| The pathogenesis of brain ischemia-reperfusion injury results from the generation of many active substances, including free radicals and platelet-activating factor. It is of significance against ischemia reperfusion injury to develop effective neuroprotectors through scavenging free radicals and antagonizing platelet-activating factor.Ginkgolide B, one of the main active component of the Ginkgo biloba extract, is a specific antagonist of platelet-activating factor. Experimental studies demonstrated that ginkgolide B could inhibit inflammatory reaction, improve energy metabolism and the pathological changes after ischemia reperfusion injury through antagonism of platelet-activating factor. In present study the brain histomorphology, the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), the generation capacity of hydroxyl radical, the expression of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in brain were taken to evaluate the neuroprotective effects and the mechanisms of ginkgolide B action on the transient focal cerebral ischemia in rats.Objective: To evaluate the neuroprotective effects of ginkgolide B on focal cerebral ischemia-reperfusion injury.Methods: The rats were randomly divided into 6 groups, which were solvent group, sham group, GinB 1mg·kg-1 group, GinB 2mg·kg-1 group, GinB 4mg·kg-1 group and ginaton 12mg·kg-1 group. Focal cerebral ischemia-reperfusion was produced by introducing a nylon suture above the bifurcation of the internal carotid artery (ICA) to block the blood flow to the middle cerebral artery (MCA) for 2 hours and drawing the nylon suture and keeping the reperfusion for 4 hours. The rats in sham group underwent the same surgical procedure without inserting nylon suture into the ICA. The rats in other groups were injected intravenously solvent, ginaton or different dosages of ginkgolide B (GinB)immediately after inserting nylon suture. The infarct volume, the histological changes of brain tissue, the neurological score, the brain water content, the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), the generation capacity of hydroxyl radical, the expression of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in cerebral cortex were determined respectively at 6 hour after the middle cerebral artery occlusion (MCAO) to evaluate the effects of ginkgolide B.Results:1. At 6 hour after MCAO, the neurological score of sham group was 0. The rats in solvent group showed apparente neurologic deficits with the highest neurological score. Ginaton, GinB 1mg·kg-1, 2mg·kg-1 and 4 mg·kg-1 could ameliorate the neurologic deficits and the neurological score were reduced to (6.38±0.92), (8.38±0.52), (7.50±0.53) and (6.38±1.06) respectively, as compared to (9.50±0.53) in solvent group (P<0.01, P<0.05).2. All the rats developed obvious infarction in the ischemic hemisphere after MCAO except those in sham operation group. GinB 1, 2 and 4 mg·kg-1 showed a 25.31%, 29.75% and 37.86% reduction in cerebral infarct volume, compared with solvent group (P<0.01). A similar effect of reducing cerebral infarct volume was also observed in ginaton-treated group.3. The brain water content was(81.1±1.2)% in solvent group after MCAO, significantly higher than that in sham group (P<0.01). GinB4 mg·kg-1 could remarkedly reduce the brain water content(P<0.01). A reduction of brain water content were observed in the other group, but there was no significant difference compared to solvent group (P>0.05).4. The activity of superoxide dismutase(SOD)in serum was (148±33)U·ml-1 in solvent group after MCAO, much lower than (401±87) U·ml-1 in sham group, (315±79) U·ml-1 in ginaton-treated group and (250±78) U·ml-1 in GinB 4mg·kg-1 -treated group (P<0.01). GinB 1 and 2mg·kg-1 could raise SOD activity too, but there was no significant difference compared with solvent group (P>0.05).5. MDA content in sham group was 14.77±1.80 nmol·ml-1. After MCAO, the MDA content in serum raised to 20.97±2.92 nmol·ml-1 in solvent group. Ginaton and different dosages of GinB could significantly decreased the MDA content (17.20±2.20nmol·ml-1 in ginaton group with 17.98% reduction, 18.24±3.12 mol·ml-1 in GinB 1mg·kg-1 group with 13.02% reduction, 17.95±2.81 mmol·ml-1 in GinB 2mg·kg-1 group with 14.40% reduction and 16.12±2.24 mmol·ml-1 in GinB 4mg·kg-1 group with 23.13% reduction) in serum, compared with solvent group(P<0.05,P<0.01).6. A generation capacity of hydroxyl radicals in serum was 145±52 U·ml-1 in sham group, lower than 294±40 U·ml-1 in solvent group(P<0.01). Ginaton and GinB1, 2 and 4 mg·kg-1 could inhibit the generation of hydroxyl radical after MCAO and showed 35.71%, 13.95%, 29.25% and 31.63% reduction of hydroxyl radical in serum, respectively. Ginaton, GinB 2mg·kg-1 and 4mg·kg-1 showed significant effects on the clearance of hydroxyl radical as compared with solvent group(P<0.01), but GinB 1mg·kg-1 did not.7. In HE staining, cerebral tissue structure in solvent group showed ischemic damage both in cortex and striatum characterized by the degeneration and necrosis of neurons. Ginaton and GinB could improve the pathological changes induced by ischemia-reperfusion although the neuronal degeneration was observed. The number of necrotic cell in ginaton and GinB-treated group was less than that in solvent group. No pathological changes were observed in sham group.8. The immunochemical positive expression for Mn-SOD appeared in cytoplasm. A weak positive expression for Mn-SOD was observed in sham group. Compared with solvent group, the positive staining remarkedly increased in ginaton and GinB group. The positive expression in GinB group was weaker than that in ginaton group.9. There was positive expression for iNOS in cytoplasm. The positive expression notably appeared in chorioidal epithelium, but was rare in neuron and glia in all experimental group. The positive staining was obvious within vascular endothelial cell in solvent group than those in sham group. The positive expression for iNOS became weak in ginaton-treated and GinB-treated group compared with solvent group, and still a little stronger than that in sham group. With the dosage increasing,the iNOS expression became weak in GinB-treated group.Conclusion: Ginkgolide B showed significant protective effects against cerebral ischemic-reperfusion injury. The mechanism maybe related to antagonizing platelet-activating factor receptor, inhibiting the production of lipid peroxidants and the generation of free radicals, enhancing endogenous antioxidant enzyme activity. |
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