Inhibition of NADPH Oxidase Mediates Protective Effect of Cardiotonic Pills against Rat Heart Ischemia/Reperfusion Injury

Abstract

Cardiotonic pill (CP) is a compound Chinese medicine currently used in China for treatment of ischemic angina pectoris. Our previous results indicated that a single dosing of CP pretreatment at 0.8 g/kg attenuates ischemia/reperfusion- (I/R-) induced myocardial injury and cardiac microcirculatory disturbance. The present study aimed to investigate the effect of CP at low dosage in a multiple dosing manner and to uncover the mechanism of antioxidative activity of CP. Male Sprague-Dawley rats were subjected to left anterior descending artery occlusion for 30 min followed by 60 min reperfusion. CP was administrated daily by gavage for six days at 0.1, 0.4, and 0.8 g/kg/day before I/R. Results showed that multiple dosing of CP at three doses significantly reduced I/R-induced myocardial injury, microcirculatory disturbance, and oxidative stress. CP dramatically inhibited I/R-induced nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase subunit gp91(phox) expression and p67(phox) and p47(phox) translocation from cytosol to cell membrane. Translocation of cytosolic subunits to membrane is required for the activation of NADPH oxidase. These data suggested that multiple dosing of CP at doses ranging from 0.1 to 0.8 g/kg/day reduced I/R-induced rat myocardial injury and microcirculatory disturbance, which was mediated by inhibition of NADPH oxidase activation.

Figure 1

CP pretreatment reduces I/R-induced infarct size. (a) Representative images of myocardial tissue slices stained with TTC in Sham group (a1), I/R group (a2), CP 0.1 + I/R group (a3), CP 0.4 + I/R group (a4), and CP 0.8 + I/R group (a5). White territory represents infarct area. (b) Quantitative results of infarct size in each group. Sham: Sham group; I/R: ischemia/Reperfusion group; CP 0.1 + I/R: CP pretreatment at 0.1 g/kg/day for 6 days plus I/R operation; CP 0.4 + I/R: CP pretreatment at 0.4 g/kg/day for 6 days plus I/R operation; CP 0.8 + I/R: CP pretreatment at 0.8 g/kg/day for 6 days plus I/R operation. The treatment of animals in each group is detailed in Section 2. Results are presented as mean ± SEM (n = 6).*P < 0.05 versus Sham group,^# P < 0.05 versus I/R group.

Figure 2

The effects of CP pretreatment on I/R-induced apoptosis and the expression of apoptosis related proteins. (a) Representative images of TUNEL stained myocardial sections in various groups. Apoptotic nuclei are indicated by TUNEL staining (green), and total nuclei are identified by Hoechst staining (blue). Arrowheads indicate normal nuclei. Arrows indicate TUNEL positive cells. Bar = 50 μm. (b) Representative photographs of immunohistochemistry staining for Caspase-3 in different groups. (c) Representative images of myocardial slices stained for Bax in different groups. (d) Representative slices of immunohistochemistry staining for Bcl-2 protein in various groups. Immunohistochemistry positive staining cells are shown in brown color. Bar = 100 μm. (e) to (h) Shown are statistical analysis of TUNEL positive nuclei, Caspase-3 expression, Bax expression, and Bcl-2 expression, respectively, in five different groups. 1, 2, 3, 4, and 5 which denotes Sham group, I/R group, CP 0.1 + I/R group, CP 0.4 + I/R group, and CP 0.8 + I/R group, respectively. Data are presented as mean ± SEM (n = 6). *P < 0.05 versus Sham group, ^# P < 0.05 versus I/R group.

Figure 3

Effect of CP pretreatment on RBC velocity and venular diameter. (a) Representative image of cardiac coronary microcirculation. Bar = 100 μm. (b) Time course of coronary venular RBC velocity in various groups. Results of RBC velocity are expressed as a percentage of baseline. (c) Quantitative results of coronary venular diameter. Results are presented as mean ± SEM (n = 6). *P < 0.05 versus Sham group, ^# P < 0.05 versus I/R group.

Figure 4

CP pretreatment prevents coronary venular albumin leakage. (a) Representative images of albumin leakage from coronary venules in Sham group (a1), I/R group (a2), CP 0.1 + I/R group (a3), CP 0.4 + I/R group (a4), and CP 0.8 + I/R group (a5). Bar = 100 µm. (b) Statistical results of albumin leakage expressed as ratio of fluorescence intensity in interstice to that in venular lumen. Results are presented as mean ± SEM (n = 6). *P < 0.05 versus Sham group, ^# P < 0.05 versus I/R group.

Figure 5

CP pretreatment improves MBF. (a) Representative MBF images acquired by Laser Doppler Perfusion Imager in Sham group (A), I/R group (B), CP 0.1 + I/R group (C), CP 0.4 + I/R group (D), and CP 0.8 + I/R group (E). For each group, images at baseline (1) and 0 min (2), 30 min (3), and 90 min of reperfusion (4) are shown, respectively. Color scale illustrates myocardial blood flow from dark blue (low flow) to red (high flow). (b) Time course of quantitative results of MBF in various groups. MBF is expressed as a percentage of baseline MBF. Data are presented as mean ± SEM (n = 6). *P < 0.05 versus Sham group, ^# P < 0.05 versus I/R group.

Figure 6

CP pretreatment diminishes I/R-induced alteration in myocardial tissue morphology. (a) Representative images of myocardial sections stained with HE. a: Disrupted myocardial fiber. b: Interstitial edema. c: Inflammatory cell infiltration. Bar = 100 μm. (b) Representative electron micrographs of myocardial capillary from various groups. d: Vascular endothelium. e: Caveolae. f: Interstitial edema. (c) Representative electron micrographs of myocardial fiber in different groups. g: Myofilament. h: Mitochondria. i: Corrupted mitochondria. Results are presented for Sham group (1), I/R group (2), CP 0.1 + I/R group (3), CP 0.4 + I/R group (4), and CP 0.8 + I/R group (5).

Figure 7

CP pretreatment alleviates I/R-induced oxidative stress. (a) Peroxide levels in peripheral blood are presented as fluorescence intensity of peroxide sensitive probe DHR in different groups. (b) MDA level of myocardial tissue in various groups. Data are presented as mean ± SEM (n = 6). *P < 0.05 versus Sham group, ^# P < 0.05 versus I/R group.

Figure 8

Effects of CP pretreatment on NF-κB activation and adhesion molecule expression after I/R. (a) Representative images of immunohistochemistry staining for IκBα. IκBα positive cells are revealed by DAB (brown). Bar = 100 μm. (b) Representative photographs of myocardial sections stained for P65 in different groups. P65 proteins are shown in brown color. Bar = 100 μm. (c) Statistical results of IκBα expression in five groups. (d) Quantitative analysis of P65 expression in various groups. 1, 2, 3, 4, and 5 denote Sham, I/R, CP 0.1 + I/R, CP 0.4 + I/R, and CP 0.8 + I/R group, respectively. (e) and (f) The expression of adhesion molecules CD18 and CD11b on rat neutrophils, respectively. The expression of adhesion molecules is presented as fluorescence intensity of FITC. Results are presented as mean ± SEM (n = 6). *P < 0.05 versus Sham group, ^# P < 0.05 versus I/R group.

Figure 9

The effect of CP pretreatment on the expression of antioxidative enzymes after I/R. (a) Myocardial SOD expression in different groups. (b) The level of myocardial CAT in various groups. (c) GSH expression of myocardial tissues in five groups. Data are presented as mean ± SEM (n = 6). *P < 0.05 versus Sham group, ^# P < 0.05 versus I/R group.

Figure 10

CP pretreatment inhibits NADPH oxidase activation induced by I/R. (a) Representative Western blot bands of gp91^phox, p67^phox, p47^phox and p40^phox on cell membrane. (b) Quantification of cell membrane expression of gp91^phox, p67^phox, p47^phox and p40^phox. All membrane protein intensities were normalized to GAPDH. (c) Representative Western blot bands of NADPH oxidase organizer subunits p67^phox and p47^phox and modulator p40^phox in cytoplasm. (d) Statistical results of cytoplasm expression of p67^phox, p47^phox and p40^phox. All band intensities were calculated based on the results from 3 independent experiments. All cytosolic protein intensities were normalized to β-tubulin. Results are presented as mean ± SEM (n = 3). *P < 0.05 versus Sham group, ^# P < 0.05 versus I/R group.