The Protective Effects Of Activin A On Primary Neuronal Injury Induced By Pilocarpine

Objective:Epilepsy is a chronic nervous system disorder disease which is caused by abnormal discharges of the neuronal networks.Because of the pathogenesis of epilepsy is complex,its cause has not be fully revealed.Glutamate is the most important excitatory neurotransmitter in the brain,it acts primarily through the glutamate receptors.Because of the ionic glutamate receptors binding to the ion channels,they are directly related to the abnormal discharges.In this study,we established an in vitro epilepsy model of primary cortical neurons induced by pilocarpine.The changes of morphological and different subunits of glutamate receptors were observed to investigate the relationship between glutamate receptors and epilepsy.Methods:The cultured primary cortical neurons from Wistar fetal rat were identified and grouped.The model group: 3m M of Pilocarpine treat the primary cortical neurons for24 h,preparation the primary cortical neurons epilepsy model.The Activin A treatment group: neurons were treated with different concentrations of Activin A(5ng/ml,10ng/ml,20ng/ml).The pilocarpine & Activin A group: treat with different concentrations of Activin A(5ng/ml,10ng/ml,20ng/ml)for 24 hours after 3m M pilocarpine treatment for 24 hours.The control group did not do any treatment.Morphological,whole-cell patch-clamp technique was used to detect neuronal changes,and the expression of glutamate receptor subunits were studied by RT-PCR,immunocytochemical staining and Western Blot.So as to explore the possibility mechanism of glutamate receptors in epileptic neurons and the protective effect of Acitivin A on neuronal injury.Results:1.Morphological results showed that Activin A increased the number and thelength of primary dendrites of primary cortical neurons cultured in vitro.2.3m M of Pilocarpine can induce primary cortical neuron injury,the number and the length of primary dendrites decreased,and showed epileptic discharge.Activin A can promote the number and the length growth of the dendrites after injury.3.The results of RT-PCR showed that,when treated with different concentrations of Activin A(5ng/ml,10ng/ml,20ng/ml),the expression of NR1,NR2 A,NR2B,Glu R1,Glu R2 and Glu R4 m RNA have little change compared with the control group;while the Glu R3 m RNA increased.The expression of NR1,NR2 A,NR2B,Glu R1,Glu R3 and Glu R4 subunits m RNA was significantly decreased after induced by pilocarpine for24 h,and the expression of Glu R2 m RNA did not change.When treated with different concentrations of Activin A(5ng/ml,10ng/ml,20ng/ml)after 3m M pilocarpine induced,the expression of NR2 A,NR2B,Glu R1,Glu R3 and Glu R4 subunits m RNA were significantly higher than those in the 3m M pilocarpine induced injury group at the concentration of Activin A 10ng/ml and 20ng/ml.4.Immunofluorescence staining showed that the expression of NR2 A,NR2B,Glu R1 and Glu R4 proteins were almost unchanged when treated with Activin A.While the proteins decreased after treated by 3m M of pilocarpine.The expression of NR2 A,NR2B,Glu R1 and Glu R4 were significantly increased when given the Activin A after injury induced by pilocarpine.5.The Western Blot results showed that: the expression of NR2 B,Glu R1 proteins have little change when treated the primary cortical neurons with Activin A,while they were decreased when treated by 3 m M pilocarpine.The expression of NR2 B and Glu R1 were significantly increased when given the Activin A after injury induced by pilocarpine.Conclusions:1.Pilocarpine can induce the damage of cultured primary cortical neurons,and establish the epilepsy model of primary cortical neurons in vitro.2.NR2 A,NR2B,Glu R1,Glu R4 subunit of Glu Rs involved in the injury process of primary cortical neuron induced by pilocarpine.3.Activin A play a protective role in pilocarpine-induced primary cortical neuroninjury through the NR2 A,NR2B,Glu R1,Glu R4 subunits of the Glu Rs.