Heme oxygenase 1, beneficial role in permanent ischemic stroke and in Gingko biloba (EGb 761) neuroprotection

Abstract

Ginkgo biloba extract, EGb 761, a popular and standardized natural extract, contains 24% ginkgo-flavonol glycosides and 6% terpene lactones. EGb 761 is used worldwide to treat many ailments, and although a number of studies have shown its neuroprotective properties, the mechanisms of action have not been elucidated fully. We hypothesize that EGb 761 and some of its bioactive components [Bilobalide (BB), Ginkgolide A (GA), Ginkgolide B (GB), and Terpene Free Material (TFM)] could provide neuroprotection in ischemic conditions through heme oxygenase 1 (HO1). Mice were subjected to permanent distal middle cerebral artery occlusion (pMCAO) and survived for 7 days. HO1 knockout (HO1⁻/⁻) mice showed significantly higher (P<0.05) infarct volume and Neurologic Deficit Scores (NDS) as compared to their wildtype (WT) counterparts. In another cohort, WT mice subjected to pMCAO and treated at 4 h of pMCAO with 100 mg/kg EGb 761, 6 mg/kg BB, GA, GB, or 10 mg/kg TFM showed significantly lower (P<0.05) infarct volumes (BB; 29.0±3.9%, GA; 31.3±4.0%, GB; 32.0±3.8%, TFM; 32.5±3.5%, and EGb 761; 27.4±4.5%) than those in the vehicle-treated mice (46.0±3.7%). Similarly, NDS were lower in BB; 7.1±1.8, GA; 7.4±2.1, GB; 7.9±1.8, TFM; 7.7±1.7, and EGb 761; 6.8±2.0 groups as compared with the vehicle-treated group (13.8±1.5). Interestingly, the protective effect of EGb 761 was essentially lost when HO1 knockout mice were treated with EGb 761. In another cohort, HO1, vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) protein levels in the brain cortices appeared to be higher in EGb 761 and BB but not in GA, GB and TFM treated groups. Together, these results suggest that HO1 plays, at least in part, an important role in the neuroprotective mechanism of EGb 761 and in delayed ischemia. Targeting this pathway could lead to neuroprotective agents against ischemic stroke.

Fig. 1

Role of HO1 against 7 d pMCAO. (A) Schematic diagram of experimental protocol. Mice were subjected to pMCAO and survived for 7 d to evaluate NDS and infarct volume. (B and C) Representative TTC stained brain slices and graph show higher infarct volume and NDS in HO1^-/- than WT mice. WT (n=8); HO1^-/- (n=6). *p<0.05 vs. corresponding control.

Fig. 2

Neuroprotective effect of EGb 761 and its constituents. (A) Schematic diagram of experimental protocol. Vehicle or test drugs were administered after 4 h of pMCAO, and mice survived for 7 d to evaluate NDS and infarct volume. (B and C) Representative TTC stained brain slices and graph show reduced infarct volumes and NDS of WT mice treated with Ginkgo biloba (EGb 761), Bilobalide (BB), Ginkgolide A (GA), Ginkgolide B (GB) and Terpene Free Material (TFM). Vehicle (n=6); BB (n=10); GA (n=9), GB (n=8), TFM (n=9), EGb 761 (n=8). *p<0.05 vs. corresponding vehicle control.

Fig. 3

EGb 761 is not protective in HO1^-/- mice. (A) Schematic diagram of experimental protocol. Vehicle or EGb 761 was administered after 4 h of pMCAO, and mice survived for 7 d to evaluate NDS and infarct volume. (B and C) Representative TTC stained brain slices and graph show EGb 761 is not protective in HO1^-/- mice as compared to vehicle treatment group (WT vehicle treatment group from experiment/Fig. 2). WT and Vehicle (n=6), EGb 761 and HO1^-/- (n=7). *p<0.05 vs. Vehicle control; ns, not significant.

Fig. 4

EGb 761 is not protective in HO2^-/- mice. (A) Schematic diagram of the experimental protocol. Vehicle or EGb 761 was administered after 4 h of pMCAO, and mice survived for 7 d to evaluate NDS and infarct volume. (B and C) Representative TTC stained brain slices and graph show EGb 761 is not protective in HO2^-/- mice as compared to vehicle treatment group. HO2^-/- and Vehicle (n=7), EGb 761 and HO2^-/- (n=7). *p<0.05 vs. corresponding control; ns, not significant.

Fig. 5

HO1, HO2, VEGF and eNOS protein expression levels in mice brain cortices. Mice were post-treated at 1.5 h of pMCAO with EGb 761 and its constituents, and Western blots were performed to measure the protein expression of HO1, HO2, VEGF and eNOS. The expression of actin was used as a loading control. (A) HO1 expression levels were observed to be significantly increased by the post-treatment of EGb 761 and BB. The expression levels of HO1 were also increased in naïve EGb 761 treated group (non surgery). (B) HO2, known to be constitutively expressed in the brain, was unaffected. (C) VEGF expression levels were significantly increased in EGB 761 and showed an increasing trend in BB treated group. (D) Similarly, eNOS protein expression levels showed significant increase in EGb 761 and BB treated groups. The histograms show the ratio of density captured from HO1, HO2, VEGF, eNOS to that of actin. Values shown are means±SEM from three independent sets of experiments. *p<0.05 vs. corresponding control.