20(S)-Ginsenoside Rg2 attenuates myocardial ischemia/reperfusion injury by reducing oxidative stress and inflammation: role of SIRT1

Abstract

Previously we demonstrated that 20(S)-ginsenoside Rg2 protects cardiomyocytes from H₂O₂-induced injury by inhibiting reactive oxygen species (ROS) production, increasing intracellular levels of antioxidants and attenuating apoptosis. We explored the protective effect of 20(S)-ginsenoside Rg2 on myocardial ischemia/reperfusion (MI/R) injury and to clarify its potential mechanism of action. Rats were exposed to 20(S)-ginsenoside Rg2 in the presence/absence of the silent information regulator SIRT(1) inhibitor EX527 and then subjected to MI/R. 20(S)-Ginsenoside Rg2 conferred a cardioprotective effect by improving post-ischemic cardiac function, decreasing infarct size, reducing the apoptotic index, diminishing expression of creatine kinase-MB, aspartate aminotransferase and lactate dehydrogenase in serum, upregulating expression of SIRT1, B-cell lymphoma-2, procaspase-3 and procaspase-9, and downregulating expression of Bax and acetyl (Ac)-p53. Pretreatment with 20(S)-ginsenoside Rg2 also resulted in reduced myocardial superoxide generation, gp91^phox expression, malondialdehyde content, cardiac pro-inflammatory markers and increased myocardial activities of superoxide dismutase, catalase and glutathione peroxidase. These results suggested that MI/R-induced oxidative stress and inflammation were attenuated significantly by 20(S)-ginsenoside Rg2. However, these protective effects were blocked by EX527, indicating that SIRT1 signaling may be involved in the pharmacological action of 20(S)-ginsenoside Rg2. Our results demonstrated that 20(S)-ginsenoside Rg2 attenuates MI/R injury by reducing oxidative stress and inflammatory responses via SIRT1 signaling.

Fig. 1. Chemical structure of 20(S)-ginsenoside Rg2.

Fig. 2. Effects of 20(S)-ginsenoside Rg2 on cardiac function in MI/R-induced injury. Echocardiographic measurement was carried out after 72 h of reperfusion. Representative M-mode images are shown. Evaluation of left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) are shown. Data are the mean ± S.D. ^##P < 0.01 vs. sham group; *P < 0.05, **P < 0.01 vs. MI/R group.

Fig. 3. Effects of 20(S)-ginsenoside Rg2 on the apoptotic index, infarct size, and serum activities of CK-MB, AST and LDH in MI/R-induced injury. Apoptotic index, infarct size, and serum levels of creatine-MB (CK-MB), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured after 6 h of reperfusion. (A) Representative images of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue fluorescence shows nuclei of total cardiomyocytes (scale bar: 100 μm). (B) Representative photographs of heart sections. Pale color indicates an ischemic region and dark-red represents a non-ischemic region. The infarct size was calculated as a percentage of the ventricular mass: weight of ischemic zone/total weight of ventricle × 100%. The size of the myocardial infarct is expressed as a percentage of the ventricle area. (C) Serum activities of CK-MB, AST and LDH after 6 h of reperfusion are shown. Data are the mean ± S.D. ^##P < 0.01 vs. sham group; *P < 0.05, **P < 0.01 vs. MI/R group.

Fig. 4. Effects of 20(S)-ginsenoside Rg2 on oxidative stress in MI/R-induced injury. The level of cardiac oxidative stress was measured after 6 h of reperfusion. (A) Cardiac superoxide generation and (B) gp91^phox expression were evaluated. (C) MDA content and the activities of SOD, CAT and GSH-PX in the myocardium were measured. Data are the mean ± S.D. ^##P < 0.01 vs. sham group; *P < 0.05, **P < 0.01 vs. MI/R group.

Fig. 5. Effects of 20(S)-ginsenoside Rg2 on the inflammatory response in MI/R-induced injury. (A) mRNA levels and (B) activities of IL-1β, IL-6 and TNF-α in the myocardium were measured after 6 h of reperfusion. Data are the mean ± S.D. ^##P < 0.01 vs. sham group; *P < 0.05, **P < 0.01 vs. MI/R group.

Fig. 6. Effects of 20(S)-ginsenoside Rg2 on expression of SIRT1, Ac-p53, procaspase-3, procaspase-9, Bax and Bcl-2 in MI/R-induced injury. SIRT1-related signaling and apoptosis-related protein levels were measured after 6 h of reperfusion. Representative blots of expression of SIRT1, Ac-p53, procaspase-3, procaspase-9, Bax and Bcl-2 are shown. Expression of the indicated proteins are shown as the mean ± S.D. ^##P < 0.01 vs. sham group; *P < 0.05, **P < 0.01 vs. MI/R group.

Fig. 7. Effects of 20(S)-ginsenoside Rg2 and EX527 on cardiac function in MI/R-induced injury. Echocardiographic measurement was carried out after 72 h of reperfusion. Representative M-mode images are shown. Evaluation of LVIDd, LVIDs, LVEF and LVFS is shown. Images of the MI/R group and MI/R + Rg2 group are shared with Fig. 2. Data are the mean ± S.D. ^#P < 0.05, ^##P < 0.01 vs. MI/R group and *P < 0.05, **P < 0.01 vs. MI/R + Rg2 group.

Fig. 8. Effects of 20(S)-ginsenoside Rg2 and EX527 on the apoptotic index, infarct size, and serum activities of CK-MB, AST and LDH in MI/R-induced injury. Apoptotic index, infarct size, and serum activities of CK-MB, AST and LDH were measured after 6 h of reperfusion. (A) Representative images of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue fluorescence shows nuclei of total cardiomyocytes (scale bar: 100 μm). (B) The infarct size was calculated as a percentage of the ventricular mass: weight of ischemic zone/total weight of ventricle × 100%. The size of the myocardial infarct is expressed as a percentage of the ventricle area. (C) The serum activities of CK-MB, AST and LDH after 6 h reperfusion are shown. Images from the MI/R group and MI/R + Rg2 group are shared with Fig. 3. Data are the mean ± S.D. ^#P < 0.05 vs. MI/R group and *P < 0.05, **P < 0.01 vs. MI/R + Rg2 group.

Fig. 9. Effects of 20(S)-ginsenoside Rg2 and EX527 on oxidative stress in MI/R-induced injury. Cardiac oxidative stress level was measured after 6 h of reperfusion. (A) Cardiac superoxide generation and (B) gp91^phox expression were evaluated. (C) MDA content and activities of SOD, CAT and GSH-PX in the myocardium were measured. Data are the mean ± S.D. ^#P < 0.05 vs. MI/R group and *P < 0.05, **P < 0.01 vs. MI/R + Rg2 group.

Fig. 10. Effects of 20(S)-ginsenoside Rg2 and EX527 on the inflammatory response in MI/R-induced injury. (A) mRNA levels and (B) activities of IL-1β, IL-6 and TNF-α in the myocardium were measured after 6 h of reperfusion. Data are the mean ± S.D. ^#P < 0.05, ^##P < 0.01 vs. MI/R group and *P < 0.05 vs. MI/R + Rg2 group.

Fig. 11. Effects of 20(S)-ginsenoside Rg2 and EX527 on expression of SIRT1, Ac-p53, procaspase-3, procaspase-9, Bax and Bcl-2 in MI/R-induced injury. SIRT1-related signaling and apoptosis-related proteins were measured after 6 h of reperfusion. Representative blots of expression of SIRT1, Ac-p53, procaspase-3, procaspase-9, Bax, and Bcl-2 and their evaluation are shown. Data are the mean ± S.D. ^#P < 0.05, ^##P < 0.01 vs. MI/R group and *P < 0.05, **P < 0.01 vs. MI/R + Rg2 group.

Fig. 12. Proposed cardioprotective signaling pathway of 20(S)-ginsenoside Rg2.