Water-soluble acacetin prodrug confers significant cardioprotection against ischemia/reperfusion injury

Figures

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  Figure 1. HPLC result for in vivo conversion of the water-soluble acacetin prodrug to the parent compound acacetin in a rat after intravenous administration. (A) Acacetin (2 μg/mL) in HPLC mobile phase. (B) HPLC with blank rat plasma (before administration of acacetin prodrug). (C) HPLC with rat plasma at 5 min after intravenous administration of acacetin prodrug (250 μg/kg/min). (D) HPLC with rat plasma at 40 min after intravenous administration of acacetin prodrug.

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  Figure 2. Anti-arrhythmic effect of prodrug in anesthetized rats with ischemia/reperfusion. (A) ECG in a rat treated with vehicle, showing ventricular fibrillation (VF) after reperfusion for 1 min. (B) ECG in a rat treated with bolus acacetin prodrug (10 mg/kg i.v. for 5 min) before ischemia, showing a brief ventricular tachycardia (VT) followed by normal rhythm after reperfusion. (C) Reduction of the ventricular arrhythmia score by acacetin prodrug. (D) Decrease of ventricular arrhythmia duration by acacetin prodrug. (E) Reduction of ventricular fibrillation (but not PVBs, premature ventricular beats) by acacetin prodrug in a concentration-dependent manner (n = 10–11 experiments, *P < 0.05, **P < 0.01 vs. vehicle).

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  Figure 3. Intravenous administration of prodrug improves the impaired heart rate and ventricular contractile function induced by ischemia/reperfusion injury in anesthetized rats. (A) Left ventricular slices with the TTC-staining, showing a reduced infarct area in animal treated with acacetin prodrug (10 mg/kg intravenous administration). (B) Acacetin prodrug antagonizes the reduction of mean femoral artery blood pressure by ischemia/reperfusion injury. (C) Acacetin prodrug slightly antagonizes the heart rate reduction by ischemia/reperfusion injury. (D) Acacetin prodrug improves the impaired systolic pressure of left ventricle (LVSP). (E) Acacetin prodrug improves the impaired developed pressure of left ventricle (LVDP). (F) Acacetin prodrug antagonizes the reduced left ventricular +dP/dT (n = 8 experiments, **P < 0.01, ***P < 0.001 vs. vehicle).

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  Figure 4. Effects of acacetin on myocardial injury by ischemia/reperfusion in ex vivo rat hearts. (A) The scheme of ischemia/reperfusion protocol in perfused ex vivo rat hearts with 30 min ischemia, followed by 2 h reperfusion. (B) Left ventricular slices of the heart with Evans blue/TCC-staining in a vehicle control heart and a heart treated with 3 μM acacetin. (C) Mean percentage of risk area and infarct area of left ventricle with ischemia/reperfusion injury, showing reduction of infarct area in hearts treated with 0.3, 1 or 3 μM in a concentration-dependent manner (n = 10–12 experiments, **P < 0.01, ***P < 0.001 vs. vehicle; #P < 0.05, ##P < 0.01 vs. 0.3 μM acacetin).

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  Figure 5. Effects of acacetin on heart rate and ventricular pressure in ex vivo rat hearts subjected to ischemia/reperfusion. (A) Percentage values of heart rate during control, ischemia and reperfusion in isolated rat hearts treated with vehicle, 0.3, 1 or 3 μM acacetin. (B) Percentage values of left ventricular pressure (LVP) during control, ischemia and reperfusion in isolated rat hearts treated with vehicle, 0.3, 1 or 3 μM acacetin (n = 9–10 for each treatment, ##P < 0.01 vs control; *P < 0.05, **P < 0.01 vs. vehicle). (C) Percentage values of +dP/dT of left ventricular pressure during control, ischemia and reperfusion in isolated rat hearts treated with vehicle, 0.3, 1 or 3 μM acacetin (n = 9–10 for each treatment, ##P < 0.01 vs control; *P < 0.05, **P < 0.01 vs. vehicle).

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  Figure 6. Effects of acacetin on endogenous anti-oxidant and inflammation response in ex vivo rat hearts with ischemia/reperfusion injury. (A) Western blots of superoxide dismutase 2 (SOD-2) and thioredoxin (Trx). (B) Mean relative levels of SOD-2 and Trx. (C) Western blots of Toll like receptor-4 (TLR-4), interleukin-6 (IL-6), and tumour necrosis factor alpha (TNFα) in rat left ventricles with ischemia/reperfusion injury in the absence and the presence of 0.3, 1 and 3 μM acacetin. (D) Mean relative levels of TLR-4, IL-6 and TNFα, showing significant reduction of TLR-4, IL-6 and TNFα in hearts treated with acacetin (0.3, 1 and 3 μM). n = 4 experiments, #P < 0.05, ##P < 0.01 vs. Sham; *P < 0.05, **P < 0.01 vs. vehicle.

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  Figure 7. Effects of acacetin on myocardial apoptosis induced by ischemia/reperfusion in ex vivo rat hearts. (A) Western blots for expression of the pro-apoptotic proteins cleaved caspase-3, Bax and the anti-apoptotic protein Bcl-2 in the absence and the presence of 0.3, 1 and 3 μM acacetin. (B) Mean relative protein levels. (C) Ratio of Bcl-2/Bax. n = 4 experiments, #P < 0.05, ##P < 0.01 vs. sham; *P < 0.05, **P < 0.01 vs. vehicle. (D) TUNEL-staining indicates significant myocyte apoptosis with dark-brown nuclei; acacetin decreases apoptosis. (E) Mean percentage of nuclei with TUNEL-staining in hearts treated with vehicle or 3 μM acacetin (n = 5 experiments, ##P < 0.01, vs. sham; **P < 0.01 vs. vehicle).

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  Figure 8. Schematic illustration shows the main cascade of molecules in myocardial ischemia/reperfusion injury. Myocardial ischemia/reperfusion (oxidative stress) triggers ROS production and inflammation response (release of pro-inflammatory cytokines TLR-4, IL-6, TNFα, which induce ventricular arrhythmias) and decreases the endogenous anti-oxidants SOD and thioredoxin (Trx). Over-production of ROS and inflammation cytokines would initiate cardiomyocyte apoptosis cell death and cause myocardial infarct. Intravenous application of acacetin prodrug (bioconversion to acacetin) prevents the ischemia/reperfusion-induced reduction of the endogenous anti-oxidants and also reduces inflammation response, and thereby inhibits ventricular arrhythmias and myocardial apoptosis and significantly reduces infarct size.