| Epilepsy is a common neurological disorder, affecting almost1%of the population and current pharmacological therapies remain insufficient to completely prevent epileptic seizures. Research in traditional medicine represents a promising strategy for developing new antiepileptic drugs and therapeutic approaches.Acori graminei Rhizoma (AGR), the dry rhizomes of Acorus gramineus Solander (Araceae), has been traditionally used in oriental prescriptions to treat epilepsy for hundreds of years. a-Asarone was shown to be the main active anticonvulsive component in AGR Although many studies have shown that a-asarone exerts antiepileptic effects, they are nearly all performed in cell cultures or animal’s attack manifestations. To our knowledge, no study has evaluated the electrophysiological effects of a-asarone in vivo. Previous studies indicate that a-asarone has antiepileptic effects in pentylenetetrazol(PTZ)-induced seizure models. Our findings represent the first attempt at studying the effects of a-asarone with in vivo electrophysiology in PTZ-induced epilepsy in conscious rats. a-Asarone have shown antioxidant effects in many studies both in vivo and vitro. The antiepileptic effect of a-asarone may attribute to its reduction and antioxidant properties. So, the aim of present study was to identify the involvement of nitric oxide (NO) in the antiepileptic effects of a-asarone on PTZ-induced epileptiform activity in rats.Epilepsy is one of the diseases in which NO is regarded as important pathogenetic factor in the mechanisms underlying seizure induction and progression. Whether in the results of animal experiments or clinical studies, it can be seen that NO involved in the development of epilepsy. The role of NO in epilepsy has been examined in a number of in vivo and in vitro studies, however, the obtained results are still contradictory, reporting both pro-and anti-convulsant properties of NO. NO is formed from arginine by the action of three different nitric oxide synthase (NOS) isozymes, two calcium-dependent forms, neuronal (nNOS) and endothelial (eNOS) and one calcium-independent inducible (iNOS). The relationship between NO and epilepsy has been a hotspot. People studied the level of NO in the earlier years, and in the recent years more research focus on detection of changes of NOS. Existing evidences have shown a lack of consistency in detection of NO and NOS level. It is believed that epileptic model, experimental designments, route of administration, attack forms and so on may all affect the results of detection. So there is need for further detailed study.The effects of systemic administration of nitric oxide synthase (NOS) inhibitors, non-selective NG-nitro-L-arginine methyl ester (L-NAME,60mg/kg, i.p.), selective neuronal nitric oxide synthase (nNOS) inhibitor,7-nitroindazole (7-NI,40mg/kg, i.p.), inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG,100mg/kg, i.p.) and NO precursor, L-arginine (ARG,500mg/kg, i.p.) on the effects of α-asarone were investigated. Considering that NO pathway may be involved in the formation of both models of epilepsy and antiepileptic effect of α-asarone, we designed three experimental programs and NO regulators were administered at different stage. All the results may be helpful to explain the relationship between NO and both PTZ and α-asarone.Experimental programs:1. To compare the characteristics of PTZ-induced epileptiform activity with two doses (50and60mg/kg) of PTZ and choose a suitable dose of PTZ to meet the observation requirements for ECoG analysis (appearance of clonic attack and discharge, appearance of interictal discharge and no less than2h for survival time).To investigate the electrophysiological effects of a-asarone with four different doses (20,40,60and80mg/kg), administered20minutes after PTZ injection.2. An effective dose of L-ARG, AG, L-NAME and7-NI was intraperitoneally administered5min after PTZ application. And animals received the effective dose of a-asarone15min after L-ARG, L-NAME, AG, and7-NI administration.3. Animals received L-NAME,7-NI, AG and LAG15min before a-asarone application. PTZ was administered intraperitoneally20min after a-asarone application.4. An effective dose of L-NAME,7-NI, L-ARG or AG was intraperitoneally administered15min before PTZ application and animals received an effective dose of a-asarone5min after PTZ administration.Results:1. There were no significant differences between the two groups with respect to the following parameters:survival time (observation time=2h), cumulative duration of tonic/clonic seizures, longest duration of a single tonic/clonic seizure, attack times of tonic/clonic seizures and latency of first tonic/clonic seizure. There was a significant difference in the latency of the first clonic seizure between the two groups (p＝0.001). The latencies were109.3±41.4sec (PTZ50mg/kg group) and49.0±22.8sec (PTZ60mg/kg group), respectively. We decided to use50mg/kg PTZ in the following experiment. a-Asarone significantly decreased the clonic frequency at doses of60and80mg/kg. The antiepileptic effects of the60mg/kg dose lasted no more than10min. The antiepileptic effects of the80mg/kg dose lasted50min. We decided to use80mg/kg a-asarone in the following experiment.2. Administration of L-NAME or7-NI5min after PTZ injection did not influence either the clonic activity or interictal discharge in both the frequency and amplitude. Administration of L-ARG significant decreased the mean frequency of interictal discharge in the first5min. The mean frequency of interictal discharge was significantly increased after AG administration, and the effect lasted for20min. a-Asarone significantly decreased the mean frequency of clonic activity and the significant effects appeared20min after a-asarone injection and lasted for50min. The administration of L-NAME reversed the anticlonic activity of a-asarone and induced significant increasement of interictal discharge. Administration of7-NI reversed the anticlonic effect of a-asarone without affecting clonic amplitude. The anticlonic effects appeared10min earlier in the L-ARG+a-asarone group compared with a-asarone group alone and lasted for60min. The administration of AG reversed the beneficial effect ofa-asarone on clonic activity, whereas the detrimental effect of AG on intericatal discharge was inhibited in aminoguanidine+a-asarone group.3. a-Asarone significantly decreased the mean frequency of clonic activity and the significant effects lasted for30min. L-NAME reversed the anticlonic activity of a-asarone and a significant increasement of clonic activity was induced70min after PTZ injection in L-NAME+a-asarone+PTZ group. Administration of7-N1also reversed the anticlonic activity of α-asarone. In the AG+α-asarone+PTZ group, similarly the mean frequency of clonic activity was decreased significantly compared to that of clonic activity in a-asarone+PTZ group. In the L-ARG+α-asarone+PTZ group, the mean frequency of clonic activity was temporally increased in the10min after PTZ injection and the anticlonic activity of a-asarone was reversed, whereas the mean frequency of interictal discharge significantly decreased in the50min after PTZ injection and lasted for20min compared with PTZ group.4. Administration of7-NI and AG significantly increased the mean frequency of clonic activity. The significant effect of7-NI occurred earlier than that of AG. Administration of L-NAME and L-ARG did not influence either clonic activity or interictal discharge. a-Asarone significantly decreased the mean frequency of clonic activity and the significant effects lasted for30min. The pro-convulsant effect of7-NI was reversed in7-NI+PTZ+α-asarone group. The pro-convulsant effect of AG was reversed in AG+PTZ+α-asarone group. In the case of using L-NAME, the antiepileptic effect of a-asarone was reversed. In the case of using L-ARG the antiepileptic effect of α-asarone was also reversed.Conclusion:Our results confirm that α-asarone, at the80mg/kg dose, significantly decreased the mean frequency of clonic epileptiform activity. All the three NOS isoforms were activated in PTZ-induced epilepsy, and NOS play an important key role in the genesis and spreading of epileptiform hyperactivity but not NO. Different NOS exhibits different effect and NO is only the mediator in realization of NOS’s effects. NO itself has neither pro-nor anti-epileptic property. NO produced by iNOS and nNOS exhibited protective effect in PTZ model. NO produced by eNOS exhibited pro-epileptic effect in PTZ model. NO may start different pathway by acting on different receptors or sites, ultimately showed similar or opposite effect. The results indicated that NOSs were activated at different stage of PTZ modeling. eNOS was activated within5min, while iNOS and nNOS were activated later, and nNOS was activated earlier than nNOS, but the action of iNOS lasted longer than that of nNOS.We could comment that both nNOS/NO and eNOS/NO pathways involved in the anticonvulsant effect of a-asarone. It can be concluded that a-asarone promoted NO synthesis and exerted anticonvulsant effects by acting on nNOS. On the other hand, a-asarone inhibited NO synthesis and exerted anticonvulsant effects by acting on eNOS. Both promoting and inhibiting synthesis of NO involved in the antiepileptic mechanism of a-asarone. It showed that eNOS and nNOS were influenced at different conditions, and this may due to the experimental programs. While iNOS was unrelated to the inhibition of a-asarone on PTZ induced epileptiform activity. |
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