Protective effect of Ginkgo biloba leaves extract, EGb761, on myocardium injury in ischemia reperfusion rats via regulation of TLR-4/NF-κB signaling pathway

Abstract

Beneficial actions of EGb 761 against ischemia/reperfusion (I/R) injury in lung, brain and renal ischemia have been described. However, the relationship between EGb 761 and signal molecules in myocardial ischemia reperfusion has not been well elucidated. In this study, we investigated the effects and mechanism of EGb 761 preconditioning on anti-myocardial I/R injuries in vivo. Meanwhile, their potential anti-oxidative stress and anti-inflammation effect were assessed. Hemodynamic parameters were monitored as left ventricular systolic pressure, LV end-diastolic pressure and maximal rate of increase and decrease of left ventricular pressure (dP/dtmax). The oxidative stress indicators and inflammatory factors were also evaluated. Western blot method was used for analysis of toll-like receptor 4 (TLR4), p-TLR4, nuclear factor-κB (NF-κB), p-NF-κB p65, Bax and Bcl-2 protein expressions. EGb 761 significantly improved cardiac function, decreased levels of creatine kinase, aspartate aminotransferase and lactate dehydrogenase. EGb 761 also restrained the oxidative stress related to myocardial ischemia injury as evidenced by decreased malondialdehyde, superoxide dismutase, catalase, glutathione-peroxidase, glutathione reductase activity. Meanwhile, the inflammatory cascade was inhibited as evidenced by decreased cytokines such as tumor necrosis factor-α, interleukin-6 and interleukin-1β. Our results still showed that EGb 761 pretreatment significantly decrease the level of cleaved Bax, and increase the level of Bcl-2 in rats subjected to I/R injury. Simultaneously, the expressions of myocardial TLR4 and NF-κB were significantly decreased. It can be concluded that EGb 761 pretreatment was protected against myocardium I/R injury by decreasing oxidative stress, repressing inflammatory cascade in vivo and inhibiting TLR4/NF-κB pathway.

Keywords: EGb761; TLR-4/NF-κB pathway; myocardial ischemia.

Figure 1. Representative UPLC- PDA -TOFMS chromatograms of EGb 761 in the negative ion mode

Figure 2. Pre-treatment with EGb 761 improve cardiac contractibility, attenuate hypercontraction and reduce diastolic pressure

Values were expressed as means ± SDs. ## P <0.01 compared with sham group. *P < 0.05 and ** P < 0.01 compared with IR group (n=10).

Figure 3. EGb 761 enhanced the antioxidant enzymes activities

Values were expressed as means ± SDs. ## P <0.01 compared with sham group. *P < 0.05 and ** P < 0.01 compared with IR group (n=10).

Figure 4. EGb 761 decreased the level of AST, CK-MB and LDH

Values were expressed as means ± SDs. ## P <0.01 compared with sham group. *P < 0.05 and ** P < 0.01 compared with IR group (n=10).

Figure 5. EGb 761 increased Na+–K+–ATPase and Ca2+–ATPase activities and percentage of infarct size

Values were expressed as means ± SDs. ## P <0.01 compared with sham group. *P < 0.05 and ** P < 0.01 compared with IR group (n=10).

Figure 6. EGb 761 inhibited inflammatory cytokines

Values were expressed as means ± SDs. ## P <0.01 compared with sham group. *P < 0.05 and ** P < 0.01 compared with IR group (n=10).

Figure 7. EGb 761 inhibited infiltration of inflammatory cells in Myocardial Histology

(A) sham group; (B) IR group; (C) IR + EGb 761 (20 mg/kg) group; (D) IR + EGb 761 (40 mg/kg) group. Scar bar: 200 μM (upper panel).

Figure 8. EGb 761 decreased Bax and increased Bcl-2 expressions

Values were expressed as means ± SDs. ## P <0.01 compared with sham group. *P < 0.05 and ** P < 0.01 compared with IR group (n=10).

Figure 9. EGb 761 inhibited TLR-4/NF-κB-mediated inflammatory signalings in the myocardium tissue

Values were expressed as means ± SDs. ## P <0.01 compared with sham group. *P < 0.05 and ** P < 0.01 compared with IR group (n=10).