Study On Neuroprotective Mechanisms Of Calmodulin Inhibitor W-7 Against Cultured Cotical Neurons Injured By NMDA And PTZ-induced Rats

Objective 1 To explore the protective effects and mechanisms of calmodulin inhibitor W-7 on cultured cortical neurons injured by NMDA.2 To explore the protective effects and mechanisms of calmodulin inhibitor W-7 on PTZ-induced rats.MethodsPartⅠPrimary cortical neurons cultured from 24h old postnatal rats for 7 days were randomly divided into 5 groups:(1)control group:(2)NMDA-injured group:(3)three-dose W-7 pretreated groups:cortical neurons were pre-cultured with regular media added W-7(concentration:25μmol/L,50μmol/L,100μmol/L)for 24h before exposed to NMDA(concentration:50μmol/L).MTT assay were used to detect cell viability.The morphological changes of cortical neurons injured by NMDA were observed under the fluorescence microscope.Apoptotic cortical neurons were examined using acridine fluorescent staining.Lactic acid dehydrogenase(LDH)in supernatant was detected.NF-κBp65 and p38MAPK protein expressions in cultured cortical neurons were detected by means of immunocytochemistry and Western blotting.PartⅡ38 Healthy adult Sprague-Dawley rats were randomly divided into 5 groups:control group(i.p.NS),PTZ group(i.p.PTZ 65mg/kg)and W-7 preteated groups(i.p.W-7 25μmol/kg,50μmol/kg,100μmol/kg,10min later,i.p.PTZ65mg/kg).After treatment with relevant reagents,the behaviour and the neuronal ultrastructure changes in hippocampal areas was observed.Brain tissue samples were taken quickly to measure the content of LDH.The expressions of caspase-3,NF-κBp65 and p38MAPK in the hippoacampus of rats were examined by means of immunohistochemistry and western bloting. ResultsPartⅠCompared to the control group,apoptotic cortical neurons were significantly increased and there was dose-dependent neuroprotective effect. (P＜0.01),the level of LDH in supernatant was significantly increased in NMDA injured group(P＜0.01).Compared with the NMDA injured group,the number of apoptotic cortical neurons was decreased in W-7 pretreared groups(P＜0.05),the level of LDH in supernatant was significantly decreased in W-7 pretreated groups(P＜0.05,P＜0.01),and the expressions of NF-κBp65 and p38MAPK in cultured neuron were remarkably up-regulated in NMDA injured group while down-regulated in W-7 pretreated groups,there were dose-dependent neuroprotective effects.(P＜0.05,P＜0.01).PartⅡⅣ-Ⅴgrade of seizure in PTZ-induced group,butⅠ-Ⅲof seizure in W-7 pretreated groups(P＜0.05).Immunohistochemical staining and western blotting showed that there were expressions of caspase-3,NF-κBp65 and p38MAPK in the hippocampus of all groups.Compared with control group,the expressions of caspase-3,NF-κBp65 and p38MAPK were increased in PTZ group(P＜0.01),while they were decreased in W-7 groups(P＜0.05,P＜0.01).Conclusion 1 NMDA could up-regulate the expressins of NF-κB,p38MAPK in cultured cotical neurons which could promote neuronal apoptosis.2 W-7 could relieve the damage of cultured corical neurons injured by NMDA,so W-7 has protective effects on cortical neurons by depressing NF-κBp65 and p38MAPK activation.3 The expressions of caspase-3,NF-κBp65 and p38MAPK were increased in the brain tissue of PTZ-induced rats which showed that seizures could lead to apoptosis of neurons in these models.4 W-7 has protective effects on PTZ induced rats by depressing expressions of caspase-3,NF-κBp65 and p38MAPK,it could interfere the occurrence and development of epilepsy.