Studies On The Molecular Mechanisms Of Antiepileptic Action Of Qingyangshenylycosides (QYS)

Qingyangshenylycosides (QYS) is a mixture of several related compounds derived from the root of a Traditional Chinese Medicine, Cynanchum Otophyllum. Pharmacological characterization of the compounds indicated that the drug had a significant antiepileptic and anticonvulsant activity on kindling, rat audiogenic seizures and ferrous sulfate-induced epilepsy. It has been shown that QYS could affect the acetylcholine concentration, 5-hydroxytryptamine concentration, c-fos expression induced by kainate acid, neuropeptide concentration and the GABA function in CNS. However the mechanism of the anticonvulsant effect of QYS has not been clearly identified.DBA/2J mouse is an inbred strain that is very sensitive to acoustic stimulus, and therefore high intensity noise causes AGS. The AGS mice exhibit continuous wild running, clonus, tonus, respiratory arrest and death. Therefore, DBA mice are often used as experimental animal models for the studies on the mechanisms of epilepsy as well as for the development of therapeutic drugs. In the present study, we first investigated the effect of QYS on the generation of audiogenic seizure (AGS) induced by high-intensity noise and PTZ-induced generalized epilepsy in DBA mice. To understand the molecular mechanism of epilepsy and the mechanism of anticonvulsant action of QYS, we performed high-density microarray, fluorescence real-time quantitative PCR (QPCR) and Western blot technologies to analyze the gene expression profiling in AGS mice as well as QYS treated mice. Our results demonstrated that the expression levels of a number of ion channels, metabolic enzymes and proteins involved in signal transduction were regulated in AGS mice. Interestingly, some of these expression changes have been reversed by QYS treatment, suggesting that these genes might be associated with the antiepileptic activity of the drug.1. Behavioral Effects of QYS on Audigenic Seizure and PTZ-induced Generalized EpilepsyAfter two consecutive intraperitoneal treatments of different dosage of QYS, we examined the effects of QYS on the audiogenic seizure(AGS) induced by the high intensity noise (113.5 dB SPL electrical bell noise) and the generalized epilepsy induced by the intraperitoneal injection of pentylenetetrazol (PTZ). DBA/2J mice were injected with different doses of QYS, and the latency of AGS onset was measured one hour after the second drug treatment. The latencies in control and 75 mg/kg of drug treatment (QY75) were 2.21±0.25s and 2.20±0.36s, there was no significant difference between these two groups of animals (student t-test, P>0.05). However, when the dose of QYS increased to 100 mg/kg (QY100), the latency was significantly longer (student t-test, P<0.05). Indeed, the latency of AGS were prolonged in a QYS dose-dependent manner with the increase of dose of QYS (one way ANOVA, P<0.05). Furthermore, twenty-five hours after the second injection, the latencies in QY100, QY125, QY150, QY160, QY175, QY200 and QY240 were still longer than that in control and QY75 (Fig 1B, P<0.05), indicating the drug has long last effects.When exposed to high-intensity noise, all the control and QY75 mice exhibited tonic seizure (Tonus% = 100). Higher doses of QYS reduced the Tonus% significantly one hour after the drug administration. The Tonus% started to decrease at 100 mg/kg of the drug. Higher doses of QYS lead more reduction of Tonus%. It has been reduced to about 20% when QYS was increased to 240 mg/kg. These results clearly showed that the drug effectively prevents the generation of AGS, exhibiting a significant anticonvulsant action.In the PTZ-induced generalized epilepsy experiment in DBA mice, we injected intraperitoneally mice with different dose of PTZ in order to well understand the effect of QYS. We found that the latencies of PTZ-induced epilepsy were gradually shortened with the increase of dosage of PTZ, from the 316 seconds in PTZ20 mice to the 70 seconds in PTZ80 mice. The epilepsy severitys were increased gradually, from less than 1 in PTZ20 mice to 6 in PTZ80 mice. After two treat...