N-acetylcysteine reduces oxidative stress, nuclear factor‑κB activity and cardiomyocyte apoptosis in heart failure

Abstract

The roles of oxidative stress on nuclear factor (NF)‑κB activity and cardiomyocyte apoptosis during heart failure were examined using the antioxidant N‑acetylcysteine (NAC). Heart failure was established in Japanese white rabbits with intravenous injections of doxorubicin, with ten rabbits serving as a control group. Of the rabbits with heart failure, 12 were not treated (HF group) and 13 received NAC (NAC group). Cardiac function was assessed using echocardiography and hemodynamic analysis. Myocardial cell apoptosis, apoptosis‑related protein expression, NF‑κBp65 expression and activity, total anti‑oxidative capacity (tAOC), 8‑iso‑prostaglandin F2α (8‑iso‑PGF2α) expression and glutathione (GSH) expression levels were determined. In the HF group, reduced tAOC, GSH levels and Bcl‑2/Bax ratios as well as increased 8‑iso‑PGF2α levels and apoptosis were observed (all P<0.05), which were effects that were attenuated by the treatment with NAC. NF‑κBp65 and iNOS levels were significantly higher and the P‑IκB‑α levels were significantly lower in the HF group; expression of all three proteins returned to pre‑HF levels following treatment with NAC. Myocardial cell apoptosis was positively correlated with left ventricular end-diastolic pressure (LVEDP), NF‑κBp65 expression and 8‑iso‑PGF2α levels, but negatively correlated with the maximal and minimal rates of increase in left ventricular pressure (+dp/dtmax and ‑dp/dtmin, respectively) and the Bcl‑2/Bax ratio (all P<0.001). The 8‑iso‑PGF2α levels were positively correlated with LVEDP and negatively correlated with +dp/dtmax and ‑dp/dtmin (all P<0.001). The present study demonstrated that NAC increased the antioxidant capacity, decreased the NF‑κB activation and reduced myocardial cell apoptosis in an in vivo heart failure model.

Figure 1

The correlation between 8-iso-PGF2α levels and cardiac function. The correlations were tested by determining Pearson correlation coefficients. 8-iso-PGF2α, 8-iso-prostaglandin F2α; LVEDP, left ventricular end-diastolic pressure; +dp/dtmax, maximal rate of rise of left ventricular pressure; −dp/dtmin, minimal rate of rise of left ventricular pressure.

Figure 2

Effects of NAC on myocardial cell apoptosis in heart failure. (A) The apoptotic index was determined using the TUNEL assay. Pair-wise multiple comparisons between groups were determined using Bonferroni’s test with α=0.017 adjustment. ^*P<0.05 indicates a statistically significant difference between the indicated group and the control group; ^†P<0.05 indicates a statistically significant difference between the indicated group and the HF group. (B) Representative images of the TUNEL analysis from each group are demonstrated (magnification, ×400). NAC, N-acetylcysteine; HF group, untreated heart failure group; TUNEL, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 3

Effects of NAC on apoptosis-associated protein expression in heart failure. (A) Bcl-2, (B) Bax, (C) Bcl-2/Bax ratio and (D) NF-κBp65 protein expression was determined by immunohistochemical analysis. The mean OD was determined using an HMIAS-2000 image analysis system; the higher OD values indicate lower protein expression. P-values are based on analysis of variance and pair-wise multiple comparisons between groups were determined using Bonferroni’s test with α= 0.017 adjustment. ^†P<0.05 indicates a significant difference between the indicated group and the control group; ^‡P<0.05 indicates a significant difference between the indicated group and the HF group. (E) Representative images of Bcl-2 (top panels), Bax (middle panels) and NF-κBp65 (bottom panels) protein expression from each group are demonstrated (magnification, ×400). NAC, N-acetylcysteine; HF group, untreated heart failure group; NF-κB, nuclear factor κB; OD, optical density.

Figure 4

Effects of NAC on NF-κBp65 expression and activity. Relative (A) NF-κBp65, (B) iNOS and (C) P-IκB expression was determined using western blot analysis following normalization to β-actin. (D) Representative blots are demonstrated. Pair-wise multiple comparisons between groups were determined using Bonferroni’s test with α=0.017 adjustment. ^*P<0.05 indicates a statistically significant difference between the indicated group and the control group; ^†P<0.05 indicates a statistically significant difference between the indicated group and the HF group. NAC, N-acetylcysteine; HF group, untreated heart failure group; NF-κB, nuclear factor κB; iNOS, inducible nitric oxide synthase.

Figure 5

Correlation of myocardial cell apoptosis with cardiac function and expression of NF-κBp65 and 8-iso-PGF2α. The correlations were tested by determining Pearson correlation coefficients. The correlations of myocardial cell apoptosis index and (A) LVEDP; (B) +dp/dtmax; (C) −dp/dtmin; (D) NF-κBp65; (E) ratio of (Bcl-2/Bax)⁻¹; (F) 8-iso-PGF2α in serum; and (G) 8-iso-PGF2α in myocardium. 8-iso-PGF2α, 8-iso-prostaglandin F2α; LVEDP, left ventricular end-diastolic pressure; +dp/dtmax, maximal rate of rise of left ventricular pressure; −dp/dtmin, minimal rate of rise of left ventricular pressure.