Cardioprotection of Ginkgolide B on Myocardial Ischemia/Reperfusion-Induced Inflammatory Injury via Regulation of A20-NF-κB Pathway

Abstract

Inflammation urges most of the characteristics of plaques involved in the pathogenesis of myocardial ischemia/reperfusion injury (MI/RI). In addition, inflammatory signaling pathways not only mediate the properties of plaques that precipitate ischemia/reperfusion (I/R) but also influence the clinical consequences of the post-infarction remodeling and heart failure. Here, we studied whether Ginkgolide B (GB), an effective anti-inflammatory monomer, improved MI/RI via suppression of inflammation. Left anterior descending (LAD) coronary artery induced ischemia/reperfusion (I/R) of rats or A20 silencing mice, as well as hypoxia/reoxygenation (H/R) induced damages of primary cultured rat neonatal ventricular myocytes or A20 silencing ventricular myocytes, respectively, served as MI/RI model in vivo and in vitro to discuss the anti-I/R injury properties of GB. We found that GB significantly alleviated the symptoms of MI/RI evidently by reducing infarct size, preventing ultrastructural changes of myocardium, depressing Polymorphonuclears (PMNs) infiltration, lessening histopathological damage and suppressing the excessive inflammation. Further study demonstrated that GB remarkably inhibited NF-κB p65 subunit translocation, IκB-α phosphorylation, IKK-β activity, as well as the downstream inflammatory cytokines and proteins expressions via zinc finger protein A20. In conclusion, GB could alleviate MI/RI-induced inflammatory response through A20-NF-κB signal pathway, which may give us new insights into the preventive strategies for MI/RI disease.

Keywords: A20; Ginkgolide B; Inflammation; Myocardial ischemia/reperfusion injury; NF-κB.

Figure 1

Chemical structure of GB.

Figure 2

Effects of GB on infarct size in MI/RI rats model. (A) The experimental procedures of in vivo MI/RI rats model. (B) Treatment with GB significantly reduced the infarct size in MI/RI rats model. Data were expressed as mean ± S.D. (n = 8). ^##P < 0.01, I/R group vs. control group; *P < 0.05, **P < 0.01, 8, 16, 32 mg/kg GB groups vs. I/R group.

Figure 3

Effects of GB on the ultrastructure of myocardial tissue, histopathological changes, histopathological scores, PMNs counting, MPO activity and ICAM-1, VCAM-1, iNOS expressions in MI/RI rats model. (A1–5) Representative transmission electron microscopy (TEM) observation of myocardial tissue injury for control group (A1), I/R group (A2), I/R + 8 mg/kg GB group (A3), I/R + 16 mg/kg GB group (A4), I/R + 32 mg/kg GB group (A5). (B1-5) Representative light microscopic appearance of rat myocardial histopathological morphology (HE staining; original magnification × 200) for control group (B1), I/R group (B2), I/R + 8 mg/kg GB group (B3), I/R + 16 mg/kg GB group (B4), I/R + 32 mg/kg GB group (B5). (C) Effect of GB on histopathological scores, (D) effect of GB on myocardial PMNs counting, (E) effect of GB on MPO activity, effect of GB on expressions of ICAM-1, VCAM-1, iNOS (F) and effect of GB on expression of A20 (G). The location of the histological images were taken in three random fields of infarcted area. Data were expressed as mean ± S.D. (n = 8). ^##P < 0.01, I/R group vs. control group; *P < 0.05, **P < 0.01, 8, 16, 32 mg/kg GB groups vs. I/R group.

Figure 4

Effects of GB on cell viability and the expressions of ICAM-1, VCAM-1, iNOS, NF-κB p65, p-IκB-α, IKK-β by Western blot in H/R ventricular myocytes model. (A) The experimental procedures of in vitro H/R ventricular myocytes model. (B) GB significantly increased the cell viability after H/R procedure. (C) GB decreased the expression of ICAM-1. (D) GB decreased the expression of VCAM-1. (E) GB decreased the expression of iNOS. GB blocked the translocation of NF-κB p65 from cytosolic (F) to nuclear (G). (H) GB down-regulated the expression of p-IκB-α. (I) GB decreased the expression of IKK-β. (J) GB increased the expression of A20. The NF-κB p65 protein levels were assayed separately in cytosolic (F) and nuclear (G) extracts. Results were expressed as Protein/reference protein ratio. Data were expressed as mean ± S.D. of three independent experiments. ^##P < 0.01 H/R group vs. control group; *P < 0.05, **P < 0.01, 1, 10, 100 μM GB groups vs. I/R group.

Figure 5

Effects of GB on infarct size in A20 gene silencing MI/RI mice model. (A) The experimental procedures of in vivo A20 gene silencing MI/RI mice model. (B) Treatment with GB significantly reduced the infarct size in A20 gene silencing MI/RI mice model. Data were expressed as mean ± S.D. (n = 8). ^##P < 0.01, I/R group vs. control group; *P < 0.05, **P < 0.01, 12, 24, 48 mg/kg GB groups vs. I/R group.

Figure 6

Effects of GB on the ultrastructure of myocardial tissue, histopathological changes, histopathological scores, PMNs counting, MPO activity and ICAM-1, VCAM-1, iNOS expressions in A20 gene silencing MI/RI mice model. (A1-5) Representative transmission electron microscopy (TEM) observation of myocardial tissue injury for control group (A1), I/R group (A2), I/R + 12 mg/kg GB group (A3), I/R + 24 mg/kg GB group (A4), I/R + 48 mg/kg GB group (A5). (B1-5) Representative light microscopic appearance of rat myocardial histopathological morphology (HE staining; original magnification × 200) for control group (B1), I/R group (B2), I/R + 12 mg/kg GB group (B3), I/R + 24 mg/kg GB group (B4), I/R + 48 mg/kg GB group (B5). (C) Effect of GB on histopathological scores, (D) effect of GB on myocardial PMNs counting, (E) effect of GB on MPO activity, effect of GB on expressions of ICAM-1, VCAM-1, iNOS (F) and effect of GB on expression of A20 (G). The location of the histological images were taken in three random fields of infarcted area. Data were expressed as mean ± S.D. (n = 8). ^##P < 0.01 I/R group vs. control group; *P < 0.05, **P < 0.01, 12, 24, 48 mg/kg GB groups vs. I/R group.

Figure 7

Effects of GB on cell viability (A) and the expressions of (B) ICAM-1, (C) VCAM-1, (D) iNOS, (E) cytoplasm NF-κB p65, (F) nucleus NF-κB p65, (G) p-IκB-α, (H) IKK-β and (I) A20 by Western blot in A20 gene silence H/R ventricular myocytes model. Results were expressed as Protein/reference protein ratio. Data were expressed as mean ± S.D. of three independent experiments. ^##P < 0.01, H/R group vs. control group; *P < 0.05, **P < 0.01, 1, 10, 100 μM GB groups vs. I/R group.

Figure 8

Schematic diagram describing the mechanism in the inhibitory effect of GB on H/R induced ventricular myocytes inflammatory injury. GB could alleviate MI/RI-induced inflammatory injury via up-regulating A20 and inhibiting IKK/IκB/NF-κB signal pathway.