Study On Antiepileptic Effect Of Scorpion Venom Neuro-nourishing Peptide

Objective:The pathogenesis of epilepsy, one of the common multiple chronic brain diseases, is not very clear now. However, the electrophysiological nature of the epilepsy onsets is that brain neurons synchronized over-discharge, causing temporary dysfunction of the brain. It's a long-term recurrent disease. Patients suffer not only physically, but to some extent, also have mental and psychosocial obstacles, with their intelligence and personality aspects damaged. It is because of the complexity and refractoriness of epilepsy, the discovery of antiepileptic drugs needs a breakthrough urgently. In the screening of drug, we found Scorpion (BmK) show a significant efficacy on refractory epilepsy. Scorpion has been used for medicine treatment of epilepsy for many years. Its efficacy lies in the venom component of scorpion, scorpion venom (SV). SV has a significant antiepileptic effect. However, the elements are complex, highly toxic, so that its clinical application is limited. Our laboratory has been engaged in the research for discovering scorpion poison for a long time and has extracted active peptide which has the anti-epileptic effect from scorpion venom. Through a special process, we have obtained the scorpion venom neuro-nourishing peptide (svNNP), which has the anti-epileptic effect. Compared with SV venom, not only the toxicity of svNNP is significantly reduced, but aslo the anti-epileptic efficacy is enhanced. The purpose of this study is to further explore the neuroprotective antiepileptic mechanism of svNNP on the general and whole cell level, in order to provide new experimental evidences for the discovery of Scorpion Poison.Method:Using whole cell patch clamp, immunohistochemistry, immunocytochemistry, MTT and Western-Blot technique, the mechanism of antiepileptic effect of svNNP was approached by epilepsy model in vivo and in vitro.Results:Section 1 (In vivo experiment)Using lithium chloride(LiCl)-pilocarpine (PILO) epilepsy rat model, the effects of svNNP were observed by behavior, immunohistochemistry. The results show that1. The times of seizures within 25min after treatment of svNPP were decreased than the control group, but the grade of seizures has no significance (n=5, P>0.05). The escape latencies and times to pass through the platform for each group animals were not significant among each other (n=5,P> 0.05)2. GFAP immunohistochemistry slices results show that svNNP treatment group could increase GFAP immunoreactive cells, The morphology of astrocytes showed irregular processes, many short curved branches and not smooth surface which demonstrated the type of most astrocytes belong to protoplasmic astrocyte.3. Map2 immunohistochemistry staining show that the number of fractured dendrites and lossing of neuron for post-treatment svNNP group were significantly reduced compared with the model control group.Section 2 (In vitro experiment)1. Brain derived neurotrophic factor (BDNF) immunostaining for cultured hippocampal neurons and glial cells which were pre-incubated with svNNP for 24hrs shows that①the number of BDNF immunoreactive glial cells of post-treatment svNNP group increases significantly and the staining becomes deeper compared with those of the post-treatment SV venom group, suggesting that svNNP could increase the expression of BDNF of glial cells.②hippocampal neuronal morphology of post-treatment svNNP group is intact with tight connections among cells, and BDNF immunoreactive staining and immunoreactive cells increased significantly (P<0.01) compared with the control group.2. The effects of svNNP on the survival of the primary cultured hippocampal neurons were observed by MTT assay. It was found that in SV venom group the OD value decreased significantly (P<0.05) compared with the normal control group; in svNNP group the OD value increased significantly (P<0.05) compared with the normal control group, suggesting that svNNP could increase neuron survival-rate, and protecte them from damage, while the SV venom has toxic effects and decreases the survival of the primary cultured hippocampal neurons3. NMDA-activated current (ⅠNMDA) were induced from cultured hippocampal neurons. The effects of different concentrations of svNNP (10-3μg/ml,10-4μg/ml,10-5μg/ml,10-6μg/ml,10-7μg/m,10-8μg/ml) onⅠNMDA were observed. The results show that the five different concentrations of svNNP all could reduceⅠNMDA of hippocampal neurons (P<0.01, n=8), and appered dose-dependent manner.4. Voltage-gated sodium channel currents from primary cultured hippocampal neurons were recorded using patch clamp technique in kainic acid (KA) excitotoxic injury model which incubated with svNNP for 24hrs. The results show that①svNNP could inhibit voltage-gated sodium channel peak currents from KA-induced hippocampal epilepsy model neurons (P<0.05).②svNNP could shift toward depolarzied potentials in steady-state activation curve of voltage-gated sodium channel on hippocampal neurons compared with those of normal control group. After treatment of 10μmol/L KA, the voltage of half-activation (V1/2) was-40.1±1.80 (n=20), post-treatment 2μg/ml svNNP+10μmol/L KA the V1/2 became-36.26±1.44 (n=19). The V1/2 was significant (P<0.05) between post-treatment KA group with post-treatment KA+svNNP group.③The curve for steady-state inactivation of sodium channel on hippocampal KA-induced epilepsy model neurons was shifted toward negative potential after treament of svNNP. The half inactivation voltage (V1/2) of the normal sodium channel was-59.03±0.29 (n= 16);post-treatment 10μmol/L KA the V1/2 became-61.59±0.58 (n=28); post-treatment 10μmol/L KA+2μg/ml svNNP the V1/2 became-64.24±0.97 (n=16). It is suggested that post-treatment 2μg/ml svNNP+10μmol/L KA the sodium channel half inactivation voltage shifted to the left, compared with the control group and KA model group. The differences were significant (P<0.05).④After svNNP treatment, the sodium channels recovered slowly from inactivation. The difference was significant (P<0.05) compared with the control group.Conclusion:1. svNNP could make epileptic rats'astrocytes change from the fibrous type into the protoplasmic type after the treatment.2. svNNP could reduce the dendritic fracture and the neuron-loss in the epileptic attack process.3. svNNP could improve the survival of primary cultured hippocampal neurons,while SV venom toxicity has a contrary effect.4. svNNP could increase the secretion of BDNF of neurons and glial cells, playing the role of nutrition and protection of neurons.5. svNNP could inhibit the NMDA receptor (NMDAR)-activated currents of the hippocampal neurons in a dose-dependent manner and reduce the nerve injury caused by excessive activation of NMDAR during seizures.6. svNNP could inhibit sodium channel current in vitro KA-induced epileptic hippocampal neuronal, slow the activation of sodium channels, speed up the inactivation of sodium channels and slow the recovery of sodium channels, thereby reducing the excessive activation of neurons in the epileptic process.