The Study Of MRTF-A Alleviating Synaptic Plasticity Injury Mediated By Cerebral Ischemia-reperfusion

Objective:To investigate the molecular mechanism of the myocardin-related transcription fanctor-A alleviating synaptic plasticity damage in rats induced by cerebral ischemia-reperfusion,and to provide theoretical evidence for finding new clinical diagnosis,treatment and prevention targets for patients with cerebral infarction.Methods:Eigty-eight SD rats were divided into 4 groups by random number table method:sham group,cerebral ischemia/reperfusion model group,high expression MRTF-A group and suppression expression MRTF-A group.With the concentration of 1.6×10~6TU for each rat,the sham group was injected with lentivirus vector,the high expression group was injected with high expression MRTF-A lentivirus,following that the suppressed-expression group was injected with suppression of MRTF-A lentivirus expression.The rat was treated by a middle cerebral artery occlusion method that was cerebral ischemia 2h and reperfusion 24h at 7 days after lentivirus injection.1.The effect of MRTF-A on synaptic plasticity injury mediated by cerebral ischemia-reperfusion:(1)Longa’s 5-point neurological function score and 2,3,5-Triphenyltetrazolium chloride(TTC)staining were used to evaluate nerve injury;(2)Transmission electron microscopy was used to observe ultrastructural changes of synapses in the cerebral cortex;(3)Golgi staining was selected to observe the morphological changes of dendritic spines in cerebral cortex.Based on all above methods,we could determine the effect of MRTF-A on synaptic plasticity damage mediated by cerebral ischemia-reperfusion.2.The effect of MRTF-A on expression of synaptic plasticity-related proteins mediated by cerebral ischemia-reperfusion:At this part,western blot assay was used to observe expression levels of synapse-related proteins,such as Ca MKII,GERB,Glu R1,Synaspin1,PSD95,p38,and p-p38,in order to explore the molecular mechanism of MRTF-A alleviating the damage of synaptic plasticity.Results:1.MRTF-A could effectively rescue synaptic damage mediated by cerebral stroke:(1)By detecting degree of brain injury and cerebral infraction volume,we found that infraction volume of the ischemic side increased significantly,as well as severe neurological impairment in the model group(P<0.05).Compared with the model group,high expression MRTF-A significantly decreased neurological function and cerebral infraction volume(P<0.05),while the group of inhibition MRTF-A significantly increased neurological function and cerebral infraction volume(P<0.05).(2)By observing the structure of dendritic spines,it was found that compared with the sham group,some of the primary branches of neurons in the model group were broken,following with significantly decrease on the number of dendritic spines,and the length of dendritic spines was also abundantly shortened(P<0.05);Compared with the model group,the neuron structure of the high expression MRTF-A group was improved,the number and length of dendritic spines increased significantly(P<0.05);after MRTF-A was inhibited,the number and length of dendritic spines decreased(P<0.05).(3)Detection of myelin ultrastructure showed that myelin sheath structure of the model group was abnormal,including the loosening and damage of the myelin membrane,various pathological changes of the axon skeleton structure and cytoplasmic structure.Compared with the model group,the myelin sheath of the high expression MRTF-A group was basically normal,the myelin membrane was restored to a tight state,and the axon structure in the myelin was clear.while after inhibiting the expression of MRTF-A,the myelin membrane was abnormally loose and disordered,and the myelin shaft was broken(P<0.05).(4)By observing the ultrastructure of synapses,it was found that compared with the sham group,the pre-and post-synaptic membrane boundaries of the model group were blurred,the number of synapses was significantly reduced,the length and thickness of PSD were significantly shortened(P<0.05),and the surrounding structures were disordered.Compared with the model group,the number of synapses increased and the synaptic structure returned to normal,the pre-and post-synaptic membrane was clearly visible,and the length and thickness of PSD were significantly increased in the high expression MRTF-A group(P<0.05).However,inhibition of the expression of MRTF-A further aggravated synaptic damage(P<0.05).2.MRTF-A could alleviate synaptic plasticity injury induced by cerebral ischemia/reperfusion by regulating expression of synapse-associated proteins:In the model group,the expression levels of synapse-related proteins(Ca MKII,GERB,Glu R1,Synaspin1 and PSD95)were significantly decreased compared with the sham group(P<0.05);the phosphorylation level of p38 protein in model group was significantly increased(P<0.05).When MRTF-A was over-expressed among brain tissue,expression level of Ca MKII,GERB,Glu R1,Synaspin1 and PSD95 was significantly increased(P<0.05);expression level of p-p38/p38 was significantly decreased(P<0.05).When MRTF-A was inhibited,the opposite phenomenon appeared(P<0.05).Conclusions:1.During the ischemic stroke reperfusion,MERF-A can effectively inhibit injury by restoring infract volume,relieving synaptic structural abnormalities,while inhibiting the expression of MRTF-A aggravate synaptic damage.These results suggest that MRTF-A can effectively inhibite the synaptic damage mediated by ischemia stroke.2.Over-expression of MRTF-A in the ischemic area can significantly upregulate the expression of Ca MKII,GERB,Glu R1,Synaspin1,PSD95,and p-p38(P<0.05),while the expression of these proteins were significantly decreased when inhibition of MRTF-A(P<0.05).These findings indicated that MRTF-A inhibit cerebral ischemia-reperfusion mediated synaptic injury by regulating the expression of synapse-related proteins,and may be related to the p38 MAPK signaling pathway.