Salvianolic Acid B Improves Postresuscitation Myocardial and Cerebral Outcomes in a Murine Model of Cardiac Arrest: Involvement of Nrf2 Signaling Pathway

Figures

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  Figure 1. (a) Schematic diagram indicating the time course of the experimental procedure. (b) Flow chart of the experimental groups. KCl: potassium chloride; CA: cardiac arrest; CPR: cardiopulmonary resuscitation; Echo: echocardiography; TEM: transmission electron microscopy; Vehicle: mice subjected to CA/CPR treated with normal saline; Sal B: salvianolic acid B; n: number of mice; ROSC: return of spontaneous circulation; h: hours; min: minutes; EF: ejection fraction; FS: fractional shortening; DHE: dihydroethidium; H&E: hematoxylin-eosin; TUNEL: terminal dUTP nick-end labeling; NADPH: nicotinamide adenine dinucleotide phosphate.

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  Figure 2. Salvianolic acid B treatment improved cardiac dysfunction and increased survival following cardiac arrest and cardiopulmonary resuscitation (CA/CPR). (a) Cardiac function was evaluated by echocardiography 3 hours following resuscitation. (b, c) Quantitative analysis of LVEF and LVFS in the indicated groups (n = 8 per group). Statistical significance was determined using one-way ANOVA followed by post-hoc Bonferroni correction.##p < 0.01 vs. sham-operated controls; ∗∗p < 0.01 vs. vehicle group. (d) Kaplan-Meier survival curve of WT and Sal B-treated WT mice after successful CA/CPR within 72 hours (n = 20 per group). The number of surviving animals is shown to the right in brackets. ∗p < 0.05 vs. Vehicle by log-rank test. WT: wild type; LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional shortening.

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  Figure 3. Salvianolic acid B treatment improved cardiovascular hemodynamics following cardiac arrest. Hemodynamic parameters of vehicle and salvianolic acid B-treated groups from baseline to 3 hours after cardiac arrest and cardiopulmonary resuscitation (CA/CPR) were determined by a Millar catheter. (a) Maximum left ventricular systolic pressure (LVSP); (b) dp/dtmax; (c) dp/dtmin; (d) heart rate; (e) stroke volume; (f) cardiac output (n = 15 per group). ##p < 0.01 vs. sham-operated controls; ∗∗p < 0.01 vs. vehicle group. Statistical significance was determined using repeated-measures two-way ANOVA followed by Bonferroni comparison post-hoc test. Mins: minutes; bpm: beats per minute; ROSC: return of spontaneous circulation.

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  Figure 4. Salvianolic acid B treatment inhibited myocytolysis and myocardial apoptosis, and preserved mitochondrial morphology when challenged with cardiac arrest and cardiopulmonary resuscitation (CA/CPR). (a) H&E staining revealed less myocytolysis and weaving in the myocardium of Sal B-treated mice (top panel, n = 6 per group). Transmission electron microscopy was performed to observe the ultrastructure of post-CA myocardial tissues. CA/CPR damaged the myocardial ultrastructure with mitochondrial swelling, outer membrane rupture, and disarrangement of the inner membrane cristae, whereas these changes were reversed by administration of Sal B (bottom panel, n = 6 per group). (b) The apoptotic cell death 3 hours post-ROSC was determined using TUNEL. TUNEL (green), apoptotic nuclei; DAPI (blue), and total nuclei. (c) The apoptosis index was quantified and compared between vehicle- and Sal B-treated groups (n = 5 − 6 per group). (d) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 from the indicated groups (n = 6 per group). (e) The ratio of Bcl-2/Bax was determined from the Bcl-2 and Bax protein band densities and converted to fold change relative to the sham-operated group. (f)…

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  Figure 5. Salvianolic acid B treatment suppressed the superoxide production in the heart following cardiac arrest and cardiopulmonary resuscitation (CA/CPR). (a) ROS steady-state levels in the myocardium were determined by in situ detection of superoxide with dihydroethidium fluorescence among different groups (n = 3 − 6 per group). (b) NADPH oxidase activity was assessed using a lucigenin assay 3 hours following CA/CPR (n = 5 − 6 per group). (c, d) gp91phox expression was determined using Western blot analysis. GAPDH was used as a loading control and results were converted to the fold change relative to the sham-operated group (n = 6 per group). Statistical significance was determined using one-way ANOVA followed by post-hoc Bonferroni correction. ##p < 0.01 vs. sham-operated controls; ∗p < 0.05 or ∗∗p < 0.01 vs. vehicle group. ROS: reactive oxygen species; DHE: dihydroethidium; NADPH: nicotinamide adenine dinucleotide phosphate.

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  Figure 6. Salvianolic acid B treatment activated the Nrf2 antioxidative pathway after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). (a) Expression of Keap1, nuclear and cytosolic Nrf2, HO-1, and NQO1 in the myocardium 3 hours following CA/CPR with or without Sal B treatment. (b–f) Western blot analysis and quantification of Keap1, nuclear and cytosolic Nrf2, HO-1, and NQO1 in the indicated groups. Histone H3 and GAPDH served as loading controls and results were converted to fold change relative to the sham-operated group (n = 6 per group). #p < 0.05 or ##p < 0.01 vs. sham-operated controls; ∗∗p < 0.01 vs. vehicle group.

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  Figure 7. Knockdown of Nrf2 abolished the antioxidative and antiapoptotic effects of Sal B treatment in vitro. (a, b) Protein expression of Nrf2 was assessed using immunoblot 72 hours after Nrf2 siRNA or NC siRNA transfection in H9c2 cardiomyocytes. ##p < 0.01 vs. NC siRNA. (c) Representative fluorescent images of DHE and TUNEL staining in the presence of Nrf2 siRNA or NC siRNA with or without Sal B treatment (10 μM) in H9c2 cardiomyocytes after H/R insult. (d) Quantification of the number of TUNEL-positive cells in the indicated groups. (e, f) gp91phox expression was determined using Western blot analysis. (g) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 from the indicated groups. (h) The ratio of Bcl-2/Bax was determined from the Bcl-2 and Bax protein band densities and converted to fold change relative to Blank. (i) Quantitative value of protein expression levels of cleaved caspase-3. GAPDH served as the internal control and results were converted to fold change relative to Blank. #p < 0.05 or ##p < 0.01 vs. Blank; ∗p < 0.05 or ∗∗p < 0.01 vs. NC+Sal B. NC: negative control siRNA; H/R: hypoxia/reoxygenation.

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  Figure 8. The neurological function score was assessed in the surviving mice within 72 hours at indicated time points after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). (a) Six hours after ROSC. (b) 24 hours after ROSC. (c) 48 hours after ROSC. (d) 72 hours after ROSC (n = 20 per group). Each circle indicates an individual animal scored after resuscitation. Dead mice, indicated by score 0, were excluded from the statistical analysis. ∗p < 0.05 or ∗∗p < 0.01 between the two groups (unpaired t-test). ROSC: return of spontaneous circulation.