⁺-K⁺-ATPase activities were measured as described in the section of Materials and Methods. (a) SOD activity; (b) MDA content; (c) Na⁺-K⁺-ATPase activity. *P < 0.05 for comparisons of I/R₁ and LX₁ groups with C₁ group, P < 0.05 for comparisons of I/R₂ and LX₂ with C₂ group; ^# P < 0.05 for comparisons of LX₁ group with I/R₁ group, P < 0.05 for comparisons of LX₂ group with I/R₂ group; ^Δ P < 0.05 for comparisons of LX₂ group with LX₁ group.

Figure 3

Transmission electron microscopy ((a)–(f)) and histological study ((g)–(l), He ×400) of cardiac tissues from rats of different groups at T₂ time point. (a) Group C₁ and (b) group C₂: regular myocardial myofilament arrangement, clear intercalated disk structure, normal mitochondrial morphology, and structure with endoplasmic reticulum embedded in myofilament matrix; (c) group I/R₁ and (d) group I/R₂: disordered arrangement of myofilament, a large number of myofilament fractured, intercalated disk structure unclear, and abnormal mitochondrial morphology (a high degree of swelling, membrane lysis, fuzzy ridge structure, formation of vacuoles, and endoplasmic reticulum vacuolization); (e) group LX₁ and (f) group LX₂: myofilament arrangement is a little sparse, the intercalated disk structure is slightly fuzzy, and slightly swollen mitochondria; (g) group C₁ and (h) group C₂: normal size cardiomyocyte, no hemorrhage, or neutrophil granulocyte infiltration; (i) group I/R₁ and (j) group I/R₂: Cardiomyocyte degeneration, hemorrhage, edema, and significant interstitial neutrophil granulocyte infiltration; (k) group LX₁ and (l) group LX₂: no significant cardiomyocyte degeneration, no obvious bleeding, slight edema between the myocardial fibers, and mild neutrophil granulocyte infiltration.

Figure 4

Immunohistochemical staining and IOD of Cx43 in cardiac tissues from rats of different groups at T₂. (a) Group C₁ and (b) group C₂: the Cx43 positive particle intensively and regularly was distributed and was clustered in the intercalated disk; (c) group I/R₁ and (d) group I/R₂: sparsely distributed Cx43 positive particles; (e) group LX₁ and (f) group LX₂: Slightly decreased Cx43 positive particles, mostly still clustered in the intercalated disk. (g) For IOD of Cx43. *P < 0.05 for comparisons of I/R₁ and LX₁ groups with C₁ group, P < 0.05 for comparisons of I/R₂ and LX₂ with C₂ group; ^# P < 0.05 for comparisons of LX₁ group with I/R₁ group, P < 0.05 for comparisons of LX₂ group with I/R₂ group; ^Δ P < 0.05 for comparisons of LX₂ group with LX₁ group.

Figure 5

mRNA levels of Cx43 and Na⁺-K⁺-ATPase of cardiac tissues from rats of different groups at T₂. Samples were collected right after I/R procedure. (a) Cx43 mRNA; (b) Na⁺-K⁺-ATPase mRNA. *P < 0.05 for comparisons of I/R₁ and LX₁ groups with C₁ group, P < 0.05 for comparisons of I/R₂ and LX₂ with C₂ group; ^# P < 0.05 for comparisons of LX₁ group with I/R₁ group, P < 0.05 for comparisons of LX₂ group with I/R₂ group; ^Δ P < 0.05 for comparisons of LX₂ group with LX₁ group.

Figure 6

Protein levels of α ₁, α ₂, and α ₃ and β ₁ subunits of Na⁺-K⁺-ATPase of cardiac tissues from rats of different groups at T₂. Samples were collected right after I/R procedure was over and western blot was performed as described in the section of Materials and Methods, and β-Actin was used as a loading control. Representative blots of three independent experiments were shown. (a) Western blotting: (A) α ₁ subunit of Na⁺-K⁺-ATPase; (B) α ₂ subunit; (C) α ₃ subunit; (D) β ₁ subunit. (b) Quantitative data of western blot: (A) α ₁ subunit of Na⁺-K⁺-ATPase; (B) α ₂ subunit; (C) α ₃ subunit; (D) β ₁ subunit. *P < 0.05 for comparisons of I/R₁ and LX₁ groups with C₁ group, P < 0.05 for comparisons of I/R₂ and LX₂ with C₂ group; ^# P < 0.05 for comparisons of LX₁ group with I/R₁ group, P < 0.05 for comparisons of LX₂ group with I/R₂ group; ^Δ P < 0.05 for comparisons of LX₂ group with LX₁ group.