Neuroprotective Effect And Mechanisms Of Sinomenine On Brain Ischemia

Previous studies have revealed that Sinomenine (Sin), a bioactive alkaloid derived from the Chinese medicinal plant sinomenium acutum which was widely used in anti-inflammatory clinical therapy in China, exhibits cytoprotection against myocardial ischemia by lowering intracellular Ca²⁺ accumulation or dopaminergic (DA) neurotoxicity in Parkinson's disease (PD) by the inhibition of microglial NADPH oxidase. Although calcium overload and pro-inflammatory factors are closely associated with the pathogenesis of ischemic brain injury, in the present study, we have investigated the neuroprotective effect and mechanisms of Sin against brain ischemia. We have found that Sin inhibited OGD-R induced PC12 cell death in vitro and administration of Sin (10 and 30 mg/kg, i.p.) significantly decreased brain infarction and water contents in rats subjected to 2-h ischemia followed by 24-h reperfusion. Down-regulation of pro-inflammatory cytokines and apoptosis-associated genes expression after ischemia and the inhibition of oxidative stress induced calcium overload were involved in the neuroprotection of Sin. These results suggested that Sin might have potential therapeutic roles in stroke.Part 1 Neuronprotective effects of Sinomenine (Sin) in vitroObjective: Sinomenine (Sin) is a bioactive alkaloid derived from the Chinese medicinal plant sinomenium acutum. The present study investigated the effects of Sin on oxidative stress-induced cytotoxicity.Methods: (1) PC12 cells were cultured to study the role of Sinomenine on the injury induced by oxygen-derived free radicals. (2) Investigate the function of Sinomenine in OGD-R model. (3) Research LDH level of PC12 cells treated with Sinomenine. (4) Observe the the effect of Sinomenine by HOECHST. (5) Study the effect of Sinomenine on their intracellular calcium overload with calcium imaging equipment.Results: (1) Sinomenine can protect cell damage caused by oxygen-derived free radicals. (2) Sinomenine inhibited OGD-R induced neuron death. (3) Sinomenine reduced the LDH release after OGD-R. (4) Sinomenine can protect PC12 cells from free radicals. (5) Sinomenine can inhibit the increase of intracellular calcium in PC12 cells.Conclusion: Sin has protective effects against H₂O₂-induced cytotoxicity in a dose-dependent manner.Part 2 Pharmacokinetics and tissue distribution of Sin in rats by HPLC/LC-MSObjective: To explore Sinomenine pharmacokinetics in rats by determination of plasma concentration and drug tissue distribution.Methods: (1) Determination of the plasma concentration after the administration of Sinomenine to rats by HPLC. (2) Determination of the drug concentration in the brain, liver and heart by LC-MS.Results: (1) The concentration curves of rats'plasma based on concentration-time curve was presented by one-compartment model, tmax was 10 min and Cmax was 154.12μg/ml; (2) The concentration in the liver was higher than that in the hearts and brains. Based on the concentration-time curve in brain, Tmax=13 min, Cmax=1.23μg/ml. The concentration of Sin in rat's brain was above 1μM 5 hours after administration Sin.Conclusion: Sin can penetrate BBB (Blood-Brain Barrier) to result in the neuronproctective effec.Part 3 Effects of Sinomenine on brain ischemia of rats and the underlying mechanismObjective: To investigate the effect of Sin on cerebral ischemia in rats and study the mechanism by western blot.Methods: (1) Treated the brain slices which were incubated with sinomenine by OGD-R, to observe the recovery of EPSP under different circumstances. (2) To invistigate the cerebral ischemic injury through MCAO model in rats. (3) To study the mechanism of sin and analyse the level of apoptosis-related proteins, inflammatory factors and calmodulin protein kinaseâ…¡.Results: (1) Sin protected brain slices anti OGD-R injury. (2) Sin 10 mg/kg and 30 mg/kg reduced the edema volume 70.30±6.29%, 83.81±9.62% respectively after MCAO. (3) Sin down-regulated pro-inflammatory cytokines, apoptosis-associated genes expression and CaMKâ…¡after ischemia.Conclusion: The mechanism anti-brain ischemia of Sin was related with inhibiting intracellular calcium, reducing the production of inflammatory factor and reducing the level of apoptosis proteins.