Baicalin protects neural cells from cerebral ischemia reperfusion injury by scavenging peroxynitrite

Ischemic stroke is the leading cause of death and disability in human diseases all around the world. As effective treatment for ischemic stroke is still absent, seeking for new therapy is of great interest. Currently, several key pathological cascades following cerebral ischemia have been explored to develop further therapies. Among them, reactive nitrogen species (RNS) has been indicated to play a critical role in cerebral ischemia reperfusion injury. As one of the RNS, peroxynitrite contributes to the neural cell death and subsequent brain dysfunction in the process. Thus, development of antioxidants targeting on peroxynitrite could be an important strategy for the treatment of cerebral ischemia-reperfusion injury. Baicalin is a polyphenolic compound isolated from roots of Scutellaria baicalensis. Baicalin exerted protective effects against cerebral ischemia-reperfusion injury but the mechanisms are not clear yet. In this study, we investigated the free radical scavenging ability and neuroprotective effects of baicalin. According to our results, baicalin neutralized DPPH radicals effectively. By using electron paramagnetic resonance (EPR) spin trapping technology and fluorescent probe DAF-2DA, we found that baicalin dose-dependently scavenged superoxide, but had very low effect on elimination of nitric oxide. The immunofluoresent results revealed that baicalin at the concentration of 50 M completely suppressed the nitrotyrosine formation induced by 3-morpholinylsydnoneimine chloride (SIN-1, a peroxynitrite donor) in neuroblastoma SH-SY5Y cells. Mass spetrum provided direct evidence of the peroxynitrite scavenging ability of baicalin. Using MTT assays, we found that baicalin totally reversed peroxynitrite-induced cytotoxicity in SH-SY5Y cells and protected SH-SY5Y cells in oxygen glucose deprivation (OGD) and following reoxygenation injury. Furthermore, in vivo experiments revealed that intravenous injection of baicalin exerted better neuroprotective effect than intraperitoneal administration in rats underwent middle cerebral artery occlusion (MCAO). After cerebral ischemia reperfusion, rats treated with 3 mg/kg of peroxynitrite decomposition catalyst (FeTMPyP) or 25 mg/kg of baicalin revealed a smaller size of infarction volume, suppressed neural cell death and reduced nitrotyrosine formation than MCAO rats. However, baicalin did not alter the expression of tight junction proteins, claudin-5 and ZO-1, in brain endothelial bEnd3 cell line treated with OGD following reoxygenation. In cerebral ischemia reperfusion rats, administration of FeTMPyP at the dosage of 3 mg/kg diminished the Evans blue leakage caused by blood brain barrier disruption, whereas treatment of baicalin did not show significant effect. In conclusion, this study suggests that baicalin can scavenge peroxynitrite and protect neural cells from peroxynitrite-induced injury. Furthermore, baicalin could prevent brains from cerebral ischemia-reperfusion injury and the neuroprotective mechanisms are associated with the scavenging effects on peroxynitrite. These findings provide new insights into the antioxidant and neuroprotective properties of baicalin and indicate the potential application of baicalin for the treatment of ischemic stroke.published_or_final_versionChinese MedicineMasterMaster of Philosoph