Inhibition of nuclear factor-κB by 6-O-acetyl shanzhiside methyl ester protects brain against injury in a rat model of ischemia and reperfusion

Background: Recent studies have demonstrated an inflammatory response associated with the pathophysiology of cerebral ischemia. The beneficial effects of anti-inflammatory drugs in cerebral ischemia have been documented. When screening natural compounds for drug candidates in this category, we isolated 6-O-acetyl shanzhiside methyl ester (ND02), an iridoid glucoside compound, from the leaves of Lamiophlomis rotata (Benth.) Kudo. The objectives of this study were to determine the effects of ND02 on a cultured neuronal cell line, SH-SY5Y, in vitro, and on experimental ischemic stroke in vivo. Methods: For TNF-alpha-stimulated SH-SY5Y cell line experiments in vitro, SH-SY5Y cells were pre-incubated with ND02 (20 mu M or 40 mu M) for 30 min and then incubated with TNF-alpha (20 ng/ml) for 15 min. For in vivo experiments, rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion for 23 h. Results: ND02 treatment of SH-SY5Y cell lines blocked TNF-alpha-induced nuclear factor-kappa B (NF-kappa B) and I kappa B-alpha phosphorylation and increased Akt phosphorylation. LY294002 blocked TNF-alpha-induced phosphorylation of Akt and reduced the phosphorylation of both I kappa B-alpha and NF-kappa B. At doses higher than 10 mg/kg, ND02 had a significant neuroprotective effect in rats with cerebral ischemia and reperfusion (I/R). ND02 (25 mg/kg) demonstrated significant neuroprotective activity even after delayed administration 1 h, 3 h and 5 h after I/R. ND02, 25 mg/kg, attenuated histopathological damage, decreased cerebral Evans blue extravasation, inhibited NF-kappa B activation, and enhanced Akt phosphorylation. Conclusion: These data show that ND02 protects brain against I/R injury with a favorable therapeutic time-window by alleviating cerebral I/R injury and attenuating blood-brain barrier (BBB) breakdown, and that these protective effects may be due to blocking of neuronal inflammatory cascades through an Akt-dependent NF-kappa B signaling pathway.