Purified Herba Leonuri and Leonurine protect middle cerebral artery occluded-rats from brain injury through antioxidative mechanism and mitochondrial protection

Abstract

Ischemic stroke refers to the condition caused by the occlusion of blood vessels, causing the injury of the territory of the supplied brain region. With ample evidences showed that free radicals play a role in ischemic cascade, studies focusing on traditional Chinese medicines (TCMs) have shown its promising effects in neuroprotection due to their antioxidant properties. In this research, therapeutic potentials of purified Herba leonuri (pHL) and one of its active ingredients Leonurine on ischemic stroke were investigated. Rodent model of permanent focal ischemia by left middle cerebral artery occlusion (MCAO) in Wistar rats was employed in this research. Left MCAO model resulted in infarct spanning left cortical and striatal tissues with neurological impairment, oxidative stress, apoptosis and mitochondrial dysfunction. In experiment I, MCAO-induced rats pretreated with pHL for 14 days prior to the induction of stroke, followed by another 7 days of post-operation treatment was demonstrated to have significant reduction in infarct volume. The pHL-administered rats had also improvement of neurological outcome. Comparing to stroke group, total plasma antioxidant concentration was increased while level of DNA oxidative damage was reduced significantly. Therefore, therapeutic effect of pHL is believed to act through antioxidant effects. In addition, experimental results also showed that pHL might interfere with apoptosis signaling as apoptosis level was reduced as shown by TUNEL assay. In experiment II, lowered mitochondrial ROS generation level was observed in the isolated mitochondrial treated with pHL, in a dose-dependent manner, suggesting that pHL prevents the contribution of mitochondria to oxidative stress. This is accompanied with the lowered ATP biosynthesis which could be the effect of metabolic arrest, leading to the cytoprotective barrier to the mitochondria. A slight increase of state 4 respiration by the treatment of pHL suggests that pHL might also have a mild uncoupling effect which has been shown to be a cytoprotective strategy. Similarly, as mitochondria were pre-treated with H2O2 as ROS inducer, pHL suppressed both mitochondrial ROS generation and ATP biosynthesis, indicating that protective effects of pHL could be executed in both physiological and pathological conditions. In vivo mitochondrial studies showed that mitochondrial dysfunction by MCAO was rescued by the treatment of pHL as mitochondrial respiration, particularly state 3 respiration was greatly enhanced from. In addition, balanced GSH pool was observed under the treatment of pHL, in both healthy and MCAO-induced rats. In experiment III, the study was extended to the research to the investigation of the therapeutic potential of Leonurine, a key and unique compound found in both Herba leonuri (HL) and pHL, on ischemic stroke model. Animal was pretreated with Leonurine orally for 7 days before the middle cerebral artery occlusion (MCAO) was done. This study demonstrated that one day after surgery, Leonurine treatment at 60mg/kg/day could significantly reduce infarct volume, improve neurological deficit in stroke group. As compared to stroke group, increased activities of antioxidant enzymes SOD and GPx, and decreased MDA level were also observed in stroke group treated with Leonurine, indicating the antioxidative mechanisms of Leonurine. In vitro studies showed that Leonurine also inhibited mitochondrial ROS production and ATP biosynthesis, dose-dependently, indicating the similar protective effect was observed between pHL and Leonurine. Mitochondrial function was also rescued by Leonurine treatment in rats subjected to MCAO. Mitochondrial state 3 respiration was enhanced in stroke groups treated with Leonurine. To summarize, both pHL and Leonurine carry a potential effects on stroke therapy, at least via antioxidant effects and modulation of mitochondrial function.